钠碘同向转运体上游增强子序列的特异性结合蛋白文库的构建及初步筛选

Construction of Yeast One-Hybrid Library for Preliminary Screening Sodium Iodide Symporter Upstream Enhancer Sequences Specific Binding Protein Genes

目的:构建酵母单杂交文库,筛选出与钠碘同向转运体上游增强子(NUE)特异性结合的蛋白质分子,进一步研究其与NUE相互作用的分子机制。方法:应用酵母单杂交系统,以NUE为酵母单杂交的"诱饵",对甲状腺癌K1细胞的c DNA文库进行初步筛选,采用DNA测序技术及生物信息学技术对筛选的克隆进行进一步功能分析。结果:对文库中的阳性克隆测序,获得53条可读序列,Blast X比对分析及基因功能注释发现其中11条具有DNA结合功能,包括Rho GTP酶激活蛋白35(ARHGAP35)、锌指蛋白394(ZNF394)、上游刺激因子2(USF2)、SOX9等。结论:应用酵母单杂交技术初步筛选出与甲状腺癌K1细胞NUE相互作用的候选蛋白,并对这些蛋白质进行了初步的功能分析。

Objective： A yeast one-hybrid library was constructed to screen the DNA-binding proteins specifical- ly recognizing sodium iodide symporter upstream enhancer（NUE） for further investigating the molecular mecha- nisms of gene. Methods： Yeast-one-hybrid system was adapted in screening a human papillary thyroid cancer K1 cell cDNA library using NUE as bait. The cDNA clones obtained were sequenced and analyzed with bioinformat- ics tools. Results： The positive clones were sequenced, 53 sequences were identified. Whereafter the sequences were annotated by Blast X and gene functional analysis showed that there were 11 proteins with DNA binding function, and this protein contained Rho GTPase activating protein 35 （ARHGAP35）, Zinc finger protein 394 （ZNF394）, upstream stimulatory factor 2（USF2）, SOX9（SRY-related high mobility group-box gene 9）. Conclu- sion： The yeast one-hybrid system provide candidate genes for NUE specific-bingding proteins, then the primary function of the proteins was analyzed.