Prokaryotic Expression Of GP5 And M Proteins Of Porcine Reproductive And Respiratory Syndrome Virus Study On The Function Of Pig PBMC

In order to study the activation function of GP5 and M proteins of porcine reproductive and respiratory syndrome virus(Peripheral blood mononuclear cell,PBMC).Using the PRRSV PC vaccine strain RNA as a template,RT-PCR was used to amplify GP5 and M gene fragments and cloned into the prokaryotic expression vector p ET-32a(+)to construct recombinant plasmids p ET-32a-GP5 and p ET-32a-M.After restriction enzyme digestion and sequencing,the positive recombinant plasmid was transformed into E.coli BL21(DE3)competent cells,and IPTG was added to induce expression at low temperature.The PRRSV-GP5 and PRRSV-M proteins were expressed using affinity chromatography.The results showed that GP5 and M recombinant proteins were expressed in two forms,soluble and inclusion bodies,with a molecular mass of about 30 KDa.The purified protein was immunized with big-eared white rabbits,and antiserum was prepared according to conventional methods and used in ELISA(ELISA)To test the antiserum titer.The results show that the purified protein can specifically bind to PRRSV positive serum;the titer of the prepared rabbit antiserum is as high as 1:64000.The peripheral blood of pigs not infected with porcine reproductive and respiratory syndrome virus is collected aseptically,and the mononuclear is isolated.Cells were cultured in vitro with GP5 and M recombinant proteins at a final concentration of 10 μg/m L,and the binding of the two recombinant proteins to PBMC was observed by immunofluorescence antibody technology(IFA).The IFA results showed that PBMCs showed red fluorescence.It shows that both expressed proteins bind to PBMC.Two recombinant proteins of different concentrations were further used to stimulate pig PBMC in vitro,and the secretion of IL-10,IFN-γ,TGF-β1 and TNF-α in lymphocytes was detected by ELISA method,and the CCK-8 cell proliferation detection kit was used to determine the secretion of IL-10,IFN-γ,TGF-β1 and TNF-α The effects of the two recombinant proteins on the proliferation of PBMC,and the two recombinant proteins at different concentrations stimulate PBMC to secrete NO.The results show that the two recombinant proteins at different concentrations significantly promote the PBMC cytokines IL-10,IFN-γand TNF-αsecretion(p<0.05): and can stimulate PBMC to cause PBMC proliferation and increase NO secretion(p<0.05).