Establishment Of An Indirect ELISA For The Detection Of Antibodies And Development Of Monoclonal Antibodies Against PRRSV GP5 Protein

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease,which is caused by porcine reproductive and respiratory syndrome virus(PRRS V).The main symptoms of the disease are respiratory disorders of piglets,reproductive failure of pregnant sows,high fever and diarrhea in pigs of other ages,and cyanosis and blueness in some pig ears.According to amino acid sequence analysis,PRRSV can be divided into two serotypes:European type(PRRSV-1)and American type(PRRSV-2).The strains that are prevalent in China are mainly American types.GP5 protein is the most important structural protein encoded by PRRSV,which has a good immunogenicity and can induce the pig to produce neutralizing antibody.1.Prokaryotic expression of PRRSV GP5 recombinant proteinBased on the known sequence of PRRSV RD2007 strain in our laboratory,the signal peptide region and transmembrane region of GP5 protein were predicted by DNAstar.Two pairs of primers were designed to amplify the target gene in sections,and then the entire GP5 gene was spliced by Overlap PCR.The complete GP5 gene was cloned into pET-32a prokaryotic expression vector and transformed into E.coli DH5a.A single clone was selected to culture and identify,and the positive plasmids were sequenced.The results showed that the complete GP5 gene were identical with the published sequence.The correctly sequenced plasmid was transformed into E.coli BL21,and the best induction conditions were explored:28℃,5h,1mM IPTG.SDS-PAGE analysis of the GP5 recombinant protein showed that a corresponding band appeared at 30KDa,which was not found in the negative control Western-blot analysis showed that GP5 recombinant protein had a specific reaction with PRRSV positive serum,indicating that GP5 recombinant protein was expressed successfully2.Establishment and preliminary application of an indirect ELISA antibody detection method for PRRSV GP5 proteinThe GP5 recombinant protein was purified,and then coated into a 96-well ELISA microtiter plate.An indirect ELISA antibody detection method for PRRSV GP5 protein was established successfully by square titration test.The best antigen coating concentration was 1.25μg/mL.The optimum sealing buffer and time were 5%skim milk and 2h.The dilution with 1:500 was the best for serum while the reaction time was 45min.The best time of HRP-goat anti-porcine IgG enzyme antibody was 1h and the optimum reaction time of substrate was 20min.Positive value of OD450nm was not less than 0.325 while negative was less than 0.295,or it was judged suspicious when 0.295<OD450nm<0.325.Specificity test showed that GP5 protein had no cross-reaction with positive serum of other 4 swine diseases(PRV,PCV-2,PEDV,TGEV),which indicated that the indirect ELISA method had a good specificity.The sensitivity experiment showed that this method could detect 1:12800 dilution of PRRSV standard positive serum.Coefficients of variation within and between batches were less than 10%.96 clinical serum samples were detected by indirect ELISA method,which were gathered from JiangSu region.Comparing with the total coincidence,this method was 82.29%with IDEXX PRRSV kit,while the IFA was 90.63%.3.Preparation of monoclonal antibodies against PRRSV GP5 proteinGP5 recombinant protein was emulsified with adjuvant and then immunized 6-8 weeks old BALB/c mice.After three immunizations,the serum antibody titer of the mice was detected,and the mice with higher antibody titer was used to strengthen immunization.Spleen lymphocytes were fused with SP2/0 myeloma cells after boosting immunization for 72-96h.The Marc-145 cells were fixed after infecting with PRRSV,which were used to screen hybridoma cells by IFA.After 2-3 times subcloning,five hybridoma cell strains that could secret stable monoclonal antibodies against GP5 protein were obtained,which were named PRRSV-2H3,PRRSV-1G8,PRRSV-2E9,PRRSV-2D5 and PRRSV-2F9,respectively.All of the mAbs Ig subtype were determined as IgGl.The IFA titers of abdominal fluid of PRRSV-2H3,PRRSV-1G8,PRRSV-2E9,PRRSV-2D5 and PRRSV-2F9 were 1:3200,1:1600,1:6400,1:6400 and 1:6400,respectively.Western-blot analysis showed that the mAbs were all specific to the purified GP5 recombinant protein.The further research showed that the binding site of mAbs to GP5 gene were located at 130aa to 201aa.