结核分支杆菌HSP70原核表达载体的构建和表达及蛋白纯化

目的：构建结核分支杆菌HSP70原核表达载体并诱导其表达和纯化及鉴定目的蛋白。方法：利用PCR技术从牛型结核分支杆菌基因组中扩增出Mtb HSP70 DNA序列，构建重组质粒pGEM-Mtb HSP70，经酶切、PCR和测序鉴定后，将Mtb HSP70基因亚克隆到原核表达质粒PQE30，构建重组表达质粒PQE30-Mtb HSP70，在大肠杆菌M15中诱导表达。用镍凝胶亲和层析的方法纯化目的蛋白，Western blotting杂交鉴定纯化蛋白。结果：经测序证实，获得的目的基因与GenBank中公布的结核杆菌Mtb HSP70基因序列一致。构建的原核表达载体PQE30-Mtb HSP70在大肠杆菌M15中经1 mmol•L^-1 IPTG诱导后表达出相对分子质量约为70 400的蛋白，镍柱纯化后经抗组氨酸单克隆抗体进行Western blotting,在相对分子质量约70 400处可见特异性着色带。结论：成功构建原核表达载体PQE30-Mtb HSP70，并成功诱导Mtb HSP70蛋白的表达，通过镍凝胶亲和层析法获得纯度较高的Mtb HSP70蛋白。

Construction, expression, purfication and identification of prokaryotic expression plasmid of mycobacterium tuberculosis HSP70 protein

Objective To clone the heat shock protein 70(HSP70) gene of mycobacterium tuberculosis bovis,and construct prokaryotic plasmid of mycobacterium tuberculosis HSP70(Mtb HSP70) and express it in E.coli,further more,to obtain high pure Mtb HSP70.Methods The whole Mtb HSP70 DNA sequence was amplified from mycobacterium tuberculosis bovis genome by polymerase chain reaction(PCR).Then the sequence was cloned into plasmid pGEM-T easy and sequenced.The recombinant expression plasmid PQE30-Mtb HSP70 was constructed and expressed in E.coli M15,and Mtb HSP70 was purified by Ni-NTA affinity chromatography column.Results The sequence of Mtb HSP70 was amplified and identical with that published in GenBank.The prokaryotic expresstion plasmid of Mtb HSP70 was constructed successfully.With induction of IPTG,a new fusion protein with relative molecular mass of 70 400 was expressed and mainly located in inclusion bodies,the expressed(6×his)-Mtb HSP70 fusion proteins were identified by Western blotting with antiHis monoclonal antibody.(Conclusion A) confirmed Mtb Hsp70 gene is cloned and expressed in E.coli successfully,and high pure Mtb HSP70 is obtained.