Hybrid expression of neisserial proteins

[0001] All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] This invention is in the field of protein expression. In particular, it relates to the heterologous expression of proteins from Neisseria (e.g. N. gonorrhoeae or, preferably, N. meningitidis).

BACKGROUND ART

[0003] International patent applications WO99/24578, WO99/36544, WO99/57280 and WO00/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhoeae. These proteins are typically described as being expressed in E. coli (i.e. heterologous expression) as either N-terminal GST-fusions or C-terminal His-tag fusions, although other expression systems, including expression in native Neisseria, are also disclosed.

[0004] It is an object of the present invention to provide alternative and improved approaches for the heterologous expression of these proteins. These approaches will typically affect the level of expression, the ease of purification, the cellular localisation of expression, and/or the immunological properties of the expressed protein.

DISCLOSURE OF THE INVENTION

[0005] In accordance with the invention, two or more (e.g. 3, 4, 5, 6 or more) proteins of the invention are expressed as a single hybrid protein. It is preferred that no non-Neisserial fusion partner (e.g. GST or poly-His) is used.

[0006] This offers two advantages. Firstly, a protein that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem. Secondly, commercial manufacture is simplified—only one expression and purification need be employed in order to produce two separately-useful proteins.

[0007] Thus the invention provides a method for the simultaneous heterologous expression of two or more proteins of the invention, in which said two or more proteins of the invention are fused (i.e. they are translated as a single polypeptide chain).

[0008] The method will typically involve the steps of: obtaining a first nucleic acid encoding a first protein of the invention; obtaining a second nucleic acid encoding a second protein of the invention; ligating the first and second nucleic acids. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.

[0009] Where just two proteins are joined, the hybrid protein can be represented simply by the formula NH2-A-B—COOH. A and B can each be selected from any Neisserial proteins, and in particular those represented by SEQ#s 1-4326. The method is well suited to the expression of proteins orf1, orf4, orf25, orf40, Orf46146.1, orf83, 233, 287, 292L, 564, 687, 741, 907, 919, 953, 961 and 983.

[0010] The 42 hybrids indicated by ‘X’ in the following table of form NH2-A-B—COOH are preferred:

1B→↓AORF46.1287741919953961983ORF46.1XXXXXX287XXXXXX741XXXXXX919XXXXXX953XXXXXX961XXXXXX983XXXXXX

[0011] Preferred proteins to be expressed as hybrids are thus ORF46.1, 287, 741, 919, 953, 961 and 983. These may be used in their essentially full-length form, or poly-glycine deletions (ΔG) forms may be used (e.g. ΔG-287, ΔGTbp2, ΔG741, ΔG983 etc.), or truncated forms may be used (e.g. Δ1-287, Δ2-287 etc.), or domain-deleted versions may be used (e.g. 287B, 287C, 287BC, ORF461-433, ORF46433-608, ORF46, 961c etc.) and so on.

[0012] Particularly preferred are: (a) a hybrid protein comprising 919 and 287; (b) a hybrid protein comprising 953 and 287; (c) a hybrid protein comprising 287 and ORF46.1; (d) a hybrid protein comprising ORF1 and ORF46.1; (e) a hybrid protein comprising 919 and ORF46.1; (f) a hybrid protein comprising ORF46.1 and 919; (g) a hybrid protein comprising ORF46.1, 287 and 919; (h) a hybrid protein comprising 919 and 519; and (i) a hybrid protein comprising ORF97 and 225.

[0013] Further embodiments are shown in the drawings and include ΔG287-919, ΔG287-953, ΔG287-961, ΔG983-ORF46.1, ΔG983-741, ΔG983-961, ΔG983-961C, ΔG741-961, ΔG741-961C, ΔG741-983, ΔG741-ORF46.1, ORF46.1-741, ORF46.1-961, ORF46.1-961C, 961-ORF46.1, 961-741, 961-983, 961C-ORF46.1, 961C-741, 961C-983, 961CL-ORF46.1, 961CL-741, and 961CL-983.

[0014] Where 287 is used, it is preferably at the C-terminal end of a hybrid; if it is to be used at the N-terminus, if is preferred to use a ΔG form of 287 is used (e.g. as the N-terminus of a hybrid with ORF46.1, 919, 953 or 961).

[0015] Where 287 is used, this is preferably from strain 2996 or from strain 394/98.

[0016] Where 961 is used, this is preferably at the N-terminus. Domain forms of 961 may be used.

[0017] Alignments of polymorphic forms of ORF46, 287, 919 and 953 are disclosed in WO00/66741. Any of these polymorphs can be used according to the present invention.

[0018] Preferably, the constituent proteins (A and B) in a hybrid protein according to the invention will be from the same strain.

[0019] The fused proteins in the hybrid may be joined directly, or may be joined via a linker peptide e.g. via a poly-glycine linker (i.e. Gn where n=3, 4, 5, 6, 7, 8, 9, 10 or more) or via a short peptide sequence which facilitates cloning. It is evidently preferred not to join a ΔG protein to the C-terminus of a poly-glycine linker.

[0020] The fused proteins may lack native leader peptides or may include the leader peptide sequence of the N-terminal fusion partner.

[0021] Host

[0022] It is preferred to utilise a heterologous host. The heterologous host may be prokaryotic or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmtonella typhi, Salmonenna typhimiurium, Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea, Mycobateria (e.g. Mtuberculosis), yeast etc.

[0023] Vectors, Hosts etc.

[0024] As well as the methods described above, the invention provides (a) nucleic acid and vectors useful in these methods (b) host cells containing said vectors (c) proteins expressed or expressable by the methods (d) compositions comprising these proteins, which may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions (e) these compositions for use as medicaments (e.g. as vaccines) or as diagnostic reagents (f) the use of these compositions in the manufacture of (1) a medicament for treating or preventing infection due to Neisserial bacteria (2) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria, and/or (3) a reagent which can raise antibodies against Neisserial bacteria and (g) a method of treating a patient, comprising administering to the patient a therapeutically effective amount of these compositions.

[0025] Sequences

[0026] The invention also provides a protein or a nucleic acid having any of the sequences set out in the following examples. It also provides proteins and nucleic acid having sequence identity to these. As described above, the degree of ‘sequence identity’ is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).

[0027] Nomenclature Herein

[0028] The 2166 protein sequences disclosed in WO99/24578, WO99/36544 and WO99/57280 are referred to herein by the following SEQ# numbers:

2ApplicationProtein sequencesSEQ# hereinWO99/24578Even SEQ IDs 2-892SEQ#s 1-446WO99/36544Even SEQ IDs 2-90SEQ#s 447-491WO99/57280Even SEQ IDs 2-3020SEQ#s 492-2001Even SEQ IDs 3040-3114SEQ#s 2002-2039SEQ IDs 3115-3241SEQ#s 2040-2166

[0029] In addition to this SEQ# numbering, the naming conventions used in WO99/24578, WO99/36544 and WO99/57280 are also used (e.g. ‘ORF4’, ‘ORF40’, ‘ORF40-1’ etc. as used in WO99/24578 and WO99/36544; ‘m919’, ‘g919’ and ‘a919’ etc. as used in WO99/57280).

[0030] The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [Science (2000) 287:1809-1815] are referred to herein as SEQ#s 2167-4326 [see also WO00/66791].

[0031] The term ‘protein of the invention’ as used herein refers to a protein comprising:

[0032] (a) one of sequences SEQ#s 1-4326; or

[0033] (b) a sequence having sequence identity to one of SEQ#s 1-4326; or

[0034] (c) a fragment of one of SEQ#s 1-4326.

[0035] The degree of ‘sequence identity’ referred to in (b) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants [e.g. see WO00/667,41]. Identity is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.

[0036] The ‘fragment’ referred to in (c) should comprise at least n consecutive amino acids from one of SEQ#s 1-4326 and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragment comprises an epitope from one of SEQ#s 1-4326. Preferred fragments are those disclosed in WO00/71574 and WO01/04316.

[0037] Preferred proteins of the invention are found in N. meningitidis serogroup B.

[0038] Preferred proteins for use according to the invention are those of serogroup B N. meningitidis strain 2996 or strain 394/98 (a New Zealand strain). Unless otherwise stated, proteins mentioned herein are from N. meningitidis strain 2996. It will be appreciated, however, that the invention is not in general limited by strain. References to a particular protein (e.g. ‘287’, ‘919’ etc.) may be taken to include that protein from any strain.

[0039] It will be appreciated that references to “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

BRIEF DESCRIPEON OF DRAWINGS

[0040] FIGS. 1 to 26 show hybrid proteins according to the invention.

MODES FOR CARRYING OUT THE INVENTION

EXAMPLE 1

Hybrids of ORF46

[0041] The complete ORF46 protein from N. meningitidis (serogroup B, strain 2996) has the following sequence:

31LGISRKISLI LSILAVCLPM HAHASDLAND SFIRQVLDRQ HFEPDGKYHL51FGSRGELAER SGHIGLGKIQ SHQLGNIMIQ QAAIKGNIGY IVRFSDHGHE101VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY151PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH NAGSMLTQGV201GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE IVGAGDAVQG251ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA AIRDWAVQNP301NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP351KGKSAVSDNF ADAAYAKYPS PYHSPNIRSN LEQRYGKENI TSSTVPPSNG401KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTKLDIQEL SGGGIPKAXP451VSDAKPRWEV DRKLNKLTTR EQVEENVQEI RNGNKNSNFS QHAQLEREIN501KLKSADEINF ADGMGKFTDS MNDKAFSELV KSVKENGFTN PVVEYVEING551KAYIVRGNNR VFAAEYLGRI HELKFKKVDF PVPNTSWKNP TDVLNESGNV601KRPRYRSK*

[0042] The leader peptide is underlined.

