Expression Of The ORF5 Gene Of Porcine Reproductive And Respiratory Syndrome Virus In HeBei

Porcine reproductive and respiratory syndrome is a new infectious disease. It causes abortions in sows and respiratory problems in piglets. It was caused by porcine reproductive and respiratory syndrome virus(PRRSV). Since it's appearance, PRRS has been causing tremendous economic losses to the swine industry throughout the world. It is difficult to control PRRS without an effective vaccine. It is evident that the current vaccines are not effective in protecting against infections with the genetically diverse field strains of PRRSV.The porcine reproductive and respiratory syndrome virus isolated from the clinic suspected lung material, named HB-3(cz). Post inoculation many times, the CPE in Mare-145 cell appeared in rounding, gathering shedding features stably. According to the complete nucleotide sequence of PRRSV CH-1a in GenBank, a pair of primers were designed to amplify ORF5 gene of HB-3(cz) strain. The specific ORF5 gene amplified by RT-PCR was cloned into pMD-T vector. The recombinant plasmid was sequenced and compared with other strains. The hydrophobicity, transmembrane helices, signal peptide and latent antigen site of GP5 protein are homologous of all Hebei strains. The ORF5 gene have 89.6% and 89.1% homologies with VR-2332(a North American isolate) in nucleotide and amine acid level, whereas it has only 63.7% and 53.2% homologies with LV (an Europen isolate); The HB-3(cz) strain belong to North American genotype; The phylogenetic trees revealed that the strains isolated in recent years are closed with HB-1(sh) strain.The dORF5 gene segment deleting N-terminal hydrophobic sequence was amplified from ORF5 gene of HB-3(cz) strain by PCR, then the gene was cloned into prokaryotic expression vector pGEX-6P-1. The recombinant fusion proteins dGP5 were highly expressed in E-coli BL21 in the forms of inclusion bodies induced by IPTG Western-blotting showed that the recombinant protein could react with the porcine polyclonal antibody against PRRSV, indicating that the protein could be used as antigen in diagno sticassay for the detection of antibodies against PRRSV.