Molecular Epidemiological Study On GP5 Gene Of Porcine Reproductive And Respiratory Syndrome Virus And Development Of Monoclonal Antibody Against NSP2 Protein

Porcine reproductive and respiratory syndrome virus(PRRSV)is an important pathogen of swine causing significant economic losses in the swine industry worldwide.It mainly leads to abortion,premature birth and stillbirth in sows and respiratory diseases in mummy piglets and fattening pigs.GP5 is a major membrane protein containing about 200 amino acids.It has an amino acid signal peptide,glycosylation sites and antigenic epitopes that can induce neutralizing antibodies.Because of its immunological significance and polymorphism,GP5 has been used to analyze the diversity of PRRSV gene.NSP2 has multiple B cell epitopes,with strong immunogenicity,can stimulate the body to produce specific antibodies.In the study,strains from 12 provinces of China from2015 to 2016 were analyzed by GP5 gene sequencing.NSP2 genes were cloned into the prokaryotic expression vector by the PCR for expression and one strain of monoclonal antibody against the NSP2 protein was obtained.Specific contents are as follows:1.Molecular epidemiological investigation on GP5 gene of porcine reproductive and respiratory syndrome viruses from 2015 to 2016 in chinaA total of 362 clinical lung samples were collected from piglets from different swine herds from Jiangsu,Zhejiang,Sichuan,Anhui,Shandong,Hebei,Henan,Shanghai,Guangdong,Guangxi,Fujian and Jiangxi from 2015 to 2016.Of these samples,191(60.82%)were positive for PRRSV detection by RT-PCR.The length of ORF5 of 34 strains of PRRSV was 603bp,and the homology was 88.9%-99.4%.Phylogenetic analysis showed that all the isolates belonged to the North America genotype,divided into 4 subtypes.Among them,2 strains and VR2332 strain belonged to the subtype II,6 strains and MN184C strain belonged to subtype III,2 strains belong to subtype IV and the other strains belonged to the subtype I.This proves the genetic diversity of PRRSV in china and provides a theoretical basis for the prevention and control of the disease.2.Prokaryotic expression of NSP2 protein of porcine reproductive and respiratory syndrome virus and development of monoclonal antibodies against NSP2 proteinAll the genes of NSP2 were amplified from the recombinant plasmid pCMV-TJM by PCR and cloned into prokaryotic expression vector pET32a.Four recombinant proteins pET32a-NSP2-1/NSP2-2/NSP2-3/NSP2-4 were expressed in Escherichia coli BL21with molecular weight of 65kDa.The four recombinant proteins could react with HIS monoclonal antibody in Western blot assay.In this study,six to eight-week-old Balb/c mice were hypodermically immunized with purified the recombinant NSP2 protein one strain of monoclonal antibody against the NSP2 protein was obtained,named 5D2.The titer of cell culture supernatant was 1:160 by indirect ELISA and the titers of the 5,10,15,and 20 generations were also the same.The result showed that the McAb belonged to IgG1 isotype,and the light chain belonged to ?type.Indirect immunofluorescence(IFA)showed that the 5D2 reacted with the PRRSV S1 and TJM but could not react with the BB0907 and FJ1402.The McAb could be used for functional analysis of NSP2 in the future.