[0043] The sequences of ORF46 from other strains can be found in WO00/66741.

[0044] ORF46 has been fused at its C-terminus and N-terminus with 287, 919, and ORF1. The hybrid proteins were generally insoluble, but gave some good ELISA and bactericidal results (against the homologous 2996 strain):

4ProteinELISABactericidal AbOrf1-Orf46.1-His850256919-Orf46.1-His12900512919-287-Orf46-Hisn.d.n.d.Orf46.1-287His1508192Orf46.1-919His28002048Orf46.1-287-919His320016384

[0045] For comparison, ‘triple’ hybrids of ORF46.1, 287 (either as a GST fusion, or in ΔG287 form) and 919 were constructed and tested against various strains (including the homologous 2996 strain) versus a simple mixture of the three antigens. FCA was used as adjuvant:

52996BZ232MC58NGH38F6124BZ133Mixture8192256 5121024>2048 >2048ORF46.1-287-16384 256409681928192 8192919hisΔG287-919-8192 6440968192819216384ORF46.1hisΔG287-4096128 2568192 512 1024ORF46.1-919his

[0046] Again, the hybrids show equivalent or superior immunological activity.

[0047] Hybrids of two proteins (strain 2996) were compared to the individual proteins against various heterologous strains:

61000MC58F6124 (MenA)ORF46.1-His<44096<4ORF1-His8256128ORF1 - ORF46.1-His10245121024

[0048] Again, the hybrid shows equivalent or superior immunological activity.

EXAMPLE 2

Hybrids of ΔG287

[0049] The deletion of the (Gly)6 sequence in 287 was found to have a dramatic effect on protein expression. The protein lacking the N-terminal amino acids up to GGGGGG is called ‘ΔG287’. In strain MC58, its basic sequence (leader peptide underlined) is:

7 SPDVKS ADTLSKPAAP VVSEKETEAK EDAPQAGSQGQGAPSAQGSQ DMAAVSEENT GNGGAVTADN PKNEDEVAQNDMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQPANQPDMANAA DGMQGDDPSA GGQNAGNTAA QGANQAGNNQAAGSSDPIPA SNPAPANGGS NFGRVDLANG VLIDGPSQNITLTHCKGDSC SGNNFLDEEV QLKSEFEKLS DADKISNYKKDGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSPARFRRSARSRRSLPAE MPLIPVNQAD TLIVDGEAVS LTGHSGNIFAPEGNYRYLTY GAEKLPGGSY ALRVQGEPAK GEMLAGAAVYNGEVLHFHTE NGRPYPTRGR FAAKVDFGSK SVDGIIDSGDDLHMGTQKFK AAIDGNGFKG TWTENGSGDV SGKFYGPAGEEVAGKYSYRP TDAEKGGFGV FAGKKEQD*

[0050] ΔG287, with or without His-tag (‘ΔG287-His’ and ‘ΔG287K’, respectively), are expressed at very good levels in comparison with the ‘287-His’ or ‘287untagged’.

[0051] On the basis of gene variability data, variants of ΔG287-His were expressed in E. coli from a number of MenB strains, in particular from strains 2996, MC58, 1000, and BZ232. The results were also good—each of these gave high ELISA titres and also serum bactericidal titres of >8192. ΔG287K, expressed from pET-24b, gave excellent titres in ELISA and the serum bactericidal assay.

[0052] Deletion of poly-Gly sequences is also applicable to Thp2 (NMB0460), 741 (NMB 1870) and 983 (NMB1969). When cloned in pET vector and expressed in E. coli without the sequence coding for their leader peptides and without poly-Gly (i.e. as “ΔG forms”), the same effect was seen—expression was good in the clones carrying the deletion of the poly-glycine stretch, and poor or absent if the glycines were present in the expressed protein.

[0053] ΔG287 was fused directly in-frame upstream of 919, 953, 961 (sequences shown below) and ORF46.1:

8ΔG287-919ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAATGATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCATCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTACCCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGCATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTACCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTCGCCAAAAGCCTGCAATCCTTCCGCCTCGGCTGCGCCAATTTGAAAAACCGCCAAGGCTGGCAGGATGTGTGCGCCCAAGCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAGTTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGCAACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCCCGCTTCCCGATTTACGGTATTCCCGACGATTTTATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAQCCCTTGTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATCGACAATACCGGCGGCACACATACCGCCGACCTCTCCCGATTCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGGTTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATACTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGCTCGGGCCGTCTGAAAACCCCGTCCGGCAAATACATCCGCATCGGCTATGCCGACAAAAACGAACATCCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGCACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACCGCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGCGAACTTGCCGGCAAACAGAAAACCACGGGTTACGTCTGGCAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCGTAACTCGAG1MASPDVKSAD TLSKPAAPVV AEKETEVKBD APQAGSQGQG APSTQGSQDM51AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS101DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL151LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI201IYKDKSASSS SARFRRSAPS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG251HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE301VLHFRTENGR PYPTRGFPAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI351DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG401KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA451VYTVVPHLSL PHWAAQDRAK SLQSFRLGCA NLKNRQGWQD VCAQAFQTPV501HSFQAKQFFE RYFTPWQVAG NGSLAGTVTG YYEPVLKGDD RRTAQARPPI551YGIPDDFISV PLPAGLRSGK ALVRIRQTGK NSGTIDNTGG THTADLSRFP601ITARTTAIKG RFEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM651HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYHADKGY LKLGQTSMQG701IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG751AVDRHYITLG APLFVATMIP VTEXALNRLI MAQDTGSAIK GAVRVDYFWG801YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP*AG287-953ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAATGATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCATCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTACCCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATGATCGACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAATATCACGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGTAAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCGGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGACATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCCGTCAAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGCCCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGCACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTCGTTAACGTTGGTATGACCAAAAGCGTCCGCATCGACATCCAAATCGAGGCAGCCAAACAATAACTCGAG1MASPDVKSAD TLSKPAAPVV AEKETBVKED APQAGSQGQG APSTQGSQDM51AAVSAENTGN GGAATTDKPK NEDEGPQNDM PWNSAESANQ TGNNQPADSS101DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL151LDEEAPSKSE FENLNESERI KKYKKDGKSD KFTNLVATAV QANGTNKYVI201IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG251HSGNIFAPEG NYRYLTYGAB KLPGGSYALR VQGEPAKGEM LAGTAVYNGE301VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI351DGNGFXGTWT ENGGGDVSGR FYGPAGEBVA GKYSYRPTDA EKGGFGVFAG401KKEQDGSGGG GATYKVDEYH ANARFAIDHF NTSTNVGGFY GLTGSVEFDQ451AXRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTKFNF501NGKXLVSVDG NLTHHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID551RTKWGVDYLV NVGMTKSVRI DIQIEAAKQ*AG287-961ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAATGATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCATCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTACCCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAACTCGAG1HASPDVKSAD TLSKPAAPVV AEXETEVKED APQAGSQGQG APSTQGSQDM51AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSABSANQ TGNNQPADSS101DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL151LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI201IYKDKSASSS SARFRRSARS RRSLPAENPL IPVNQADTLI VDGEAVSLTG251HSGNIFAPEG NYRYLTYGAB KLPGGSYALR VQGEPAKGEM LAGTAVYNGE301VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI351DGNGFKGTWT ENGGGDVSGR PYGPAGEEVA GKYSYRPTDA EKGGFGVFAG401KKEQDGSGGG GATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE451DGTITKKDAT AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE501SEIEKLTTKL ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV551KIDEKLEAVA DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE601TKQNVDAKVK AAETAAGKAE AAAGTANTAA DKABAVAAKV TDIKADIATN651KDNIAKKANS ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH701DTRLNGLDKT VSDLRXETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK751SESAVAIGTG FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEW*

[0054]

9

ELISA
Bactericidal

ΔG287-953-His
3834
65536

ΔG287-961-His
108627
65536

[0055] The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens (using 287-GST) for 919 and ORF46.1:

10Mixture with 287Hybrid with ΔG28791932000128000ORF46.112816000

[0056] Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained:

11919ORF46.1StrainMixtureHybridMixtureHybridNGH38102432000—16384MC585128192—512BZ232512512——MenA (F6124)51232000—8192MenC (C11)>2048>2048——MenC (BZ133)>409664000—8192

[0057] The hybrid proteins with ΔG287 at the N-terminus are therefore immunologically superior to simple mixtures, with ΔG287-ORF46.1 being particularly effective, even against heterologous strains. ΔG287-ORF46.1K may be expressed in pET-24b.

