摘要： 目的 筛选人星状病毒（HAstV）衣壳蛋白ORF2与宿主细胞互作的蛋白质。方法 构建HAstV ORF2蛋白的重组载体pGBKT77-ORF2，以其作为诱饵质粒，采用酵母双杂交方法，从人结肠癌细胞Caco-2 cDNA文库中筛选与ORF2蛋白互作的蛋白。以HAstV-1病毒株的衣壳蛋白为模板，通过PCR扩增定向克隆构建诱饵载体pGBKT7-ORF2。通过菌落PCR和测序验证质粒在酵母细胞Y2H中正确表达。明确ORF2蛋白对酵母细胞无毒性，无自激活活性。将含有pGBKT7-ORF2的酵母菌株与靶标Caco-2 cDNA文库进行杂交、筛选、验证及测序。结果 获得15个与ORF2互作的宿主蛋白，分别为FN1、COMMD1、RASAL2、BMP4、RPL11、RBM8A、SUMO1、WWC2、NGDN、HINFP、RPS7、IMMT、UBE2I、EEF1A1和FTL。结论 成功筛选出15个与HAstV ORF2互作的蛋白，为研究星状病毒与宿主相互作用、ORF2蛋白功能及病毒的致病机制奠定了基础。

酵母双杂交筛选与星状病毒衣壳蛋白ORF2互作的宿主蛋白

李香雨, 于漫, 聂梓陶, 陈彦如, 王嘉会, 韩喜彬, 赵微

锦州医科大学基础医学院实验动物学教研室, 辽宁 锦州 121200

收稿日期:2020-10-14出版日期:2021-06-30发布日期:2021-05-28
通讯作者:赵微E-mail:zhaowei-v@jzmu.edu.cn
作者简介:李香雨(1997-),男,本科在读.
基金资助:国家自然科学基金（81201285）；辽宁省自然科学基金（20170540346）；辽宁省教育厅青年育苗项目（JYTQN201925）；辽宁省大学生创新创业计划（201810160004）


关键词: 星状病毒, 酵母双杂交, ORF2蛋白, 蛋白互作, cDNA文库筛选
Abstract: Objective Screening of host proteins that interact with human astrovirus (HAstV) capsid protein ORF2. Methods The recombinant vector pGBKT7-ORF2 of the HAstV ORF2 was constructed and used as bait plasmid to screen the proteins interacting with ORF2 protein from the cDNA library of human colon adenocarcinoma cell line using the yeast two-hybrid method. HAstV-1 virus was used as a template and amplified via PCR to obtain the ORF2 gene and the bait plasmid pGBKT7-ORF2 was constructed via directed cloning. Plasmids verified through colony PCR and sequencing were transformed into the yeast strain Y2H Gold. The expression of ORF2 was confirmed and it showed no self-activation and cytotoxicity,and therefore,was suitable for this screen. Results There was no toxicity and self-activation of bait protein in yeast hybridization system,and 15 proteins (FN1,COMMD1,RASAL2,BMP4,RPL11,RBM8A,SUMO1,WWC2,NGDN,HINFP,RPS7,IMMT,UBE2I,EEF1A1,and FTL) interacting with ORF2 protein were identified. Conclusion This study has laid a foundation for further study on the function of ORF2 protein and the pathogenic mechanism of human astrovirus.
Key words: astrovirus, yeast two-hybrid, ORF2 protein, protein interaction, screening of cDNA library
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