Construction Of The Vaccine Strain Of The Attenuated Salmonella Typhimurium Strain Expressing TGEV S And M Gene

Transmissible gastroenteritis (TGE) is a highly infectious enteritis disease caused by Transmissible gastroenteritis virus (TGEV), and it makes a great economic loss of pig raising industry. S and M genes are the two important functional proteins of TGEV, and in this article, recombinant attenuated salmonella typhimurium vaccine carried S and M genes of TGEV was constructed, as well as did preliminary immune evaluation based on mice models.1. Construction of eukaryotic expression vector of S and M genesBased on gene sequences of TGEV purdue in GenBank, a pair of specific primers were designed. With RT-PCR, M gene (989bp) was amplified, and then its vector T cloned plasmid pMD19T-M was constructed, by inserting M gene into vector pVAX, vector pVAX-M was constructed. Sub-cloning M gene into bicistron eukaryotic expression plasmid pIRES and insuring M gene was on the polyclonal sites of downstream sequence of IRES, intermediate expression plasmid pIRES-M was constructed. A1639bp IRES-M segment was obtained double digested plasmid pIRES-M with EcoR I and Not I, put it into pVAX-S and insured it was in the downstream of S gene, bicistron eukaryotic expression plasmid pVAX-S-M was constructed and identified by PCR and double digestion.Transfected HEK293cells with expression plasmids pVAX-M, pVAX-S and pVAX-S-M as well as null plasmid pVAX, and detected the result with RT-PCR at72h after transfection. The result showed that except control group pVAX, groups pVAX-M, pVAX-S and pVAX-S-M all detected the transcription of target genes.2. Construction and identification of S and M gene oral vaccine mediated by attenuated salmonella typhimurium Electric transformed eukaryotic plasmid pVAX-M and pVAX-S-M pVAX into attenuated salmonella typhimurium SL7207and constructed recombinant strains SL7207(pVAX-M) and SL7207(pVAX-S-M). Mice were intragastric administration with the two recombinant strains in the dose of5×108CFU,1×109CFU and2×109CFU and boosted immunization2weeks after first-immune. Randomly killed3mice every2weeks after first-immune, and observed the pathological changes of organs and count colony.1-4weeks after first-immune, bacteria could be detected in every group, at5th week, there’s no bacteria in the two groups with the dose of5×108CFU, in the6th week, all recombinant groups had no bacteria to be detected which showed that recombinant attenuated salmonella typhimurium did well in security.3Preliminary immune valuation of S and M gene oral vaccine mediated by attenuated salmonella typhimuriumOral immunized mice with SL7207(pVAX-S), SL7207(pVAX-M), SL7207(pVAX-S-M) and SL7207(pVAX-S)+SL7207(pVAX-M) with the dose of1×109CFU, every2weeks a time,3times in total.2weeks,4weeks,6weeks and8weeks after immunization, detected serum IgG and intestine mucosa IgA antibody with indirect ELISA and serum IFN-y and IL-4with kit respectively. The results indicated that,2weeks after first-immune, serum IgG antibody (P<0.05) and mucosa IgA antibody could be detected. All the groups grew to the peak in the6th week, and began to reduce in the8th week.Detected the content of IFN-y and IL-4in mice’s serum with indirect ELISA, and the result showed that,①since2nd week after first-immune, content of IL-4in each recombinant strain group increased apparently, while content in the two control groups always kept at a low level. Meanwhile the content in double gene group SL7207(pVAX-S-M) kept an increasing tendency from2nd to8th week, and reached99.308pg/mL in the8th week.②since2nd week after first-immune, content of IFN- Y in each group increased apparently, while content in the two control groups always kept in a low level. Content of IFN-y in mixed group SL7207(pVAX-S)+SL7207(pVAX-M) was a little higher than other groups in the former6weeks after immunization, and reached729.622pg/mL in the6th week. In the8th week, content in each group reduced, but double gene group SL7207(pVAX-S-M) reduced less than other groups, the content of IFN-y was also relatively higher than other group, it could reach507.739pg/mL.The result indicated that the recombinant oral immunized mice had a better immunogenicity, it could induce immunized mice produce specific serum IgG and intestine mucosa IgA antibody as well as apparent cellular immunologic response.