耻垢分枝杆菌调节蛋白TrpD的原核表达及纯化

发布时间：2018-01-23 10:55

本文关键词： 耻垢分枝杆菌 TrpD 原核表达多克隆抗体　出处：《潍坊医学院学报》2015年02期 　论文类型：期刊论文

【摘要】：目的克隆编码耻垢分枝杆菌调节蛋白TrpD的基因并在大肠杆菌中进行蛋白表达及纯化。方法利用PCR技术从耻垢分枝杆菌中扩增TrpD基因,构建重组表达质粒pET-32a(+)-TrpD;以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌株,IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行镍离子亲和层析纯化,通过SDS-PAGE和Westem blot鉴定目的蛋白的表达及反应原性。结果扩增了TrpD基因,克隆于表达载体pET-32a(+)中,PCR筛选和酶切鉴定获得阳性克隆,测序证实正确。经诱导在大肠杆菌中表达出相对分子质量(Mr)为56000的目的蛋白。结论已成功构建TrpD基因的原核表达载体,并在大肠杆菌中获得高效蛋白表达及纯化,为进一步研究TrpD蛋白在结核病中的致病机制提供了依据。
[Abstract]:Objective to clone the gene encoding the regulatory protein TrpD of Mycobacterium smeareus and to express and purify the protein in E. coli. Methods the TrpD gene was amplified from Mycobacterium smeareus by PCR technique. The recombinant expression plasmid pET-32a (pET-32a) was constructed. The recombinant plasmid was transformed into Escherichia coli BL21 and DE3, and the target protein was induced by IPTG. The target protein was purified by nickel ion affinity chromatography under denaturing conditions. The expression and reactivity of the target protein were identified by SDS-PAGE and Westem blot. Results the TrpD gene was amplified and cloned into the expression vector pET-32a (). Positive clones were obtained by PCR screening and restriction endonuclease digestion. The target protein with relative molecular weight of 56000 was induced to express in E. coli. Conclusion the prokaryotic expression vector of TrpD gene has been constructed successfully. High efficient protein expression and purification were obtained in Escherichia coli, which provided a basis for further study of the pathogenicity of TrpD protein in tuberculosis.
【作者单位】：潍坊医学院医学检验学系;潍坊医学院病原生物学教研室;
【基金】：国家自然科学基金资助课题(课题编号:81100006) 山东省自然科学基金项目资助课题(课题编号:ZR2010HM073)
【分类号】：R392
【正文快照】： 耻塘分枝杆菌(Mycobacterium Smegmatis,Ms)与成),得到重组菌pET-32a(+)-TrpD。结核分枝杆菌(A/yco6ocferium tuterciifosii,MTB)同属 1.2.3重组蛋白的诱导表达及鉴定将重组质粒于分枝杆菌属,在进化上亲缘关系接近,蛋白序列同源 pET-32a(+)-TrpD转化感受态大肠杆菌BL21性高,

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