Preparation Of Monoclonal Antibody Against PRRSV NADC30-like Nsp2 Protein And Establishment Of Indirect ELISA

Porcine reproductive and respiratory syndrome(PRRS)is an infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which causes reproductive disorders such as miscarriage of pregnant sows,stillbirth,mummified fetus,weak piglets and respiratory diseases of piglets.At the end of 2014,PRRSV NADC30-like strain became widespread in China,and its Nsp2 region was different from that of HP-PRRSV strain in that of Nsp2,with discontinuous deletion of multiple amino acids.The epidemic has caused huge economic losses to Chinese pig industry.Epidemiological investigation found that after 2014 in China,the positive rate of HP-PRRSV in China decreased year by year,while the positive rate of NADC30-like strain increased year by year,and the epidemic strain of PRRSV in China gradually changed from HP-PRRSV strain to NADC30-like strain.In order to prevent and control the disease and to further differentiate and diagnose subtypes of the disease,this study took Nsp2 of PRRSV NADC30-like QHD1 strain as the research object,and carried out related studies on the preparation of monoclonal antibody against Nsp2.Main research contents include:1.Preparation of monoclonal antibody against Nsp2 proteinOn the basis of the QHD1 strain isolated in our laboratory,Nsp2 gene was amplified by designing specific primers,and the recombinant plasmid pET-32a(+)-Nsp2 was constructed by prokaryotic expression system.Balb/c mice were immunized with the purified Nsp2 protein.The titer of serum was detected and analyzed,and routine cell fusion was performed.Indirect ELISA,IFA and Western blot were used to detect the titer of Nsp2 After subcloningt,two monoclonal antibody hybridoma cell lines against NADC30-like Nsp2 protein was finally obtained,which were named as9C2 and 9C8,respectively.Antibody subtypes were identified as lgG1.2.Establishment of indirect ELISAUsing recombinant protein as coating antigen,the optimal coating concentration,coating time,substrate action time and specificity test were studied,and an indirect Nsp2-ELISA was established.The method has the advantages of good repeatability,high sensitivity,strong specificity and convenience,which provides technical support and convenience for the clinical diagnosis of anti-PRRSV NADC30-like strain antibody.