大肠杆菌外膜蛋白OmpW的克隆及在外膜上高表达

中国生物工程杂志

2011, Vol. 31

Issue (12): 46-50

研究报告

大肠杆菌外膜蛋白OmpW的克隆及在外膜上高表达

邹海杰, 吴贤斌, 潘建义, 赵辅昆

浙江理工大学生命科学学院杭州 310018

Cloning and Expression of ompW Location in Outer Membrane of Escherichia coli

ZOU Hai-jie, WU Xian-bin, PAN Jian-yi, ZHAO Fu-kun

School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China

全文: PDF(840 KB) HTML

摘要：

目的: 利用表达载体pLLP-OmpA实现大肠杆菌K12外膜蛋白OmpW在外膜上高表达。方法: PCR扩增ompW基因,构建重组表达载体pLLP-OmpA-ompW,然后转化大肠杆菌K12,得到在外膜上高表达的菌株。提取该菌外膜蛋白,利用免疫小鼠制备得到的抗血清进行Western blot分析验证高表达的OmpW是否定位于外膜。结果: 成功构建了重组表达载体,经转化后成功筛选到高表达菌株,并经Western blot证实高表达的OmpW定位在外膜上。结论: 首次成功获得OmpW在外膜上的高表达,该高表达菌株可为深入研究OmpW在细菌致病机制中的作用及其它功能提供研究基础。

关键词： 大肠杆菌K12; OmpW; 重组载体; 抗血清制备

Abstract:

Objective:To achieve the mutant stains of Escherichia coli K12 which up-expressed OmpW located in outer membrane by vector pLLP-OmpA. Methods:The complete sequence of ompW from E. coli K12 was cloned by PCR. The cloned DNA fragments were linked with the plasmid to construct the vector pLLP-OmpA-ompW. The recombinant vector was then transferred into E. coli K12 to obtain the engineering strain. Western bolt analysis with primary antibody against OmpW was used to verify whether the highly expressed OmpW of the strain located in outer membrane or not. The polyclonal antibody was prepared by immunizing mice with OmpW protein extracted from strain of pET-28a-ompW. Results: The recombinant expression vector of OmpW was successfully constructed and the high expression strains were also obtained after transformed into the host. The strains which the high-expression of OmpW located in the outer membrane were further confirmed by Western blot analysis. Conclusion: The strain of high-expression proteins location in outer membrane was achieved for the first time. The result will contribute to investigation the function of OmpW.

Key words: Escherichia coli K12 OmpW Recombination plasmid Antiserum preparation

收稿日期: 2011-08-03 出版日期: 2011-12-25

ZTFLH:

Q786

基金资助:

浙江省自然科学基金(Y2090396)、浙江省公益技术研究社会发展项目(2011C23067)资助项目

通讯作者: 潘建义,电子信箱:panjy@zstu.edu.cn E-mail: panjy@zstu.edu.cn

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引用本文:

邹海杰, 吴贤斌, 潘建义, 赵辅昆. 大肠杆菌外膜蛋白OmpW的克隆及在外膜上高表达[J]. 中国生物工程杂志, 2011, 31(12): 46-50.

ZOU Hai-jie, WU Xian-bin, PAN Jian-yi, ZHAO Fu-kun. Cloning and Expression of ompW Location in Outer Membrane of Escherichia coli. China Biotechnology, 2011, 31(12): 46-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I12/46

[1] 李碧华, 杨正时. 革兰氏阴性菌外膜蛋白的结构、功能及遗传学. 国外遗传学:微生物学分册, 1991, 14(5): 193-196. Li B H, Yang Z S. Foreign Medicine: Microbiology Section, 1991, 14(5): 193-196.

[2] Tsolis R M. Comparative genome analysis of the alphaproteobacteria: relationships between plant and animal pathogens and host specificity. Proc Natl Acad Sci USA, 2002, 99(20): 12503-12505.

[3] 居尔毅, 宁喜斌. 副溶血性弧菌外膜蛋白的提取与免疫鉴定. 安徽农业科学, 2008, 36(32): 14116-14111. Ju E Y, Ning X B. Journal of Anhui Agricultural Sciences, 2008, 36(32): 14116-14111.

[4] Stahlberg H, Fotiadis D, Scheuring S, et al. Two-dimensional crystal:a powerful approach to access structure, function and dynamics of membrane protein. FEBS Lett, 2001, 504(3): 166-172.

[5] Hong H, Patel D R, Tamm L K, et al. The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel. J Biol Chem, 2006, 281(11): 7568-7577.

[6] Sharma A, Chaturvedi A N. Prevalence of virulence genes (ctxA, stn, OmpW and tcpA) among non-O1 Vibrio cholerae isolated from fresh water environment. Int J Hyg Environ Health, 2006, 209(6): 521-526.

[7] McPhee J B, Tamber S, Bains M, et al. The major outer membrane protein OprG of Pseudo monas aeruginosa contributes to cytotoxicity and forms an anaerobically regulated, cation-selective channel. FEMS Microbiol Lett, 2009, 296(2): 241-247.

[8] Singh R, Shasany A K, Aggarwal A, et al. Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics. Clin Exp Immunol, 2007, 148(3): 486-493.

[9] Albrecht R, Zeth K, Söding J, et al. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2006, 62(Pt 4): 415-418.

[10] 刘明智, 叶星, 田园园, 等. 嗜水气单胞菌外膜蛋白W基因的表达及其免疫原性分析. 微生物学通报, 2011, 38(3): 437-445. Liu M Z, Ye X, Tian Y Y, et al. Microbiology China, 2011, 38(3): 437-445.

[11] Qian R, Xiao Z, Zhang C, et al. Expression and purification of two major outer membrane proteins from Vibrio alginolyticus. Acta Biochim Biophys Sin, 2007, 39(3):194-200.

[12] 苏宁, 韩成贵, 李大伟, 等. 小麦黄花叶病毒RNA2 28kDa蛋白基因的表达及表达产物抗血清的制备. 中国病毒学, 1999, 14(3): 217-221. Su N, Han C G, Li D W, et al. Virologica Sinica, 1999, 14(3): 217-221.

[13] 鱼艳荣, 刘希成. 革兰氏阴性菌外膜蛋白研究进展. 动物医学进展, 2000, 21: 35-39. Yu Y R, Liu X C. Progress in Veterinary Medicine, 2000, 21: 35-39.

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