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Prokaryotic expression, identification and bioinformatics analysis of fbpB-esxA fusing gene from Mycobacterium tuberculosis

文献类型： 外文期刊

作者： Wu, Qiang 1 ; Fu, Qiongyao 2 ; Chen, Qian 4 ; Cai, Qunfang 2 ; Fan, Zhigang 2 ; Zhan, Zhinong 2 ; Niu, Lina 2 ; Pei, Hua; 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Anal & Testing Ctr, Haikou 571101, Peoples R China

2.Hainan Med Univ, Sch Tropt & Lab Med, Haikou 571101, Peoples R China

3.Hainan Univ, Sch Agr, Haikou 570228, Peoples R China

4.Hainan Med Univ, Affiliated Hosp, Haikou 571102, Peoples R China

关键词： Mycobacterium tuberculosis;Fusing gene;Protein expression;Identification;Bioinformatics

期刊名称：ASIAN PACIFIC JOURNAL OF TROPICAL MEDICINE （ 影响因子：1.226； 五年影响因子：2.285 ）

ISSN： 1995-7645

年卷期： 2011 年 4 卷 7 期

页码：

收录情况： SCI

摘要： Objective: To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis (MTB), express the encoded fusing protein in Escherichia call (E. coli), identify protein acquired, and predict the structure and function of the protein utilizing methods of bioinformatics. Methods: fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR. The fbpB-esxA fusing gene ligated by (Gly(4)Ser)(3) linker was gained by means of Gene Splicing by Overlapping Extension PCR (SOE-PCR), and fusing gene was cloned into expression vector pET-30a. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3). The protein was identified by Western blot using anti HIS antibody. Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics. Results: The DNA sequences fbpB-esxA were identical with that published by GenBank. The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E. coli BL21(DE3) under the induction of IPTG. Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes. Conclusions: The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag. Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly. The existence of linker doesn't affect immunogenicity of Ag85B and ESAT-6. It will allow for characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent..

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