Vectors and hosts with increased expression of HBCAG

Abstract

The invention relates to vectors and hosts with increased expression of HBcAg.

Description

FIELD OF THE INVENTION

The invention is in the field of molecular biology. In particular, the invention relates to new sequences and improved expression of hepatitis B core protein.

BACKGROUND OF THE INVENTION

Hepatitis B virus is the most thoroughly characterized pathogen of the hepatitis diseases. The hepatitis B virus is associated with a wide spectrum of liver disease, from a subclinical carrier state to accute hepatitis, chronic hepatitis, postnecrotic (posthepatitic) cirrhosis, and hepatocellular carcinoma. It also has a poorly understood association with several primary non-hepatic disorders including polyarteritis nodesa and other collagen vascular diseases, membraneous glomerulonephritis, essential mixed cryoglobulinemina and papular acrodermatitis of childhood. Hepatitis symptoms and signs vary from minor flu-like illnesses to fulminant, fatal liver failure.

Groups at risk for contracting hepatitis B virus include certain hospital and dentist staff (e.g., oncology, hemodialysis-transplantation, gastroenterology, intensive care units, diagnostic laboratories, and surgical units), staff in institutions for the mentally handicapped, patients receiving blood and blood products, drug addicts, male homosexuals, and the families of chronic carriers.

The infective "DANE" particle consists of an inner core plus an outer surface coat. The inner core contains DNA and DNA polymerase. The DNA replicates within the nuclei of infected hepatocytes. The core antigen (HBcAg) is associated with the viral inner core. It can be found in infected liver cells but is not detectable in serum except by special techniques which disrupt the DANE particle. The hepatitis B virus is present in the cytoplasm of parenchymal liver cells of individuals with hepatitis B and constitutes the infective virus. The core particle displays HBcAg. The core of this particle is found in the nucleus of parenchymal cells, but as it passes through the cytoplasm, it acquires a surface coat.

Antibody to the core antigen appears promptly in the blood of infected individuals and persists indefinitely. High titers of IgM anti-HBc is found in patients with accute disease and may be the only marker of accute hepatitis B in some situations.

Serological detection of anti-HBc is accepted diagnostic evidence of hepatitis B viral infection. Therefore, it is desirable to have substantial quantities of HBcAg for use as an immunogen in development of monoclonal and polyclonal antibodies to the HBcAg, for preparing vaccines, and for use in detection of the viral infection in patients.

SUMMARY OF THE INVENTION

The present invention provides new HBcAg sequences with improved expression. The invention also provides nucleotide sequences which enhance expression of HBcAg. The invention also provides new HBcAg protein sequences. In addition, the invention provides methods for increasing expression of HBcAg.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Enzyme linked immunosorbent Assay (ELISA) data showing increased expression of HBcAg with sequences of the invention.

DETAILED DESCRIPTION

The present invention provides new HBcAg sequences and methods for increasing expression of HBcAg. When Sequence ID No: 1 is altered to include additional nucleotides between the ribosome binding site and start codon, (also referred to as the initiation codon), the expression level is about ten (10) times higher than the same sequence unmodified. When the modification involves changing the second codon (first codon after the start codon) from GAC to GCT, plus nucleotides between the ribosome binding site and first ATG codon, increases of about twenty-five (25) times higher expression are obtained over the native sequence.

The present invention also provides DNA molecules described in Sequence ID No: 3, Sequence ID No: 5, Sequence ID No: 7, Sequence ID No: 9, and Sequence ID No: 11.

The invention also provides hosts, and methods for increasing expression of HBcAg which comprises transforming a host with the disclosed sequences.

The invention also provides amino acid sequences described in Sequence ID No: 2, Sequence ID No: 4, Sequence ID No: 6, Sequence ID No: 8, Sequence ID No: 10, and Sequence ID No: 12 and kits comprising the sequences.

Sequence ID No: 1 is referred to as the "native" sequence. Preferably Sequence ID No: 1 has an eight base pair spacer between the ribosome binding site and start codon. Most preferably the eight base pair spacer is Sequence ID No: 14 (AACAGACC). Sequence ID No: 3 is the same as Sequence ID No: 1 (silent mutations from a G to A at bp 39 and a C to T at bp 249 are present). Preferably Sequence ID No: 3 has a 10 base pair spacer between the ribosome binding site and the start codon. Most preferably the ten base pair spacer is Sequence ID No: 13 (AACAGAATTC). Sequence ID No: 5 is the same as Sequence ID No: 1 but has the alterations of the second codon change, bp 467 changes from G to A and deletion of bp 468, causing a frameshift. Preferably Sequence ID No: 5 has a ten base pair spacer between the ribosome binding site and start codon. Most preferably the ten base pair spacer is Sequence ID No: 13. Sequence ID No: 7 is the same as Sequence ID No: 1 but has the second codon change. Preferably Sequence ID No: 7 has an eight base pair spacer between the ribosome binding site and start codon. Most preferably the eight base pair spacer is Sequence ID No: 14. Sequence ID No: 9 is the same as Sequence ID No: 7 with a transition at bp 359 from a T to a C (resulting in a Val to Ala change in the protein). Preferably Sequence ID No: 9 has a ten base pair spacer between the ribosome binding site and start codon. Most preferably the ten base pair spacer is Sequence ID No: 13. Sequence ID No: 11 is the same as Sequence ID No: 7 with alterations of the second codon change, and deletion of bp 455, causing a frameshift. Preferably an eight base pair spacer is between the ribosome binding site and start codon. Most preferably the eight base pair spacer is Sequence ID No: 14.

Sequence ID No: 2, 4, 6, 8, 10, and 12 are predicted HBcAg protein sequences of the invention. Sequence ID No: 1 encodes the protein of Sequence ID No: 2. Sequence ID No: 3 also encodes the protein sequence in Sequence ID No: 2 (or 4). Sequence ID No: 5 encodes the protein in Sequence ID No: 6. Sequence ID No: 7 encodes the protein in Sequence ID No: 8. Sequence ID No: 9 encodes the protein in Sequence ID No: 10 Sequence ID No: 11 encodes the protein in Sequence ID No: 12. Since many amino acids are selected by more than one codon (degeneracy), DNA sequences can vary without corresponding changes in the amino acid sequences.

Tables 1 through 11 depict nucleotide and amino acid sequences of the invention (nucleotide sequences left to right are 5' to 3' and amino acid sequences left to right are amino terminus to carboxy terminus).

