结核分枝杆菌Rv1729c基因编码的S-腺苷甲硫氨酸依赖性甲基转移酶的表达及生物信息学分析

Expression and bioinformatics analysis of S-adenosylmethionine-dependent methyltransferases encoded by Rv1729c gene in Mycobacterium tuberculosis

目的:探讨结核分枝杆菌(H37Rv)Rv1729c基因编码的S-腺苷甲硫氨酸依赖性甲基转移酶(S-adenosylmethionine-dependent methyltransferase,SAM-MTs)的结构功能及抗原表位等生物信息学特征。方法:采用PCR方法扩增H37Rv菌株Rv1729c基因,构建重组质粒转化大肠杆菌BL21菌株并用Western blot方法优化表达条件。通过在线Protparam和Protscale软件分析SAM-MTs的理化性质;在线SignaIP 4.1和TMHMM软件预测其信号肽及跨膜区;在线SSPro和MotifScan软件分析蛋白质二级结构;在线Expasy分析工具SWISS_MODEL建立蛋白的三级结构模型;应用ABCpred、NetMHC等程序分析SAM-MTs的B细胞及T细胞抗原表位。结果:获得的Rv1729c基因全长939 bp,基因序列与GenBank上公布的MTB标准株的核苷酸同源性为100%。该基因诱导表达的最佳IPTG浓度为0.4 mmol/L,最佳表达时间为7 h,最适表达温度为20℃。Western blot显示SAM-MTs的分子量为35 kDa。生物信息学分析显示SAM-MTs(Rv1729c)共有312个氨基酸,分子质量为33.654 ku,理论等电点4.75,脂溶性系数91.41,不稳定系数31.41,为稳定蛋白。SAM-MTs蛋白无信号肽,其二级结构中无规则卷曲占47.4%,α螺旋占40.7%,β折叠占11.9%,结构疏松。三级结构同源建模与putative S-adenosyl-l-methionine-dependent methyltransferase ML2640具有高度相似的序列。SAM-MTs氨基酸序列中有5个潜在的B细胞抗原表位,8个Th细胞表位,6个磷酸化位点,4个酰基化位点。结论:成功扩增了结核分枝杆菌Rv1729c基因,并对其表达产物进行了表达纯化,生物信息学获得了其编码蛋白SAM-MTs的结构功能及抗原表位等生物信息学的基本特征。

Objective:To investigate the structure,function,antigenic epitopes and other bioinformatics characteristics of S-adenosylmethionine-dependent methyltransferase(SAM-MTs)encoded by Rv1729c gene in M.tuberculosis.Methods:The Rv1729c gene was amplified from M.tuberculosis strain H37Rv by PCR.The recombinant plasmid was constructed and transformed into E.coli strain BL21,and the expression conditions were optimized by Western blot.The physicochemical properties of SAM-MTs(Rv1729c)were analyzed by Protparam and Protscale software.SignalaIP 4.1 and TMHMM software were employed to predict signal peptides and transmembrane regions of SAM-MTs(Rv1729c).Protein secondary structure was analyzed by SSPro and MotifScan software.Tertiary structure model of SAM-MTs(Rv1729c)was established by the online Expasy analysis tool-SWISS_MODEL.B cell and T cell epitopes of SAM-MTs(Rv1729c)were mainly predicted by ABCpred,NetMHC and other programs.Results:The obtained Rv1729c gene was 939 bp in length,and its nucleotide homology with the f MTB standard strain published in GenBank was 100%.The optimum concentration of IPTG for expressing Rv1729c gene was 0.4 mmol/L,the optimum induction time was 7 h,and the best induction temperature was 20℃.Western blot showed that the molecular weight of SAM-MTs was 35 kDa.Bioinformatics analysis showed that SAM-MTs(Rv1729c)had 312 amino acids with a molecular mass unit of 33.654 ku,a theoretical isoelectric point of 4.75,a fat solubility coefficient of 91.41,and an instability coefficient of 31.41.It was predicted to be a stable,hydrophobic,non-transmembrane and loosely structured protein.Tertiary structure homology modeling showed that SAM-MTs(Rv1729c)had a highly similar sequence with putative S-adenosylmethionine-dependent methyltransferase ML2640.There were 5 potential B-cell epitopes,8 T-cell epitopes,6 phosphorylation sites and 4 acylation sites in the amino acid sequence of SAM-MTs(Rv1729c).Conclusions:The Rv1729c gene of M.tuberculosis is amplified and its expression product is purified.The structure,function and antigenic epitopes of the protein SAM-MTs(Rv1729c)are obtained by bioinformatics analysis.