禾谷镰孢菌β_2-微管蛋白在大肠杆菌中的可溶性表达及纯化  

Soluble Expression and Purification of Fusarium graminearum β_2-tubulin in Escherichia coli

作　　者：徐建强[1,2] 周裕军[1] 张聪[1] 周明国[1] 

机构地区：[1]南京农业大学植物保护学院/农业部作物病虫害监测及防控重点开发实验室,南京210095 [2]河南科技大学林学院,河南洛阳471003

出　　处：《中国农业科学》2012年第6期1084-1092,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30971891);国家"973"计划项目(2009CB118906);国家"863"计划项目(2008AA10Z414)

摘　　要：【目的】在大肠杆菌中表达禾谷镰孢菌(Fusarium graminearum)的β2-微管蛋白,筛选使β2-微管蛋白可溶性表达的诱导因子,并对可溶性蛋白进行纯化。【方法】以构建好的质粒(pET32a+-β2-tubulin)为模板,PCR扩增出β2-微管蛋白基因,将其克隆到pET30a+表达载体上,并转化到表达宿主菌BL21(DE3)及Rossatta(DE3)pLysS,筛选阳性克隆,进行蛋白诱导表达,并筛选蛋白可溶性表达的诱导因子;对纯化后的蛋白进行SDS-PAGE和Western blot验证。【结果】获得了在大肠杆菌中表达效率高的阳性克隆;为了克服常规诱导条件下表达的β2-微管蛋白主要以包涵体形式存在的问题,通过对诱导温度、时间、诱导物浓度、初始诱导时的菌液浓度、培养液及宿主菌等六因子的综合分析,获得了β2-微管蛋白可溶性表达的优化条件;利用HisTrap HP预装柱对β2-微管蛋白进行纯化,可获得纯度很高的可溶性β2-微管蛋白;SDS-PAGE确认表达出的融合蛋白分子量为51.6 kD;Western blot证实表达的融合蛋白能与6×His及β-微管蛋白的单克隆抗体发生特异性反应。【结论】本研究为外源基因在大肠杆菌中的可溶性表达提供了参考;获得的微管蛋白为研究微管蛋白靶标类药物的抗性机制以及生产中开发新的以微管蛋白为靶标的杀菌剂提供了物质基础。【Objective】 The objective of this study is to express the β2-tubulin of Fusarium graminearum in E.coli in soluble form and purify it by HisTrap？ HP columns.【Method】 β2-tubulin gene contained in the plasmid（pET32a＋-β2-tubulin） was amplified,cloned to the vector pET30a＋,and then transformed into the hosts Rossatta（DE3） pLysS and BL21（DE3）.After the positive clones were screened by the colony PCR and double enzymatic digestion,the induced fusion proteins were obtained and verified by SDS-PAGE and Western blot.In order to express the fusion protein in soluble form,the inducing factors,including temperature,induction time,IPTG（Isopropyl β-D-Thiogalactoside） concentration,cell density,medium composition and hosts were screened.【Result】The positive clones which could express more fusion protein after induced were screened,however,the fusion proteins formed inclusion bodies.The molecular weight of fusion proteins were confirmed to be 51.6 kD by SDS-PAGE,which also showed specific activity to anti-6×His monoclonal antibody.After the optimization of imidazole concentration in binding and washing buffer,the soluble fusion protein was purified and its structural integrity was preserved through the purification process by the verification of Western blot.【Conclusion】 The methods described here can be used to express and purify other recombinant proteins in soluble form in E.coli.The purified fusion tubulin can be used in the studies of tubulin target drug resistant mechanisms as well as high throughout screening of new fungicide.

关 键 词：禾谷镰孢菌 β2-微管蛋白 包涵体 可溶性表达 纯化 

分 类 号：S482.2[农业科学—农药学]