TABLE 1__________________________________________________________________________ATGGACATTG ACCCTTATAA AGAATTTGGA GCTACTGTGG AGTTACTCTC GTTTTTGCCT 60TCTGACTTCT TTCCTTCCGT CAGAGATCTC CTAGACACCG CCTCAGCTCT GTATCGGGAA 120GCCTTAGAGT CTCCTGAGCA TTGCTCACCT CACCATACCG CACTCAGGCA AGCCATTCTC 180TGCTGGGGGG AATTGATGAC TCTAGCTACC TGGGTGGGTA ATAATTTGGA AGATCCAGCA 240TCAAGGGACC TAGTAGTCAA TTATGTTAAT ACTAACATGG GTTTAAAAAT TAGGCAACTA 300TTGTGGTTTC ATATATCTTG CCTTACTTTT GGAAGAGAGA CTGTACTTGA ATATTTGGTC 360TCTTTCGGAG TGTGGATTCG CACTCCTCCA GCCTATAGAC CACCAAATGC CCCTATCTTA 420TCAACACTTC CGGAAACTAC TGTTGTTAGA CGACGGGACC GAGGCAGGTC CCCTAGAAGA 480AGAACTCCCT CGCCTCGCAG ACGCAGATCT CAATCGCCGC GTCGCAGAAG ATCTCAATCT 540CGGGAATCTC AATGTTAG 558__________________________________________________________________________ TABLE 2__________________________________________________________________________ATGGACATTG ACCCTTATAA AGAATTTGGA GCTACTGTAG AGTTACTCTC GTTTTTGCCT 60TCTGACTTCT TTCCTTCCGT CAGAGATCTC CTAGACACCG CCTCAGCTCT GTATCGGGAA 120GCCTTAGAGT CTCCTGAGCA TTGCTCACCT CACCATACCG CACTCAGGCA AGCCATTCTC 180TGCTGGGGGG AATTGATGAC TCTAGCTACC TGGGTGGGTA ATAATTTGGA AGATCCAGCA 240TCAAGGGATC TAGTAGTCAA TTATGTTAAT ACTAACATGG GTTTAAAAAT TAGGCAACTA 300TTGTGGTTTC ATATATCTTG CCTTACTTTT GGAAGAGAGA CTGTACTTGA ATATTTGGCC 360TCTTTCGGAG TGTGGATTCG CACTCCTCCA GCCTATAGAC CACCAAATGC CCCTATCTTA 420TCAACACTTC CGGAAACTAC TGTTGTTAGA CGACGGGACC GAGGCAGGTC CCCTAGAAGA 480AGAACTCCCT CGCCTCGCAG ACGCAGATCT CAATCGCCGC GTCGCAGAAG ATCTCAATCT 540CGGGAATCTC AATGTTAG 558__________________________________________________________________________ TABLE 3__________________________________________________________________________ATGGCTATTG ACCCTTATAA AGAATTTGGA GCTACTGTGG AGTTACTCTC GTTTTTGCCT 60TCTGACTTCT TTCCTTCCGT CAGAGATCTC CTAGACACCG CCTCAGCTCT GTATCGGGAA 120GCCTTAGAGT CTCCTGAGCA TTGCTCACCT CACCATACCG CACTCAGGCA AGCCATTCTC 180TCGTGGGGGG AATTGATGAC TCTAGCTACC TGGGTGGGTA ATAATTTGGA AGATCCAGCA 240TCAAGGGACC TAGTAGTCAA TTATGTTAAT ACTAACATGG GTTTAAAAAT TAGGCAACTA 300TTGTGGTTTC ATATATCTTG CCTTACTTTT GGAAGAGAGA CTGTACTTGA ATATTTGGTC 360TCTTTCGGAG TGTGGATTCG CACTCCTCCA GCCTATAGAC CACCAAATGC CCCTATCTTA 420TCAACACTTC CGGAAACTAC TGTTGTTAGA CGACGGGACC GAGGCAATCC CCTAGAAGAA 480GAACTCCCTC GCCTCGCAGA CGCAGATCTC AATCGCCGCG TCGCAGAAGA TCTCAATCTC 540GGGAATCTCA ATGTTAGAAG CTTCCGACAA AACCGCCTAC TCTCTTCTAA AAGTCGGACT 600ATGTCTAATT TAGTCTTGCG TCTTCGCCAG ACTATTTTGT CTTAA 645__________________________________________________________________________ TABLE 4__________________________________________________________________________ATGGCTATTG ACCCTTATAA AGAATTTGGA GCTACTGTGG AGTTACTCTC GTTTTTGCCT 60TCTGACTTCT TTCCTTCCGT CAGAGATCTC CTAGACACCG CCTCAGCTCT GTATCGGGAA 120GCCTTAGAGT CTCCTGAGCA TTGCTCACCT CACCATACCG CACTCAGGCA AGCCATTCTC 180TGCTGGGGGG AATTGATGAC TCTAGCTACC TGGGTGGGTA ATAATTTGGA AGATCCAGCA 240TCAAGGGACC TAGTAGTCAA TTATGGTAAT ACTAACATGG GTTTAAAAAT TAGGCAACTA 300TTGTGGTTTC ATATATCTTG CCTTACTTTT GGAAGAGAGA CTGTACTTGA ATATTTGGTC 360TCTTTCGGAG TGTGGATTCG CACTCCTCCA GCCTATAGAC CACCAAATGC CCCTATCTTA 420TCAACACTTC CGGAAACTAC TGTTGTTAGA CGACGGGACC GAGGCAGGTC CCCTAGAAGA 480AGAACTCCCT CGCCTCGCAG ACGCAGATCT CAATCGCCGC GTCGCAGAAG ATCTCAATCT 540CGGGAATCTC AATGTTAG 558__________________________________________________________________________ TABLE 5__________________________________________________________________________ATGGCTATTG ACCCTTATAA AGAATTTGGA GCTACTGTGG AGTTACTCTC GTTTTTGCCT 60TCTGACTTCT TTCCTTCCGT CAGAGATCTC CTAGACACCG CCTCAGCTCT GTATCGGGAA 120GCCTTAGAGT CTCCTGAGCA TTGCTCACCT CACCATACCG CACTCAGGCA AGCCATTCTC 180TGCTGGGGGG AATTGATGAC TCTAGCTACC TGGGTGGGTA ATAATTTGGA AGATCCAGCA 240TCAAGGGACC TAGTAGTCAA TTATGTTAAT ACTAACATGG GTTTAAAAAT TAGGCAACTA 300TTGTGGTTTC ATATATCTTG CCTTACTTTT GGAAGAGAGA CTGTACTTGA ATATTTGGCC 360TCTTTCGGAG TGTGGATTCG CACTCCTCCA GCCTATAGAC CACCAAATGC CCCTATCTTA 420TCAACACTTC CGGAAACTAC TGTTGTTAGA CGACGGGACC GAGGCAGGTC CCCTAGAAGA 480AGAACTCCCT CGCCTCGCAG ACGCAGATCT CAATCGCCGC GTCGCAGAAG ATCTCAATCT 540CGGGAATCTC AATGTTAG 558__________________________________________________________________________ TABLE 6__________________________________________________________________________ATGGCTATTG ACCCTTATAA AGAATTTGGA GCTACTGTGG AGTTACTCTC GTTTTTGCCT 60TCTGACTTCT TTCCTTCCGT CAGAGATCTC CTAGACACCG CCTCAGCTCT GTATCGGGAA 120GCCTTAGAGT CTCCTGAGCA TTGCTCACCT CACCATACCG CACTCAGGCA AGCCATTCTC 180TGCTGGGGGG AATTGATGAC TCTAGCTACC TGGGTGGGTA ATAATTTGGA AGATCCAGCA 240TCAAGGGACC TAGTAGTCAA TTATGTTAAT ACTAACATGG GTTTAAAAAT TAGGCAACTA 300TTGTGGTTTC ATATATCTTG CCTTACTTTT GGAAGAGAGA CTGTACTTGA ATATTTGGTC 360TCTTTCGGAG TGTGGATTCG CACTCCTCCA GCCTATAGAC CACCAAATGC CCCTATCTTA 420TCAACACTTC CGGAAACTAC TGTTGTTAGA CGACGGACCG AGGCAGGTCC CCTAGAAGAA 480GAACTCCCTC GCCTCGCAGA CGCAGATCTC AATCGCCGCG TCGCAGAAGA TCTCAATCTC 540GGGAATCTCA ATGTTAGAAG CTTCCGACAA AACCGCCTAC TCTCTTCTAA AAGTCGGACT 600ATGTCTAATT TAGTCTTGCG TCTTCGCCAG ACTATTTTGT CTTAA 645__________________________________________________________________________ TABLE 7__________________________________________________________________________ ##STR1## ##STR2## ##STR3## ##STR4## ##STR5## ##STR6## ##STR7## ##STR8## ##STR9## ##STR10## ##STR11## ##STR12##__________________________________________________________________________ TABLE 8__________________________________________________________________________ ##STR13## ##STR14## ##STR15## ##STR16## ##STR17## ##STR18## ##STR19## ##STR20## ##STR21## ##STR22## ##STR23## ##STR24##__________________________________________________________________________ TABLE 9__________________________________________________________________________ ##STR25## ##STR26## ##STR27## ##STR28## ##STR29## ##STR30## ##STR31## ##STR32## ##STR33## ##STR34## ##STR35## ##STR36##__________________________________________________________________________ TABLE 10__________________________________________________________________________ ##STR37## ##STR38## ##STR39## ##STR40## ##STR41## ##STR42## ##STR43## ##STR44## ##STR45## ##STR46## ##STR47## ##STR48## ##STR49## ##STR50##__________________________________________________________________________ TABLE 11__________________________________________________________________________ ##STR51## ##STR52## ##STR53## ##STR54## ##STR55## ##STR56## ##STR57## ##STR58## ##STR59## ##STR60## ##STR61## ##STR62## ##STR63## ##STR64##__________________________________________________________________________

