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Gene Cloning, Expression, Purification, and in vitro Adhesion Activity Determination of Amino-terminal Fragment of Helicobacter pylori BabA Protein |
GAO Han1, XUE Li-jun2, LIU Xiao-bei2, MAO Xiao-bei2, REN Li-li2, GENG Jian2, DAI Ting-ting2, YU Feng1, CHU Xiao-yuan 2 |
1. College of Pharmacy, China Pharmaceutical University, Nanjin 211198, China; 2. Department of Oncology, Nanjing General Hospital of Nanjing Military Command, Nanjin 210002, China |
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Abstract Objective: To clone the gene of amino-terminal fragment of Helicobacter pylori BabA protein ( BabA1 ) and construct a prokaryotic expression plasmid. A fusion protein of 6譎is-BabA1 was then acquired and evaluated its adhesion activity in vitro. Methods: BabA1 gene was amplified by PCR with genomic DNA of H. pylori J99 strain as the template and inserted into pET-32a(+)vector. The pET32a-BabA1 plasmid was confirmed through restriction enzyme analysis and DNA sequencing and then transformed E. coli BL21 (DE3) cell. The 6譎is-BabA1 protein was induced by IPTG, purified through a Ni2+-NTA column, and identified by SDS-PAGE and Western blot. Then a bacterial colony count assay was adopted to determine the cellular adhesion activity of 6譎is-BabA1. Results: BabA1 gene was correctly cloned and recombinant pET32a-BabA1 plasmid was obtained. The 6譎is-BabA1 protein was efficiently induced and successfully purified under optimized conditions. Moreover, 6譎is- BabA1 protein enhanced the adhesion of E. coli BL21 (DE3) strain to gastric cancer MFC cells in vitro. Conclusion: The acquired 6譎is-BabA1 protein has significant adhesion activity, which facilitates further studies on its immunological and biological functions.
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Received: 27 November 2012
Published: 25 March 2013
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Cite this article:
GAO Han, XUE Li-jun, LIU Xiao-bei, MAO Xiao-bei, REN Li-li, GENG Jian, DAI Ting-ting, YU Feng, CHU Xiao-yuan. Gene Cloning, Expression, Purification, and in vitro Adhesion Activity Determination of Amino-terminal Fragment of Helicobacter pylori BabA Protein. China Biotechnology, 2013, 33(3): 34-40.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2013/V33/I3/34
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