Study On Double-gene Co-expression Of Fimbrial Subunit Gene K88/K99 And 987P/F41 Of Enterotoxigenic Escherichiacoli

The EnterotoxigenicEscherichia coli(ETEC) is an important pathogenic bacteria causing Colibacillus diarrhea of piglet. Adhesions gene plays an important role in the pathogenesis of disease.There are serveral kinds adhesions for example K88,K99,987P,F41,F42,F165,F17and F18 ,especially K88,K99,987P,F41 are more important.Both K88-K99 and 987P-F41 obtained co-expression on the base of single- expressed the four adhesions.It indicated that co-expression fusion-proteins had good reactivity and immunity activity.It established the foundation to manufacturing the gene engineering vaccine of preventing diarrhea of piglet.The results are as follows:1. The DNA fragments of K88 \ K99\987P\F41 without signal peptide sequence were amplified by PCR from the plasmid DNA of E.coli C83907 \C83644\ C83710 and from the DNA of E.coli C83707. Then these amplified genes were inserted respectively into pMD18-t, obtained the recombinant plasmid pMD18-K88\ pMD18-K99\ pMD18-987P\ pMD18-F41 by identification. Then, the four recombinant plasmids and pET30a expression-carrier were digested separately by restriction endonuclease, were linked with T4 DNA Ligase, were recombined to be pET K88\ pET K99\ pET 987P\ pET F41. The analysis of restriction endonuclease-digesting and PCR and nucleotide sequence analysis confirmed that the fusion genes were constructed successfully. The result of SDS-PAGE indicated that the four target genes contained expression2.The K88 gene which had no termination cipher was amplified on the base of cloning K99 gene . The k99 gene was set to the K88 gene downstream end by DNA recombined technology.The recombinant plasmid pET K88-K99 was constructed.The same to F41-987P.The difference was that the 987P gene which had no termination cipher was amplified .It was called 987p-2.The 987p-2 gene was set to the upstream end of F41 by DNA recombined technology. The recombinant plasmid pET 987P-F41 was constructed on the base. 3.The result of western-blotting indicated that the co-expression protein could be recognized by the antiserum against K88 and K99 and 987P and F41. By the reverse indirect hemagglutination assay,K88 potency was 2⁸～2¹⁰ and K99 potency was 2⁷～2⁸ and F41 potency was 2⁶～2⁸ and 987P potency was 2⁸～2¹⁰ .It indicated that the co-expression protein had the favourable antigenicity.It indicated that the constructed recombinant strain may be taken as the candidate strain to prevent the colibacillus diarrhea of piglet with Escherichia coli gene engineering vaccine.