Molecular Epidemiological Analysis Of PRRSV In Parts Of China And Prokaryotic Expresstion Of GP5 Protein Of NADC30-like Strain

Porcine reproductive and respiratory syndrome(PRRS)is a widely disseminated and highly pathogenic disease,which is characterized by the failure of sow breeding and swine respiratory symptoms,can cause serious economic loss to the global pig industry.Because of the virus that causes this disease have strong variability,in order to understand the epidemic situation and variation characteristics of PRRS in China in the past two years,also to have a deeper understanding about the epidemic strains.In this study,the molecular epidemiology of PRRS in some areas of China was analyzed,the isolation of the epidemic virus and the prokaryotic expression of GP5 protein were completed.The main contents are as follows:1.PRRSV pathogen detection was performed on PRRS suspected samples from 23 provinces in China from 2015 to 2016.The results showed that 889 positive samples were detected in 3078 suspected samples,the positive rate was 28.88%.Gene sequence analyzing on the obtained ORF5 gene sequence showed that,177 strains were belonged American-type strains.In the 177 strains,90 were highly homologous to JXA1,the representative strains of HP-PRRSV subgroups,34 were highly homologous to NADC30.Compared with 2015,China's the main epidemic PRRSV strains still are HP-PRRSV,while the proportion of NADC30-like strains in the epidemic strains increased 12.04%.And the popularity of PRRSV does not have obvious geographical characteristics.The deduced amino acid sequence of ORF5 gene was analyzed.The results showed that some NADC30-like strains lacked the glycosylation site at position N34,suggesting that the production of neutralizing antibody may be affected.Reminds that it is necessary to continue the PRRSV molecular epidemiological monitoring,to provide the basis for the next step prevention and control.2.PRRSV-positive tissue samples were grinded and seeded into Marc-145 cells,then the virus was isolated and the cells were exposed to the third to forth generation.Through RT-PCR test,measurement of the titer virus and gene sequencing we confirmed that the 3isolated strains were PRRSV,name them as JX1?JX2?SD1.Analysis of the sequence of the strain showed that the three strains belonged to the NADC30 strain,The viral titers of the 5thgeneration cell culture were determined.The results showed that the viral contents were 10-6TCID50 / 0.1 m L,10-5.33 TCID50 / 0.1 mL,10-5.67 TCID50 / 0.1 mL,respectively.3.Through amplify the GP5 gene of a isolated PRRSV strain(JX1),and amplify the recombinant plasmid vector with all GP5 gene as template,get two amplified fragments.Through fusion PCR obtain gene fragment that lacks GP5 signal peptide and transmembrane region(dORF5).The prokaryotic expression vector pGEX-6P-1 was transformed into E.coli BL21,with 1mmol/L concentration of IPTG at 37 ° C.SDS-PAGE electrophoresis showed that the molecular weight of the expressed protein was about 38 kDa,and through induction time optimization we found that the expression level reached the maximum at 5 h.Identification of protein solubility showed that some of the expressed protein had good solubility.Western Blot results showed that the expressed protein was react with PRRSV positive sera,indicating that the expressed protein had good immunoreactivity.