Cloning And Expression Of Hemagglutinin-neuraminidase Gene From Newcastle Disease Virus B95 Strain

Newcastle Disease(ND) is published one of the sixteen very virulent infectious disease by OIE. In recent years ,the outbreak of ND appear some new characters ,it is reported that the amount of immunity defeat on ND increased ,even chicks with high HI titer can be infected by NDV virulent strain .The gene-engineering vaccine is badly needed to control ND outbreak .So Newcastle disease virus strain B95, a naturally avirulent and heat stable strain ,was introduced from Australia. A surface antigen hemagglutinin-neuraminidase(HN) gene of the NDV B95 strain ,was selected to study as target gene .Referred to the reported sequence ,rwo primers were designed and synthesized .HN gene of NDV B95 strain was amplified by RT-PCR,The PCR products were checked by agrose gel electrophoresis and purified by agrose gel fraction method. The purified products were cloned into PGEM-T-easy vector successfully, the cloned plasmids were transformed into E.coli.TGl. The specific recombinant plasmid was identified by molecular weight, PCR and restriction endonuclease analysis. The results indicated that the resultant construct contained the gene of interest HN at right orientation of the insert. The positive cloned was sequenced by Sanger ' s dideoxy sequencing method. The results demonstrated the size of HN gene was 1930bp in length and included an open reading frame encoding a protein of 578 amino acid residues. The cDNA of HN gene from B95 strain, shares a high identity between 88% and 95.1% with that of reported HN genes .The amino acid residues predicted, shares an identity between 92.1% and 96.1% with that of other strains. Major difference of HN proteins lies in No.18-No.75 amino acid residues on N-terminal which includes three active sites of HN protein.The influence on biological action,caused by the difference of HN protein ,is needed to study further.HN gene has only four glycosylation sites, which is 1 or 2 less on amount than that of other strains. So, this difference can be take on as a natural mark of NDV B95 strainFurthermore, the fragment encoding HN gene was excised from the positive clone PGEM-HN with Sail and Saci enzyme, and purified by agrose gel fraction method. Then the fragment was subcloned into the PET-28c expressing vector digested by Sail and Saci restriction enzyme. The recombinant expressing plasmids were identified by PCR and restriction enzymes analysis. The results indicated that the fragment was correctly inserted into the PET-28c expressing vector and conformed to a reading frame. The fusion protein was expressed in E.coli BL21 (DE3) host with a predicted molecular-47-weight of 66KDa .Peaks of target protein was achieved at 8h after adding IPTG. The target protein purified by Ni-NTA metal chelate affinity presented one major band in the SDS-PAGE and reacted with rabbit serum raised against the NDV B95 strain in Western blotting, suggested that the protein could be used as one of candidated antigenes. It provided the basis for further studying on recombinant vaccine against NDV.