Prokaryotic Expression , Subcloning Of GP5 Gene Of PRRSV And An Indirect ELISA For The Detection Of Viral Antibodies With The Expression Produce

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious and infections disease, and has caused the reproductive failure in sows, serious respiratory illness in the nursery-age pigs and high mortality rates. To a certain extent, it could prevent this diease to establish quick diagnostic method and take appropriate control measures. An indirect ELISA assay was constructed with fusion protein as coating antigen.In this study, a pair of specific primer was designed and synthesized according to the sequence of GP5 protein complete gene of PRRSV published in GenBank.Then a fragment of GP5 gene was amplified from RNA of the lung materials of six swines. The specific GP5 gene obtained by RT-PCR was cloned into pMD18-T vector. And then the recombinant plasmid was transformed into competent cells of Escherichia coli DH5α. The recombinant plasmid was indentified by PCR and restriction endonuclease analysis.The results showed that the gene fragment of GP5 at length of 600 bp was amplified and cloned into the vector pMD18-T. The homology is 99 %. The dORF5 gene segment deleting N-terminal hydrophobic sequence was cloned into prokaryotic expression vector pGEX-6p-1 and the recombinant plasmid named pGEX-6p-dGP5 was transformed into E.coli cell BL21, after the analysis of gene fragment was proved exactly. The recombinant plasmid was induced by 1mmol/L IPTG, then SDS-PAGE indicatesd that the recombinant fusion protein was about 40 ku, which was the same as the expected results and expressed in the form of inclusion bodies. The protein dGP5 showed a strong immunological reaction to the PRRSV-positive sera in Western-blotting analysis. The purified recombinant protein was injected to mice, and then collected serum. It could induce the serum antibody against PRRSV which confirmed with Western-blotting, but viral neutralization test showed that the serum antibody was not neutralizing antibody.An indirect ELISA assay was constructed and optimized to detect antibodies against PRRSV with fusion protein as coating antigen. The optimal concentration of antigen was 1.25μg /mL, the sealing buffer was PBST with 5 % fetal bovine serum, and the sealing time was 37℃for 2 h, then 4℃12 h. The serum sample was diluted by 1:80, and incubated at 37℃for 1 h. The dilution of conjugate was 1:400, and reaction time was at 37℃for 1 h. The TMB substrate was add for 15 min before terminated with stopping solution. Compared with IDEXX company, the specificity was 87.0 %, sensitivity was 90.3 %, and coincidence rate was 88 %. Moreover, dGP5 fusion protein had no cross-reaction with positive sera of other 4 swine diseases (PEDV, CSFV, PRV and TGEV). The difference value among wells in a plate and among plates for ELISA was both less than 10 %. The results showed that the indirect ELISA assay has good specificity and repetition.The results could provide a quick and easy serodiagnosis for the diagnosis of PRRSV, the detection of antibodies against PRRSV and epidemiological investigation.