Agriculture School of Hainan University, Haikou 570228, PR China.
Asian Pac J Trop Med. 2011 Apr;4(4):266-70. doi: 10.1016/S1995-7645(11)60083-7. Epub 2011 May 29.
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.
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.
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 α-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.
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.
在有利条件下，将结核分枝杆菌（Mycobacterium tuberculosis）的 Rv3265c 基因在大肠杆菌（Escherichia coli）中进行克隆和表达，获得并鉴定表达的蛋白质，利用生物信息学方法分析蛋白质的结构和特性，为未来的应用提供参考。
采用聚合酶链反应（PCR）扩增结核分枝杆菌 H37Rv 中的 Rv3265c 基因，经纯化回收后克隆至 pET-30a 载体。对重组质粒进行测序，并在大肠杆菌 BL21（DE3）中表达，然后通过 Western blot 进行鉴定和纯化。利用生物信息学工具预测蛋白质的基本理化性质，包括亚细胞定位、二级结构、结构域、抗原表位等。基于同源建模构建蛋白质的三级结构，并进行多序列同源性比对和系统发育分析。
在携带 pET30-Rv3265c 质粒的大肠杆菌 BL21（DE3）表达系统中，获得了可溶形式的重组蛋白，且 Rv3265c 基因表达正确。生物信息学分析表明该蛋白不含信号肽和跨膜螺旋，定位于膜外。二级结构分析显示，该蛋白含有α-螺旋、延伸链和无规则卷曲，分别占 46.8%、14.6%和 38.6%。此外，它具有 6 个潜在的抗原表位和一个糖基转移酶结构域。通过 Swiss-model 服务器构建了该蛋白的简单三维模型。无论是在基因还是在蛋白质水平上，该序列和结构都具有保守性和特异性。
Rv3265c 基因可能是抗结核药物和疫苗的理想分子靶标。从表达产物中纯化的蛋白质将用于研究 L-岩藻糖基转移酶的动力学，并开发用于筛选疫苗或药物的酶活性测定法。
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