突变型脑红蛋白原核表达载体的构建与表达鉴定  

Construction and expression of a prokaryotic vector for mutant human neuroglobin

作　　者：李伟光[1] 吴永红[1] 任长虹[1] 高艳[1] 张成岗[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所蛋白质组学国家重点实验室,北京100850

出　　处：《军事医学》2011年第1期25-29,共5页Military Medical Sciences

基　　金：国家973计划项目(2006CB504100);国家科技重大专项课题(2009ZX09503-002;2009ZX09301-002;2009ZX09103-616);国家自然科学基金(81070741;30973107;30772293)

摘　　要：目的为了研究脑红蛋白(neuroglobin,Ngb)发挥神经保护功能的作用机制,拟构建Ngb中关键氨基酸His突变的原核表达载体,并对其进行诱导表达与鉴定。方法利用引物突变法和PCR技术,扩增出突变型Ngb的cDNA,并克隆到原核表达载体pBV220,经测序鉴定正确转化到大肠杆菌HB101;对突变型Ngb蛋白进行诱导表达,产物经SDS-PAGE和Western印迹进行鉴定。结果与结论成功构建了pBV220-Ngb(H64V)和pBV220-Ngb(H96A)原核表达载体,SDS-PAGE和Western印迹检测结果显示突变型Ngb能够正常表达,为进一步揭示脑红蛋白神经保护功能与作用机制奠定了重要基础。Objective To construct the prokaryotic expression vector for expressing mutant neuroglobin（Ngb） protein in order to study the function of Ngb.Methods The mutant Ngb gene was amplified by PCR with primer-directed site-specific mutagenesis.The amplified DNA fragment was then sub-cloned into the prokaryotic expression vector pBV220.The recombinant plasmid was transferred into E.coli HB101.Expression of the mutant Ngb protein was detected with SDS-PAGE and Western blot.Results Two mutant Ngb prokaryotic expression vectors,pBV220-Ngb（H64V） and pBV220-Ngb（H96A）,were successfully constructed.The SDS-PAGE and Western blot analyses showed that the mutant Ngb proteins could be induced for expression as expected.Conclusion The preparation of the mutant Ngb protein will facilitate studies on the function as well as the mechanism for the neuroprotective effect of Ngb.

关 键 词：脑红蛋白 神经保护 突变 原核表达 引物突变法 氨基酸类 聚合酶链反应 大肠杆菌 基因表达 

分 类 号：Q786[生物学—分子生物学]