【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)。更多还原

【Abstract】 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 MIC2CW/A/pGEX-4T-1.The mutant protein GST-MIC2CW/A was expressed in E.coli upon IPTG induction.Glutathione sepharose beads were incubated with GST-MIC2CW/A and GSTMIC2 Crespectively,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 products coming from GST pull-down of GST-MIC2 C,but not in pull-down products coming from GST-MIC2CW/A.With mutation of MIC2CW767 to A,MIC2 protein lost the binding ability to aldolase.Tryptophan(W767)was the protein interaction site of MIC2 and aldolase in T.gondii.更多还原