Cloning, Expression Of Hemagglutinin-neuraminidase & Fusion Genes Of Newcastle Disease Virus And Preliminary Development Of ELISA Antibody Decteting

Newcastle disease (ND) is a highly contagious disease caused by Newcastle disease virus (NDV). NDV can infect chicken, turkey, and many other avian species, and even human being. It is one of the List A animal infectious disease which cause sever avian epidemic worldwide, and one of the severe infections to poultry breeding which we insist to prevent for several decades years. Since the disease has broken out, it has brought huge economic lose. In this research, we clone the Hemagglutinin-neuraminidase and Fusion genes from NDV (La Sota strain) by RT-PCR. Then two proteins were expressed in the E.coli. The purified HN-GST fusion protein was used to establish an indirect ELISA method for the detection of antibodies to NDV. This work provided foundations for genetically engineered vaccine and rapid diagnosis on Newcastle disease. The following researches were explored:1. Cloning and sequence analysis of Hemagglutinin-neuraminidase and Fusion genesAccording to published F and HN gene sequence of NDV strain La Sota, primers with restriction sites were designed and synthesized. 1677 bp and 1734 bp fragments were amplified from Newcastle disease virus RNA (La Sota strain) by RT-PCR and cloned into the pMD18-T vector. The recombinant plasmid was proved by enzyme analysis. The sequence analysis showed that Fand HN genes consisted of 1677 bp and 1734 bp respectively, which corresponded to the length of published F and HN genes sequence, and their homology were up to 99%.2. Subcloning and expression of NDV F and HN genes in E.coliThe signal peptide and some other fragments of F and HN genes were removed by using PCR. Then the fragments of F and HN were subcloned into prokaryotic expression vector pGEX-KG and were expressed in E.coli B121. Analyzed by SDS-PAGE and western blotting, F-GST and HN-GST fusion proteins presented a 78 KDa and 84 KDa proteins with immunogenicity.3. Development of indirect HN-ELISA for Diagnosis of NDV antibodiePurified HN-GST fusion protein was used to establish an indirect ELISA for detection of antibodies to NDV La Sota strain. The indirect ELISA method was established by optimizing the conditions. The results showed that the optimal coating...