By only varying the number of bases (i.e., single nucleotides or base pairs referring to pairs of complementary nucleotides) between the ribosomal binding site and the start codon without the second codon change, an increase in expression is also obtainable. Preferably the spacing (i.e. spacer) between the ribosomal binding site and the start codon is between 8 and 20 nucleotides. Most preferably 10 to 15 nucleotides are between the ribosomal binding site and the start codon. Sequence ID No: 13 and Sequence ID No: 14 are good examples of a 10 base pair and 8 base pair spacer, respectively. The particular nucleotide composition chosen to vary the distance between the ribosomal binding site and the start codon can vary. Preferably the nucleotides are chosen to have a high degree of homology with the same region between the ribosomal binding sites and the start codons of the host organisms.

Although the particular modification of the present invention at the second codon utilizes GCT as the second codon, the particular modification of the second codon is not limited to only GCT. For example, any codon utilized by a large number of the host organisms can be engineered into the second codon region. Preferred E. coli codons in the second position are AAA, AGC, GCG, AAC, TCT, AAT, ACC, AGT, ACA, GCT, ACT, CAA, GCA, GAA, GGT and ATC. It is unexpected that varying the second codon alone results in increased expression. As evident from FIG. 1, changing the second codon results in a two-fold increase in expression over the native sequence.

Since the modification of the gene at the second codon is especially beneficial for enhancement and expression in the E. coli host, an E. coli host is preferred.

Although any single alteration of the invention (e.g., spacer between the ribosomal binding site and start codon, second codon change, and deletion of basepair 455 or 468) in the DNA molecule results in increased expression of about two (2) fold over the native sequence, if any two alterations are made in combination, about a ten (10) fold increase in expression is obtained. If all three alterations are made in combination, about a twenty-five (25) fold increase in expression is obtained. Importantly, the antigenicity of the HBcAg is not compromised by the alterations. Even the deletion of base pair 455 or 468, which creates a frame shift at the C-terminus of the HBcAg protein, does not alter the antigenicity of the protein.

Sequence ID No: 1 was originally amplified from an HBsAg positive plasma using appropriate primers and standard protocols. Now that the sequence is known, the HBcAg sequence can be prepared in a variety of ways and therefore is not limited to any particular preparation means.

For example, the nucleotide sequences of the invention can be prepared by use of recombinant DNA technology or, alternatively, by automated synthesis. The sequences of the invention can also be cloned in a suitable vector and amplified in a suitable host.

Also, sequences of the invention can be synthesized using commercially available methods and equipment. For example, the solid phase phosphotriester method can be used to produce the sequences of the invention. The sequences can be conveniently synthesized by the modified phosphotriester method using fully protected DNA building blocks. Such synthetic methods can be carried out in substantial accordance with the procedure of Itakura, et al., 1977, Science 198:1056 and Crea, et al., 1978, Proc. Nat. Acad. Sci. U.S.A., 75:575, and Narang, et al., 1980, Methods in Enzymology, 68:90. In addition to manual procedures the sequences can be synthesized using automated synthesizers.

