结核分枝杆菌异柠檬酸裂解酶aceA基因原核表达载体的构建及融合蛋白的研究

发布时间：2018-12-08 16:48

【摘要】： 目的结核分枝杆菌长期处于持续感染状态已成为目前在全世界范围内消灭结核病的最大阻碍之一。异柠檬酸裂解酶(ICL)是结核分枝杆菌在体内持续感染时所进行的乙醛酸途径中的关键酶,对结核分枝杆菌在巨噬细胞内生存及持续感染起着重要的作用。编码异柠檬酸裂解酶的基因有两个:icl和aceA,我们已经成功地克隆表达了icl基因,并对其表达的重组酶进行了深入的研究。本研究拟克隆结核分枝杆菌异柠檬酸裂解酶的aceA基因,并构建表达重组体,通过表达条件的优化,获得AceA可溶性蛋白。纯化该蛋白,并对其结构、分子量及性质等进行研究,为以异柠檬酸裂解酶为靶点的抗结核药物筛选奠定基础。 方法根据aceA基因编码序列设计引物,并在引物中设计HindⅢ和BamHI酶切位点,以MTB基因组为模板,PCR扩增编码异柠檬酸裂解酶的aceA基因,并将其克隆入pUC18质粒进行扩增,再亚克隆入原核表达载体pET28a(+),构建pET28a(+)-aceA重组体;用重组体转化大肠杆菌BL21(DE3)plysS菌,在IPTG诱导下进行表达;表达产物经SDS-PAGE鉴定,再经镍离子螯合型琼脂糖凝胶亲和层析柱纯化,以获得纯化的结核分枝杆菌异柠檬酸裂解酶重组蛋白;以时实波长扫描对重组蛋白的酶活性进行测定分析,通过圆二色及质谱分析对其结构及分子质量进行测定。 结果以MTB基因组DNA为模板,以Pfu Tag DNA聚合酶扩增得到2.3kb的片段,并克隆入pUC18质粒;经测序证实与GenBank中的aceA基因序列一致。进而构建出pET28a(+)-aceA表达质粒,该重组质粒能在大肠杆菌BL21(DE3)plysS中高效表达;以0.1mM的IPTG诱导6hrs后重组蛋白表达量最高。表达蛋白以可溶性非包涵体形式存在于胞浆中,经Ni-NTA琼脂糖亲和柱一步纯化得到了90KD左右的纯化蛋白产物。通过酶学方法检测该重组蛋白证明其具有异柠檬酸裂解酶活性;重组蛋白经高效液相色谱及质谱鉴定,测得相对分子质量为89660Da。经圆二色仪分析,重组ICL的二级结构中相对有32.4 %的α螺旋,27.5%β转角,40.1 %无规卷曲。 结论本研究成功地克隆表达并纯化了具有生物学活性的结核分枝杆菌异柠檬酸裂解酶AceA蛋白,并对其分子质量,结构及酶活性进行了研究。该研究对于结核病药物靶点的研究有着重要的价值,为结核病的治疗及抗结核药物的开发奠定了基础。
[Abstract]:Objective Mycobacterium tuberculosis has become one of the biggest obstacles to the worldwide eradication of tuberculosis. Isocitrate lyase (ICL) is a key enzyme in glyoxylic acid pathway of Mycobacterium tuberculosis, which plays an important role in the survival and persistent infection of Mycobacterium tuberculosis in macrophages. There are two genes encoding isocitrate lyase: icl and aceA,. We have successfully cloned and expressed icl gene and studied its recombinant enzyme. In this study, the aceA gene of isocitrate lyase of Mycobacterium tuberculosis was cloned, and the expression recombinant was constructed. The soluble protein of AceA was obtained by optimizing the expression conditions. The protein was purified, and its structure, molecular weight and properties were studied, which laid a foundation for the screening of antituberculous drugs targeting isocitrate lyase. Methods the primers were designed according to the coding sequence of aceA gene, and Hind 鈪，

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