结核杆菌DnaA蛋白的原核表达及免疫血清的制备和检测  

Prokaryotic expression of Mycobacterial Tuberculosis DnaA as well as the preparation and identification of immune serum against DnaA protein

作　　者：周爱萍[1] 韩蕾[1] 王娟[1] 段艳 吴晓康[1] 徐纪茹[1] 

机构地区：[1]西安交通大学医学院免疫学与病原生物学系,陕西西安710061 [2]西安市结核病院,陕西西安710067

出　　处：《西安交通大学学报（医学版）》2011年第1期50-53,88,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：陕西省卫生厅科学研究基金项目(No.08E04)~~

摘　　要：目的在大肠杆菌中表达重组结核杆菌DnaA蛋白,对表达产物进行纯化、鉴定后制备DnaA蛋白特异性免疫血清并进行Western blot检测。方法以结核分枝杆菌标准菌株H37Rv基因组为模板,PCR扩增DnaA基因,构建DnaA原核表达质粒pET-DnaA,转化表达宿主大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达,非变性条件下镍琼脂糖凝胶亲和层析纯化重组蛋白pET-DnaA,并用Western blot检测和鉴定重组蛋白,用纯化得到的重组蛋白制备特异性免疫血清。结果成功构建了结核杆菌pET-DnaA重组质粒,并在大肠杆菌中得到高效表达;用分离纯化后的重组蛋白免疫动物,获得高效价的特异性免疫血清。结论结核杆菌DnaA蛋白及其特异性抗体的成功制备,为进一步研究该蛋白在结核菌DNA复制机制中的作用奠定了基础。Objective To express the recombinant Mycobacterium tuberculosis（Mtb） DnaA protein in E.coli and obtain the specific immune serum against it by purifying and identifying of the expressed product.Methods The DnaA gene of Mtb was amplified by PCR from the Mtb genome and cloned into the expression vector pET-22a（＋） to generate the pET-DnaA.The recombinant protein was induced by IPTG and purified via Ni-NTA affinity chromatography.Rabbit was immunized with preliminary purified protein.The immune serum was collected and identified with Western blot.Results The pET-DnaA recombinant for Mtb was successfully constructed and the recombinant DnaA protein was expressed in E.coli at a relatively high level.The antiserum against DnaA was obtained and its titer was more than 1∶3000,which was confirmed by Western blot.Conclusion The successful preparation of the purified Mtb DnaA protein and the specific antiserum against it will be very helpful for further study on the replication mechanism of DNA of Mtb.

关 键 词：结核分枝杆菌 DnaA蛋白 DNA复制起始 原核表达 

分 类 号：R378.91[医药卫生—病原生物学]