Patent Application
20080008722
The feature of the present invention is based on a finding that, when most structure proteins in ORF5 and ORF6 were removed, leaving dozens of N-terminal amino acids of ORF5 and ORF6, by which constructing a fusion peptide chain PQGAB, and then inserting the peptide chain between PE and KDEL3 sequence was possible, it was confirmed that the fusion protein PE-PQGAB-KDEL3 had serum neutralization titers by mice and porcine immunization tests.
The following examples are proposed to explain the present invention, but not set forth as to limit the scope thereof.
Protein sequences of ORF5 and ORF6 of PRRSV were obtained from the National Center for Biotechnology Information (NCBI, USA) database. It had been known based on aforementioned mechanisms of viral infections that regions of PRRSV having neutralization titers are each N-terminus of ORF5 and ORF6. That is, amino acids No. 2 to 26 of ORF6 structural protein (SEQ ID NO.13), and amino acids No. 30 to 63 of ORF5 structural protein (SEQ ID NO.12). The fused amino acid sequence of the two peptides are illustrated as follows:
The sequence of PRRSV-ORF6-2˜26-ORF5-31˜63 fusion peptide was the combination of (ORF6)-G2SSLDDFCYDSTAPQKVLLAFSITY26 (SEQ ID NO.13) and (ORF5)-A31SNDSSSHLQLIYNLTLCELNGTDWL ANKFDWA63 (SEQ ID NO.12) peptides, wherein the fragment GSSLDDFC is designated “P”, fragment YDSTAPQKVLLAFSITY “Q”, fragment ASNDSSSHLQLIYNLTLC “A”, and ELNGTDWLANKFDWA “B”. Fragment PQ is a portion of ORF6, and Fragment AB is a portion of ORF5. G is the gap or bridge of PQ and AB ploypeptides. G can be the 27th animo acid of ORF6 or any polypeptide fragment of ORF6 from 27th to any linked codons. The position G can also be not added any amino acid within the polypeptides of PQ and AB.
The example employs the PQGAB fusion peptide region to construct a key protein (epitope) capable of inducing neutralization titers and immune protection in order to obtain effects of inducing immune protection in vivo. The schematic illustration of PE-PQGAB-K3 fusion protein and the flowcharts of plasmids construction of PE(ΔIII)-PQGAB and PE(ΔIII)-PQGAB-K3 are shown in
The preparation of nucleic sequence encoding PQGAB peptide is illustrated below. An amino acid corresponds to various sets of nucleotide triplets, so it is preferable to obtain corresponding nucleotide triplets from literature (such as http://www.kazusa.orjp/codon) that is suitable to be expressed in the E. coli system instead of the corresponding nucleotide triplets not easy to be recognized and expressed by E. coli. Likewise, if the sequence encoding PQGAB peptide is to be expressed in yeast systems, the appropriate nucleotide triplets for expression in yeast systems (such as Saccharomycesor Pichia spp.) are preferred.
A corresponding sequence with nucleotide triplets suitable to be expressed in E. Coli system was designated according to the amino acid sequence of PQGAB fusion protein. The 5′ and 3′ ends of the corresponding sequence were added by restriction sites for subsequent cloning. To improve efficiency of digestion and facilitate designing PCR primers, both ends of the sequence could be added to with nucleotide triplets with replicating bases, such as CCC, AAA, GGG, or TTT. The nucleic acid sequence encoding PQGAB fusion protein is illustrated in SEQ ID NO.1.
There are totally 207 nucleotides in SEQ ID NO.1, and when it was cloned into a plasmid by restriction enzymes, some of the nucleotide triplets were cut off, leaving 180-186 nucleotides linked to the plasmid.
When the target nucleic acid sequence encoding PQGAB fusion protein was identified, the restriction map of the nucleic acid sequence was analyzed by DNA Strider before synthesis, and then each end of the target sequence was linked to restriction site sequences for subsequent cloning, in accordance with the restriction map. The synthesized product of the target sequence must be digested by certain restriction enzymes before cloning, so it is preferable that any restriction site susceptible to the enzymes used be avoided in the structural region of the sequence. If the restriction sites subjected to cloning enzymes exist in the structural region of the target sequence, the target sequence must be re-designated such that different codons of the same amino acids were used, to eliminate restriction sites that were identical for cloning in the structural region of the target sequence.
