柔嫩艾美耳球虫MIC1与MIC2相互作用的鉴定

Identification of interaction between Eimeria tenella MIC1 and MIC2

目的观察柔嫩艾美耳球虫基因微线蛋白1(EtMIC1)和微线蛋白2(EtMIC2)之间的相互作用,为研究微线蛋白之间相互影响的分子机制以及该互作在鸡球虫入侵宿主细胞过程中的作用提供参考依据。方法采用PCR方法克隆柔嫩艾美耳球虫EtMIC1和EtMIC2基因,构建不同的重组表达载体。将构建的酵母双杂交诱饵质粒pGBKT7-EtMIC1及捕获质粒pGADT7-EtMIC2转化至Y2HGold酵母感受态,涂布于对应的营养缺陷培养基进行毒性和自激活活性检测。表达GST-EtMIC1和His-EtMIC2两种融合蛋白,纯化后通过GST-Pull down验证二者在体外的相互作用。将构建的pcDNA3.1-His-EtMIC1和pcDNA3.1-HA-EtMIC2重组质粒转染HEK-293T细胞,RIPA裂解后取上清进行免疫共沉淀试验,鉴定二者在体内的相互作用。将真核表达载体pEtMIC1-Myc-LC151和pEtMIC2-HA-KN151共转染HEK-293T细胞,激光共聚焦显微镜观察转染后的细胞内发出的红色荧光。结果pGBKT7-EtMIC1和pGADT7-EtMIC2单转化对酵母细胞无明显毒性作用,且自身无自激活活性,而pGBKT7-EtMIC1/pGADT7-EtMIC2共转化后能够在显色板上长出蓝色菌落,说明二者在细胞内可相互作用;GST-Pull down试验表明二者可在体外发生相互作用;免疫共沉淀和双分子荧光互补试验证实EtMIC1和EtMIC2在细胞内相互作用。结论EtMIC1和EtMIC2在体内、外均能够产生相互作用,为进一步探索微线蛋白之间相互影响的分子机制以及该互作在鸡球虫入侵宿主细胞过程中的作用提供参考依据。

Objective The molecular mechanism of interaction between Eimeria tenella MIC1(EtMIC1)and MIC2(EtMIC2)were investigated,thus providing a reference for the invasion of chiken coccidian into host cells.Methods The EtMIC1 and EtMIC2 genes were cloned by PCR using the cDNA of Eimeria tenella sporozoite as templates.The yeast two-hybrid bait plasmid pGBKT7-EtMIC1 and prey plasmid pGADT7-EtMIC2 were transformed into Y2 HGold,cultured on the auxotrophic selection medium for investigating the toxicity and self-activation.For GST-Pull down assay,the GST-EtMIC1 and His-EtMIC2 fusion proteins were expressed and purified.The recombinant plasmids pcDNA3.1-His-EtMIC1 and pcDNA3.1-HA-EtMIC2 were transfected into HEK-293 T cells to certify the interaction in vivo with co-immunoprecipitation using the RIPA lysate supernatant.The pEtMIC1-Myc-LC151 and pEtMIC2-HA-KN151 were co-transfected into HEK-293 T cells for the observation of red fluorescence by confocal laser scanning microscopy.Results EtMIC1 and EtMIC2 were not toxic and self-activating to yeast cells,while the co-transformation of pGBKT7-ETMIC1/PGADT7-ETMIC2 could activate the yeast two-hybrid reporting system,which showed that EtMIC1 and EtMIC2 could interact in vivo.While the GST-Pull down results showed that EtMIC1 and EtMIC2 could interact in vitro.The interaction between EtMIC1 and EtMIC2 in 293 T cells was furtherly confirmed by co-immunoprecipitation and bimolecular fluorescence complementation assays.Conclusion EtMIC1 and EtMIC2 could interact each other in vitro and in 293 T cells,which provided a reference for exploring the molecular mechanism of interaction and the invasion of chiken coccidian into host cells.