结核分支杆菌phoS2重组质粒的构建、表达、纯化及生物信息学预测  

Construction,prokaryotic expression,purification and bioinformatic predication of the recombinant plasmid carrying phoS2 gene from Mycobacterium tuberculosis

作　　者：丁淑琴[1] 徐文锦[1] 杨园园[2] 杨风琴[1] 

机构地区：[1]宁夏医科大学检验学院,宁夏银川750004 [2]银川市第一人民医院感染性疾病科,宁夏银川750001

出　　处：《西安交通大学学报（医学版）》2014年第3期306-310,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：宁夏自然科学基金资助项目(No.NZ1196)~~

摘　　要：目的构建结核分支杆菌phoS2表达重组质粒、原核表达及纯化重组蛋白，并对重组蛋白进行免疫学特性的初步鉴定。方法将目的基因亚克隆到pET32a表达载体，成功构建的重组质粒双酶切鉴定并测序。加IPTG诱导表达，表达产物经SDS-PAGE鉴定后用含Ni^2＋的His-bind树脂柱亲和层析纯化phoS2，并用Westernblot对其进行鉴定。应用DNAstar等生物分析软件对该蛋白的理化特性、跨膜区域、二级结构、三维结构进行预测。结果双酶切鉴定及测序分析表明，成功构建了基因工程菌株高效表达质粒phos2／pET32a。IPTG诱导表达的SDS-PAGE鉴定结果显示，上清液中只见极少量的特异蛋白表达带，沉淀部分可见明显的特异表达带，说明phoS2主要是包涵体表达。经免疫印迹分析获得的phoS2分子质量为37953ku。生物信息学预测该蛋白分子质量为37953．1ku，理论等电点为5．75，具有1个跨膜区，位于1-19位，结构域位于1-370位。结论成功构建了结核分支杆菌phoS2／pET32a重组质粒，phoS2能够高效表达。蛋白结构功能的预测对本实验的实施及进一步的研究提供了理论依据。Objective To construct a recombinant plasmid carrying phoS2 from Mycobacterium tuberculosis, to express prokaryotically, to purify the recombinant protein and to identify the immunogenicity of its recombinant protein. Methods phoS2 gene was subcloned into expression vector pET32a. The constructed recombinant plasmid phoS2/pET32a was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and then purified by His-bind affinity chromatography with Ni2＋. Results of the induced expression level were detected by SDS-PAGE. The recombinant protein was purified by affinity chromatography. The biological activity of phoS2 was tested by Western blot; its secondary structure and 3-D structure were predicted with software packages such as DNAstar and Rasmol. Results The recombinant plasmid phoS2/pET32a of Mycobacterium tuberculosis was constructed successfully and confirmed by restriction endonuclease analysis and DNA sequencing analysis. SDS-PAGE analysis showed that the expressed proteins were mainly insoluble; Western blot analysis showed that the molecular weight of phoS2 recombinants protein was 37 953 u. The recombinant protein was purified by affinity chromatography. Analysis of the predicted protein indicated that the molecular mass was 37 953.1 ku, PI was 5.75, there was one transmembrane region and function sites were 1 to 370. Conclusion The recombinant plasmid phoS2/pET32a of Mycobaeterium tuberculosis was successfully constructed and phoS2 gene could be expressed in BL21 with high efficiency. Predicting protein structure and function can provide some theoretical basis for conducting the present experiment and selecting further research.

关 键 词：结核分支杆菌 phoS2 重组质粒 表达 纯化 生物信息学 预测 

分 类 号：Q78[生物学—分子生物学]