Wei sheng wu xue bao = Acta microbiologica Sinica · 2012
[Cloning, expression and activity of K99 fimbrial operon gene from enterotoxigenic Escherichia coli].
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Yang Yang, Hou Huayan, Yu Lei, Zhu Guoqiang
Аннотация
OBJECTIVE: To clone and express fan operon gene clusters of K99 fimbriae in enterotoxigenic Escherichia coli (ETEC) in vitro, and study the activity of the recombinant E. coli expressing K99 fimbriae.
METHODS: K99 fimbriae gene clusters were amplified by long-PCR method, using the genomic DNA of K99-fimbriae E. coli C8307 as the DNA template. The 5.7Kb PCR products were inserted into expressing vector pBR322 with restriction endonuclease, then positive clones were screened. The positive recombinant plasmid was transformed into non-fimbriae E. coli SE5000 strains, and pBR322 plasmid was also transformed into SE5000 for negative control strain.
RESULTS: The recombination E. coli expressing K99 fimbriae was tested with agglutination assay, using monoclonal antibody serum and brush border vesicles from the piglet small intestinal epithelia cells. The expressed fimbriae on the surface of the recombinant E. coli SE5000 were observed by transmissible electromicroscope. Heat extraction method was employed to isolate and purify K99 fimbriae, which was exerted SDS-PAGE, and 18.5 kDa protein band was detected. The mouse sera produced from recombinant fimbriae was used to test K99-fimbriae strains C83907, C83914, C83260 with positive agglutination results, while negative results were found with E. coli contain other kinds of fimbriae. The assays of SDS-PAGE, Western blot, agglutination assay were used to evaluate antigenicity and biologic activity between C83907 and recombinant strain. Adhesion test with HeLa cell line demonstrated the recombinant strain and wild type have the similar adherence ability, and this adhesion can be inhibited with mouse serum containing polyclonal antibody against recombinant K99 fimbriae.
CONCLUSION: This study has laid a good foundation for further study on bioactivity of K99.
METHODS: K99 fimbriae gene clusters were amplified by long-PCR method, using the genomic DNA of K99-fimbriae E. coli C8307 as the DNA template. The 5.7Kb PCR products were inserted into expressing vector pBR322 with restriction endonuclease, then positive clones were screened. The positive recombinant plasmid was transformed into non-fimbriae E. coli SE5000 strains, and pBR322 plasmid was also transformed into SE5000 for negative control strain.
RESULTS: The recombination E. coli expressing K99 fimbriae was tested with agglutination assay, using monoclonal antibody serum and brush border vesicles from the piglet small intestinal epithelia cells. The expressed fimbriae on the surface of the recombinant E. coli SE5000 were observed by transmissible electromicroscope. Heat extraction method was employed to isolate and purify K99 fimbriae, which was exerted SDS-PAGE, and 18.5 kDa protein band was detected. The mouse sera produced from recombinant fimbriae was used to test K99-fimbriae strains C83907, C83914, C83260 with positive agglutination results, while negative results were found with E. coli contain other kinds of fimbriae. The assays of SDS-PAGE, Western blot, agglutination assay were used to evaluate antigenicity and biologic activity between C83907 and recombinant strain. Adhesion test with HeLa cell line demonstrated the recombinant strain and wild type have the similar adherence ability, and this adhesion can be inhibited with mouse serum containing polyclonal antibody against recombinant K99 fimbriae.
CONCLUSION: This study has laid a good foundation for further study on bioactivity of K99.