[0058] The same hybrid proteins were made using New Zealand strain 394/98 rather than 2996:

12AG2B7NZ-919ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGGCGGTCGCTTCCGGCCGAGkTGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAkGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAGGAGCAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGCATGCAAACCTTTCCGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTACCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTCGCCAAAAGCCTGCAATCCTTCCGCCTCGGCTGCGCCAATTTGAAAAACCGCCAAGGCTGGCAGGATGTGTGCGCCCAAGCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAGTTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGCAACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCCCGCTTCCCGATTTACGGTATTCCCGACGATTTTATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTTGTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATCGACAATACCGGCGGCACACATACCGCCGACCTCTCCCGATTCCCCATCACCGCGCGCACAACGGCAATCAAAGGGAGGTTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATACTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGCTCGGGCCGTCTGAAAACCCCGTCCGGCAAATACATCCGCATCGGCTATGCCGACAAAAACGAACATCCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGCACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACCGCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGCGAACTTGCCGGCAAACAGAAAACCACGGGTTACGTCTGGCAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCGTAAAAGCTT1HASPDVKSAD TLSKPAAPVV SEKETEAXED APQAGSQGQG APSAQGGQDM51AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM101PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG151TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL201THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV251GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AENPLIPVNQ301ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP351SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS401GDGLHHGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY451RPTDAEKGGF GVFAGKKEQD GSGGGGCQSK SIQTFPQPDT SVINGPDRPV501GIPDPAGTTV GGGGAVYTVV PHLSLPHWAA QDFAKSLQSF RLGCANLKNR553QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV601LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNGSTI651DNTGGTHTAD LSRFPITART TAIKGRFEGS RPLPYHTRNQ INGGALDGKA701PILGYAEDPV ELFFMHIQGS GRLKTPSGKY IRIGYADKNB HPYVSIGRYM751ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG801PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAHPVTEKA LNRLIMAQDT851GSAIKGAVRV DYFWGYGDEA GELAGKQKTT GYVWQLLPNG MKPEYRP*ΔG287NZ-953ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAACAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAGACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAATATCACGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGTAAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCGGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGACATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCCGTCAAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGCCCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGCACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTCGTTAACGTTGGTATGACCAAAAGCGTCCGCATCGACATCCAAATCGAGGCAGCCAAACAATAAAAGCTT1NASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM51AAVSEBNTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM101PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG151TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL201THCKGDSCSG NNFLDEEVQL KSEPEKLSDA DKISNYKKDG KNDGKNDKFV251GLVADSVQMK GINQYIIFYK PKPTSFAPFR RSARSRRSLP AEMPLIPVNQ301ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP351SKGEMLAGTA VYNGEVUIFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS401GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY451RPTDAEKGGF GVFAGKKEQD GSGGGGATYK VDEYHANARF AIDHFNTSTN501VGGFYGLTGS VEFDQAKRDG KIDITIPVAN LQSGSQHFTD HLKSADIFDA551AQYPDIRFVS TKFNFNGKKL VSVDGNLTMH GKTAPVKLKA EKFNCYQSPM601AKTEVCGGDF STTIDRTKWG VDYLVNVGMT KSVRIDIQIE AAKQ*ΔG287NZ-961ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAAAAGCTT1MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM51AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM101PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG151TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL201THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV251GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ301ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP351SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS401GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY451RPTDAEKGGF GVFAGKKEQD GSGGGGATND DDVKKAATVA IAAAYNNGQE501INGFKAGETI YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV551NENKQNVDAK VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG601ENITTFAEET KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD651EAVKTANEAK QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA701VAAKVTDIKA DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK751LDTRLASAEK SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY801NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY851HVGVNYEW*

EXAMPLE 3

Hybrids of ΔG983

[0059] Protein 983 has the following sequence:

13983

ΔG983

1MRTTPTFPTK TFKPTAMALA VATTLSACLG GGGGGTSAPD FNAGGTGIGS51NSRATTAKSA AVSYAGIKNE HCKDRSNLCA GRDDVAVTDR DAKINAPPPN101LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE151LYGRKERGYN ENYKNYTAYN RKEAPEDGGG KDIEASFDDE AVIETEAKPT201DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM251MVAAIRNAWV KLGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL301DYSGGDKTDE GIRIMQQSDY GNLSYHIRNK NNLFIFSTGN DAQAQPNTYA351LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA401MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN451LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK ANNGPASFPF GDFTADTKGT501SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN551KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ601LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS651AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY YVRRGNAART701ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT751DMPGIRPYGA TFRAAAAVQR ANAADGVRIF NSLAATVYAD STAAHADHQG801RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKNR GSTQTVGIAA851KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL901FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE951GGLRYDLLKQ DAPAEKGSAL GWSGNSLTBG TLVGLAGLKL SQPLSDKAVL1001FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE1051FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*

[0060] ΔG983 thus has the following basic sequence:

14 TSAPDFNAGGTGIGS NSRATTAKSA AVSYAGIKNE MCKDRSMLCAGRDDVAVTDR DAKINAPPPN LHTGDFPNPN DAYKNLINLKPAIEAGYTGR GVEVGIVDTG ESVGSISFPE LYGRKEHGYNENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPTDIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIMNTNDETKNEM MVAAIRNAWV KLGERGVRIV NNSFGTTSRAGTADLFQIAN SEEQYRQALL DYSGGDKTDE GIRLMQQSDYGNLSYHIRNK NMLFIFSTGN DAQAQPNTYA LLPFYEKDAQKGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITAMWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLLQKYPWMSNDN LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGKAMNGPASFPF GDFTADTKGT SDIAYSFRND ISGTGGLIKKGGSQLQLHGN NTYTGKTIIE GGSLVLYGNN KSDMRVETKGALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQLDGKGTLYTR IGKLLKVDGT AIIGGKLYHS ARGKGAGYLNSTGRRVPFLS AAKIGQDYSF FTNIETDGGL LASLDSVEKTAGSEGDTLSY YVRRGNAART ASAAAHSAPA GLKHAVEQGGSNLENLMVEL DASESSATPE TVETAAADRT DMPGIRPYGATFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQGRRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMRGSTQTVGIAA KTGENTTAAA TLGMGRSTWS ENSANAKTDSISLFAGIRHD AGDIGYLKGL FSYGRYKNSI SRSTGADEHAEGSVNGTLMQ LGALGGVNVP FAATGDLTVE GGLRYDLLKQAFABKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVLFATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTRLVAGLGADVE FGNGWNGLAR YSYAGSKQYG NHSGRVGVGYRF*

[0061] ΔG983 was expressed as a hybrid, with ORF46.1, 741, 961 or 961c at its C-terminus:

15AG983-ORF46.1ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGAQGGAGGCGGTAAAGAATTGAAGCTTCTTTCGACGATGAAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGOGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAkGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGACGGTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCPTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV101GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE151ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD201ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD251LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF301IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP351GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451PASFPFGDFT WMTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT501GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT551DQSGANETVH IKGSLQLDGR GTLYTRLGKL LKVDGTAIIG GKLYMSARGK601GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE651GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE701SSATPETVET AAADRTDNPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA751ATVYADSTAA HADMQGRBLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG801VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF851AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL901GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR1001NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD1051GGGGTGSSDL ANDSFIRQVL DRQHFEPDGK YHLFGSRGEL AERSGHIGLG1101KIQSHQLGNL MIQQAAIKGN IGYIVRFSDH GHBVHSPFDN HASHSDSDEA1151GSPVDGFSLY RIHWDGYEHH PADGYDGPQG GGYPAPKGAR DIYSYDIKGV1201AQNIRLNLTD NRSTGQRLAD RFHNAGSMLT QGVGDGFKRA TRYSPELDRS1251GNAAEAFNGT ADIVKNIIGA AGEIVGAGDA VQGISEGSNI AVMHGLGLLS1301TENKMARIND LADMAQLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI1351PIKGIGAVRG KYGLGGITAH PIKRSQMGAI ALPKGKSAVS DNFADAAYAK1401YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGKNVKLAD QRHPKTGVPF1451DGKGFPNFBK HVKYDTLEHH HHHH*ΔG983-741ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV101GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE151ASFDDEAVIE TEAKPTDIRH VKBIGHIDLV SHIIGGRSVD GRPAGGIAPD201ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD251LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF301IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKRBMYGEP351GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT501GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT551DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK601GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE651GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVBQGGSNLE NLMVELDASE701SSATPETVET AAADRTDHPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA751ATVYADSTAA HADMQGRBLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG801VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF851AGIPHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLNQLGAL901GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR1001NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE1051GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ1101GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ1151SHSALTAPQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT1201YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD1251GK~HAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL1301AAKQLEHHHH HH*ΔG983 -961ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCCATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGGGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA1MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVBV101GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMBKEA PEDGGGKDIE151ASFDDEAVIE TEAKPTDIRH VKEIGHIDDV SHIIGGRSVD GRPAGGIAPD201ATLHIMNTND ETKNBNMVAA IRNAWVKLGE RGVRIVNNSF GTTSRSGTAD251LFQIANSEEQ YRQALLDYSG GDKTDEGIHL MQQSDYGNLS YHIRNKIMLP301IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP351GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401TAALLLQKYP WHSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT501GKTIIEGGSL VLYGNNKSDH RVETKGALIY NGAASGGSLN SDGIVYLADT551DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK601GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE651GDTLSYYVRR GNAARTASAA AHSAPAGLKE AVBQGGSNLE NLMVELDASE701SSATPETVET AAADRTDHPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA751ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWBQGG801VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF851AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL901GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAkTGKTGAR1001NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE1051GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI1101TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE1151KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFABE TKTNIVKIDE1201KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN1251VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAMCVTDIK ADIATNKDNI1301AKKANSADVY TREBSDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTEL1351NGLDKTVSDL RKETRQGLAE QAALSGLPQP YNVGRFNVTA AVGGYKSESA1401VAIGTGFRPT ENFAAKAGVA VGTSSGSSAA YHVGVNYEWL EHHHHHH*AG983 -961cATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAkTGACGCATACIAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGGTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTQAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGkCGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAkTTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA1MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIB AGYTGRGVEV101GIVDTGESVG SISFPELYGR KEEGYNENYK NYTAYMRKEA PEDGGGKDIE151ASFDDEAVIE TEAKPTDIBH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD201ATLHINNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD251LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF301IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP351GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT501GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT551DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYHSARGK601GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETPGGLLASL DSVEKTAGSE651GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE701SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAP DGVRIFNSLA751ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG801VEGKMRGSTQ TVGIAAKTGE NTTAAATLGN GRSTWSENSA NAKTDSISLF851AGIEHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLNQLGAL901GGVNVPFAkT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951LAGLKLSQPL SDKAVLPATA GVERDLNGRD YTVTGGFTGA TAATGKTGAP1001NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE1051GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI1101TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE1151KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE1201KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN1251VDAKVKAAET AAGKABAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI1301AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL1351NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGLEHHHH HH*

EXAMPLE 4

Hybrids of ΔG741

[0062] Protein 741 has the following sequence:

161VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL51QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRPDFIRQ101IEVDGQLITL ESGBFQVYKQ SHSALTAFQT EQIQDSBHSG KMVAKRQFRI151GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI201EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA251QEVAGSAEVK TVNGIRHIGL AAKQ*

[0063] ΔG741 thus has the following basic sequence:

17 VAAD IGAGLADALTAPLDHKDKGL QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGDSLNTGKLKND KVSRPDFIRQ IEVDGQLITL ESGEFQVYKQSHSMJTAPQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSFDKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKIEHLKSPBLNV DLAAADIKPD GKRHAVISGS VLYNQAEKGSYSLGIFGGKA QEVAGSAEVK TVNGIRHIGL AAKQ*

[0064] ΔG741 was fused directly in-frame upstream of proteins 961, 961c, 983 and ORF46.1:

18ΔG741-961ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGGCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGkGCACCACCACCACCACCACTGA1MVAJWIGAGL ADALTAPLDH KDKGLQSLTL DQSVRRNBKL KLAAQGAEKT51YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL101TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA151PGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA201VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL251EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT301ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI351EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID401EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ451NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN501IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR551LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGRFDVT AAVGGYKSES601AVAIGTGFRF TENFAAKAGV AVGTSSGSSA AYHVGVNYEW LEHHHHHH*ΔG741-961cATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAkCGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAkTCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGAGAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA1MVAADIGAGL ADALTAPLDH KDKGLGSLTL DQSVRKNEKL KLAAGGAEKT51YGNGDSLNTG KLKNDKVSRF DFIRGIEVDG GLITLESGEF GVYKGSHSAL101TAFGTEGIGD SEHSGKMVAK RGFRIGDIAG EETSFDKLPE GGRATYRGTA151FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA201VISGSVLYNG AEKGSYSLGI FGGKAGEVAG SAEVKTVNGI RHIGLAAKGL251EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG GEINGFKAGE TIYDIDEDGT301ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKGNVD AKVKAAESEI351EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID401EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKGTAEETKG451NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN501IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR551LNGLDKTVSD LRKETRGGLA EGAALSGLFG PYNVGLEHHH HHH*ΔG741-983ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGGAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAkGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGkTGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA1MVAADIGAGL ADALTAPLDH KDKGLGSLTL DGSVRKNEKL KLAAGGAEKT51YGNGDSLNTG KLKNDKVSRF DFIRGIEVDG GLITLESGEF GVYKGSHSAL101TAFGTEGIGD SBHSGKNVAK RGFRIGDIAG EHTSFDKLPE GGRATYRGTA151FGSDDAGGKL TYTIDFAAKG GNGKIEHLKS PELNVDLAAA DIKPDGKRHA201VISGSVLYNG AEKGSYSLGI FGGKAGEVAG SABVKTVNGI RHIGLAAKGL251EGSGGGGTSA PDFNAGGTGI GSNSRATTAR SAAVSYAGIK NEHCKDRSML301CAGRDDVAVT DRDAKINAPP PNLHTGDFPN PNDAYKNLIN LKPAIEAGYT351GRGVEVGIVD TGESVGSVGF PELYGRKEHG YNENYKNYTA YMRKEAPEDG401GGKDIEASFD DEAVIETEAK PTDIRHVKEI GHIDLVSHII GGRSVDGRPA451GGIAPDATLH IMNTNDETKN EMMVAAIRNA WVKLGERGVR IVNNSFGTTS501RAGTADLFGI LNSEEGYRGA LLDYSGGDKT DEGIRLMQQS DYGNLSYHIR551NKNMLFIFST GNDAGAGPNT YALLPFYEKD AGKGIITVAG VDRSGEKFKR601EMYGEPGTEP LEYGSNHCGI TAMWCLSAPY EASVRFTRTN PIGIAGTSFS651APIVTGTAAL LLGKYPWMSN DNLRTTLLTT AGDIGAVGVD SKFGWGLLDA701GKAHNGPASF PFGDFTADTK GTSDIAYSFR NDISGTGGLI KKGGSGLGLH751GNNTYTGKTI IEGGSLVLYG NNKSDMRVET KGALIYNGAA SGGSLNSDGI801VYLADTDGSG ANETVHIKGS LGLDGKGTLY TELGKLLKVD GTAIIGGKLY851MSARGKGAGY LNSTGRRVPF LSAAKIGGDY SFFTNIETDG GLLASLDSVE901KTAGSEGDTL SYYVRRGNAA RTASAAAHSA PAGLKHAVEG GGSNLENLMV951ELDASESSAT PETVETAAAD RTDMPGIRPY GATFRAAAAV GHANAADGVR1001IFNSLAATVY ADSTAAHADN GGRPLKAVSD GLDHNGTGLR VIAGTGGDGG1051TWEGGGVBGK MRGSTGTVGI AAKTGENTTA AATLGMGRST WSENSANAKT1101DSISLFAGIR HDAGDIGYLK GLFSYGRYKN SISRSTGADE HAEGSVNGTL1151MGLGALGGVN VPFAATGDLT VEGGLRYDLL KGDAFAEKGS ALGWSGNSLT1201EGTLVGLAGL KLSGPLSDKA VLFATAGVER DLNGRDYTVT GGFTGATAAT1251GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSKG YGNHSGRVGV1301GYRFLEHHHH HH*ΔG741-ORF46.1ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGACGGTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATAGCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCGGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTGAAACATTGCTGTGATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGGACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGGGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1MVAADIGAGL ADALTAPLDH KDKGLGSLTL DGSVRKNEKL KLAAGGAEKT51YGNGDSLNTG KLKNDKVSRF DFIRGIEVDG GLITLESGEF GVYKGSHSAL101DAFGTEGIGD SEHSGKMVAK RGFRIGDIAG EHTSFDKLPE GGRATYRGTA151FGSDDAGGKL TYTIDFAAKG GNGRIEHLKS PELNVDLAAA DIKPDGKRHA201VISGSVLYNG AEKGSYSLGI FGGKAGEVAG SAEVKTVNGI RHIGLAAKGL251DGGGGTGSSD LANDSFIRGV LDRGHFBPDG KYHLFGSRGE LAERSGHIGL301GKIGSHGLGN LMIGGAAIKG NIGYIVRFSD HGHEVHSPFD NHASHSDSDE351AGSPVDGPSL YRIHWDGYEH HPADGYDGPG GGGYPAPKGA RPIYSYDIKG401VAGNIRLNLT DNRSTGQRLA DRFHNAGSML TGGVGDGFKR ATRYSPELDR451SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN IAVHHGLGLL501STENKMARIN DLADMAGLKD YAAAAIRDWA VGNPNAAGGI EAVSNIFNAA551IPIKGIGAVR GKYGLGGITA HPIKRSGMGA IALPKGKSAV SDNFADAAYA601KYPSPYHSRN IRSNLEGRYG KENITSSTVP PSNGKNVKLA DGRHPKTGVP651FDGKGFPNFE KHVKYDTLRH HHHHH*

EXAMPLE 5

Hybrids of 287

[0065] Expression of 287 as full-length with a C-terminal His-tag, or without its leader peptide but with a C-terminal His-tag, gives fairly low expression levels. Better expression is achieved using a N-terminal GST-fusion. As an alternative to using GST as an N-terminal fusion partner, 287 was placed at the C-terminus of protein 919 ('919-287′), of protein 953 (‘953-287’), and of proteins ORF46.1 (‘ORF46.1-287’). In both cases, the leader peptides were deleted, and the hybrids were direct in-frame fusions.

[0066] To generate the 953-287 hybrid, the leader peptides of the two proteins were omitted by designing the forward primer downstream from the leader of each sequence; the stop codon sequence was omitted in the 953 reverse primer but included in the 287 reverse primer. For the 953 gene, the 5′ and the 3′ primers used for amplification included a NdeI and a BamHI restriction sites respectively, whereas for the amplification of the 287 gene the 5′ and the 3′ primers included a BamHI and a XhoI restriction sites respectively. In this way a sequential directional cloning of the two genes in pET21b+, using NdeI-BamHI (to clone the first gene) and subsequently BamHI-XhoI (to clone the second gene) could be achieved.

[0067] The 919-287 hybrid was obtained by cloning the sequence coding for the mature portion of 287 into the XhoI site at the 3′-end of the 919-His clone in pET21b+. The primers used for amplification of the 287 gene were designed for introducing a SalI restriction site at the 5′- and a XhoI site at the 3′- of the PCR fragment. Since the cohesive ends produced by the SalI and XhoI restriction enzymes are compatible, the 287 PCR product digested with SalI-XhoI could be inserted in the pET21b-919 clone cleaved with XhoI.

[0068] The ORF46.1-287 hybrid was obtained similarly.

[0069] The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens:

19Mixture with 287Hybrid with 287919320001600095381928192ORF46.11288192

[0070] Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained for 919-287 and 953-287:

20919953ORF46.1StrainMixtureHybridMixtureHybridMixtureHybridMC58 5121024 5121024 —1024NGH381024204820484096 —4096BZ232 512 1281024 16——MenA (F6124) 51220482048 32—1024MenC (C11)>2048 n.d.>2048 n.d.—n.d.MenC (BZ133)>4096 >8192 >4096 <16—2048

[0071] Hybrids of ORF46.1 and 919 were also constructed. Best results (four-fold higher titre) were achieved with 919 at the N-terminus.

[0072] Hybrids 919-519His, ORF97-225His and 225-ORF97His were also tested. These gave moderate ELISA fitres and bactericidal antibody responses.

