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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GST pull-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Western blot,分析GST pull-down产物与目标蛋白抗体的相互作用;分别用GSTMIC8CTD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Western blot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GST pull-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 functional protein of the carboxy terminal cytoplasmic tail (MIC8CTD) of microtubule protein 8 from Toxoplasma gondii lysate. Methods GST-MIC8CTD protein and GST protein (control) were used as probe proteins. The target protein was screened from Toxoplasma lysate by GST pull-down technique and analyzed by SDS-PAGE. The target protein was transferred to PVDF membrane and sequenced , The similar protein sequence was searched by BLAST2 in-line and the target protein was initially identified; the interaction between the GST pull-down product and the target protein antibody was analyzed by Western blot with the antibody of the target protein as the primary antibody; the polyclonal antibody of GSTMIC8CTD and rabbit pre-immune Serum-bound sepharose and Toxoplasma lysate were co-immunoprecipitated and the pellet was analyzed by SDS-PAGE and Western blot. PAGE showed that there was a protein band in the pull-down product of GST-MIC8CTD protein (about 35-45 ku in terms of molecular mass), and no such protein was found in the pull-down product of GST protein. BLAST2 was used to compare the amino acid sequence of the target protein with that of Toxoplasma gondii The protein components of the co-immunoprecipitation product of GST-MIC8CTD polyclonal antibody detected by ECM were identifiable by the corresponding antibodies. There was no identifiable protein in the co-immunoprecipitation products of pre-immune serum. Conclusion GST pull-down technology and co-immunoprecipitation screening screening, identification, Toxoplasma gondii MIC8CTD the role of aldolase protein.