Expression And Identification Of Main Antigen Region In Hemagglutinin-Neuraminidase Gene Of Newcastle Disease Virus Xx08Strain

Newcastle disease, caused by Newcastle disease virus (NDV), is an acute, highly contagious disease, potentially infects most species of birds, and for susceptible poultry and fatal. NDV also known as avian Paramyxovirus serotype-1(APMV-1), a member of the genus Avulavirus within the family Paramyxoviridae. The HN is one of the main structural glycoproteins and immune protective antigens, which locates on Newcastle disease virus membrane surface. related closely to the virulence of NDV, plays an extremely important role in the pathogenic and immune response process.The purpose of this study was to obtain the HN glycoprotein with antigenicity and immunogenicity, from the system of prokaryotic expression. The main antigen region (523～1101bp) of HN gene, named tNH (579bp) was truncated from the pMD-HN template of xx08strain of NDV through the specific primer and restriction site, EcoR I,Xho I. Then the tHN fragment was subcloned into pMD18-T-simple vector to construct the clone plasmid, pMD-tHN.The tHN fragment, which obtained from EcoR I and Xho I and digested pMD-tHN clone plasmid was cloned into the pET-32a (+) expression vector to construct expression plasmid of pET32a-tHN. Then, the pET32a-tHN plasmid was transformed into the E. coli BL21(DE3), in order to expression the taget protein of tHN, which induced with the best induction condition of28℃、0.1mmol/L IPTG、4h and expression level of tHN recombinant protein was analysised by SDS-PAGE. Specificity of expressed productions of the tHN recombinant protein was identified in Western-blot assay. The tHN protein was purified by cutting gel recovery and immunized to BALB/c mice in order to develop polyclonal antibody of anti-tHN protein. The anti-tHN protein polyclonal antibody was identified in Western-blot and indirect ELISA assays. Result showed that the tHN recombinant protein was overly expressed in form of inclusion body in E. coli BL21(DE3), with molecular weight about38kDa, and reacted specifically with anti-NDV antiserum. The tHN protein murine anti-serum could react tHN and NDV.It is indicated that the tHN recombinant protein was obtained from the prokaryotic expression system, with good antigenicity and immunogenicity, which lay the foundation for establishing the way of detect antibody titer of NDV, the preparation of the polypeptide vaccine and monoclonal antibody in the future.