Methods for solution and solid phase synthesis are widely known, and various commercially available automatic synthesizers can be used in accordance with known protocols. See, for example Stewart & Young, Solid Phase Peptide Synthesis 2d Edition, Pierce Chemical Company, 1984; Tam, et al., J. Am. Chem. Assoc. 105:6442 (1983) and Merrifield, et al., Biochemistry 21:5020 (1982), which are incorporated herein by reference. The sequences of the invention can be produced by a number of procedures, including synthetic DNA synthesis, cDNA cloning, genomic cloning, polymerase chain reaction (PCR) technology, or a combination or these approaches. See, e.g., Maniatis, MOLECULAR CLONING, A LABORATORY MANUAL, 1982. Mutagenesis procedures to produce "deletion mutants" that encode the desired sequence can also be employed. Procedures suitable for producing such mutants include polymerase chain reaction technology and variations thereof, or site specific mutagenesis procedures similar to those of Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985) or Eckstein, et al., Nucleic Acids Res. 13:8764 (1985).

For cloning, a selected DNA sequence of the invention will be inserted into a suitable vector. A number of suitable vectors may be used, including cosmids, plasmids, bacteriophage and viruses. The principal requirements for such a vector are that it be capable of reproducing itself and stably transforming a host cell. Most preferably, the vector will comprise an "expression vector" which is capable of directing "expression" or cellular production of a peptide encoded by the DNA sequence of the invention. Typical expression vectors comprise a promoter region, a 5' untranslated region, a coding sequence, a 3' untranslated region, an origin of replication, a selective marker and a transcription termination site.

Suitable vectors for use in practicing the invention include pKK233-2 (Pharmacia), pKK223-2 (Pharmacia), pTTQ18 (Amersham, Arlington Heights, Ill.), pBTacl (Boehringer Mannheim), pET expression systems (Novagen, Madison, Wis.), and pPL-.lambda. (Pharmacia).

Promoters for use in expression vectors include, lactose (lac) control elements, lambda (PL) control elements, arabinose control elements, tryptophan (trp) control elements, and hybrids thereof. In addition, the vector may contain any one of a number of various markers facilitating selection of a transformed host cell. Such markers include genes associated with temperature sensitivity, drug resistance (e.g., resistance to ampicillin, chloramphenicol or tetracycline), or enzymes associated with visual characeristics of the host organism.

The vector may be prepared for insertion of the DNA sequence in a number of ways. Most simply, both the DNA sequence and the vector are digested with an appropriate set of restriction enzymes to generate sites suitable for ligation of the selected DNA sequence into the vector in an orientation and position suitable to allow expression of the peptide encoded by the sequence. Then, the DNA sequence is integrated into the vector by any of a number of suitable procedures, including treatment with a selected ligase.

After the sequence has been integrated into the vector, the vector may be used to transform a host cell. In general, the host cell may comprise any cellular organism including a prokaryotic cell or eukaryotic cell that is transformed with or competent of becoming transformed with the vector comprising the sequences of the present invention. Suitable host cells include, for example, bacterial cells such as E. coli or B. subtilis, mammalian cells (Kaufman, High Level Production of Proteins in Mammalian Cells, Genetic Engineering Principles and Methods, ed. J. K. Setlow Plenum Press, 9:155, 1988), yeast (Barr, et al., Yeast Genetic Engineering, eds. Butterworth, Boston, 1989), and insect cells (Maeda, Expression of Foreign Genes in Insects using Baculovius Vectors, T. E. Mittler eds. Annual Review of Entomology 34:351, 1989). A number of transformation techniques suitable for use with the particular vector-host cell combination may be employed. See, e.g., Maniatis, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories (1982).

For production of HBcAg product, the transformed host cell is cultured in a suitable medium under conditions designed to allow maximal expression with the particular combination of host and vector employed. By transforming hosts with sequences of the invention, greater amounts of HBcAg product is obtained.

Sequences of the invention can be used in methods and kits designed to detect the presence of antibodies in humans and therefore recognize Hepatitis B virus (HBV) infected humans and blood samples which have been infected by the hepatitis virus.

For example, the HBcAg produced by hosts transformed by recombinant DNA molecules of this invention or synthetically, can be used in the immunological diagnostic tests currently available for hepatitis B virus detection, such as radioimmunoassay or ELISA (enzyme linked immunosorbent assay). In one type of radioimmunoassay anti-core antigen antibody, raised in a laboratory animal, is attached to a solid phase, such as the inside of a test tube. HBcAg is then added to the tube so it can bind with the antibody. To the tube coated with the antigen-antibody complex is added a sample of the patient's serum, together with a known amount of HBV anti-core antibody labelled with a radioactive isotope such as radioactive iodine. Any HBV antibody in the patient's serum will compete with the labelled antibody for the free binding sites on the antigen-antibody complex. Once the serum has been allowed to interact, the excess liquid is removed, the test tube washed, and the amount of radioactivity measured. A positive result (i.e., that the patient's serum contains HBV antibody) is indicated by a low radioactive count. In one type of ELISA test, a microtitre plate is coated with HBcAg and a sample of a patient's serum is added. After a period of incubation permitting interaction of any antibody with the antigen, the plate is washed and a preparation of anti-human antibodies, raised in a laboratory animal, and which are linked to an enzyme label, is added, incubated to allow reaction to take place, and the plate rewashed. Thereafter, enzyme substrate is added to the microtitre plate and incubated for a period of time to allow the enzyme to work on the substrate, and the adsorbance of the final preparation is measured. A large change in adsorbance indicates a positive result.

The following examples illustrate the specific embodiments of the invention described in this document. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.

EXAMPLES

Materials and Preparation

Amplification of Core Gene

Polymerase chain reactions (PCR) are set up as follows:

______________________________________H.sub.2 O 21.5 .mu.lPCR 10X Buffer* 10.0 .mu.l1.25 mM dNTPs 16.0 .mu.l20 .mu.M Forward Primer 1.0 .mu.l20 .mu.M Reverse Primer 1.0 .mu.lHBV DNA 50.0 .mu.lP/E Amplitaq 0.5 .mu.l 100.0 .mu.l*PCR 10X Buffer: 30 cycles: 95 degrees C. - 2.0 minutes500 mM KCl 50 degrees C. - 1.5 minutes100 mM Tris-HCl (pH 8.3) 72 degrees C. - 1.5 minutes15 mM MgCl.sub.20.1% gelatin______________________________________

Oligonucleotide Primer Design and Synthesis

Oligonucleotide primers are synthesized on an Applied Biosystems, Inc. (ABI) DNA Synthesizer Model 381-A (Foster City, Calif.), or equivalent, according to manufacturer's specifications. "Trityl-On" oligonucleotides are purified using ABI "OPC" purification columns or equivalent according to manufacturer's protocol. One ml of eluent is dried in a Savant Speed-Vac, or equivalent, and resuspended in 100 .mu.l of 10 mM Tris-HCl (pH 7.2), 1 mM EDTA. A 20 .mu.M Stock solution of each primer is made for use in the PCR.

