刚地弓形虫MIC8 CTD作用蛋白的筛选及鉴定

Identification of the protein interacting with the C-terminal cytosolic tail domain of MIC8 in Toxoplasma gondii

目的从刚地弓形虫速殖子裂解液中筛选并鉴定微线体蛋白8羧基端胞质尾（MIC8CTD）的作用蛋白。方法分别以GST—MIC8CTD蛋白和GST蛋白（对照）作为探针蛋白，采用GSTpull—down技术从弓形虫裂解液中筛选目标蛋白并进行SDS-PAGE分析；将目标蛋白转印至PVDF膜，测序，通过BLAST2在线对比搜索相似蛋白序列，初步确定目标蛋白；以目标蛋白抗体为一抗进行Westernblot，分析GSTpull—down产物与目标蛋白抗体的相互作用；分别用GST—MIC8CTD多克隆抗体和兔免疫前血清结合的sepharose与弓形虫裂解液进行免疫共沉淀试验，沉淀物进行SDS—PAGE和Westernblot分析。结果PAGE显示GST—MIC8CTD蛋白的pull—down产物中有一蛋白条带（分子质量单位约35～45ku），GST蛋白的pull—down产物中无此蛋白；BLAST2比对目标蛋白的氨基酸序列与弓形虫醛缩酶序列一致；ECM检测GST—MIC8CTD多克隆抗体的免疫共沉淀产物中有蛋白组分可被相应的抗体识别，免疫前血清的免疫共沉淀产物中无可被识别的蛋白。结论经GSTpull—down技术和免疫共沉淀试验筛选、鉴定，弓形虫MIC8CTD的作用蛋白为醛缩酶。

Objective To screen and identify the protein from tachyzoite lysates interacting with the C-terminal cytosolic tail domain of microneme protein 8 （MICSCTD） in To.roplasrna gondii. Methods Glutathione Sepharose beads were incubated with GST-MICSCTD protein and GST protein （as a control protein）, respectively, and then incubated with tachyzoite lysates. Bound proteins were eluted using sample buffer. Eluants were resolved using SDS-PAGE and trans ferred onto polyvinylidene difluoride （PVDF） membranes. Protein bands cut from the membrane were subjected to N-terminal sequencing. Analysis of the sequences was performed online using BLAST2 to determine primarily the target protein. Anti-GST-MIC8CTD antibody or preimmune rabbit sera was added to protein G-coated Sepharose beads and parasite lysates were added to the beads, which were then incubated for 12 h. Beads were washed and resolved in protein loading buffer, and precipitates were subjected to SDS-PAGE and Western blotting. Results There was a protein band （molecular weight; 35-45 ku） in the pull-down products of GST-MIC8CTD protein but not in the pull-down products of GST protein. The N-terminal sequence of the target protein was SGYGLPISQE, coinciding with the sequence of aldolase of T. gondii. The target protein band on NC membranes was specifically recognized by anti aldolase antibody. Protein bands were found in co-precipitates yielded by an immunoprecipitation （IP） experiment with anti-GST-MIC8CTD antibody and were not found in co-precipitates of preimmune sera. Conclusion The protein interacting with MIC8CTD in T. gondii is aldolase.