Construction Baculovirus Isolation And Structure Of Recombinant Porcine Transmissible Gastroenteritis Virus

Transmissible gastroenteritis virus (TGEV) is the etiological agent of transmissible gastroenteritis (TGE), a condition associated with high morbidity in animal of all ages and high mortality in sucking piglets It occurs at almost every swine-raising farm and results in severe economic losses. Transmissible gastroenteritis virus is a member of the Coronaviridae family, it possesses a large28.5kb single-stranded sense RNA genome, and is comprised of four structural proeins encoded by the spike (S), membrane (M), envelope (E), and nucleoprotein (N) genes. The S protein forms the peplomers on the virion envelope and features major antigenic site.The M proein is embedded in the lipid virion envelope, taking part in VLP assembly and the N protein associates with the genomic RNA to form the nucleocapsid, inducing cell immunity of infection animals. The small E protein is localized in the perinuclear region of infected cells, and is expressed on the cell surface. Strudes demonstrated that baculovirus can deliver exogenous antigen to host cells. This provides the foundation for researching recombiant baculovirus expressing structural genes of transmissible gastroenteritis virus.A transmissible gastroenteritis virus designated CQ strain was isolated from suspect feces of piglets in Chongqing and identified by ST cell culture, direct fluorescent antibody test (FA), TME examinatin and RT-PCR methods. The M gene were amplifed from the infected cells, and cloned into PMD18-T vector and further sequenced. The results of sequencing showed that homolgy of the M gene of CQ strain is more than96.0%compared with the published strains in GenBank. A phylogenetic tree constructed on the M sequences revealed that CQ strain is more closely relate to the TGEV PUR46-MAD (GenBank accession number NC002306), and formed a separated part.M, sM, N genes of transmissible gastroenteritis virus were amplified, then cloned into PMD18-T vector, after enzyme identification, inserted into donor plasmid pFastBacTM Dual to obtain recombinant donor plamid pFastBacTM Dual-M、pFastBacTM Dual-M-sM and pFastBacTM Dual-N. Plasmid pFastBacTM Dual-M、pFastBacTM Dual-M-sM and pFastBacTM Dual-N were transformed into E.coli DHlOBac respectively and recombinant plasimid Bacmid-M、Bacmid-M-sM and Bacmid-N were obtained. Recombinant baculovirus rBac-M、rBac-M-sM and rBac-N was obtained by transfection of the sf9cells with Bacmid-M、Bacmid-M-sM and Bacmid-N. Western blot and indirect immunofluorescence assay (IFA) was carried and the resulted showed that recombinant M protein, sM protein and N protein were expressed successfully.In vitro expetiments of VLP assembly of TGEV was performed by infection of insect cell with rBac-M and rBac-M-sM. It is showed that expression M proteins alonely and co-expression of M and sM proteins in insect cell Sf9infected by recombinant baculovirus could result in the occurrence of morphologically similar to virion of transmissible gastroenteritis virus. Diameter of VLP is variable from60nm to120nm. This study provide the foundation for researching the VLP assembly of TGEV.