Cloning And Expression Of Fusion Protein And Hemagglutinin-neuraminidase Gene From Newcastle Disease Virus La Sota Strain And Analysis Of Immunogenicity

Newcastle Disease (ND) is widespread all over the world and published one of the main pathogens affecting poultry industry seriously disease by OIE. In recent years, the outbreak of ND appear some new characters , it was 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.Referred to the reported sequence, two pairs of oligonucleotide primers were designed and synthesized. F and HN gene of NDV La Sota strain were amplified by RT-PCR. 1700bp and 1800bp DNA fragments were amplified and cloned into PGEM-T easy vector respectively. And then transformed into E.coli.DH5a. The two specific recombinant plasmids were identified by molecular weight, PCR and restriction endonuclease analysis. The results indicated that the resultant constructs contained the gene of interest F and HN at right orientation of the insert. The nucleotide sequence of the F and HN gene of La Sota were sequenced and amino acid sequence was inferred and compared. The result showed the homology of nucleotide sequence and amino acid sequence of F genes with those of reported strains were 84.7%~99.9% and 88.1 %~99.6%. The homology of nueleotide sequence and amino acid sequence of HN gene with those of reported strains were 82.5%~99.6% and 88.1 %~99.0%.The cloned genomic DNA were subcloned into prokaryotic expression vector PET-32a(+). The two recombinant expressing plasmids(PET-F, PET-HN) were identified by PCR and restriction enzymes analysis. The results indicated that the fragment were correctly inserted into the PET-32a expressing vector and conformed to the reading frame 83kd and 86kd fusion protein were produced in E. coli BL21(DE3) under the induction of IPTQ and the expression quantity and immunoreactivity of the fusion protein were detected by SDS-PAGE electrophoresis and Western -blotting.After the conditions were optimized, the recombinant protein was expressed on a larger scale. Kunming rats were immuned with purified recombinant protein, and all the rats produced relatively supernal antibody. The results showed that therecombinant protein expressed in E.coli still maintained some antigenicity of NDV. antigenes. It provided the basis for further studying on recombinant vaccine against NDV.