The following is the list of oligo primers for amplifying the core gene for cloning and expression.

__________________________________________________________________________Forward primers for expression in pKK233"native" (Seq ID No: 15) GGCC ATG GAC ATT GAC CCT TAT AAA2nd codon ##STR65## GGAForward primers for expression in pKK223 EcoRI10 bp spacer (Seq ID No: 17) CCGAATTC ATG GAC ATT GAC CCT TAT AAA2nd + 10 bp ##STR66##11 bp (Seq ID No: 19) CGGAATTCG ATG GAC ATT GAC CCT TAT AAA12 bp (Seq ID No: 20) CGGAATTCGG ATG GAC ATT GAC CCT TAT AAA13 bp (Seq ID No: 21) CGGAATTCGGA ATG GAC ATT GAC CCT TAT AAA14 bp (Seq ID No: 22) CGGAATTCGGAT ATG GAC ATT GAC CCT TAT AAA15 bp (Seq ID No: 23) CGGGAATTCGGATC ATG GAC ATT GAC CCT TAT AAAReverse primer for both pKK233 and pKK223 HindIII (Seq ID No: 24) GGAAGCTT CTA ACA TTG AGA TTC CCG AGA TTG AGA TCT TCT GCG__________________________________________________________________________

EXAMPLE 1

Isolation of Hepatitis B DNA

Using a protocol in substantial accordance with I. Baginski, et. al. "Detection of Hepatitis B Virus", In PCR Protocols, edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, 348-355, Academic Press, Inc. (1990). Hepatitis B virus (HBV) DNA is isolated from an HBsAg positive serum (sample #2791-19A obtained from Interstate Blood Bank, Inc., Memphis, Tenn.) as follows:

To about 60 .mu.l of HBsAg positive serum is added about 75 .mu.l of 250 .mu.g/ml proteinase K (Boehringer Mannheim, Indianapolis, Ind.) in: 0.25% SDS, 5 mM EDTA, 10 mM Tris-HCl (pH 8.0). This solution is incubated for about 2 hours at about 56 degrees C. The proteinase K is then heat inactivated at about 95 degrees C. for about 10 minutes. The total volume is brought to 1215 .mu.l in 1X Taq Polymerase buffer: 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 2.5 mM MgCl.sub.2, 0.01% gelatin (w/v), 0.5% Tween 20, 0.5% NP 40. Fifty microliters of this prepared DNA is used in subsequent polymerase chain reactions (PCRs) for generating HBcAg clones.

EXAMPLE 2

Cloning of Amplified HBcAg Gene Fragment

A PCR is set up using primers Sequence ID No: 15 and Sequence ID No: 24 to generate the first core gene fragment to be cloned. The resulting .about.570 bp fragment and the expression vector pKK233-2 (Pharmacia) are doubly digested with NcoI/HindIII (BRL). A ligation is set up with T4 ligase (BRL). Using a method in substantial accordance witin the teaching of D. H. Hanahan, "Techniques for Transformation of E. coli", In DNA cloning volume I, a practical approach, edited by D. M. Glover, 109, IRL Press Limited (1985), frozen competent E. coli strain XL1Blue (Stratagene) is transformed with the ligation, spreading 100 .mu.l of the transformation mix on LB (100 .mu.g/ml ampicillin) plates. Transformants are screened for inserts and those containing the desired fragment are used in expression experiments.

EXAMPLE 3

Expression of Core Protein

Cultures (12.5 ml) LB (100 .mu.g/ml ampicillin) are grown to mid log then induced with isopropylthio-.beta.-D-galactoside (IPTG) to a final concentration of 1 mM. Cultures are allowed to grow overnight, approximately 16 hours. 1.5 ml of the culture is removed, spun down at full speed in a Brinkman microfuge or equivalent. The pellet is resuspended in 100 .mu.l 1X sample prep buffer (U. K. Laemmli, Nature 227:680 (1970), boiled about 5 minutes and 10 .mu.l run on a 12% SDS PAGE gel at 40 mA for about 11/4 hour. A Western blot is prepared by transferring proteins from the gel to Immobilon-P membrane (Millipore, Bedford, Mass.), or equivalent at 100 mA in a Hoefer Semidry Transfer apparatus or equivalent for 1 hour using Towbin buffer (25 mM Tris (pH 8.3), 192 mM Glycine, 15% methanol). After transfer, membrane is blocked with 5% BSA in 1X Tris buffered saline (TBS) (10 mM Tris-HCl, (pH 8.0), 0.9% NaCl) for 30 minutes. An anti-HBcAg polyclonal antibody (this antibody solution in phosphate buffered saline (PBS)+a carrier protein, is a component of a kit, but is sold separately, this antibody concentration is unknown) (BioGenex Laboratories, Dublin, Calif.--catalog #PA082-5P) is incubated with the membrane for 1 hour. Membrane is washed 5X with TBS. Membrane was then incubated with a goat anti-rabbit horseradish peroxidase conjugated polyclonal antibody (Cappel Organonteknika, West Chester, Pa.) at 1:1000 in the 5% bovine serum albumin (BSA)/TBS solution for one hour. Membrane is washed 5X with TBS then developed with 4-chloro-1-naphthol and hydrogen peroxide. Clones expressing HBcAg show the appropriate size reactive band at .about.21 KDa. However, all show very low levels of expression.

EXAMPLE 4

Increasing Expression Levels

Oligonucleotide primers are synthesized (see "Materials and Preparations" above) to allow about 10 to 15 bp between the ribosome binding site and the ATG start codon. PCRs are set up with the following primer sets using the method previously described: Sequence ID No: 17--Sequence ID No: 24, Sequence ID No: 19--Sequence ID No: 24, Sequence ID No: 20--Sequence ID No: 24, Sequence ID No: 21--Sequence ID No: 24, Sequence ID No: 22--Sequence ID No: 24, and Sequence ID No: 23--Sequence ID No: 24. All PCRs generate the appropriate .about.570 bp fragment. These fragments are doubly digested with EcoRI and Hind III and directionally cloned into pKK223-3 (Pharmacia, Piscataway, N.J.) as described above. Transformations are set up as previously described.