[0073] As hybrids of two proteins A & B may be either NH2-A-B—COOH or NH2-B-A—COOH, the “reverse” hybrids with 287 at the N-terminus were also made, but using ΔG287. A panel of strains was used, including homologous strain 2996. FCA was used as adjuvant:

21287 & 919287 & 953287 & ORF46.1StrainΔG287-919919-287ΔG287-953953-287ΔG287-46.146.1-287299612800016000655368192163848192BZ232256128128<4<4<410002048<4<4<4<4<4MC5881921024163841024512128NGH38320002048>20484096163844096394/9840963225612812816MenA (F6124)320002048>20483281921024MenC (BZ133)64000>8192>8192<1681922048

[0074] Better bactericidal titres are generally seen with 287 at the N-terminus.

[0075] When fused to protein 961 [NH2-ΔG287-961-COOH—sequence shown above], the resulting protein is insoluble and must be denatured and renatured for purification. Following renaturation, around 50% of the protein was found to remain insoluble. The soluble and insoluble proteins were compared, and much better bactericidal titres were obtained with the soluble protein (FCA as adjuvant):

222996BZ232MC58NGH38F6124BZ133Soluble655361284096>2048>20484096Insoluble 8192 <4 <4 16n.d.n.d.

[0076] Titres with the insoluble form were, however, improved by using alum adjuvant instead:

23Insoluble327681284096>2048>20482048

[0077] 961c was also used in hybrid proteins (see above). As 961 and its domain variants direct efficient expression, they are ideally suited as the N-terminal portion of a hybrid protein.

EXAMPLE 23

Further Hybrids

[0078] Further hybrid proteins of the invention are shown in the drawings and have the sequences set out below. These are advantageous when compared to the individual proteins:

