Location Of B-cell Epitopes And T-cell Epitopes In Glycoprotein 2 Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome?PRRS?is commonly known as blue-ear pig disease and is defined the second class animal epidemic diseases,which can be transmitted through personal contact,contaminants,and airborne spread.PRRS is characterized by reproductive problems in pregnant sows?abortion,stillbirth,mummy?and respiratory diseases in pigs of all ages,especially piglets.It has now become one of the major diseases in large-scale pig farms and has caused great economics losses to porcine industry in the world.PRRSV is a positive-sense,single-stranded RNA virus with a total genome length of approximately 15 kb.The virus encodes at least 11 functional open reading frames?ORFs?that surrounded by envelopes and PRRSV virions appear as ellipsoid particles ranging in diameter from50 nm to 74 nm.According to the differences in viral gene sequences and antigenicity,they were divided into genotype I and genotype II.Because of its antibody dependent enhancement?ADE?and ability to suppress innate immune responses,PRRSV is still one of the most important pathogens that seriously harm the swine industry.Studies have shown that the acquired immune response plays a key role in the immune protection of host against PRRSV infection,and PRRSV GP2 is not only an essential structural protein for the virus to infect the host cell,but also can cause the host to produce a significant acquired immune response.In this study,there B-cell epitopes and six T-cell epitopes of Glycoprotein 2 were located by indirect ELISA and IFN-?ELISPOT methods using overlapping peptides,which may provide assistance for the research of novel epitope vaccines.In this study,PRRSV JXA1-R strain,which was kept in the laboratory in the past,was expanded.According to the Reed-Muench's method,the virus titer of the strain was calculated to be 10⁶.6.6 TCID₅₀/mL.Total RNA of JXA1-R strain was extracted and reverse-transcribed into cDNA.The full length of ORF2 gene sequence was amplified by using cDNA as a template,and the pMD19-T-ORF2 cloning vector was constructed.According to the sequencing results of ORF2 gene,the amino acid sequence of Glycoprotein 2 of JXA1-R strain was deduced.The gene sequence of Glycoprotein 2 extracellular region was amplified by using pMD19-T-ORF2 as a template.The gene sequence was cloned into the pET-32a expression vector.And then,the pET-32a-GP2 expression plasmids was transformed into the Rosetta?DE3?prokaryotic expression bacterium.PRRSV GP2 recombinant protein was expressed by inducing recombinant expression bacteria.The molecular weight of the target protein was determined to be approximately 36 kDa,mainly in the form of inclusion bodies in the precipitate after cell disruption.The result of Western Blot showed that the purified GP2 protein had good reactogenicity.Eight pigs were determined to be negative for porcine reproductive and respiratory syndrome virus by PRRSV antibody test strips.Five pigs in the experimental group were then inoculated intramuscularly with 3×10⁶TCID₅₀0 of the collected PRRSV JXA1-R virus fluid.Three pigs in the control group were intramuscular inoculated with supernatant liquid of cell disruption.These experimental animals were immunized twice successively.The immunization interval was two weeks and the immunization dose was 3 mL.The experimental pigs were bled weekly for 12 weeks,and the serum samples were collected.The peripheral blood mononuclear cells were collected at 12th weeks post-infection.PRRSV specific antibodies of five pigs in the experimental group can be detected at 3 weeks post-infection already existed,which indicated that specific immune response has been generated in the animals which were immunized with the virus.By indirect ELISA assay,the anti-Glycoprotein 2 antibody of 5 pigs in the experimental group began to exhibit at 4th weeks post-infection.At 8th and 9th weeks post-infection,the antibody peaked and then stabilized.According to the overlapped peptide method,Twenty-two overlapped polypeptides with a length of 18 amino acid residues were designed and synthesized.The adjacent peptide overlapped 9 amino acids.The synthetic overlapping peptides were used as the coating antigen and the pig positive serums which were isolated from experimental animals at 12 weeks post-infection were used as primary antibody.The B-cell epitopes of glycoprotein 2 were located by indirect ELISA.Three immunodominant B-cell epitopes of glycoprotein 2 were screened out,which were77⁹4 aa,113¹30 aa,and 131¹48 aa.Alignments of epitope sequences revealed that the three B-cell epitopes on glycoprotein 2 were more than 92%homologous among the North American-type strains.The peripheral blood mononuclear cells?PBMCs?were cultured in vitro.PBMCs were stimulated with overlapping polypeptides.The IFN-gamma ELISPOT method was used to detect the spots associated with IFN-gamma secretion from peripheral blood mononuclear cells.The number of spots presented in different experimental groups was recorded.If the number of spots was greater than or equal to the positive determination threshold,the polypeptide was scored as positive.After two rounds of peptide library and single peptide screening,6 T-cell epitopes of GP2protein were screened out,which were 86¹03 aa,95¹12 aa,104¹21 aa,113¹30aa,140¹57 aa,and 185²02 aa.