Cloning, expression, identification and bioinformatics analysis of Rv3265c gene from Mycobacterium tuberculosis in Escherichia coli

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• 作者：Qiang Wu^a ; ^b ; ^c ; Peng Zhou^a ; ^b ; ^{zhp6301@126.com} ; Shiyun Qian^c ; Xi Qin^d ; Zhigang Fan^c ; Qiongyao Fu^c ; Zhinong Zhan^c ; Hua Pei^c
• 关键词：Mycobacterium tuberculosis ; L-rhamnosyltransferase ; Expression ; Identification ; Bioinformatics
• 刊名：Asian Pacific Journal of Tropical Medicine
• 出版年：2011
• 出版时间：April 2011
• 年：2011
• 卷：4
• 期：4
• 页码：266-270
• 全文大小：367 K

文摘

Objective

To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E. coli) under optimistic conditions, obtain and identify protein expressed, analyze the structure and characteristics of the protein using bioinformatics methods for future applications.

Methods

Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction, and was cloned into the pET-30a vector after purification and recovery. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3), and then purified and identified by western blotting. The essential physical-chemical properties of the protein were predicated by bioinformatics tools, including subcellular location, secondary structure, domains, antigenic epitopes, etc. Tertiary structure of the protein based on homology modeling was established, while multi-sequence homological alignment and phylogenetic analysis were proformed.

Results

The recombinant protein was obtained in soluble fraction from expression system in E. coli BL21(DE3) carrying pET30- Rv3265c plasmid, and Rv3265c gene was expressed correctly. Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices, located outside of membrane. Secondary structure analysis revealed it contained ¦Á-helix, extended strand and random coil, 46.8 % , 14.6 % , 38.6 % , respectively. Furthermore, it possessed six potential antigenic epitopes, one glycosyl transferase domain. A simple three-dimensional model of this protein was constructed by Swiss-model sever. Both sequences and structures were conservative and especial either in gene or in protein.

Conclusions

Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine. The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.