Subsequently, the method disclosed in Taiwan Patent No. 1-2289933 (also as U.S. patent publication no. 2004/02147617) is used to modify the corresponding nucleotides codons of wild type amino acids sequence such that the wild type protein was mass expressed in by E. coli system. The essence of modification is to modify wild type nucleic acid sequence such that the normally expressed amino acids were not affected, and expression in E. coli was kept effective. The modified nucleic acid sequence can be synthesized by PCR using a variety of primer pairs. The primers are numbered as shown in Table 1.
The sequences of forward and reverse primers are shown as follows:
Forward primer F1 (SEQ ID No.2) corresponds to the amino acid 81st-124th of SEQ ID No.1, namely
Forward primer F2 (SEQ ID No.3) corresponds to the amino acid 48th-96th of SEQ ID No.1, namely
Forward primer F3 (SEQ ID No.4) corresponds to the amino acid 22nd-65th of SEQ ID No.1, namely
Forward primer F4(SEQ ID No.5) corresponds to the amino acid 1st-41st of SEQ ID No.1, namely
Reverse primer R1(SEQ ID No.6) corresponds to the amino acid 148th-106th of SEQ ID No.1, namely
Reverse primer R2(SEQ ID No.7) corresponds to the amino acid 176th-133rd of SEQ ID No.1, namely
Reverse primer R3(SEQ ID No.8) corresponds to the amino acid 204th-164th of SEQ ID No.1, namely
wherein R1, R2 and R3 were reversely complementary sequences of a gene sequence.
The fragment synthesized with no DNA template was performed firstly. Forward primer F1 and reverse primer R1 were hybridized to each other, wherein 10-18 bases at 3′ ends of each primer were designed complementary to each other, and the resultant complex was read and complemented by polymerase so as to obtain a double-stranded DNA template product.
After the first round of PCR, 0.01˜4 μl of the PCR product was taken as the template DNA of the second round of PCR, adding therein the second primer pair, i.e. forward primer F2 and reverse primer R2, 0.01˜4 μl each, in conjunction with needed dNTPs, reagents and Pfu polymerse, and the second round PCR was performed. Likewise, primer pair F3 and R3 were added therein and PCR was performed again; the procedures were repeated with primer pair F4 and R3, and thereby a modified PQGAB nucleic acid sequence having 207 bp was obtained.
The synthesized nucleic acid fragments were subjected to electrophoresis and confirmed that they had the expected sizes. PQGAB-1(207 bp), as shown in
The design of the fusion protein in example 1 and 2 aimed at American strain PRRSV, but apart from American strain PRRSV, European strain and Australian strain is also very prevalent globally. Similarity of structural amino acids is not high, only 60-80%, so designs of other ORF5&ORF6 fusion proteins can be done in the same manner as example 1 and 2 to design and synthesize primers.
Taking PQGAB of PRRSV European strain as the example, the amino acid sequence of the fusion domains is shown in SEQ ID NO.11. It contains ORF6-M1˜I28+ (SEQ ID NO.15), and ORF5-F31˜A64 (SEQ ID NO.14) of the PRRSV European strain.
After the sequence is confirmed, preparation of PRRSV European strain fusion proteins can be performed in the same manner as examples 1-2. The modified nucleic acid sequence can be synthesized by PCR using a variety of primer pairs. The primers are numbered as shown in Table 2.
The target nucleic acid sequence encoding PQGAB-EP fusion protein can be synthesized with those primers shown above in vitro, by following the procedure described in example 2. To improve efficiency of digestion and facilitate designing PCR primers, both ends of the sequence could be added to with nucleotide triplets with replicating bases, such as CCC, AAA, GGG, or TTT. The nucleic acid sequence encoding PQGAB-EP fusion protein is illustrated in SEQ ID NO.10.
Taking the product from example 2 as illustration. The synthesized 207-bp DNA fragment was digested with EcoR1 and Xho1, linked to a E.coli plasmid containing a peptide sequence having functions of binding and translocation, and a carboxyl terminus peptide, and the resultant plasmid was pPE-PQGAB-K3.
The pET15 plasmid having a T7 promoter and an antibiotic resistance(ampicillin) marker constructed therein can express the fusion protein of PRRSV PQGAB fragment and detoxified Pseudomonas exotoxin (Pseudomonas exotoxin A without domain III). The vector map is shown in
Finally, the above-mentioned plasmid was transformed into bacterial strains or cells capable of expressing the fusion proteins.