LB plates are spread with 50 .mu.l of 1M IPTG and a membrane sandwich placed on the surface of the agar. The sandwich is prepared by placing a nitrocellulose membrane (BA 85; Schleicher & Schuell, Inc., Keene, N.H.) or equivalent, on top of the agar medium and then a cellulose acetate membrane (OE67; Schleicher & Schuell), or equivalent, on top of that. Transformations are then plated as before and plates incubated at about 37 degrees C. overnight. The nitrocellulose filters are removed and blocked for about 1 hour in 5% BSA/TBS. An anti-HBcAg monoclonal antibody is added at 20 .mu.g/ml and incubated for about 1 hour. Membranes are washed with TBS a secondary goat anti-mouse horseradish conjugated antibody (Cappel) is added at 1:2000 in 5% BSA/TBS for about 1 hour. Membranes are washed with TBS and developed with 4-chloro-1-naphthol and hydrogen peroxide. Reactive colonies are patched out for a second direct colony immunoblot (DCI) and again chosen for reactivity. The 10 bp spacer clones are chosen for further analysis and quantitation in a sandwich ELISA.

Two oligo primers Sequence ID No: 16 and Sequence ID No: 18 (see "materials and Preparation",) are designed to change the HBcAg second codon from GAC to GCT. Primer Sequence ID No: 16 changes the second codon with the fragment to be cloned into pKK233-2 while primer Sequence ID No: 18 allows for the 10 bp spacer as well as the second codon change. PCRs are set up and fragments cloned as previously mentioned. DCI are set up for screening transformants. Reactive colonies are chosen for further analysis and quantitation in a sandwich ELISA.

EXAMPLE 5

Quantitation for Increased Expression of HBcAg

Strains tested by ELISA

______________________________________Strain Vector Fragment______________________________________Sequence ID No: 1 pKK233-2 HBcAg DNA sequenceSequence ID No: 3 pKK223-3 10 bp spacerSequence ID No: 5 Pkk223-3 10 bp spacer + 2nd codon changeControl pKK223-3 No insert______________________________________ 3 ml overnight cultures (LB--100 .mu.g/ml Ampicillin) of each of the above stains are grown at about 37 degrees C. 250 .mu.l of the overnight culture (1:50) is used to inoculate 12.5 ml cultures (same medium). Cultures are grown to 0.6-0.8 A.sub.600 then induced by adding 1M IPTG to .about.1 mM. Cultures are allowed to grow overnight (about 19 hours). One O.D. unit (A.sub.600) of cells from each culture is removed and spun down and resuspended in 975 ul of PBS to which 10 .mu.l of 10 .mu.g/ml lysozyme (Sigma, St. Louis, Mo.) in PBS was added. Each is frozen in liquid nitrogen and thawed at about 37 degrees C. 3 times. 1M MgCl.sub.2 is added to 5 mM (5 .mu.l) and 10 .mu.l of 1 mg/ml DNase (Sigma) is added and then the solution is incubated at room temperature for about 10 minutes. Lysates are spun at .about.7000 xg in microfuge for about 10 minutes. The supernatant fraction is removed and used in the ELISA.

One hundred microliters of "capture" antibody is coated onto plates at 20 .mu.g/ml in phosphate buffered saline (PBS) (10 mM Phosphate (pH 7.2), 130 mM NaCl) at about 4 degrees C. overnight. Capture antibody solution is removed and the wells blocked with 2.5% BSA in PBS for about 1 hour. Fifty microliters of serial two fold dilutions of E. coli lysates (in 2.5% BSA/PBS) from 1:8 down to 1:16,000 are placed in wells and incubated for about 1 hour. Wells are washed 5 times with PBS+0.05% Tween 20 (PBS-Tween). Fifty microliters of biotinylated detector antibody is added at 5 .mu.g/ml in 2.5% BSA/PBS to each well and incubated for about 1 hour. Plates are washed 5 times with PBS-Tween. Fifty microliters of Avidin-HRP (1 mg/ml) (Sigma) diluted 1:2000 in 2.5% BSA/PBS are added to each well and incubated for about 1 hour. Plates are washed 5 times with PBS Tween, and developed with 50 .mu.l of o-phenylenediamine dihydrochloride (OPD) (20 mg OPD, 100 mM citrate (pH 5.5), 7 .mu.l 30% hydrogen peroxide). After two minutes the reaction is stopped by adding about 50 .mu.l of 4.5M sulphuric acid. Plates are read at A.sub.490. Relative quantities of HBcAg in each lysate is calculated against a standard curve generated with purified HBcAg from 20 .mu.g to 0.02 ng per well (2 fold serial dilution).

Although the invention has been described with respect to specific modifications, the details thereof are not to be construed as limitations, for it will be apparent that various equivalents, changes and modifications may be resorted to without departing from the spirit and scope thereof, and it is understood that such equivalent embodiments are to be included therein.

