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

In the research,sites A and D of S gene(named S1 gene in the research) in Procine transmissible gastroenteritis virus(TGEV) and LTB in ETEC were cloned to construct recombinant expression vectors pYA3342-S1 and pYA3342-LTB-S1.Constructed recombinant plasmids were respectively electroporated into S.typhimurium X3730 and X4550,then the two recombinant products in X4550 which could express interest protein with high stability in vitro were obtained.It makes a great contribution to the development of oral live vaccine against TGEV.1.Cloning of TGEV S1 gene and LTBA pair of specific primers were designed and synthesized on published S gene cDNA sequence of TGEV strain SC-H,with which a DNA fragment of 964 bp coding S protein epitopes A and D of TGEV was amplified from plasmid pMD19T-S by PCR.A pair of specific primers were designed and synthesized on published LTB gene sequence of ETEC strain M17873 in Genbank,with which a DNA fragment of 395bp was amplified from plasmid LT of ETEC strain 196 by PCR.The sequencing result is the same as published sequence of M17873 without any differences in nucleotide level.2.Constrnction of attenuated S.typhimurium vaccine strain expressing TGEV S proteinLTB gene was cloned into pVAX between enzyme sites of EcoRâ… and BamHâ… to construct vector pVAX-LTB.Then the digested S1 gene with BamH经and Hindâ…¢was inserted into pVAX-LTB to construct vector pVAX-LTB-S1.LTB-S1 digested from vector pVAX-LTB-S1 was cloned between enzyme sites EcoRâ… and Hindâ…¢of pYA3342 to construct vector pYA3342-LTB-S1.S1 gene digested from pMD19-S1 with BamHâ… and Hindâ…¢was inserted into pYA3342 to construct vector pYA3342-S1.pYA3342-S1 and pYA3342-LTB-S1 were electroporated into S.typhimurium X3730 and X4550 by turns,respectively,by which S.typhimurium X4550(pYA3342-S1)and X4550(pYA3342-LTB-S1)were constructed.The expression protein of recombinants were analysised by SDS-PAGE induced by IPTG with a dose of 1 mmol/L.The result indicated that the expression S1 protein and LTB-S1 fusion protein were about 37kDa and 49kDa,respectively. 3.Bionomics rudiment experiment of two vaccine strainsOral vaccination of attenuated S.typhimurium X4550(pYA3342-LTB-S1) or X4550 (pYA3342-S1) with a dose of 1×10¹¹CFU is safe for 6～8week-old mice.There was no diference in virulence between the original atenuated Salmonella and the bacteria that had been cultured 10 generations.Moreover,the recombinant plasmid was also very.There is no significant difference among X4550(pYA3342-LTB-S1),X4550(pYA3342-S1) and X4550(pYA3342);They were stable after being cultured 10 generations in LB(DAP+).6～8week-old mice were vaccinated orally with either 1010 or 1011CFU of X4550(pYA3342-LTB-S1),X4550(pYA3342-S1).Persistence of Salmonella in spleen and liver detection up to 3 weeks post inoculation was experienced.These mice inoculated were no abnormal symptom in 30 days.