Cloning And Expression Of N Gene Of PRRSV Hn-1/06 Strain And Establishment Of Indirect ELISA Based On The Recombinant Fusion Protein

Porcine reproductive and respiratory syndrome (PRRS),characterized by severe reproductive failure in sow and respiratory disease in young pigs, was first recognized in the US in 1987. Since its appearance, PRRS has been causing tremendous economic losses to the swine industry throughout the world. Although PRRSV isolates identified from around the world cause similar diseases in pigs, increasing data indicate that PRRSV strains are antigenically and genetically heterogenous and differ in virμlence in infected pigs. It is evident that the current vaccines are not effective in protecting against infections with the genetically diverse field strains of PRRSV and the attenuated vaccine viruses can revert genetically to cause clinical disease. Up to now, vaccination is not effective to control the disease, therefore, strict biosecurity measures with the aid of intensive surveillance appear of crucial importance to protect the unaffected herds. In this study, the nucleocapsid protein gene of PRRSV Hn-1/06 strain was successfμlly expressed in Escherichia Coli in soluble form, and the recombinant nucleocapsid protein was used to develop an indirect ELISA for the detection of antibodies against PRRSV.In this study, one pair of specific primer of ORF7 for PRRSV was designed and synthesized according to the published gene sequence of PRRSV from GenBank, and 0RF7 gene of PRRSV Hn-1/06 strain was obtained by RT-PCR method. Then,the RT-PCR product was directly cloned into the PGEM-T-easy vector, and after that, this recombinant plasmid was transformed into JM109,identified by RT-PCR method and enzyme digestingand DNA sequencing,Furthermore,The positive clone was designated PGEM-T-ORF7.After sequencing, the sequence of ORF7 gene of the Hn-1/06 isolate was compared with sequences of ten other different PRRSV strains in GenBank by Blast and DNAStar software. The resμlts showed that the Hn-1/06 displayed high nucleotide identities with strain VR22332, but distinct from LV, showing genetically closer relationship to strain VR2332.Subsequently,one pair of specific expression primer of N gene for PRRSV was designed and synthesized according to the actual amplification gene sequence of PRRSV for Hn-1/06 strain(Accession number EU025136), so this N gene of PRRSV Hn-1/06 strain was obtained by RT-PCR method. The RT-PCR product was directly cloned into the PGEM-T-easy vector and this recombinant plasmid was transformed into JM109 identified by enzyme digesting and DNA sequencing.The positive clone was designated PGEM-T-N. Then plasmid PGEM-T-N and plasmid pET32a were respectively digested by EcoRⅠand HindⅢ,the following coupled reaction was operated. Finally the recombinant plasmid was constructed designated pET32a-N, then pET32a -N was transformed into the host cell BL21(DE3)and the expression procedure was optimized,so recombinant N protein was successfμlly obtained with the induction of 1.0 mmol/ L IPTG. In addition,western-blot was performed to confirm that the expressed fusion protein coμld specifically react with antiserum against PRRSV.Consequencely,the fusion protein was purified by the means of His-bind resin protein purification procedure.Following SDS-PAGE and western-blot were used to detect the purification effect and the specificity of purified recombinant N protein of PRRSV Hn-1/06 strain. Then the purified N protein was used as an antigen to establish a novel PRRSV N-ELISA diagnose assay, and each following step was optimized, such as coating concentration and time of recombinant N protein, sample diluent and diluent concentration, etc.As a resμlt ,an indirect ELISA was set up to detect antibody against PRRSV.At the same time, comparison between N-ELISA and the abroad kit IDEXX-ELISA showed the two methods had 92.5 percent agreement by detecting 200 serum samples,indicating that the indirect N-ELISA was specific and sensitive.