摘要
目的利用基因定点突变的方法将刚地弓形虫微线体蛋白2(MIC2)的767位氨基酸的碱基定点突变,蛋白相互作用技术分析突变体的特性。方法利用基因定点突变的方法,将MIC2蛋白羧基端(MIC2C)的767位色氨酸(tryptophan,W)的碱基突变为丙氨酸(alanine,A)的碱基,PCR扩增MIC2C W/A突变体基因片段;构建MIC2C W/A/p GEX-4T-1重组原核表达系统,IPTG诱导表达GST-MIC2C W/A突变体蛋白。以该蛋白和MIC2C蛋白(未突变蛋白)为探针蛋白与弓形虫裂解液进行GST pull-down实验,SDS-PAGE及Western blot分析。结果获得了MIC2C W/A突变体基因片段,制备了GST-MIC2C W/A突变体蛋白;GST pull-down实验产物分析显示GST-MIC2C W/A蛋白的产物中未见蛋白条带,而未突变蛋白的产物中有一蛋白条带,且能被抗醛缩酶抗体识别。结论在体外获得了MIC2突变体;MIC2突变体不能沉降弓形虫裂解液中的醛缩酶,即MIC2突变体失去了与醛缩酶作用的特性。
Objective To construct and protein activity analysis of microneme protein 2（ MIC2） mutant in Toxoplasma gondii. Methods The tryptophan at sites 767（ W767） of carboxyl terminus of MIC2（ MIC2C） was mutated into alanine（ A） by site- directed mutagenesis to construct plasmid MIC2 C W / A / p GEX- 4T- 1.The mutant protein GST- MIC2 C W / A was expressed in E. coli upon IPTG induction. Glutathione sepharose beads were incubated with GST- MIC2 C W / A and GST- MIC2 C respectively,then incubated with tachyzoite lysates,and bound proteins were eluted using sample buffer. Eluants were resolved by SDS- PAGE and Western blot. Results MIC2 C W / A gene was amplified and the mutant protein GST- MIC2 C W / A was obtained;Protein band was not detected in products coming from GST pull- down experiment of GST- MIC2 C W / A,compared with the unmutated protein（ GST- MIC2C）. Conclusions The mutant of MIC2 was prepare in Vitro; MIC2 mutant protein lost the binding ability to aldolase.
出处
《医学动物防制》
2016年第2期126-128,共3页
Journal of Medical Pest Control
基金
河南省科技攻关计划项目(112102310209)
河南省教育厅自然科学研究项目(2012B310019)
新乡医学院博士科研启动基金(2010)
