Purification Of Newcastle Disease Virus F48E9 Strain And Expression Of HN Gene In The Prokaryotic Expression Systems

Ultracentrifugation and density gradient are usually used to purify virus in the laboratory, but these methods can hardly used in industrial production. Ultra filtration and affinity chromatography are usually used in the purification of human vaccine, which can not be generally used for animals'vaccine for its cost. Ammonium sulfate, cold alcohol and pET6000 were used in this research for the preliminary sedimentation of NDV. Erythrocyte hemagglutination was also used to detect concentration result. The result showed that when the final concentration of ammonium sulfate reached 40% it had a best effectiveness, the HA valence was 32 times as initial; when the final concentration of cold alcohol reached 30% it had a best effectiveness, the HA valence was 8 times as initial; and when the final concentration of PEG6000 reached 20% it had a best effectiveness, the HA valence was 16 times as initial. These results will be helpful to purify the vaccine for animals.Newcastle disease (ND) is an acute and highly contiguous avian infectious disease brought by Newcastle disease virus (NDV). ND is a violent contagion classified by OIE. We still have not any efficacious drugs for ND. Vaccine was the best way to against its outbreak. Among all kinds of vaccines, the gene-engineering vaccine of NDV has a bright outlook. Two parts of hemagglutinin-neuraminidase (HN) gene were selected to study as target gene, which contain most parts epitopes of HN gene. These two fragments were amplified by RT-PCR. The PCR products were checked by agrose gel electrophoresis and purified by agrose gel fraction method. The size of these two fragments is 790bp and 579bp.The purified products were cloned into pGEM-T vector. Recombinant endonuclease analysis and sequencing indicated that the fragments were successfully inserted into the pGEM-T vector.Furthermore, the fragments was subcloned into the pET-28a (+) expressing vector. The recombinant expressing plasmids were identified by PCR and restriction enzymes analysis. The results indicated that the fragments were correctly inserted into the pET-28a(+) expressing vector. The fusion proteins were expressed in E.coli BL21 (DE3) host with the predicted molecular weight of 28.8KDa and 19KDa. The target protein purified by Ni-NTA metal chelate affinity presented one major band in the SDS-PAGE.The protein could be used for further studying on gene-engineering vaccine and monoclonal antibody of NDV.