__________________________________________________________________________SEQUENCELISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 24(vi) CURRENT APPLICATION DATA:(A) APPLICATION NUMBER:(B) FILING DATE:(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 558 base pairs(B) TYPE: nucleic acid(C ) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..558(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC48MetAspIleAspProTyrLysGluPheGl yAlaThrValGluLeuLeu151015TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC96SerPheLeuProSerAspPhePheProS erValArgAspLeuLeuAsp202530ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC144ThrAlaSerAlaLeuTyrArgGluAlaLeu GluSerProGluHisCys354045TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAA192SerProHisHisThrAlaLeuArgGlnAlaIleLeu CysTrpGlyGlu505560TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspPr oAla65707580TCAAGGGACCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAA288SerArgAspLeuValValAsnTyrValAsnThrAsnMetG lyLeuLys859095ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThr PheGlyArg100105110GAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACT384GluThrValLeuGluTyrLeuValSerPheGlyValTrpIle ArgThr115120125CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPr o130135140GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg145 150155160AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528ArgThrProSerProArgArgArgArgSerGlnSerProArgArgArg 165170175AGATCTCAATCTCGGGAATCTCAATGTTAG558ArgSerGlnSerArgGluSerGlnCys180 185(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 185 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu1 51015SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp202530ThrAlaSerAlaLeuTyrArgGlu AlaLeuGluSerProGluHisCys354045SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu505560LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys 859095IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100105110GluThrValLeuG luTyrLeuValSerPheGlyValTrpIleArgThr115120125ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro130135 140GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg145150155160ArgThrProSerProArgArgArgArgSerGlnSerPro ArgArgArg165170175ArgSerGlnSerArgGluSerGlnCys180185(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 558 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..558(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTAGAGTTACTC48MetAspIleAs pProTyrLysGluPheGlyAlaThrValGluLeuLeu151015TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC96SerPheLeuP roSerAspPhePheProSerValArgAspLeuLeuAsp202530ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC144ThrAlaSerAla LeuTyrArgGluAlaLeuGluSerProGluHisCys354045TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAA192SerProHisHisThrAla LeuArgGlnAlaIleLeuCysTrpGlyGlu505560TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240LeuMetThrLeuAlaThrTrpValGl yAsnAsnLeuGluAspProAla65707580TCAAGGGATCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAA288SerArgAspLeuValValAsnT yrValAsnThrAsnMetGlyLeuLys859095ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336IleArgGlnLeuLeuTrpPhe HisIleSerCysLeuThrPheGlyArg100105110GAGACTGTACTTGAATATTTGGCCTCTTTCGGAGTGTGGATTCGCACT384GluThrValLeuGluTyrLeuAla SerPheGlyValTrpIleArgThr115120125CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432ProProAlaTyrArgProProAsnAlaPr oIleLeuSerThrLeuPro130135140GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480GluThrThrValValArgArgArgAspArgGlyArgS erProArgArg145150155160AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528ArgThrProSerProArgArgArgArgSerGln SerProArgArgArg165170175AGATCTCAATCTCGGGAATCTCAATGTTAG558ArgSerGlnSerArgGluSerGlnCys 180185(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 185 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLe uLeu151015SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp202530ThrAla SerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu50 5560LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580SerArgAspLeuValValAsnTyrValAsnThr AsnMetGlyLeuLys859095IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100105 110GluThrValLeuGluTyrLeuAlaSerPheGlyValTrpIleArgThr115120125ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro130 135140GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg145150155160ArgThrProSerProArgArg ArgArgSerGlnSerProArgArgArg165170175ArgSerGlnSerArgGluSerGlnCys180185(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 645 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..645(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:ATGGCTATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC 48MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu151015TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC 96SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp202530ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC 144ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTCGTGGGGGGAA192 SerProHisHisThrAlaLeuArgGlnAlaIleLeuSerTrpGlyGlu505560TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240LeuMetTh rLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580TCAAGGGACCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAA288SerA rgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys859095ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336Ile ArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100105110GAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACT384GluThr ValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr115120125CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432ProProAlaTy rArgProProAsnAlaProIleLeuSerThrLeuPro130135140GAAACTACTGTTGTTAGACGACGGGACCGAGGCAATCCCCTAGAAGAA480GluThrThrValValArgA rgArgAspArgGlyAsnProLeuGluGlu145150155160GAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGAA528GluLeuProArgLeu AlaAspAlaAspLeuAsnArgArgValAlaGlu165170175GATCTCAATCTCGGGAATCTCAATGTTAGAAGCTTCCGACAAAACCGC576AspLeuAsnLeuGly AsnLeuAsnValArgSerPheArgGlnAsnArg180185190CTACTCTCTTCTAAAAGTCGGACTATGTCTAATTTAGTCTTGCGTCTT624LeuLeuSerSerLysSe rArgThrMetSerAsnLeuValLeuArgLeu195200205CGCCAGACTATTTTGTCTTAA645ArgGlnThrIleLeuSer 210215(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 214 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu 151015SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp202530ThrAlaSerAl aLeuTyrArgGluAlaLeuGluSerProGluHisCys354045SerProHisHisThrAlaLeuArgGlnAlaIleLeuSerTrpGlyGlu5055 60LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580SerArgAspLeuValValAsnTyrValAsnThrAsnM etGlyLeuLys859095IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100105110 GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr115120125ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro130 135140GluThrThrValValArgArgArgAspArgGlyAsnProLeuGluGlu145150155160GluLeuProArgLeuAlaAspAlaAs pLeuAsnArgArgValAlaGlu165170175AspLeuAsnLeuGlyAsnLeuAsnValArgSerPheArgGlnAsnArg180185 190LeuLeuSerSerLysSerArgThrMetSerAsnLeuValLeuArgLeu195200205ArgGlnThrIleLeuSer210(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 558 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..558(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:ATGGCTATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC 48MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu151015TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC 96SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp202530ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC1 44ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAA192Ser ProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu505560TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240LeuMetThrLe uAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580TCAAGGGACCTAGTAGTCAATTATGGTAATACTAACATGGGTTTAAAA288SerArgA spLeuValValAsnTyrGlyAsnThrAsnMetGlyLeuLys859095ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336IleArg GlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100105110GAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACT384GluThrVal LeuGluTyrLeuValSerPheGlyValTrpIleArgThr115120125CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432ProProAlaTyrAr gProProAsnAlaProIleLeuSerThrLeuPro130135140GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480GluThrThrValValArgArgA rgAspArgGlyArgSerProArgArg145150155160AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528ArgThrProSerProArg ArgArgArgSerGlnSerProArgArgArg165170175AGATCTCAATCTCGGGAATCTCAATGTTAG558ArgSerGlnSerArgGlu SerGlnCys180185(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 185 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:MetAlaIleAspProTyrLysGluPheGl yAlaThrValGluLeuLeu151015SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp2025 30ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu 505560LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580SerArgAspLeuValVal AsnTyrGlyAsnThrAsnMetGlyLeuLys859095IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100 105110GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr115120125ProProAlaTyrArgProProAsnAlaProIleLeuSerTh rLeuPro130135140GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg145150155160ArgThr ProSerProArgArgArgArgSerGlnSerProArgArgArg165170175ArgSerGlnSerArgGluSerGlnCys180185(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 558 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..558(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:ATGGCTATTGACCCTTATAAAGAATTTGGAGCT ACTGTGGAGTTACTC48MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu151015TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTC AGAGATCTCCTAGAC96SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp202530ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTC TCCTGAGCATTGC144ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCT GGGGGGAA192SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu505560TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA 240LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580TCAAGGGACCTAGTAGTCAATTATGTTAATACTAACATGGGTTTA AAA288SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys859095ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGG AAGA336IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100105110GAGACTGTACTTGAATATTTGGCCTCTTTCGGAGTGTGGATTCGCA CT384GluThrValLeuGluTyrLeuAlaSerPheGlyValTrpIleArgThr115120125CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG 432ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro130135140GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480GluT hrThrValValArgArgArgAspArgGlyArgSerProArgArg145150155160AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528 ArgThrProSerProArgArgArgArgSerGlnSerProArgArgArg165170175AGATCTCAATCTCGGGAATCTCAATGTTAG558 ArgSerGlnSerArgGluSerGlnCys180185(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 185 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:MetAlaIleAs pProTyrLysGluPheGlyAlaThrValGluLeuLeu151015SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp20 2530ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045SerProHisHisThrAlaLeuArgGlnAlaIleL euCysTrpGlyGlu505560LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580 SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys859095IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg 100105110GluThrValLeuGluTyrLeuAlaSerPheGlyValTrpIleArgThr115120125ProProAlaTyrArgProProAs nAlaProIleLeuSerThrLeuPro130135140GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg145150155 160ArgThrProSerProArgArgArgArgSerGlnSerProArgArgArg165170175ArgSerGlnSerArgGluSerGlnCys180 185(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 645 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..645(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:ATGGCTATTGACCCTT ATAAAGAATTTGGAGCTACTGTGGAGTTACTC48MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu151015TCGTTTTTGCCTTCT GACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC96SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp202530ACCGCCTCAGCTCTGTAT CGGGAAGCCTTAGAGTCTCCTGAGCATTGC144ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045TCACCTCACCATACCGCACTCAG GCAAGCCATTCTCTGCTGGGGGGAA192SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu505560TTGATGACTCTAGCTACCTGGGTGGGTAATA ATTTGGAAGATCCAGCA240LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580TCAAGGGACCTAGTAGTCAATTATGTT AATACTAACATGGGTTTAAAA288SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys859095ATTAGGCAACTATTGTGGTTTCATATA TCTTGCCTTACTTTTGGAAGA336IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100105110GAGACTGTACTTGAATATTTGGTCTCTTT CGGAGTGTGGATTCGCACT384GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr115120125CCTCCAGCCTATAGACCACCAAATGCCCCTATCT TATCAACACTTCCG432ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro130135140GAAACTACTGTTGTTAGACGACGGACCGAGGCAGGTCCCCTA GAAGAA480GluThrThrValValArgArgArgThrGluAlaGlyProLeuGluGlu145150155160GAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGC GTCGCAGAA528GluLeuProArgLeuAlaAspAlaAspLeuAsnArgArgValAlaGlu165170175GATCTCAATCTCGGGAATCTCAATGTTAGAAGCTTCCG ACAAAACCGC576AspLeuAsnLeuGlyAsnLeuAsnValArgSerPheArgGlnAsnArg180185190CTACTCTCTTCTAAAAGTCGGACTATGTCTAATTTAGTCT TGCGTCTT624LeuLeuSerSerLysSerArgThrMetSerAsnLeuValLeuArgLeu195200205CGCCAGACTATTTTGTCTTAA 645ArgGlnThrIleLeuSer210215(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 214 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:MetAlaIleAspProTy rLysGluPheGlyAlaThrValGluLeuLeu151015SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp20 2530ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys354045SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysT rpGlyGlu505560LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla65707580SerArg AspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys859095IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg100 105110GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr115120125ProProAlaTyrArgProProAsnAlaPr oIleLeuSerThrLeuPro130135140GluThrThrValValArgArgArgThrGluAlaGlyProLeuGluGlu145150155 160GluLeuProArgLeuAlaAspAlaAspLeuAsnArgArgValAlaGlu165170175AspLeuAsnLeuGlyAsnLeuAsnValArgSerPheArgGlnAsnArg 180185190LeuLeuSerSerLysSerArgThrMetSerAsnLeuValLeuArgLeu195200205ArgGlnThrIleLeuSer210(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:AACAGAATTC10(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:AACAGACC8(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 25 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:GGCCATGGACATTGACCCTTATAAA25(2) INFORMATION FOR SEQ ID NO:16:( i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:GGCCATGGTCATTGACCCTTATAAAGAATTTGGA34(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:CCGAATTCATGGACATTGACCCTTATAAA29(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:CCGAATTCATGGTCATTGACCCTTATAAA29(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:CGGAATTCGATGGACATTGACCCTTATAAA30(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:CGGAATTCGGATGGACATTGACCCTTATAAA31(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:CGGAATTCGGAATGGACATTGACCCTTATAAA32(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:CGGAATTCGGATATGGACATTGACCCTTATAAA33(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 35 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D ) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:CGGGAATTCGGATCATGGACATTGACCCTTATAAA35(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 44 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GGAAGCTTCTAACATTGAGATTCCCGAGATTGAGATCTTCTGCG44

