Expression Of Porcine OASL Protein And Preparation Of Monoclonal Antibody And Preliminary Study On Anti-PRRSV Effect

Interferon(IFN)plays a key role in the body’s antiviral process,OAS is an antiviral protein induced by interferon when the body is infected by the virus,porcine OAS protein family has OAS1,OAS2,OASL three members,OASL as a potential interferon-stimulated gene(ISG),in order to facilitate subsequent research on natural immunity,In this experiment,porcine OASL prokaryotic expression and eukaryotic expression vectors were constructed,porcine OASL protein was expressed and purified,monoclonal antibodies were prepared,and the inhibitory effect of porcine OASL on PRRSV on Marc-145 cells was verified,as follows:1.Prokaryotic expression and purification of porcine OASL proteinThe experiment was conducted by inserting the synthetic porcine OASL gene sequence into the p ET-21 a expression vector to construct the p ET-21a-OASL recombinant plasmid.The optimal induction conditions were determined to be 37℃,220 r/min,8 h.The final concentration of isopropyl-β-d-galactoside(IPTG)was 0.5 mmol/L,and was identified as inclusion body by SDSPAGE.Porcine OASL protein was purified by affinity chromatography(Ni column)after dissolution and renaturation.2.Preparation of monoclonal antibody to porcine OASLA 6-week-old female BABL/c mouse was selected and the purified porcine OASL protein was injected intraperitoneally.The immune protein of each mouse was 50 μg and immunized three times,each time at an interval of 14 days.After three times of immunization,the blood was collected and the titer reached 1:125000 by ELISA.Three days after immunization enhancement,spleen cells were fused and screened to obtain positive clone cells.After two subclones,a hybridoma cell strain was screened and named 7D7E3.The titer of ascites preparation reached1:500,000.Western blot indicated that monoclonal antibody could react specifically with porcine OASL protein,but not with P30 protein.Indirect immunofluorescence assay showed that the monoclonal antibody had good reactivity with eukaryotic expression protein.The results showed that 7D7E3 monoclonal cell line was successfully screened and could secrete anti-porcine OASL antibody stably with high specificity.3.Application of antiviral effect of porcine OSAL proteinTo verify the inhibitory effect of porcine OASL protein on replication of porcine reproductive and respiratory syndrome virus(PRRSV)in Marc-145 cells.Porcine OASL gene was cloned into the eukaryotic expression vector p CMV-3×Flag,and the recombinant expression plasmid p CMV-3×flag-OASL was constructed.Western blot detected the overexpression of porcine OASL gene in Marc-145 cells.At the same time,the overexpression of porcine OASL inhibited PRRSV replication in Marc-145 cells by detecting the change of PRRSV copy number and the titer of PRRSV virus 48 h after exposure.