Expression Of GP5 Protein Of Porcine Reproductive And Respiratory Syndrome Virus In E.coli And Establishment Of The Indirect ELISA Antibody Detection Assay

Porcine reproductive and respiratory syndrome(PRRS)is a common acute and highly contagious disease in pig herds.It mainly causes reproductive and respiratory symptoms,causing big harm to the pig all over the world.Porcine reproductive and respiratory syndrome virus(PRRSV)is a single-stranded positive-strand RNA virus with a membrane that encodes 13 non-structural proteins and 6 structural proteins.Among them,GP5 protein is the most important immunoprotective protein of the virus,and neutralizing antibodies against GP5 will be produced 4 to 5 weeks after infection of PRRSV in pigs.At present,there is no commercial GP5 ELISA antibody detection kit in China.In this study,a novel antigen molecule(GP5-3m2)was designed by GP5 gene modification.The GP5 recombinant protein was obtained by prokaryotic expression system,and the ELISA method for GP5 protein ELISA was established.The specific content of this study is as follows:1.Expression,purification and identification of GP5 recombinant protein of PRRSVIn this study,the GP5 gene fragment deleted transmembrane region of highly pathogenic PRRSV BB0907 strain,was optimized as E.coli biophagy and fused with two different length gene sequences.They was amplified from pET-28a-GP5-3m by PCR and cloned into pET-28a and pET-32a(M)for constructing four prokaryotic expression vectors.Then these four prokaryotic expression vectors were transformed into BL21(DE3).By inducing with IPTG,the four recombinant GP5 proteins were expressed and identified by Western blot.The results showed that the recombinant protein has good immunogenicity and reactogenicity.2.Establishment of an indirect ELISA to detect the antibody against PRRSV GP5An indirect ELISA was established by using the punfied GP5-3m2 protein as the coating antigen,and optimizing the reaction conditions by square test.The optimizing antigen concentration was 2 ?g/mL,the optimal dilution of serum was 1:100.It has no cross reaction with the antibodies against porcine circovirus type 2,classical swine fever virus,pseudorabies virus,foot-and-mouth disease virus,encephalomyocarditis virus and parssuis haemophilus.Compared with the commercial PRRSV ELISA kit by IDEXX Company,it has the relative sensitivity of 90.4%and specificity of 90.3%.The coincidence rate of the two methods was 90.4%.In summary,in this study the GP5-3m2 recombinant protein was prepared.And it has good antigenicity with PRRSV GP5.The established indirect ELISA method for detecting PRRSV antibody has good specificity,sensitivity and reproducibility.It should be used to detect the antibody against PRRSV in the future.