禽流感病毒AM2蛋白相互作用宿主蛋白的筛选及初步验证  

Screening and preliminary identification of host proteins interacting with avian influenza virus AM2 protein

作　　者：金新[1,2] 于洪敏[2] 张茂林[2] 段铭[2] 李影[1] 关振宏[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林大学人兽共患病研究所人兽共患病教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2017年第5期866-870,共5页Chinese Journal of Veterinary Science

基　　金：国家"973"计划重点基础研究发展计划资助项目(2012CB518901)

摘　　要：采用酵母双杂交系统筛选与禽流感病毒AM2相互作用的宿主蛋白,可为研究AM2蛋白的生物学新功能提供依据。将检测无毒性与自激活作用的诱饵质粒pGBKT7-AM2与淋巴文库质粒进行融合杂交,然后对经选择性培养基筛选到的阳性克隆进行酶切鉴定和测序分析,阳性克隆进行酵母回转试验验证。经酵母双杂交筛选与验证最终获得2个与AM2相互作用的蛋白。选择线粒体细胞色素C氧化酶亚单位Ⅱ(COXⅡ)蛋白进行进一步研究,构建真核表达载体pcDNA3.0-HA-COXⅡ,并进行GST-pull down和免疫荧光试验验证。结果表明,COXⅡ与AM2存在相互作用,且在细胞质中存在共定位现象,为进一步研究禽流感病毒的致病机理和病毒-宿主相互作用机制提供了理论基础。In order to screen and confirm the host proteins interacting with avian influenza virus AM2 protein using yeast two-hybrid system and provide scientific basis for the new biological function of AM2. After exclusion of self-activation capacities of bait protein, yeast cells were co- transformed with bait plasmid pGBKT7 AM2 and human lymphatic cDNA library plasmids and were grown in selective SD/-Trp-I.eu-His-Ade medium. Subsequently, positive clones were se- quenced,analyzed with bioinformatics methods and further conformed by retransformation assay. Finally,two positive clones were obtained by yeast two-hybrid screening and preliminary identifi- cation. The mitochondrial protein cytochrome C oxydase subunit Ⅱ （COX Ⅱ ） was selected for fur- ther research. The eukaryotic expression vector pcDNA3.0-HA-COX Ⅱ was constructed,and then GST pull-down and immunofluorescence assay were performed to further confirm the interaction. These results demonstrated that COX Ⅱ could specifically interact with AM2,and AM2 could be co localized with COX Ⅱ protein in the cytoplasm by immunofluorescenee assay. The study may provide a theoretical basis for further research of the pathogenic mechanism of avian influenza vi- rus,as well as the interaction mechanism between virus and host.

关 键 词：禽流感病毒 AM2蛋白 酵母双杂交 COXⅡ蛋白 共定位 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]