Secretory Expression Of The Spike Glycoprotein Of Transmissible Gastroenteritis Virus In Yeast Pichia Pastoris And Immunogenicity Analysis Of The Recombinant Protein

Transmissible gastroenteritis (TGE) has been reported in many parts of the world, including America, Asia and Europe. The disease is spread by the faeco-oral route and is characterised by vomiting, diarrhoea and high mortality in piglets. The causative agent is a coronavirus (TGEV), which is a member of the Coronaviridae family and belongs to the order Nidovirales. This study includes three parts.Partâ… The acquaintance of the proliferation of TGEV in ST cellsAs the host of the TGEV, ST cell was revived and cultured. At fist, the tissue culture infective dose (TCID50) of the TGEV was determinated to measure the cytotoxicity. To confirmed the proliferation of TGEV, cytopathic effect (CPE) and Immunoperoxidase monolayer assay was observed by the Optical Microscopy. The CPE result showed that nucleus had been denatured or integrity was fully destroied. The IPMA confirmed the place of the sythesis of the protein was cytoplasm.Partâ…¡Experimental study on anthocyanin resisting transmissible gastroenteritis virus in vitroIn the patrtâ… , the TCID50 of TGEV had been determinated. By the observation of the morphological changes, the cytotoxic influence, directly inactivited action and treatment action of the procyanidins were studied. The results showed that the safe concentration was not more than 0.002 gram per milliliter, in fact 1 milligram per milliliter. The optimal dose of directly inactivited action was confirmed to be 500 microliter and the treatment dose be 500-375 microliter. This part is only about the study in vitro, since the result is not identical to the study of animals. More animal experiment on procyanidins resisting TGEV needs to be done further. Partâ…¢Secretory expression of the spike glycoprotein of transmissible gastroenteritis virus in yeast Pichia pastoris and analysis on Immunogenicity of the recombinant proteinIn this study, A pair of primers were disigned according to TGEV S gene sequence in Genbank. After amplified by Polymerase Chain Reaction, a 2.2 kb fragment of the amino terminal half of the S gene containing all four major antigenic sites (A, B, C and D) was sub-cloned into pBS-T vector, then S gene was ligated with vector pPIC9K after cleaved from T-vector,.the recombinant shuttle plasmids pPIC9k-S were linearized by SalI and transformed into competent Pichia pastoris GS115 by electroporation and positive clones were screened with G418 and PCR amplification and induced with 1% methanol. At last, the supernatant of medium was analyzed by SDS-PAGE and Western-blotting and purified by ion exchange chromatography and ultrafiltration. A group of 2-week old BALB/c mouse were injected with the purified protein and the serum was detected by ELISA. The results showed that the partial fragment of spike protein (S), a approximately molecular weight 82 000 soluble protein, was secreted into the culture supernatant. The western-blotting results indicated that the protein could be recognized by the positive swine's serum of TGEV. After diluted into 1:100, the specific antibody against S protein could be detected by ELISA.