十二指肠贾第虫端粒酶逆转录酶RNA结合域TRBD互作蛋白的筛选及验证

Screening and verification of interaction protein of telomerase reverse transcriptase RNA binding domain in Giardia duodenalis

目的利用酵母双杂交系统筛选十二指肠贾第虫端粒酶逆转录酶RNA结合域TRBD的互作蛋白,并验证其是否存在互作关系。方法通过分子克隆技术构建诱饵质粒pGBKT7-TRBD,应用酵母双杂交技术将诱饵质粒与贾第虫酵母双杂交cDNA质粒文库共转化至Y2H Gold酵母感受态细胞中,通过不同营养缺陷型培养基筛选杂交成功的菌落。对筛选出的阳性克隆进行回返试验,并通过双分子荧光互补试验及GST-Pull down试验进一步验证二者存在的互作关系。结果诱饵质粒pGBKT7-TRBD经双酶切和测序鉴定证明构建正确。该诱饵质粒在贾第虫酵母双杂交cDNA质粒文库中初步筛选得到5个阳性克隆,即histone acetyltransferase type B subunit 2、bZIP family protein、WD-repeat protein、NHL repeat-containing protein、heat shock protein 90(Hsp90),经回返验证、双分子荧光互补和GSTPull down试验确定阳性基因Hsp90与TRBD存在互作关系。结论利用酵母双杂交技术成功筛选出与十二指肠贾第虫端粒酶TRBD存在相互作用的Hsp90,为进一步探索贾第虫端粒酶调控机制奠定了基础。

Objective To screen the interaction proteins of telomerase reverse transcriptase RNA binding domain(TRBD)in Giardia duodenalis by using yeast two-hybrid system and verify their interactions.Methods The bait plasmid pGBKT7-TRBD was constructed by molecular cloning technology and co-transformed to competent Y2H Gold yeast cells by yeast two-hybrid system with Giardia yeast two-hybrid cDNA plasmid library.The successfully hybridized colonies were screened through different auxotrophic media and subjected to return test.The interaction between the bait plasmid pGBKT7-TRBD and Giardia yeast two-hybrid cDNA plasmid library was verified by bimolecular fluorescence complementation and GSTPull down tests.Results Bait plasmid pGBKT7-TRBD was constructed correctly as proved by restriction analysis and sequencing,by which five positive clones named as histone acetyltransferase type B subunit 2,bZIP family protein,WDrepeat protein,NHL repeat-containing protein and heat shock protein 90(Hsp90)were screened in Giardia yeast twohybrid cDNA plasmid library.Return verification,bimolecular fluorescence complementation test and GST-Pull down test confirmed the interaction between positive genes Hsp90 and TRBD.Conclusion The Hsp90 interacted with Giardia telomerase TRBD was successfully screened by using yeast two-hybrid system,which provided a basis for further investigation on the regulatory mechanism of Giardia telomerase.