24ORF46.1-741.ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1HSDLANDSFI RGVLDRWHFE PDGKYHLFGS RGELAERSGH IGLGKIGSHG51LGNLMIGGAA IKGNIGYIVR FSDHGHBVHS PFDNHASHSD SDEAGSPVDG101FSLYRIHWDG YEHHPADGYD GPGGGGYPAP KGARDIYSYD IKGVAGNIRL151NLTDNRSTGG RLADRFHNAG SMLTGGVGDG FKRATRYSPE LDRSGNAAFA201FNGTADIVKN IIGAAGEIVG AGDAVGGISE GSNIAVNHGL GLLSTENKMA251RINDLADMAG LKDYAAAAIR DWAVGNPNAA GGIRAVSNIF MAAIPIKGIG301AVRGKYGLGG ITAHPIKRSG MGAIALPKGK SAVSDNFADA AYAKYPSPYH351SBNIRSNLEG RYGKENITSS TVPPSNGKNV KLADGRHPKT GVPFDGKGFP401NFBKHVKYDT GSGGGGVAAD IGAGLADALT APLDHKDKGL GSLTLDGSVR451KNEKLKLAAG GAEKTYGNGD SLNTGKLKND KVSRPDFIRG IEVDGGLITL501ESGEFGVYKG SHSALTAPGT EGIGDSEHSG KMVAKRGFRI GDIAGEHTSF551DKLPEGGRAT YRGTAPGSDD AGGKLTYTID FAAKGGNGKI EHLKSPELNV601DLAAADIKPD GKRHAVISGS VLYNGAEKGS YSLGIFGGKA GEVAGSAEVE651TVNGIRHIGL AAKGLBHHHH HH*IRF46.1-961ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCAGTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA1MSDLANDSFI RGVLDRGHFE PDGKYHLFGS RGELABRSGH IGLGKIGSHG51LGNLHIGGAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG101FSLYRIHWDG YEHHPADGYD GPGGGGYPAP KGARDIYSYD IKGVAGNIRL151NLTDNRSTGG RLADRFHNAG SMLTGGVGDG FKRATRYSPB LDRSGNAAEA201FNGTADIVKN IIGAAGEIVG AGDAVGGISE GSNIAVMHGL GLLSTENKMA251RINDLADNAG LKDYAAAAIR DWAVGNPNAA GGIEAVSNIF MAAIPIKGIG301AVRGKYGLGG ITAHPIKRSG MGAIMaPKGK SAVSDNFADA AYAKYPSPYH351SRNIRSNLBG RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP401NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGGE INGFKAGETI451YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKGNVDAK501VKAABSEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET551KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK601GTAEETKGNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA651DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK701SIADEDTRLN GLDKTVSDLR KETRGGLAEG AALSGLFGPY NVGRFNVTAA751VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY HVGVNYEWLE801HHHHHH*ORF46.1-961cATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGGCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA1MSDLANDSFI RGVLDRGHPE PDGKYHLFGS RGELAERSGH IGLGKIGSHG51LGNLMIGGAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG101FSLYRIHWDG YEHHPADGYD GPGGGGYPAP KGARDIYSYD IKGVAGNIRL151NLTDNRSTGG RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA203FNGTADIVKN IIGAAGEIVG AGDAVGGISE GSNIAVHHGL GLLSTENKMA251RINDLADMAG LKDYAAAAIR DWAVGNPNAA GGIEAVSNIF MAAIPIKGIG301AVRGKYGLGG ITAHPIKRSG NGAIALPKGK SAVSDNFADA AYAKYPSPYH351SRNIRSNLBG RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP401NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGGB INGFKAGETI451YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKGNVDAK501VKAABSEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET551KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK601GTAEETKQNV DAKVKAAETA AGKABAAAGT ANTAADKAEA VAAKVTDIKA651DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK701SIADHDTELN GLDKTVSDLR KETRGGLAEG AALSGLFGPY NVGLEHHHHH751H*961-ORF46.1ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACAGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTGGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGGCTCACATTCCGATTCTGATGAAGCCGGTAGTCCGGTTGACGGATTTAGCGTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGGATAAGCGAAGGGTCAAACATTGCTGTCATGCACGGCTTGGGTGTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGGAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCGGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGGCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1HATNDDDVKK AATVAIAAAY NNGGEINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKG NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151DTVDKHAEAP NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301VSDLRKETRG GLAEGAALSG LFGPYNVGRF NVTAAVGGYK SESAVAIGTG351FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGS DLANDSFIRG401VLDRGHFBPD GKYHLFGSRG ELAERSGHIG LGKIGSHGLG NLMIGGAAIK451GNIGYIVRFS DHGHEVHSPF DNHASHSDSD EAGSPVDGFS LYRIHWDGYE501HEPADGYDGP GGGGYPAPKG ARDIYSYDIK GVAGNIBLNL TDNRSTGGRL551ADRFHNAGSM LTGGVGDGFK EATRYSPELD RSGNAAEAFN GTADIVKNII601GAAGEIVGAG DAVGGISEGS NIAVMHGLGL LSTENKHARI NDLADMAGLK651DYAAAAIRDW AVGNPNAAGG IEAVSNIFNA AIPIKGIGAV RGKYGLGGIT701AHPIKRSGMG AIALPKGKSA VSDNFADAAY AKYPSPYHSR NIRSNLEGRY751GKENITSSTV PPSNGKNVKL ADGRHPKTGV PFDGKGFPNF EKHVKYDTLE801HHHHHH*961-741ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTCTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCTCGGTATCTTTGGCGGAAAAGCCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1MATNDDDVKK AATVAIAAAY NNGGBINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKG NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDBKEHAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKGTAEE TKGNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301VSDLRKBTRG GLAEGAALSG LFGPYNVGRF NVTAAVGGYK SESAVAIGTG351FRPTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGV AADIGAGLAD401ALTAPLDHKD KGLGSLTLDG SVRKNEKLKL AAGGAEKTYG NGDSLNTGKL451KNDKVSRFDF IRGIEVDGGL ITLESGEFGV YKGSHSALTA FGTEGIGDSE501HSGKMVAKRG FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY551TIDFAAKGGN GKIEHLKSPE LNVDLAAADI KPDGKEHAVI SGSVLYNGAE601KGSYSLGIFG GKAGEVAGSA EVKTVNGIRE IGLAARGLEH HHHHH*961-983ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAADCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA1HATNDDDVKK AATVAIAAAY NNGGEINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKG NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKGTAEE TKGNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAARV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301VSDLRKETRG GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG351FRFTENFAAR AGVAVGTSSG SSAAYHVGVN YEWGSGGGGT SAPDFNAGGT401GIGSNSRATT AKSAAVSYAG IKNEMCKDRS MLCAGRDDVA VTDRDAKINA451PPPNLHTGDF PNPNDAYKNL INLKPAIEAG YTGRGVEVGI VDTGESVGSI501SFPELYGRKE HGYNENYKNY TAYMRKEAPE DGGGKDIEAS FDDEAVIETE551AKPTDIRHVK EIGHIDLVSH IIGGRSVDGR PAGGIABDAT LHIMNTNDET601KNEMMVAAIR NAWVKLGERG VRIVNNSFGT TSRAGTADLF GIANSEEGYR651QALLDYSGGD KTDEGIRIMG GSDYGNLSYH IRNKNNLFIF STGNDAGAGP701NTYALLPFYE KDAGKGIITV AGVDRSGEKF KPEMYGEPGT EPLEYGSNHC751GITAMWCLSA PYEASVRFTR TNPIGIAGTS FSAPIVTGTA ALLLGKYPWM801SNDNLRTTLL TTAGDIGAVG VDSKFGWGLL DAGKAMNGPA SFPFGDFTAD851TKGTSDIAYS FRNDISGTGG LIKKGGSGLG LHGNNTYTGK TIIEGGSLVL901YGNNKSDMRV ETKGALIYNG AASGGSLNSD GIVYLADTDG SGANETVHIK951GSLGLDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGKGA GYLNSTGRRV1001PFLSAAKIGG DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSYYVRRGN1051AARTASAAAH SAPAGLKHAV EQGGSNLENL HVELDASESS ATPETVETAA1101ADRTDMPGIR PYGATFRAAA AVGHANAADG VRIFNSLAAT VYADSTAAHA1151DMGGRRLKAV SDGLDHNGTG LRVIAGTGGD GGTWEGGGVE GKMRGSTGTV1201GIAAKTGENT TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY1251LKGLFSYGRY KNSISRSTGA DEHAEGSVNG TLMGLGALGG VNVPFAATGD1301LTVEGGLRYD LLKGDAFAEK GSALGWSGNS LTEGTLVGLA GLKLSGPLSD1351KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG1401ADVEFGNGWN GLARYSYAGS RQYGNHSGRV GVGYRFLEHH HHHH*961c-ORF46.1ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACTAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAATGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1HATNDDDVKK AATVAIAAAY NNGGEINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKG NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKGTAEE TKGNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNThKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301VSDLRKETRG GLAEGAALSG LFQPYNVGGS GGGGSDLAND SFIRQVLDRG351HFEPDGKYHL FGSRGELAER SGHIGLGKIG SHQLGNLMIG GAAIKGNIGY401IVRFSDHGHE VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD451GYDGPGGGGY PAPKGARDIY SYDIKGVAGN IELNLTDNRS TGGELADRFH501NAGSMLTGGV GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE551IVGAGDAVGG ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAGLKDYAAA601AIRDWAVGNP NAAGGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK651RSGMGAIALP KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEGRYGKENI701TSSTVPPSNG KNVKLADGRH PKTGVPFDGK GFPNFEKHVK YDTLEHHHHH751H*961c-741ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1MATNDDDVKK AATVAIAAAY NNGGEINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDERLEAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKGTAEE TKGNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTEL ASAEKSIADH DTRLNGLDKT301VSDLEREThG GLAEGAALSG LFQPYNVGGS GGGGVAADIG AGLADALTAP351LDHKDKGLGS LTLDGSVRKN EKLKLAAGGA EKTYGNGDSL NTGKLKNDKV401SRPDFIRGIE VDGGLITLES GEFGVYKGSH SALTAPGTEG IQDSEHSGKM451VAKRGFRIGD IAGEHTSFDK LPEGGRATYR GTAFGSDDAG GKLTYTIDFA501AKGGNGKIEH LKSPELNVDL AAADIKPDGK REAVISGSVL YNGAEKGSYS551LGIFGGKAGE VAGSAEVKTV NGIRHIGLAA KGLEHHHHHH *961c-983ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCCGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAATCAATGCCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCTATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA1MATNDDDVKK AATVAIAAAY NNGGEINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKG NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKGTAEE TKGNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTBL ASAEKSIADH DTRLNGLDKT301VSDLRKETRG GLAEGAALSG LFGPYNVGGS GGGGTSAPDF NAGGTGIGSN351SRATTAKSAA VSYAGIKNEM CKDRSHLCAG RDDVAVTDRD AKINAPPPNL401HTGDFPNPND AYKNLINLKP AIEAGYTGRG VEVGIVDTGE SVGSISFPEL451YGRKEHGYNB NYKNYTAYMR KEAPEDGGGK DIEASFDDEA VIETEAKPTD501IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDETKNEMM551VAAIRNAWVK LGERGVRIVN NSFGTTSRAG TADLFGIANS EEGYRGALLD601YSGGDKTDEG IRLMGGSDYG NLSYHIRNKN MLFIFSTGND AGAGPNTYAL651LPFYEKDAGK GIITVAGVDR SGEKFKRBHY GEPGTEPLEY GSNHCGITAM701WCLSAPYEAS VRFTRTNPIG IAGTSFSAPI VTGTAALLLG KYPWMSNDNL751RTTLLTTAGD IGAVGVDSKF GWGLLDAGKA MNGPASFPPG DFTADTKGTS801DIAYSFRNDI SGTGGLIKKG GSGLGLHGNN TYTGKTIIEG GSLVLYGNNK851SDNRVETKGA LIYNGAASGG SLNSDGIVYL ADTDGSGANE TVHIKGSLGL901DGKGTLYTRL GKLLKVDGTA IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA951AKIGGDYSFF TNIETDGGLL ASLDSVEKTA GSBGDTLSYY VRRGNAARTA1001SAAAHSAPAG LKHAVEGGGS NLENLMVELD ASESSATPET VETAAADRTD1051MPGIRPYGAT FRAAAAVGHA NAADGVRIFN SLAATVYADS TAAHADMGGR1101RLKAVSDGLD HNGTGLRVIA GTGGDGGTWE GGGVEGKMRG STGTVGIAAK1151TGENTTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIRHDA GDIGYLKGLF1201SYGRYRNSIS RSTGADEHAE GSVNGTLMGL GALGGVNVPF AATGDLTVEG1251GLRYDLLKGD AFAEKGSALG WSGNSLTEGT LVGLAGLKLS GPLSDKAVLF1301ATAGVEEDLN GEDYTVTGGF TGATAATGKT GAPNMPHTRL VAGLGADVEF1351GNGWNGLARY SYAGSKGYGN HSGRVGVGYR FLEHHHHHH*961cL-ORF46.1ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGTAACTCGAG1MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGGEING51FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN101KGNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI151TTFAEETKTN IVKIDEKLEA VADTVDKHAE APNDIADSLD ETNTKADEAV201KThNEAKGTA EETKGNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA251KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT301RLASAEKSIA DHDThLNGLD KTVSDLRKBT RQGLAEQAAL SGLPGPYNVG351GSGGGGSDLA NDSFIRGVLD RGHFEPDGKY HLPGSRGELA ERSGHIGLGK401IGSHGLGNLM IQQAAIKGNI GYIVRFSDHG HEVHSPFDNH ASHSDSDHAG451SPVDGFSLYR IHWDGYBHHP ADGYDGPGGG GYPAPKGARD IYSYDIKGVA501GNIRLNLTDN RSTGQRLADR FHNAGSMLTQ GVGDGFKRAT RYSPELDRSG551NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV GGISEGSNIA VMHGLGLLST601ENKMARINDL ADMAGLKDYA AAAIRDWAVG NPNAAGGIEA VSNIFMAAIP651IKGIGAVRGK YGLGGITAHP IKRSGMGAIA LPKGKSAVSD NFADAAYAKY701PSPYHSRNIR SNLEGRYGKE NITSSTVPPS NGKNVKLADG RHPKTGVPFD751GKGFPNFEKH VKYDT*961cL-741ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGGEING51FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN101KGNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI151TTFAEETKTN IVKIDEKLEA VADTVDKHAB AFNDIADSLD ETNTKADEAV201KTANEAKGTA EETKGNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA251KVTDIKADIA TNKDNLAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT301RLASABKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFGPYNVG351GSGGGGVAAD IGAGLADALT APLDHKDKGL GSLTLDGSVR KNEKLKLAAG401GAEKTYGNGD SLNTGKLKND KVSRFDFIRG IEVDGGLITL ESGEFGVYKG451SHSALTAPGT EGIGDSEHSG KMVAKRGFRI GDIAGEHTSF DKLPEGGRAT501YRGTAPGSDD AGGKLTYTID FAAKGGNGKI EHLKSPELNV DLAAADIKPD551GKRHAVISGS VLYNGAEKGS YSLGIFGGKA GEVAGSAEVK TVNGIRHIGL601AAKGLEHHHH HH*961cL-983ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGAGGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCTGACTCGAG1MKHFPSKVLT TAILATFCSG MJAATNDDDV KKAATVAIAA AYNNGGEING51FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN101KGNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI151TTFAEETKTN IVKIDEKLEA VADTVDKHAB AFNDIADSLD ETNTKADEAV201KTANEAKGTA EETKGNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA251KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT301RLASAEKSIA DHDTRLNGLD KTVSDLRKET RGGLAEGAAL SGLFGPYNVG351GSGGGGTSAP DFNAGGTGIG SNSRATTAKS AAVSYAGIKN EMCKDRSMLC401AGRDDVAVTD RDAKINAPPP NLHTGDFPNP NDAYKNLINL KPAIEAGYTG451RGVEVGIVDT GESVGSISFP ELYGRKEHGY NENYKNYTAY MRKEAPEDGG501GKDIEASFDD EAVIETEAKP TDIRHVKEIG HIDLVSHIIG GRSVDGRPAG551GIAPDATLHI MNTNDETKNE MMVAAIRNAW VKLGERGVRI VNNSFGTTSR601AGTADLPGIA NSEEGYRGAL LDYSGGDKTD EGIRLHGGSD YGNLSYHIRN651KNMLFIFSTG NDAGAGPNTY ALLPFYEKDA GKGIITVAGV DRSGEKFKRE701MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVRPTRTNP IGIAGTSFSA751PIVTGTAALL LGKYPWMSND NLRTTLLTTA GDIGAVGVDS KFGWGLLDAG801KAMNGPASFF FGDFTADTKG TSDIAYSFRN DISGTGGLIK KGGSGLGLHG851NNTYTGKTII EGGSLVLYGN NKSDMRVETK GALIYNGAAS GGSLNSDGIV901YLADTDGSGA NETVHIKGSL GLDGKGTLYT RLGKLLKVDG TAIIGGKLYM951SARGKGAGYL NSTGRRVPFL SAAKIGQDYS FFTNIETDGG LLASLDSVEK1001TAGSEGDTLS YYVRRGNAAR TASAAAHSAP AGLKHAVEGG GSNLENLMVE1051LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVG HANAADGVRI1101FNSLAATVYA DSTAAHADMG GRRLKAVSDG LDHNGTGLRV IAGTGGDGGT1151WEGGGVEGKM RGSTGTVGIA AKTGENTTAA ATLGMGRSTW SENSANAKTD1201SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTLM1251GLGALGGVNV PFAATGDLTV EGGLRYDLLK GDAFAEKGSA LGWSGNSLTB1301GTLVGLAGLK LSGPLSDKAV LFATAGVERD LNGRDYTVTG GFTGATAATG1351KTGAR~MPRT RLVAGLGADV EFGNGWNGLA RYSYAGSKGY GNHSGRVGVG1401YRF*

[0079] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. For instance, the use of proteins from other strains is envisaged [e.g. see WO00/66741 for polymorphic sequences for ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953].

