酵母双杂交筛选人肝细胞文库戊肝病毒pORF3结合蛋白

Screening of the pORF3 binding protein of HEV in human liver cells by using yeast double hybridization system

目的筛选人肝细胞文库戊肝病毒pORF3结合蛋白,为HEVORF3蛋白的功能研究提供依据。方法构建pSOS-baitORF3诱饵重组载体,诱饵蛋白的自激活检测以及表达验证后,筛选cDNA文库中HEVORF3相互作用蛋白。结果经验证pSOS-baitORF3诱饵重组载体构建成功,诱饵蛋白的自激活检测以及表达验证均符合预期结果,共筛选出八种相互作用蛋白。结论筛选出的蛋白基本上为功能性蛋白,其中包括免疫信号通路激酶p110δ以及线粒体膜蛋白,和补体系统C3等,有进一步研究的价值。

HEV is a positive strand RNA virus containing 3 open reading frames（ORFs）, while the function of its binding protein pORF3 is poorly understood. In our study, the interactive protein of the pORF3 from human liver cDNA library was screened by the SOS recruitment system, and the construction of the pSOS bait-ORF3 plasmid was identified. The expression of the pORF3 was analyzed by Western blotting, and the detection result of the self activation of bait protein was obtained as expected. Totally, 8 putative interacting proteins had been successively screened. Some of these cellular binding proteins of pORF3, especially the important immunological pathway kinase p110δ, may advance our understanding of the role in ORF3 protein of HEV, and the possible implications of these candidate proteins for the further research are discussed.