Preparation Of Monoclonal Antibodies To Nsp2 And Nsp4 Proteins Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS)is one of the diseases that current ly endanger the global pig breeding industry,and is commonly known as porcine blue ear dise ase.The disease mainly causes miscarriage,premature delivery,stillbirth or mummified fetus in sows in late pregnancy,and obvious respiratory symptoms in piglets.The pathogen caused by this disease is PRRSV,an envelope RNA virus,a genome size of about 15 k B,a total of 7structural proteins and 12 non-structural proteins.Nsp2 is the largest non-structural protein in PRRSV,and Nsp2 gene homology is large between PRRSV-1 PRRSV-2-type poisonous strai ns,and also contains rich B cell epitopes,which is an ideal goal of monitoring genetic variatio n and identifying diagnostic trials.Nsp4 is a 3C-like protease that can cleave other non-structu ral proteins,containing rich B cell epitopes.Antibodies against NSP2 and NSP4 in the serum of infected pigs appeared earliest in clinical practice.Thus,these two proteins and their mono clonal antibodies can be used as an ideal target for PRRSV infection.In this study,PRRSV Nsp2 and PRRSV Nsp4 recombinant proteins were first expressed in prokaryotic cells.The expression product was verified and analyzed by SDS-PAGE,and th e recombinant protein of PRRSV Nsp2 was expressed insoluble(existing in inclusion bodies),and the recombinant protein of PRRSV Nsp4 was expressed soluble.The recombinant protei n was purified by Ni-NTA affinity chromatography column,and the purified PRRSV Nsp2 re combinant protein was refolded by urea gradient dialysis.The two purified recombinant protei ns were mixed and emulsified with Freund’s complete adjuvant to immunize 7-week-old fema le BALB/C mice.Blood was collected from the tail of the immunized mice to separate serum10-14 days after the third immunization.IFA and ELISA detect the level of antibodies in mic e.A booster immunization was performed on mice whose antibody titers reached the level of cell fusion.After immunization,spleen cells of the mice and resuscitated SP2/0 cells were use d for routine cell immunization,Fusion.The recombinant protein was used as the antigen coating,the fusion cell culture supernat ant was detected by ELISA,and the positive wells of hybridoma cells that could secrete antib odies were screened out.The hybridoma cells were subcloned by the limiting dilution metho d.After two cell subcloning,a monoclonal antibody against PRRSV Nsp2 was finally prepare d,named 3-A8;a monoclonal antibody against PRRSV Nsp4,named B+D11.After expandin g the culture of the two hybridoma cell lines,they were injected into the abdominal cavity of t he mother mouse to produce a large number of hybridoma ascites.The two prepared monoclonal antibodies were applied to Western blot experiments and I FA experiments,and it was found that the best dilution of ascites prepared by the two monocl onal antibodies for Western blot experiments was 1:8000.Ascites monoclonal antibodies agai nst PRRSV Nsp2 protein(3-A8)The best dilution for IFA experiment is 1:100,and the hybri doma cell culture supernatant against PRRSV Nsp4 monoclonal antibody(B+D11)can be use d for IFA experiment.The two monoclonal antibodies prepared in this study can provide favorable tools for the research of PRRSV and the detection and diagnosis of PRRS,and have important application value.