柔嫩艾美耳球虫顶膜抗原-1与微线蛋白-2相互作用的鉴定

Identification of interaction between Eimeria tenella apical membrane antigen-1 and microneme protein-2

目的研究柔嫩艾美耳球虫顶膜抗原-1(EtAMA1)与微线蛋白-2(EtMIC2)之间的相互作用。方法以柔嫩艾美耳球虫子孢子cDNA为模板,PCR扩增EtAMA1和EtMIC2基因,构建酵母双杂交诱饵载体pGBKT7-EtAMA1及捕获载体pGADT7-EtMIC2并转化Y2HGold酵母菌中,观察酵母在营养缺陷选择培养基上的生长情况。原核表达GST-EtAMA1和His-EtMIC2融合蛋白,纯化后进行GST-Pulldown试验。同时构建真核表达载体pcDNA3.1-HisEtAMA1和pcDNA3.1-HA-EtMIC2并转染HEK-293T细胞,收集细胞裂解液,进行免疫共沉淀试验。在此基础上构建双分子荧光互补载体pEtAMA1-Myc-LC151和pEtMIC2-HA-KN151并转染HEK-293T细胞,激光共聚焦观察转染细胞荧光发生情况。结果诱饵基因和捕获基因均对酵母双杂交系统无自激活和毒性作用,pGBKT7-EtAMA1/pGADT7-EtMIC2共转化菌能激活酵母双杂交报告系统;原核表达并纯化GST-EtAMA1和His-EtMIC2融合蛋白进行GST-Pulldown试验,二者可在体外相互作用;EtAMA1和EtMIC2可在HEK-293T细胞中共表达,免疫共沉淀试验进一步证实二者间的相互作用;双分子荧光互补技术验证EtAMA1和EtMIC2在细胞内存在相互作用。结论通过酵母双杂交、GST-Pulldown、免疫共沉淀及双分子荧光互补技术验证柔嫩艾美耳球虫顶膜抗-1(EtAMA1)与微线蛋白-2(EtMIC2)之间存在相互作用,这为揭示微线蛋白在鸡球虫入侵宿主细胞过程中的分子机制提供了理论基础。

Objectives To study the interaction between apical membrane antigenV1（EtAMA1）and microneme protein-2（EtMIC2）in Eimeria tenella（E.tenella）. Methods Using the cDNA of Eimeria tenella sporozoites as template,the EtAMA1 and EtMIC2 genes were amplified by PCR.The yeast two-hybrid bait vector pGBKT7-EtAMA1 and the prey vector pGADT7-EtMIC2 were constructed and transformed into yeast Y2 HGold strain to observe the growth of yeast on auxotrophic selection medium.The GST-EtAMA1 and His-EtMIC2 fusion proteins were expressed in E.coli and purified for GST-Pull down assay.The eukaryotic expression vectors pcDNA3.1-His-EtAMA1 and pcDNA3.1-HA-EtMIC2 had been constructed and transfected into HEK-293 Tcells,and the cell lysate was collected and subjected to co-immunoprecipitation（Co-IP）.And then,we constructed the bimolecular fluorescence complementation（BiFc）vectors pEtAMA1-Myc-LC151 and pEtMIC2-HA-KN151 and transfected into HEK-293 Tcells.The confocal laser scanning microscope was used to observe the fluorescence of transfected cells. Results Both EtAMA1 and EtMIC2 had no auto-activation and toxicity on the yeast two-hybrid system,and pGBKT7-EtAMA1/pGADT7-EtMIC2 co-transformed group could activate yeast two-hybrid reporter system.The interaction between EtAMA1 and EtMIC2 has been identified by GST-Pulldown after prokaryotic expression and purification of the GST-EtAMA1 and His-EtMIC2 fusion protein.EtAMA1 and EtMIC2 protein can be co-expressed in HEK-293 Tcells,and the interaction between EtAMA1 and EtMIC2 can be identified by Co-IP.Red fluorescence was observed in the EtAMA1 and EtMIC2 co-transfected cells in the bimolecular fluorescence complementation. Conclusion The interaction between EtAMA1 and EtMIC2 was identified by yeast two-hybrid,GSTPulldown,Co-IP and BiFc.This study provides the theoretical basis for revealing the molecular mechanism of microneme protein in the invasion of chicken coccidian into host cells.