Baculovirus Expression Of The HN And F Genes Of GPMV

Goose Paramyxovirus(GPMV)is homologous with New castle disease virus(NDV) at a greatsignificance, GPMV was revealed to be a variant of NDV but not a new virus. One strain of NDVinfect waterfowl and the virulence was enhanced. However, GPMV was not absolutely differentwith NDV, hemagglutinin-neuraminidase(HN) and fusion protein(F)protein were mutant and resultin the invalidation of traditional vaccine. Therefore, the study on the virus at molecular degree wasof great significance. HN and F proteins were the primary target antigens of humoral immuneresponse against virus. HN and F proteins have become the preferred genes for production ofgenetically engineering vaccine and pathogen diagnosis.In this research, one set of primers was designed based on the sequence of HN and F gene ofGPMV of GenBank and the sequence of eukaryotic transfer vector pBlueBachis2A using primer 5.0. HN and F gene was amplified from pMD18-T-HN and pMD18-T-F respectively and designatedas pMD18-T-GPMV-HN and pMD18-T-GPMV-F. They were ligated into the transfer vectorpBlueBachis2A with Xhoâ… and EcoRâ… restriction sites, and the recombinant plasmids weredesignated as pBlue-GPMV-HN and pBlue-GPMV-F. Co-transfection of Sf9 insect cell wasperformed using purified recombinant plasmids and Bacmid-N-blue DNA. Recombiant virusnamed P1 was recovered 4d after transfection. After 3 serial passges of plaque assay screenfollowed by PCR confirmation, the recombinant virus were designated as rBv-HN and rBv-F. After3 passages of propagation of the recombinant virus, high-titer virus named P3 was used to inoculateSf9 cells followed by harvest after 4d. The F and HN proteins were confirmed to be 60ku and 66kuin length which were consistent with the speulated size as judged by SDS-PAGE seperation. HNprotein could be specifically recognized by chicken-anti-GPMV antibodies as investigated byWestern blot and Dot-ELISA assays. All the results proved the better reactionogenicity of HNprotein.In this research, HN and F proteins of GPMV/JS/1/97/Go strain were expressed withbaculovirus expression system. The expression of the antigenic sites of HN protein wasconfirmed. The results of this study provided data for fundamental research on GPMV, preventivecontrol and virus diagnosis.