Claims

1. A vector comprising a DNA molecule having a sequence consisting essentially of Sequence ID No: 3, Sequence ID No: 5, Sequence ID No: 7, Sequence ID No: 9, or Sequence ID No: 11.

2. The vector of claim 1 in which said sequences further comprises about an 8 to 20 base pair spacer before the initiation codon.

3. The vector of claim 2 in which said spacer consists essentially of Sequence ID No: 13.

4. The vector of claim 2 in which said spacer consists essentially of Sequence ID No: 14.

5. The vector of claim 1 in which the second codon of said sequence is a host preferred codon.

6. The vector of claim 2 in which the second codon of said sequence is a host preferred codon.

7. The vector of claim 3 in which the second codon of said sequence is a host preferred codon.

8. The vector of claim 4 in which the second codon of said sequence is a host preferred codon.

9. The vector of claim 5 in which said host preferred codon is an E. coli preferred codon.

10. The vector of claim 6 in which said host preferred codon is an E. coli preferred codon.

11. The vector of claim 7 in which said host preferred codon is an E. coli preferred codon.

12. The vector of claim 8 in which said host preferred codon is an E. coli preferred codon.

13. A host transformed with a vector comprising a DNA molecule having the sequence consisting essentially of Sequence ID No: 3, Sequence ID No: 5, Sequence ID No: 7, Sequence ID No: 9, or Sequence ID No: 11.

14. The host of claim 13 in which the vector further comprises about an 8 to 20 base pair spacer before the initiation codon of said sequences.

15. The host of claim 14 in which said spacer consists essentially of Sequence ID No: 13.

16. The host of claim 14 in which said spacer consists essentially of Sequence ID No. 14.

17. The host of claim 13 in which the second codon of said sequences are host preferred codons.

18. The host of claim 14 in which the second codon of said sequences are host preferred codons.

19. The host of claim 15 in which the second codon of said sequences are host preferred codons.

20. The host of claim 16 in which the second codon of said sequences are host preferred codons.

21. The host of claim 13 which is E. coli.

22. The host of claim 14 which is E. coli.

23. The host of claim 15 which is E. coli.

24. The host of claim 16 which is E. coli.