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【目的】应用原核表达体系对结核分枝杆菌PPE蛋白家族Rv1168c进行高效表达,进一步进行蛋白纯化和结构分析。【方法】以结核分枝杆菌H37Rv基因组为模板,扩增Rv1168c基因,构建pET32a-Rv1168c重组质粒;转化重组质粒到大肠杆菌DH5α并在BL21(DE3)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺电泳(SDS-PAGE)鉴定Rv1168c在大肠杆菌中的表达情况;Ni-NTAHis﹡Bind Resin纯化重组蛋白Rv1168c;SDS-PAGE和质谱分析测定相对分子量后,用圆二色光谱(CD)和同源模建方法分析和检测重组蛋白Rv1168c的二级和三级结构。【结果】成功克隆了971bp的目的基因Rv1168c,并获得了高纯度的重组蛋白Rv1168c。重组蛋白的分子量为51.5kDa(含载体蛋白)。25℃时重组蛋白Rv1168c的二级结构包括34.4%α螺旋,33.7%β转角,31.9%无规则卷曲,它的三维模型显示为(β/α)5结构。【结论】成功得到高纯度的重组目的Rv1168c蛋白,并初步进行了结构分析,为进一步对Rv1168c结构和功能研究奠定了基础。
【Objective】 The prokaryotic expression system of Mycobacterium tuberculosis PPE protein family Rv1168c was highly expressed for further protein purification and structural analysis. 【Method】 Mycobacterium tuberculosis H37Rv was used as a template to amplify Rv1168c gene and construct pET32a-Rv1168c recombinant plasmid. The recombinant plasmid was transformed into E.coli DH5α and expressed in BL21 (DE3). The recombinant plasmid was expressed by sodium dodecyl sulfate-poly The recombinant protein Rv1168c was purified by Ni-NTAHis * Bind Resin. SDS-PAGE and mass spectrometry were used to determine the relative molecular weight. The results of circular dichroism (CD) Source modeling method analysis and detection of recombinant protein Rv1168c secondary and tertiary structure. 【Result】 The 971 bp Rv1168c gene was successfully cloned and a highly purified recombinant protein Rv1168c was obtained. The recombinant protein has a molecular weight of 51.5 kDa (with carrier protein). The secondary structure of the recombinant protein Rv1168c at 25 ° C included 34.4% α-helix, 33.7% β-turn, 31.9% random coil and its three-dimensional model showed a (β / α) 5 structure. 【Conclusion】 The recombinant Rv1168c protein with high purity was successfully obtained and its structure was preliminary analyzed, which laid the foundation for the further study on the structure and function of Rv1168c.