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

作　　者：郑斌[1] 尹志奎[2] 史明珠[1] 任红斌[1] 詹希美[3] 

机构地区：[1]新乡医学院寄生虫学教研室,河南新乡453000 [2]新乡医学院药理学教研室 [3]中山大学中山医学院寄生虫学教研室,广东广州510080

出　　处：《中国病原生物学杂志》2014年第3期255-257,共3页Journal of Pathogen Biology

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项目(No.2010)

摘　　要：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.

关 键 词：刚地弓形虫 微线体蛋白8羧基端胞质尾 GST pull—down 醛缩酶 免疫共沉淀 

分 类 号：R382.5[医药卫生—医学寄生虫学]