| Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine re-productive and respiratory syndrome virus(PRRSV), which is a kind of contagious diseases and can be infected with different age and breed of pigs, but PRRSV mainly infected sows and piglets and coused late pregnancy sow preterm birth, abortion, stillbirth. Consequently, the PRRSV caused great economic losses in pig industry in the world. It was the biggest characteristic that PRRSV can make the pig immunosuppression and secondary infection, at present, PRRSV vaccines for prevention and control of PRRSV is not very ideal, so PRRSV, as one of the important pathogen, has always been a serious harm in pig industry. The cellular immunity in PRRSV played a important role in the immune protection, therefore screening PRRSV T cell epitope and studying the immune regulation mechanism have great significance to develop new generation vaccine. Previous studies showed that PRRSV structural protein (GP) 2a played an important role in the immune protection, therefore, this aims of this study were to screen the T cell epitope and B cell epitope of GP2a, which layed the foundation for the further study of PRRSV infection and regulation.T and B cell epitopes of GP2a protein were predicted by bioinformatics methods, and then the epitopes were identified by overlapping peptide. At first, total RNA was extracted and reverse transcribed into cDNA, the plasmid pMD19-T-ORF2 was constructed by using cDNA as PCR template. The CD8+T cell epitopes were predicted by using SYFPEITHI, RANKPEP and IEDB of bioinformatics soft, the CD4+T cell epitopes spectial for MHC-II molecules were predicted by Propped, and the B cell epitopes of the GP2a protein were predicted by DNAstar software, ABCpred, Bepipredl.0. The CD8+T cell epitopes were 51～59,56～64,78～86, 105～113,143～151,173～181,182～190,198～206,202～210 amino acids of GP2a protein, respectively. The CD4+T cell epitopes were 75～83,89～97,116～124, 144～152,172～180,181～189,183～191,191～199,211～220 amino acids of GP2a protein, respectively. B cell epitope area were 41～47,51～66,86～93,104～119, 122-126,150-165,195～208 amino acids of GP2a protein.The plasmid pET～32a-eORF2 was constructed by a pair of specific GP2a41～20 8aa primers which were designed by using Primer Premier 5.0 and by using Eukaryotic plasmid pcDNA3.1-ORF2 as the PCR template. Then the plasmid was transformed into the E.coli Rosetta(DE3). The results of SDS-PAGE showed that the recombinant protein was about 36 kDa and was in form of inclusion body by the induction of the 1 mmol/L IPTG. At the same time, through the denaturation and renaturation, the recombinant protein was dissolved in 20 mmol/L Tris-HCl (pH= 8.0). Then the recombinant proteins were used as immunogen to be injected subcutaneously for Balb/C mouse. After three immunization, the antibody titers were up to 1:102400. In conclusion, the ectodomain of GP2a protein was expressed successfully and had well immunogenicity.By the overlapping method, a B cell epitope peptide was identified. It was the no.18 peptide(194-208aa), and B cell epitope core region was PTPGSRPKLHDFQ (195-207aa) by ELISA method. And the preliminary appraisal about T cell epitope through the final concentration of 20 μg/mL of peptide pool or peptide in vitro stimulated spleen lymphocytes and using the Mouse IFN-gamma ELISA kit II to determine the cell supernatant of IFN-y OD450 value, the present work indicated that the peptides SPSPVGWWSFASDWFAPR which was the 41-58aa of GP2a and STLNQVFAIFPTPGSRPK which was the 185-202aa of GP2a were the T cell epitopes of GP2a protein. |
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