Immunological Activity And Prokaryotic Expression Of PRRSV Hn-1/06 GP5

Porcine reproductive and respiratory syndrome(PRRS),caused by porcine reproductive and respiratory syndrome virus(PRRSV), which is really a new viral disease of swine. Since it was first described in 1987 in the United States, PRRS has been one of the most economically important diseases of the global swine industry.The ORF5-encoded major envelope glycoprotein (GP5) is one of the major structural proteins and immunoprotection proteins of porcine reproductive and respiratory syndrome virus (PRRSV ),it can induce the production of neutralization antibody, and has the vital significance in the pathogenicity, diagnosis, prevention and control of PRRSV ,it is regarded as a good material for the designing of new type molecule diagnostic reagent and new generation vaccine. therefore, It is a base that futher study the nature and function of GP5 protein and pathogenicity of PRRS.According to He Nan province isolates Hn-1/06 strain ORF5 sequence of Porcine reproductive and respiratory syndrome virus(PRRSV), The full-length of ORF5 gene fragment ORF5-L and ORF5-W which deleted the signal peptides respectively were obtained from recombinant plasmid PTG19-T-ORF5 by PCR amplification using theirs primers. PCR amplification about 603 bp and 527 bp. Subsequently through GP5 sequence analysis software in the transmembrane domain,synthesized primer, pTG19-T-GP5 as a template, PCR amplification about 121 bp, 90bp and 250 bp of the GP5 transmembrane protein coding sequence outside the region. With three amplified products as a template to SOE-PCR (gene splicing by overlap extension PCR extended) amplified about 413bp deletion of the transmembrane domain of GP5abc gene sequencing. The constructed recombinant plasmid was confirmed by RE digestion,PCR and sequencing. After sequencing, the sequences of ORF5 genes of the Hn-1 isolate were compared with sequences of ten other different PRRSV strains in GenBank by Blast and DNAStar software. The results showed that the Hn-1 displayed high nucleotide identities with strain VR22332, but distinct from LV, showing genetically closer relationship to strain VR22332.The RT-PCR product was directly cloned into the pTG19-T easy vector and this recombinant plasmid was transformed into JM109 identified by enzyme digesting and DNA sequencing.The positive clone was designated pTG19-T-GP5-L,pTG19-T-GP5-W and pTG19-T-GP5abc. Then The three positive plasmid and plasmid pET32a were respectively digested by BamH I and Hindâ…¢,the following coupled reaction was operated. Subsequently, ORF5-L,ORF5-W and ORF5-abc gene fragments by RE digestion were inserted into prokaryotic expression vector pET-32a, resulting in the recombinant plasmids pET32a-ORF5-L,pET32a-ORF5-W and pET32a-ORF5abc , then The three positive plasmid was transformed into the host cell BL21(DE3)and the expression procedure was optimized,so recombinant GP5abc protein was successfully obtained with the induction of 1.0 mmol/ L IPTG. In addition,western-blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against PRRSV and has favourable immunogenicity.The mice were immunized with the inclusion body, denaturation protein and renaturation protein, respectively,and the murine antibody of GP5abc fusion protein was obtained. The result showed that the antibody titer is 1:20,Through the establishment of the ELISA method for detection of murine antibody. After the Marc-145 cells infected about 24h, using mouse anti-GP5bac make a anti-protein antibody, The strong fluorescence can be detected by IFA. the neutralization of mouse antibody GP5abc were tested in Marc-145 cells, and a pig anti-PRRSV antibody as positive control.The results showed that mouse antibody GP5abc have no neutralization in Marc-145 cells, while the control pig anti-PRRSV antibodies, was collected 45 days, have neutralization, and the titer was 1:40.