幽门螺杆菌尿素酶B亚单位的克隆表达及纯化

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

生物技术通报 ›› 2015, Vol. 31 ›› Issue (7): 220-225.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.034

幽门螺杆菌尿素酶B亚单位的克隆表达及纯化

闫锦锦, 闫东明, 邹雪, 刘丹, 彭超, 苏亚南

(芜湖康卫生物科技有限公司，芜湖 241000)

收稿日期:2014-10-15 出版日期:2015-07-16 发布日期:2015-07-16
作者简介:闫锦锦，男，硕士，研究方向：生物化学与分子生物；E-mail：yanjinjin222702@163.com
基金资助:
国家科技重大专项子课题项目(2014ZX09102042-002)，安徽省科技攻关计划项目(1301041020)

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

Yan Jinjin, Yan Dongming, Zou Xue, Liu Dan, Peng Chao, Su Yanan

(Wuhu Kangwei Biotechnology Co.，Ltd.，Wuhu 241000)

Received:2014-10-15 Published:2015-07-16 Online:2015-07-16

摘要/Abstract

摘要： 为获得高纯度具有生物活性的尿素酶B亚单位(UreB)蛋白，利用PCR方法扩增出ureB目的基因，将其插入到pET28a载体中，构建表达质粒pET28a-ureB。将鉴定正确的质粒转入大肠杆菌中培养，通过IPTG诱导表达获得UreB蛋白。采用Q Sepharose High Performance阴离子交换层析纯化，G-25凝胶过滤层析脱盐，并通过SDS-PAGE和免疫双扩散法对UreB蛋白进行鉴定。结果表明，该蛋白相对分子质量约为64 kD，与预期结果相符，脱盐后获得的UreB蛋白纯度为98.5%；免疫双扩散法证明该蛋白具有良好的生物活性和反应特异性。最终确定的纯化工艺，达到了一步纯化即得到高纯度、具有生物活性蛋白的目的，该纯化工艺简单、有效。

关键词: 幽门螺杆菌, 尿素酶B亚单位, UreB蛋白, 蛋白纯化

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

闫锦锦, 闫东明, 邹雪, 刘丹, 彭超, 苏亚南. 幽门螺杆菌尿素酶B亚单位的克隆表达及纯化[J]. 生物技术通报, 2015, 31(7): 220-225.

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.

参考文献

[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.

幽门螺杆菌尿素酶B亚单位的克隆表达及纯化

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

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	刘晓黎, 童真艺, 赵亮, 尹丽, 刘晨光. 非抗生素类活性物质抗幽门螺杆菌研究进展[J]. 生物技术通报, 2022, 38(9): 96-105.
[2]	覃雪晶, 王雨涵, 曹一博, 张凌云. 青杄PwHAP5基因原核表达及多克隆抗体制备[J]. 生物技术通报, 2022, 38(8): 142-149.
[3]	唐禄, 董丽平, 尹茉莉, 刘磊, 董媛, 王会岩. 成纤维细胞生长因子20单克隆抗体的制备及鉴定[J]. 生物技术通报, 2021, 37(10): 179-185.
[4]	孟利, 杜彩萍. 大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定[J]. 生物技术通报, 2020, 36(12): 98-103.
[5]	周阳, 王桃桃, 闫丹丹, 王莹莹, 施沁璇, 孙丽慧, 林锋. 类弹性蛋白作为功能纳米材料在生物工程领域研究及应用进展[J]. 生物技术通报, 2020, 36(11): 198-208.
[6]	李玲, 白娟娟, 郭梅, 王思禹, 王晓玥. 番茄果实转录因子CNR的原核表达与纯化及其抗体的制备[J]. 生物技术通报, 2019, 35(2): 1-8.
[7]	张鹏飞, 边帅, 赵月, 赵雨, 王佳雯. 人参thionins的克隆、原核表达和活性研究[J]. 生物技术通报, 2019, 35(2): 53-57.
[8]	陈建军, 刘梁涛, 曹香林. 黄孢原毛平革菌漆酶基因lac1680的克隆、表达及产酶研究[J]. 生物技术通报, 2018, 34(4): 214-220.
[9]	王鹏举,谭焕波,苏文成,张文宇,邹培建. 菌丝霉素MP1106融合蛋白的复性及纯化方法[J]. 生物技术通报, 2017, 33(9): 94-100.
[10]	袁敏,齐玉荣,王瑞菊,. 蛋白激酶BIN2的纯化及活性分析[J]. 生物技术通报, 2017, 33(7): 145-149.
[11]	王亚美,魏原杰,艾新宇,刘小宁. 棉蚜His-CYP6J1融合蛋白的分离纯化及多克隆抗体的制备[J]. 生物技术通报, 2017, 33(5): 164-169.
[12]	陈飞, 周彤, 魏宇佳, 杨晶, 戴传超. 茅苍术质膜蛋白的制备及其双向电泳蛋白质组学平台的构建[J]. 生物技术通报, 2016, 32(9): 72-82.
[13]	喻放, 杨忠华, 范汉东, 左振宇. 人IL-24基因原核表达载体的构建及蛋白的表达纯化[J]. 生物技术通报, 2016, 32(2): 84-89.
[14]	李晶泉, 徐永平, 王熙涛, 李媛, 王丽丽, 李晓宇. 抗金黄色葡萄球菌单链抗体（scFv）的原核表达及蛋白纯化[J]. 生物技术通报, 2016, 32(11): 194-201.
[15]	杨川, 胡敏. 斑马鱼SFPQ蛋白的原核表达及纯化[J]. 生物技术通报, 2016, 32(1): 163-168.