结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

doi:Q786

中国生物工程杂志

2010, Vol. 30

Issue (08): 72-75 DOI: Q786

研究报告

结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

周爱萍,陈艳炯,李薇,张旭燕,徐纪茹^**

西安交通大学医学院免疫学与病原生物学系西安 710061

Homologous Expression of M. Tuberculosis DnaA Protein in M. smegmatis

ZHOU Ai-ping,CHEN Yan-jiong,LI Wei,ZHANG Xu-yan,XU Ji-ru

Department of Medical Technology, Xi’an Jiaotong University, Xi’an 710061, China

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摘要：

目的：在耻垢分枝杆菌中表达重组结核杆菌DnaA蛋白并对表达产物进行鉴定。方法：用PCR的方法扩增结核杆菌dnaA基因并克隆至表达载体pMF406中，构建重组大肠杆菌-分枝杆菌穿梭质粒pMF-dnaA。经双酶切及测序鉴定后，用电转化的方法将重组质粒转至耻垢分枝杆菌mc2155中。用0.02%乙酰胺诱导重组耻垢分枝杆菌，对表达产物进行SDS-PAGE和Western blotting检测和鉴定。结果：重组耻垢分枝杆菌构建成功，SDS-PAGE及Western blotting结果显示该重组耻垢杆菌可以实现结核杆菌DnaA蛋白的同源高效表达。结论：结核杆菌DnaA蛋白的同源表达为结核杆菌DNA复制机制的研究奠定了基础。

关键词： 结核分枝杆菌; 耻垢分枝杆菌; DnaA蛋白; 同源表达

Abstract:

Objective To express Mycobacterium tuberculosis DnaA protein in Mycobacterium smegmatis and identify the expression product. Methods The dnaA gene of M. tuberculosis was amplified by PCR and cloned into expression vector pMF406 to generate the shuttle vector of E.coil and Mycobacterium pMF-dnaA. It was comfirmed by restriction endonuclease digestion and sequence analysis. The recombinant plasmid was transformed into M. Smegmatis mc2155 by electroporation. The recombinant M. smegmatis was induced by 0.02% acetamide and the expression product was analyzed with SDS-PAGE and Western blotting. Results The recombinant M. smegmatis was successfully constructed and the M. tuberculosis DnaA protein was expressed in the recombinant M. Smegmatis at a relatively high level identified by SDS-PAGE and Western blotting. Conclusion The successful homologous expression of the M. tuberculosis DnaA protein will be very helpful for the further study on the mechanism of M. tuberculosis DNA replication .

Key words: M.tuberculosis M.smegmatis DnaA Homologous expression

收稿日期: 2010-03-24 出版日期: 2010-08-25

基金资助:

陕西省卫生厅科学研究基金（08E04）资助项目

通讯作者: 徐纪茹 E-mail: xujiru@mail.xjtu.edu.cn

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引用本文:

周爱萍陈艳炯李薇张旭燕徐纪茹. 结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达[J]. 中国生物工程杂志, 2010, 30(08): 72-75.

ZHOU Ai-Ping, CHEN Yan-Jiong, LI Wei, ZHANG Xu-Yan, XU Ji-Ru. Homologous Expression of M. Tuberculosis DnaA Protein in M. smegmatis. China Biotechnology, 2010, 30(08): 72-75.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q786 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I08/72

［1］ Mott M L, Berger J M. DNA replication initiation: mechanisms and regulation in bacteria. Nat Rev Microbiol, 2007,5(5):343354.
［2］ Morigen Boye E, Skarstad K, LobnerOlesen A. Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region. Biochim Biophys Acta, 2001,1521(13):7380.
［3］ Ogura Y, Imai Y, Ogasawara N, et al. Autoregulation of the dnaAdnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis. J Bacteriol, 2001,183(13):38333841.
［4］ Greendyke R, Rajagopalan M, Parish T, et al. Conditional expression of Mycobacterium smegmatis dnaA, an essential DNA replication gene. Microbiology, 2002,148(Pt 12):38873900.
［5］ Kumar S, Farhana A, Hasnain S E. Invitro helix opening of M. tuberculosis oriC by DnaA occurs at precise location and is inhibited by IciA like protein. PLoS One, 2009,4(1):e4139.
［6］ Daugelat S, Kowall J, Mattow J, et al. The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization. Microbes Infect, 2003,5(12):10821095.
［7］ Triccas J A, Parish T, Britton W J, et al. An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol Lett, 1998,167(2):151156.
［8］ Triccas J A, Roche P W, Winter N, et al. A 35kilodalton protein is a major target of the human immune response to Mycobacterium leprae. Infect Immun, 1996,64(12):51715177.
［9］ Roche P W, Winter N, Triccas J A, et al. Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis. Clin Exp Immunol, 1996,103(2):226232.
［10］ Mahenthiralingam E, Draper P, Davis E O, et al. Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis. J Gen Microbiol, 1993,139(3):575583.
［11］范小勇. 分支杆菌新型表达系统的建立及其在基因重组卡介苗研究中的应用.上海：复旦大学.生命科学学院. 2008. Fan X Y. Establishment of the novel systems in mycobacteria and their application for recombinant BCG. Shanghai: Fudan University School of Life Sciences, 2008.

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