Expression Of Type Ⅶ Newcastle Disease Virus F Gene In Bombyx Mori Baculovirus Expression System

【Objective】Newcastle disease is a highly contagious avian disease with worldwide distribution.Because ofthe potential shortcomings of traditional Newcastle disease vaccine, it’s urgent to improve and enhance theeffectiveness of the Newcastle disease vaccine. In this study, antigen gene F of Newcastle disease type VIIwas expressed using silkworm baculovirus expression system, which laid a foundation for further research ofNDV antigen-antibody detection reagents and Newcastle genetically engineered vaccine.【Method】(1)In this study,the NDV-F gene sequence(GenBank accession number:AF453761) wasartificially synthesized after codon optimization. Partial fragment of NDV F gene (433bp~1083bp) wasamplified by PCR, and in this region, there were two antigenic sites encoding217amino acid residues. Thetarget fragment was connected to pET-28a (+) and induced by IPTG. The purified fusion protein wasidentified by MALDI-TOF-TOF/MS. After four immunization, we obtained the polyclonal antiserum.【Result】(1) By restriction enzyme digestion and gene sequencing, the target gene was cloned to prokaryoticexpression vector pET-28a (+).The recombinant plasmid was induced by0.5mmol/L IPTG at37°C for4h,and the protein was detected by SDS-PAGE, which was consistent to expected results. Furthermore, theprotein was identified by MALDI-TOF-MS, indicating that it’s consistent with theoretical amino acidssequence. The antiserum titer was1:3200, and it can be used to detect eukaryotic expression products.(2)Western-Blot assay showed that the target protein was detected in the hemolymphof the infected silkworm larvae and its molecular weight was about59ku.【Conclusion】(1) Polyclonal antibody was obtained, which can be used for eukaryotic expression productsdetection;(2) NDV-F gene was Successfully expressed. This study laid a foundation for the further researchof animal experiments and Newcastle genetically engineered vaccine.