[0080] Experimental Details

[0081] Cloning Strategy and Oligonucleonide Design

[0082] Genes coding for antigens of interest were amplified by PCR, using oligonucleotides designed on the basis of the genomic sequence of N. meningitidis B MC58. Genomic DNA from strain 2996 was always used as a template in PCR reactions, unless otherwise specified, and the amplified fragments were cloned in the expression vector pET21b+(Novagen) to express the protein as C-terminal His-tagged product, or in pET-24b+(Novagen) to express the protein in ‘untagged’ form (e.g. ΔG 287K).

[0083] Where a protein was expressed without a fusion partner and with its own leader peptide (if present), amplification of the open reading frame (ATG to STOP codons) was performed.

[0084] Where a protein was expressed in ‘untagged’ form, the leader peptide was omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence.

[0085] The melting temperature of the primers used in PCR depended on the number and type of hybridising nucleotides in the whole primer, and was determined using the formulae:

25Tm1 = 4 (G + C) + 2 (A + T)(tail excluded)Tm2 = 64.9 + 0.41 (% GC) − 600/N(whole primer)

[0086] The melting temperatures of the selected oligonucleotides were usually 65-70° C. for the whole oligo and 50-60° C. for the hybridising region alone.

[0087] Oligonucleotides were synthesised using a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2.0 ml NH4OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were centrifuged and the pellets resuspended in water.

26RestrictionSequencessitefu (961)-FwdCGCGGATCC-GGAGGGGGTGGTGTCGBamHI741(MC58)-HisRevCCCGCTCGAG-TTGCTTGGCGGCAAGGCXhoIfu (961)-983-HisFwdCGCGGATCC-GGCGGAGGCGGCACTTBamHIRevCCCGCTCGAG-GAACCGGTAGCCTACGXhoIfu (961)-Orf46.1-FwdCGCGGATCCGGTGGTGGTGGT-BamHIHisTCAGATTTGGCAAACGATTCRevCCCGCTCGAG-CGTATCATATTTCACGTGCXhoIfu (961 c-L)-FwdCGCGGATCC-GGAGGGGGTGGTGTCGBamHI741(MC58)RevCCCGCTCGAG-TTATTGCTTGGCGGCAAGXhoIfu (961c-L)-983FwdCGCGGATCC-GGCGGAGGCGGCACTTBamHIRevCCCGCTCGAG-TCAGAACCGGTAGCCTACXhoIfu (961c-L)-FwdCGCGGATCCGGTGGTGGTGGT-BamHIOrf46.1TCAGATTTGGCAAACGATTCRevCCCGCTCGAG-TTACGTATCATATTTCACGTGCXhoIfu-(ΔG287)-919-FwdCGCGGATCCGGTGGTGGTGGT-BamHIHisCAAAGCAAGAGCATCCAAACCRevCCCAAGCTT-TTCGGGCGGTATTCGGGCTTCHindIIIfu-(ΔG287)-953-FwdCGCGGATCCGGTGGTGGTGGT-BamHIHisGCCACCTACAAAGTGGACRevGCCCAAGCTT-TTGTTTGGCTGCCTCGATHindIIIfu-(ΔG287)-961-FwdCGCGGATCCGGTGGTGGTGGT-ACAAGCGACGACGBamHIHisRevGCCCAAGCTT-CCACTCGTAATTGACGCCHindIIIfu-(ΔG287)-FwdCGCGGATCCGGTGGTGGTGGT-BamHIOrf46.1-HisTCAGATTTGGCAAACGATTCRevCCCAAGCTT-CGTATCATATTTCACGTGCHindIIIfu-(ΔG287-919)-FwdCCCAAGCTTGGTGGTGGTGGTGGT-HindIIIOrf46.1-HisTCAGATTTGGCAAACGATTCRevCCCGCTCGAG-CGTATCATATTTCACGTGCXhoIfu-(ΔG287-FwdCCCAAGCTTGGTGGTGGTGGTGGT-HindIIIOrf46.1)-919-HisCAAAGCAAGAGCATCCAAACCRevCCCGCTCGAG-CGGGCGGTATTCGGGCTTXhoIfu ΔG287(394.98)-FwdCGCGGATCCGCTAGC-CCCGATGTTAAATCGGCNheI. . .RevCGGGGATCC-ATCCTGCTCTTTTTTGCCGGBamHIfu Orf1-(Orf46.1)-FwdCGCGGATCCGCTAGC-GGACACACTTATTTCGGCATCNheIHisRevCGCGGATCC-CCAGCGGTAGCCTAATTTGATfu (Orf1)-Orf46.1-FwdCGCGGATCCGGTGGTGGTGGT-BamHIHisTCAGATTTGGCAAACGATTCRevCCCAAGCTT-CGTATCATATTTCACGTGCHindIIIfu (919)-Orf46.1-Fwd1GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGSalIHisFwd2GGAGGCACTGGATCCTCAGATTTGGCAAACGATTCRevCCCGCTCGAG-CGTATCATATTTCACGTGCXhoIFu (orf46)-287-HisFwdCGGGGATCCGGGGGCGGCGGTGGCGBamHIRevCCCAAGCTTATCCTGCTCTTTTTTGCCGGCHindIIIFu (orf46)-919-HisFwdCGCGGATCCGGTGGTGGTGGTCAAAGCAAGAGCATCCAAACCBamHIRevCCCAAGCTTCGGGCGGTATTCGGGCTTCHindIIIFu (orf46-919)-FwdCCCCAAGCTTGGGGGCGGCGGTGGCGHindIII287-HisRevCCCGCTCGAGATCCTGCTCTTTTTTGCCGGCXhoIFu (orf46-287)-FwdCCCAAGCTTGGTGGTGGTGGTGGTCAAAGCAAGAGCATCCAAACCHindIII919-HisRevCCCGCTCGAGCGGGCGGTATTCGGGCTTXhoI(ΔG741)-961c-HisFwd1GGAGGCACTGGATCCGCAGCCACAAACGACGACGAXhoIFwd2GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAGRevCCCGCTCGAG-ACCCAGCTTGTAAGGTTGXhoI(ΔG741)-961-HisFwd1GGAGGCACTGGATCCGCAGCCACAAACGACGACGAXhoIFwd2GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAGRevCCCGCTCGAG-CCACTCGTAATTGACGCCXhoI(ΔG741)-983-HisFwdGCGGCCTCGAG-XhoIGGATCCGGCGGAGGCGGCACTTCTGCGRevCCCGCTCGAG-GAACCGGTAGCCTACGXhoI(ΔG741)-orf46.1-Fwd1GGAGGCACTGGATCCTCAGATTTGGCAAACGATTCSalIHisFwd2GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGARevCCCGCTCGAG-CGTATCATATTTCACGTGCXhoI(ΔG983)-FwdGCGGCCTCGAG-GGATCCGGAGGGGGTGGTGTCGCCXhoI741(MC58)-HisRevCCCGCTCGAG-TTGCTTGGCGGCAAGXhoI(ΔG983)-961c-HisFwd1GGAGGCACTGGATCCGCAGCCACAAACGACGACGAXhoIFwd2GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAGRevCCCGCTCGAG-ACCCAGCTTGTAAGGTTGXhoI(ΔG983)-961-HisFwd1GGAGGCACTGGATCCGCAGCCACAAACGACGACGAXhoIFwd2GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAGRevCCCGCTCGAG-CCACTCGTAATTGACGCCXhoI(ΔG983)-Orf46.1-Fwd1GGAGGCACTGGATCCTCAGATTTGGCAAACGATTCSalIHisFwd2GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGARevCCCGCTCGAG-CGTATCATATTTCACGTGCXhoI*This primer was used as a Reverse primer for all the C terminal fusions of 287 to the His-tag. §Forward primers used in combination with the 287-His Reverse primer. NB - All PCR reactions use strain 2996 unless otherwise specified (e.g. strain MC58)

[0088] In all constructs starting with an ATG not followed by a unique NheI site, the ATG codon is part of the NdeI site used for cloning. The constructs made using NheI as a cloning site at the 5′ end (e.g. all those containing 287 at the N-terminus) have two additional codons (GCT AGC) fused to the coding sequence of the antigen.

[0089] Preparation of Chromosomal DNA Templates

[0090]

N. meningitidis
strains 2996, MC58, 394.98, 1000 and BZ232 (and others) were grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% w/v sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml of lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one CHCl3/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes of ethanol, and collected by centrifugation. The pellet was washed once with 70%(v/v) ethanol and redissolved in 4.0 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA concentration was measured by reading OD260.

[0091] PCR Amplfication

[0092] The standard PCR protocol was as follows: 200 ng of genomic DNA from 2996, MC581000, or BZ232 strains or long of plasmid DNA preparation of recombinant clones were used as template in the presence of 40 μM of each oligonucletide primer, 400-800 μM dNTPs solution, 1× PCR buffer (including 1.5 mM MgCl2), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, Boerhingher Mannheim Expand™ Long Template).

[0093] After a preliminary 3 minute incubation of the whole mix at 95° C., each sample underwent a two-step amplification: the first 5 cycles were performed using the hybridisation temperature that excluded the restriction enzyme tail of the primer (Tm1). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (Tm2). Elongation times, performed at 68° C. or 72° C., varied according to the length of the Orf to be amplified. In the case of Orf1 the elongation time, starting from 3 minutes, was increased by 15 seconds each cycle. The cycles were completed with a 10 minute extension step at 72° C.

