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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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[1] Julie P,Gary D,Friedman M S,et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med,1991,325(16):1127-1131.
[2] Yoshio Y. Roles of Helicobacter pylori BabA in gastroduodenal pathogenesis. World J Gastroenterol,2008,14(27): 4265-4272.
[3] Dag I,Anna A,Johan O,et al. Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science,1988,279(5349): 373-377.
[4] 薛利军,苏全胜,刘小北 等. 幽门螺杆菌VacA、CagA及BabA蛋白的二级结构和免疫表位预测分析. 中国免疫学杂志,2011,27(12):1153-1158. Xue L J,Su Q S,Liu X B,et al. Prediction and analysis of secondary structures and immune epitopes for Helicobacter pylori VacA, CagA and BabA proteins. Chinese Journal of Immunology,2011,27(12):1153-1158.
[5] 萨姆布鲁克J,拉塞尔D W. 分子克隆实验指南. 第三版,北京:科学出版社,2005,1228-1232. Sambrook J,Russell D W. Molecular Cloning:A Laboratory Manual. 3rd ed,Beijing:Science Press,2005,1228-1232.
[6] 汪家政,范明. 蛋白质技术手册.北京:科学出版社,2000,27-33,77-90,167-183. WANG J Z,FAN M. Handbook of Protein Technology. Beijing:Science Press,2000,27-33,77-90,167-183.
[7] 白杨,陈烨,林焕健,等. 幽门螺杆菌重组粘附素的安全性、免疫原性和生物活性的体外评价. 第一军医大学学报,2003;23(9):882-884. Bai Y,Chen Y,Lin H J,et al. In vitro evaluation of the safety and biological activity of recombinant Helicobacter pylori blood group antigen-binding adhesion. Journal of First Military Medical University. 2003;23(9):882-884.
[8] International Agency for Research on Cancer. Infection with Helicobacter pylori. In: IRAC monographs on the evaluation of carcinogenic risks to humans. Schistosomes, liver flukes and Helicobacter pylori. Lyon: IRAC. 1994,61:177-244.
[9] Wilson K T,Crabtree J E. Immunology of Helicobacter pylori: insights into the failure of immune response and perspectives on vaccine studies. Gastroenterol,2007,133(1): 288-308.
[10] Rad R,Gerhard M,Lang R,et al. The Helicobacter pylori blood group antigen-binding adhension facilitates bacterial colonization and augments a nonspecific immune response. Immunol,2002,168(6):3033-3041.
[11] Molnar B,Galamb O,Sipos F,et al. Molecular pathogenesis of Helicobacter pylori jnfection: the role of bacterial virulence factors. Dig Dis,2010,28(4-5):604-608.
[12] Todoroki I,Joh T,Watanabe K,et al. Suppressive effects of DNA vaccines encoding heat shock protein on Helicobacter pylori-induced gastritis in mice. Biochem Biophys Res Commun,2000,277(1):159-63.
[13] Cynthia K L,Richard W,William D,et al. Oral Immunization with Recombinant Helicobacter pylori Urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis. Infect Dis,1995,172(1):161-172.
[14] Shahjahan K. The current status of Helicobacter pylori vaccines: a review. Helicobacter,2007,12(2): 89-102.
[15] Toller I A,Neelsen K J,Steger M,et al. Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double-strand breaks and a DNA damage response in its host cells. Proc Natl Acad Sci,2011,108(36):14944-14949. |
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