Bacterial strains confirmed having the above mentioned plasmid contained both the plasmid and PQGAB gene in 90% of the population. The strains were prepared as glycerol stocks in 2-ml aliquots and stored at −70° C. In a sterile environment, 2 ml of the stored stocks was inoculated in an autoclaved 500 ml flask containing 200 ml LB (+500 μg/ml Amp), shaken in a rotary incubator at 37° C., 150 rpm for 10˜12 hours, and a culture was obtained. The liquid was cultured until OD600 reached 1.0±0.4.
In a sterile environment, 50 ml culture liquid was inoculated in each of eight 3000-ml flasks containing 1250 ml LB (+500 μg/ml Amp +50 ml 10% Glucose), shaken at 37° C., 150 rpm for 2˜3 hours until OD 600 reached 0.3±0.1, 50 ppm IPTG was added, the culture was shaken again at 37° C., 150 rpm for 2 hours such that protein production was accomplished.
Then PE-PQGAB-K3 contained in inclusion bodies was dissolved by 8M urea extraction method, the extracted PE-PQGAB-K3 proteins are shown in
In an SPF farm, pigs were grouped randomly into 3 groups, each having five pigs. Each group was bred in an isolation room equipped with air conditioning and circulation instruments. For pigs of PE-PQGAB-K3 immunization group aged 14 to 28 days, 2 ml vaccine containing 1 ml PE-PQGAB-K3 (containing 200 μg proteins/injection) and emulsified in 1 ml ISA206 (SEPPIC®, France) was injected intramuscularly, and the procedure of immunization was performed twice. The immunization group GP5&M was immunized with PE-ORF5-K3 PE-ORF6-K3(containing 200 μg proteins/injection), respectively. The control group was bred without immunization.
Two weeks after the last inoculation, 100 mg ketamine solution was administered intramuscularly to tranquilize the pigs, then 1 ml 2% Lidocaine was dropped in the nasal cavities of the pigs to inhibit coughing reflex actions, and then the virus was inoculated in pigs nasally. Five pigs of each group were inoculated with 1 ml MD-1 strain PRRSV cultures having a 1×107 TCID50/ml dosage.
14 days after inoculation, the pigs were sacrificed to proceed with complete autopsy. Lung or liver samples were collected (from both parts of the head lobe central part and auxiliary part of caudate lobe) and fixed by 10% neutral buffered formaldehyde for subsequent tissue pathology examination. The examination was conducted in a blind fashion and evaluated on the basis of interstitial pneumonitis severity (Opriessnig T, P. G. Halbur, et al., Journal of Virology, 76(2002):11837-11844, and Halbur, P. G., P. S. Paul, et al., 1996. J. Vet. Diagn. Investig. 8:11-20) in a scale from 0 to 6, wherein the severity increases with the number.
Two weeks post second immunization, leukocytes from porcine blood were tested for PRRSV. The results indicated that viremia did not occur in any pig before PRRSV inoculating. The leukocyte samples were tested with RT-PCR at 3, 7, and 14 days post virus inoculating, respectively, and the results are shown in Table 3.
All pigs, including those that had been sacrificed and the surviving after the two-week study, were dissected. Macroscopic examinations indicated that the lungs from virus-inoculated pigs of ORF5&ORF6 vaccine group and the control group showed more extensive lesions and severe interstitial pneumonitis, whereas the PE-PQGAB-K3 vaccine group of the present invention did not show as extensive lesions and severe interstitial pneumonitis. As shown in Table 4, the PE-PQGAB-K3 vaccine group of the present invention showed less severity in terms of interstitial pneumonitis than the control group and ORF5&ORF6 vaccine group.
The above experiments clearly indicate that PE-PQGAB-K3 of the present invention not only can effectively protect pigs from PRRSV infections, but also cause slighter interstitial pneumonitis than other vaccines (such as PE-ORF5-K3, PE-ORF6-K3).
The antibody titers variation in immunized pigs are shown in table 6. The A group has good IgG ELISA titers, but the IFA and NT titers are less than that of C group. Also, from table 5, it indicates that PRRSV ORF5 or ORF6 have an antigen-specific allergy effect after immunization and virus challenged. Manifestly, it is difficult to use them as PRRS vaccine antigens.
Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the scope of the invention as hereinafter claimed.
