Cloning，Expression and Purification of Urease B Subunit of Helicobacter pylori

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.034

Abstract

Abstract: In order to obtain high-purity and bioactive urease B subunit protein(UreB), the fragments of ureB amplified by PCR was inserted into expression vector pET28a, and the recombinant plasmid pET28a-ureB was constructed successfully. The identified plasmid was transformed into Escherichia coli, which was induced to express by IPTG. The expressed protein UreB was purified by Q Sepharose High Performance anion exchange chromatography and desalted by G-25 gel filtration chromatography, then analyzed by SDS-PAGE and double immunodiffusion. The results indicated that the molecular weight of the protein was about 64 kD and its purity was 98. 5% after desalting, which was in accord with the prediction. Double immunodiffusion showed that protein UreB possessed a favorable bioactivity and specificity. The finalized purification process achieved the goal of 1-step purification may obtain high-purity and bioactive protein, and it was simpler than the existing purification process as well as effective.

Key words: Helicobacter pylori, urease B subunit, UreB protein, protein purification

Yan Jinjin, Yan Dongming, Zou Xue, Liu Dan, Peng Chao, Su Yanan. Cloning，Expression and Purification of Urease B Subunit of Helicobacter pylori[J]. Biotechnology Bulletin, 2015, 31(7): 220-225.

References

[1]Yamaoka Y. Mechanisms of disease：Helicobacter pylori virulence factors[J]. Nature Reviews Gastroenterology and Hepatology, 2010, 7(11)：629-641.
[2]Machado AM, Figueiredo C, Seruca R, et al. Helicobacter pylori infection generates genetic instability in gastric cells[J]. Biochimica et Biophysica Acta(BBA)-Reviews on Cancer, 2010, 1806(1)：58-65.
[3]Suerbaum S, Michetti P. Helicobacter pylori infection[J]. New England Journal of Medicine, 2002, 347(15)：1175-1186.
[4]Peter S, Beglinger C. Helicobacter pylori and gastric cancer：the causal relationship[J]. Digestion, 2007, 75(1)：25-35.
[5]Graham DY, Malaty HM, Evans DG, et al. Epidemiology of Helicobacter pylori in an asymptomatic population in the United States[J]. Gastroenterology, 1991, 100(6)：1495-1501.
[6] 张卫军, 郭刚, 刘开云, 等. 幽门螺杆菌双价亚单位分子内佐剂疫苗的构建表达与纯化[J]. 第三军医大学学报, 2008, 30(17)：1587-1590.
[7]Khoder G, Yamaoka Y, Fauchre JL, et al. Proteomic Helicobacter pylori biomarkers discriminating between duodenal ulcer and gastric cancer[J]. Journal of Chromatography B, 2009, 877(11)：1193-1199.
[8]Hung CT, Leung WK, Chan FK, et al. Comparison of two new rapid serology tests for diagnois of Helicobacter pylori infection in Chinese patients[J]. Digestive and Liver Disease, 2002, 34(2)：111-115.
[9]Voland P, Weeks DL, Marcus EA, et al. Interactions among the seven Helicobacter pylori proteins encoded by the urease gene cluster[J]. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2003, 284(1)：G96-G106.
[10]史彤, 刘文忠. 金属螯合亲和层析法对重组幽门螺杆菌尿素酶 B 亚单位的纯化[J]. 胃肠病学, 2002, 7(4)：199-201.
[11]袁小澎, 邹全明, 柏杨, 等. 幽门螺杆菌尿素酶 B 亚单位功能片段的纯化及活性研究[J]. 南方医科大学学报, 2007, 27(7)：959-962.
[12]王缚鲲, 邹全明. 从包涵体中纯化重组人幽门螺杆菌尿素酶 B 亚单位[J]. 中国生物制品学杂志, 2002, 15(1)：38-41.
[13]杨晓梅. 包涵体蛋白的复性技术[J]. 国外医学：临床生物化学与检验学分册, 2000, 21(2)：98-99.
[14]Patra AK, Mukhopadhyay R, Mukhija R, et al. Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli[J]. Protein Expression and Purification, 2000, 18(2)：182-192.
[15]Cho WK, Sohn U, Kwak JW. Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I[J]. Journal of Biotechnology, 2000, 77(2)：169-178.