摘要
目的确定刚地弓形虫微线体蛋白2(MIC2)与醛缩酶的作用位点。方法利用定点突变技术,将MIC2羧基端(MIC2C)的767位色氨酸(W767)突变为丙氨酸(A)。PCR扩增MIC2CW/A突变体基因片段;构建MIC2CW/A/pGEX-4T-1重组原核表达系统,IPTG诱导表达GST-MIC2CW/A突变体蛋白。分别以该蛋白和GST-MIC2C蛋白(对照蛋白)作为探针蛋白与弓形虫速殖子裂解液进行GST pull-down实验,SDS-PAGE及Western blot分析。结果获得了MIC2CW/A突变体基因片段,制备了GST-MIC2CW/A突变体蛋白;GST-MIC2C蛋白的pull-down产物中有一蛋白条带,而且可以被醛缩酶抗体识别,而GST-MIC2CW/A蛋白的pull-down产物中未见蛋白条带。结论将MIC2的W767突变为A后,MIC2失去与醛缩酶的作用,即MIC2与醛缩酶的作用位点为色氨酸(W)。
In this study ,we aim to identify the protein interaction site of microneme protein 2 （MIC2） and aldolase in Toxoplasma gondii .The tryptophan （Trp ,W） at site 767 of carboxyl terminus of MIC2 （MIC2C） was mutated into alanine （Ala ,A） by site-directed mutagenesis to construct plasmid MIC2C W/A/pGEX-4T-1 .The mutant protein GST-MIC2C W/A was expressed in E .coli upon IPTG induction .Glutathione sepharose beads were incubated with GST-MIC2C W/A and GST-MIC2C respectively ,then incubated with tachyzoite lysates ,and bound proteins were eluted using sample buffer .Eluants were resolved by SDS-PAGE and Western blot .A protein band specifically recognized by anti-aldolase antibody was detected in prod-ucts coming from GST pull-down of GST-MIC2C ,but not in pull-down products coming from GST-MIC2C W/A .With muta-tion of MIC2C W767 to A ,MIC2 protein lost the binding ability to aldolase .Tryptophan （W767 ） was the protein interaction site of MIC2 and aldolase in T .gondii .
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第7期698-700,708,共4页
Chinese Journal of Zoonoses
基金
河南省科技攻关计划项目(No.112102310209)
河南省教育厅自然科学研究项目(No.2012B310019)
新乡医学院博士科研启动基金(2010)~~