[0094] The amplified DNA was either loaded directly on a 1% agarose gel. The DNA fragment corresponding to the band of correct size was purified from the gel using the Qiagen Gel Extraction Kit, following the manufacturer's protocol.

[0095] Digestion of PCR Fragments and of the Cloning Vectors

[0096] The purified DNA corresponding to the amplified fragment was digested with the appropriate restriction enzymes for cloning into pET-21b+, pET22b+or pET-24b+. Digested fragments were purified using the QIAquick PCR purification kit (following the manufacturer's instructions) and eluted with either H2O or 10 mM Tris, pH 8.5. Plasmid vectors were digested with the appropriate restriction enzymes, loaded onto a 1.0% agarose gel and the band corresponding to the digested vector purified using the Qiagen QIAquick Gel Extraction Kit.

[0097] Cloning

[0098] The fragments corresponding to each gene, previously digested and purified, were ligated into pET21b+, pET22b+ or pET-24b+. A molar ratio of 3:1 fragment/vector was used with T4 DNA ligase in the ligation buffer supplied by the manufacturer.

[0099] Recombinant plasmid was transformed into competent E. coli DH5 or HB101 by incubating the ligase reaction solution and bacteria for 40 minutes on ice, then at 37° C. for 3 minutes. This was followed by the addition of 800 μl LB broth and incubation at 37° C. for 20 minutes. The cells were centrifuged at maximum speed in an Eppendorf microfuge, resuspended in approximately 200 μl of the supernatant and plated onto LB ampicillin (100 mg/ml) agar.

[0100] Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37° C. in 4.0 ml of LB broth +100 μg/ml ampicillln. Cells were pelleted and plasmid DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions. Approximately 1 μg of each individual miniprep was digested with the appropriate restriction enzymes and the digest loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 kb DNA Ladder, GIBCO). Positive clones were selected on the basis of the size of insert.

[0101] Expression

[0102] After cloning each gene into the expression vector, recombinant plasmids were transformed into E. coli strains suitable for expression of the recombinant protein. 1 μl of each construct was used to transform E. coli BL21-DE3 as described above. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, to give an OD600 between 0.1 and 0.2. The flasks were incubated at 30° C. or at 37° C. in a gyratory water bath shaker until OD600 indicated exponential growth suitable for induction of expression (0.40.8 OD). Protein expression was induced by addition of 11.0 mM IPTG. After 3 hours incubation at 30° C. or 37° C. the OD600 was measured and expression examined. 1.0 ml of each sample was centrifuged in a microfuge, the pellet resuspended in PBS and analysed by SDS-PAGE and Coomassie Blue staining.

[0103] Purification of His-tagged Proteins

[0104] Various forms of 287 were cloned from strains 2996 and MC58. They were constructed with a C-terminus His-tagged fusion and included a mature form (aa 18-427), constructs with deletions (Δ1, Δ2, Δ3 and Δ4) and clones composed of either B or C domains. For each clone purified as a His-fusion, a single colony was streaked and grown overnight at 37° C. on a LB/Amp (100 μg/ml) agar plate. An isolated colony from this plate was inoculated into 20 ml of LB/Amp (100 μg/ml) liquid medium and grown overnight at 37° C. with shaking. The overnight culture was diluted 1:30 into 1.0 L LB/Amp (100 μg/ml) liquid medium and allowed to grow at the optimal temperature (30 or 37° C.) until the OD550 reached 0.6-0.8. Expression of recombinant protein was induced by addition of IPTG (final concentration 1.0 mM) and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 8000 g for 15 min at 4° C. The bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8.0) for soluble proteins or (ii) buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8 and, optionally, 8M urea) for insoluble proteins. Proteins purified in a soluble form included 287-His, Δ1, Δ2, Δ3 and Δ4287-His, Δ4287MC58-His, 287c-His and 287cMC58-His. Protein 287bMC58-His was insoluble and purified accordingly. Cells were disrupted by sonication on ice four times for 30 sec at 40 W using a Branson sonifier 450 and centrifuged at 13000 g for 30 min at 4° C. For insoluble proteins, pellets were resuspended in 2.0 ml buffer C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5 and treated with 10 passes of a Dounce homogenizer. The homogenate was centrifuged at 13000 g for 30 min and the supernatant retained. Supernatants for both soluble and insoluble preparations were mixed with 150 μl Ni2+-resin (previously equilibrated with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 min. The resin was Chelating Sepharose Fast Flow (Pharmacia), prepared according to the manufacturer's protocol. The batch-wise preparation was centrifuged at 700 g for 5 min at 4° C. and the supernatant discarded. The resin was washed twice (batch-wise) with 10 ml buffer A or B for 10 min, resuspended in 1.0 ml buffer A or B and loaded onto a disposable column. The resin continued to be washed with either (i) buffer A at 4° C. or (id) buffer B at room temperature, until the OD280 of the flow-through reached 0.02-0.01. The resin was further washed with either (i) cold buffer C (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8.0) or (ii) buffer D (10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3 and, optionally, 8M urea) until OD280 of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8.0) or (ii) elution buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5 and, optionally, 8M urea) and fractions collected until the OD280 indicated all the recombinant protein was obtained. 20 μl aliquots of each elution fraction were analysed by SDS-PAGE. Protein concentrations were estimated using the Bradford assay.

[0105] Renaturation of Denatured His-fusion Proteins

[0106] Denaturation was required to solubilize 287bMC8, so a renaturation step was employed prior to immunisation. Glycerol was added to the denatured fractions obtained above to give a final concentration of 10% v/v. The proteins were diluted to 200 μg/ml using dialysis buffer I (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, 2.0M urea, pH 8.8) and dialysed against the same buffer for 12-14 hours at 4° C. Further dialysis was performed with buffer II (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was estimated using the formula:

Protein (mg/ml)=(1.55×OD280)−(0.76×OD260)

[0107] Immunization

[0108] Balb/C mice were immunized with antigens on days 0, 21 and 35 and sera analyzed at day 49.

[0109] Sera Analysis—ELISA

[0110] The acapsulated MenB M7 and the capsulated strains were plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO2. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD620. The bacteria were let to grow until the OD reached the value of 0.4-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and bacteria were washed twice with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 1 hour at 37° C. and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% Tween-20 in PBS). 200 μl of saturation buffer (2.7% polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN3 in PBS) were added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 1011 of H2O2) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl 12.5% H2SO4 was added to each well and OD490 was followed. The ELISA titers were calculated abitrarely as the dilution of sera which gave an OD490 value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD490 of 0.4 was higher than 1:400.

[0111] Sera Analysis—FACS Scan Bacteria Binding Assay

[0112] The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO2. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD620. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA in PBS, 0.4% NaN3) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD620 of 0.05. 100 μl bacterial cells were added to each well of a Costar 96 well plate. 100 μl of diluted (1:100, 1:200, 1:400) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 400 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytrin conjugated F(ab)2 goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan (Laser Power 15 mW) setting were: FL2 on; FSC—H threshold:92; FSC PMT Voltage: E 01; SSC PMr: 474; Amp. Gains 6.1; FL-2 PMT: 586; compensation values: 0.

[0113] Sera Analysis—Bactericidal Assay

[0114]

N. meningitidis
strain 2996 was grown overnight at 37° C. on chocolate agar plates (starting from a frozen stock) with 5% CO2. Colonies were collected and used to inoculate 7 ml Mueller-Hinton broth, containing 0.25% glucose to reach an OD620 of 0.05-0.08. The culture was incubated for approximately 1.5 hours at 37 degrees with shacking until the OD620 reached the value of 0.23-0.24. Bacteria were diluted in 50 mM Phosphate buffer pH 7.2 containing 10 mM MgCl2, 10 mM CaCl2 and 0.5% (w/v) BSA (assay buffer) at the working dilution of 105 CFU/ml. The total volume of the final reaction mixture was 50 μl with 25 μl of serial two fold dilution of test serum, 12.5 μl of bacteria at the working dilution, 12.5 μl of baby rabbit complement (final concentration 25%).

[0115] Controls included bacteria incubated with complement serum, immune sera incubated with bacteria and with complement inactivated by heating at 56° C. for 30′. Immediately after the addition of the baby rabbit complement, 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 0). The 96-wells plate was incubated for 1 hour at 37° C. with rotation. 7 μl of each sample were plated on Mueller-Hinton agar plates as spots, whereas 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 1). Agar plates were incubated for 18 hours at 37 degrees and the colonies corresponding to time 0 and time 1 were counted.

[0116] Sera Analysis—Western Blots

[0117] Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded onto a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., using transfer buffer (0.3% Tris base, 1.44% glycine, 20% (v/v) methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

[0118] The OMVs were prepared as follows: N. meningitidis strain 2996 was grown overnight at 37 degrees with 5% CO2 on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20 mM Tris-HCl pH 7.5, 2 mM EDTA. Heat inactivation was performed at 56° C. for 45 minutes and the bacteria disrupted by sonication for 5 minutes on ice (50% duty cycle, 50% output, Branson sonifier 3 mm microtip). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes, the supernatant containing the total cell envelope fraction recovered and further centrifuged overnight at 50000 g at the temperature of 4° C. The pellet containing the membranes was resuspended in 2% sarkosyl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA and incubated at room temperature for 20 minutes to solubilise the inner membranes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, the supernatant was further centrifuged at 50000 g for 3 hours. The pellet, containing the outer membranes was washed in PBS and resuspended in the same buffer. Protein concentration was measured by the D.C. Bio-Rad Protein assay (Modified Lowry method), using BSA as a standard.

[0119] Total cell extracts were prepared as follows: N. meningitidis strain 2996 was grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.

Number	Date	Country	Kind
0004695.3	Feb 2000	GB
0027675.8	Nov 2000	GB

Hybrid expression of neisserial proteins

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