Expression And Immunity Of Fusion Gene Of K88ac-ST Ⅱ Of Escherichia Coli In Attenuated Salmonella Typhimurium

Object:To construct a live oral Salmonella typhimurium vaccine strain expressing K88ac-STII fusion gene of enterotoxigenic Escherichia coli.Methods: Primers(P1,P2) were designed according to the nucleotide sequence of K88ac gene of enterotoxigenic Escherichia coli(ETEC) reported respectively and the expression vector pYA3334 . With the specific primers, the gene encoding the fusion protein K88ac-STII was amplified by PCR of the plasmid pET- K88ac-STâ…¡and was inserted into the linearized plasmid vector PBS-T. By enzyme analysis, PCR and sequencing, the recombinant plasmid was proved to be true. The target fragment was obtained by digestion of the plasmid pBS- KS using restrictive enzyme NcoI/BamHI and was inserted into the prokaryotic expression vector pYA3334 containing asd gene and was introduceded into the delta Cya ,delta Crp ,delta Asd attenuated Salmonella typhimurium strain X4550 by electroporation transformations , which is a balanced lethal recombinant. Analyzed the products of expression through SDS-PAGE and Western-blotting . The Immune Effect of Recombinant strain X4550(pYA3334-K88ac-STII) was indicated by the level of antibody and pathological change. Mice were then inoculated with 109 or 1010 cfu recombination strain through a stainless steel gavage tube,as experimentation vaccine, empty vector X4550(pYA3334)and PBS were used as control.Mice of three groups were immunized with each of them and challenged with virulent strain C83915(STâ…¡ï¹¢).Result: The fusion gene was coloned into prokaryotic expression vector pYA3334 with the right ORF, and can express the products expected successfully in X4550. The recombinant strain can express fusion protein, which has the same molecular weight as prospective protein.The fusion protein expressed by recombinant strain X4550(pYA3334-K88ac-STII) can be identified by anti-K88ac.No corresponding protein was expressed in X4550 transformed by pYA3334 .The stability of The recombinant strain X4550(pYA3334-K88ac-STII) in vitro asserted The foreign antigen K88ac-STâ…¡can be expressed stabily. The mice immunized with recombinant strain X4550(pYA3334-K88ac-STII)vaccine can produce anti- K88ac-STâ…¡.Recombinant strain X4550(pYA3334-K88ac-STII) has effective Immune Effect to mice. The recombinant strain is worth considering as a candidate vaccine strain against enterotoxin Escherichia coli disease and Salmonella typhimurium infections.