Expression And Identification Of The S Gene Segment Of Porcine Transmissible Gastroenteritis Virus

Porcine transmissible gastroenteritis (TGE), which is caused by Porc ine transmissible gastroenteritis virus (TGEV), is a highly contagious swi ne disease characterized by up to 100% mortality in piglets and lead to se vere damage to the global pig industry; its main clinical symptoms includ e acute diarrhea, vomiting, and dehydration. In the TGEV genome, S prot eins encoded by ORF2 are the main structural proteins capable of inducin g neutralizing antibodies in vivo, which consists of four B-cell antigen rec ognition sites A, B, C, and D in the 543 amino acid residues of N terminal sequence. Therefore, it is of great significance to express S protein for th e development of TGEV gene engineering vaccine and disease diagnosis reagent.In this study, three pairs of specific primers were designed and synth esized based on published cDNA sequence of TGEV S gene, four S gene segments containing different antigen recognition sites were obtained by PCR amplification:S2 (containing D antigen recognition site), S3 (contai ning A antigen recognition site), S(2+3)(containing A and D antigen reco gnition sites), and Sfull (containing A, B, C and D antigen recognition sit es). The gene segments were cloned into pMD19-Tsimple respectively an d sequenced. identification. The sequence was used to compare with the S sequence of TGEV in Genebank, the results showed the nucleotide homo logy was 100%.The S gene segments were cloned into baculovirus transfer vector pF BDMY-IM, then introduced into the baculovirus Bacmid by Tn7 transpos on. The recombinant Bacmids were transfected into Sf9 insect cell to obta in recombinant virus. Sf9 insect cells were infected with recombinant viru s and observed using fluorescence microscope at 72 hours post infection.Obvious cell pathological changes and red fluorescence were found, it suggests that recombinant baculoviruses were constructed successfully. The genome DNA were extracted and identified by PCR, the results conf irmed that TGEV S gene has been introduced successfully into baculovir us genome.SDS-PAGE and Western Blot analysis were used to analysis protein expression product, the results showed that S2, S3 and Sfull genes were e xpressed successfully in baculovirus expression system, the protein size were 25.9 KDa,27.5 KDa and 85.1 KDa; S(2+3)genes could not express in baculovirus expression system.Therefore, TGEV S genes containing different antigen sites were ex pressed in baculovirus expression system in this research, which laid the f oundation for the development of TGEV genetic engineering vaccines an d diagnostic reagents.