人星状病毒非结构蛋白nsP1 a／1相互作用的宿主细胞靶蛋白筛选  

Screening of the Host Target Proteins of HAstV nsP1 a/1

作　　者：刘志成[1] 苑荣亮 张成[1] 汪玉婷[1] 黄芬[1] 井申荣[1] 

机构地区：[1]昆明理工大学医学院,昆明市650500

出　　处：《医学分子生物学杂志》2016年第5期281-285,共5页Journal of Medical Molecular Biology

基　　金：资助项目：昆明理工大学分析测试基金（No.20150997）

摘　　要：目的：利用细菌双杂交技术筛选与人星状病毒非结构蛋白nsP1a／1相互作用的蛋白。方法培养人肠道细胞HT29，提取基因组，利用细菌双杂交系统载体pUT18 C构建HT29基因组文库，同时构建诱饵载体nsP1a／1-pKT25。将文库和诱饵载体共转化进入细菌BTH101，在M63培养基上进行筛选。结果通过酶切鉴定以及测序分析，诱饵载体构建正确，并且利用细菌双杂交技术成功从HT29细胞基因组文库中筛选出能与人星状病毒非结构蛋白 nsP1a／1相互作用的蛋白。结论成功构建了人星状病毒非结构蛋白nsP1a／1的诱饵载体，在HT29细胞基因组文库中筛选得到了3个与nsP1a／1相互作用的蛋白，为进一步研究人星状病毒在细胞内的增殖和防治药物的应用奠定基础。Objective To screen out the host target proteins that can interact with the human astrovirus nonstructural protein nsP1 a/1 （ HAstV nsP1 a/1 ） by utilizing the bacterial two-hybrid system. Methods The genome was extracted from cultured HT29 cells. HT29 genomic library was constructed by using pUT18 C, which was named HT29-pUT18 C. Then the carrier of bait nsP1a/1-pKT25 was established as well. Both the genomic library and the carrier of bait were transformed into BTH101 , which was followed by screening on M63 culture medium. Results The structure of the bait carrier was demonstrated to be right by the enzyme digestion method and the sequencing analy-sis. The host target proteins that had interaction with HAstV nsP1 a/1 were successfully isolated by u-tilizing the bacerial two-hybrid system. Conclusion The bait carrier of HAstV nsP1 a/1 was suc-cessfully established, and three proteins that could interact with nsP1a/1 were identified in the HT29 genomic library, which lay a foundation for further researches on the cellular proliferation of human astroviruses and application of drugs for the prevention and treatment of human astroviruse in-fections.

关 键 词：细菌双杂交 人星状病毒 nsP1a/1 HT29细胞 筛选 

分 类 号：R373[医药卫生—病原生物学]