弓形虫免疫作图蛋白1互作因子的筛选与鉴定  

Screening and identification of the interaction factors for Toxoplasma gondii immune mapped protein 1

作　　者：姚营营 张丽新 赵桂华[1] 徐超[1] 孙慧[1] 刘功振[1] 肖婷[1] 朱嵩[1] 魏庆宽[1] 黄炳成 闫歌[1] 尹昆[1] YAO Ying-ying;ZHANG Li-xin;ZHAO Gui-hua;XU Chao;SUN Hui;LIU Gong-zhen;XI-AO Ting;ZHU Song;WEI Qing-kuan;HUANG Bing-chen;YAN Ge;YIN Kun(School of Medicine and LifeSciences,University of Jinan,Shandong Academyof Medical Sciences,Shandong Institute of Parasitic Diseases,Jining,China 272033;Jining No.1People's Hospital)

机构地区：[1]济南大学,山东省医学科学院医学与生命科学学院,山东省寄生虫病防治研究所,山东济宁272033 [2]济宁市第一人民医院

出　　处：《中国病原生物学杂志》2020年第3期306-310,316,共6页Journal of Pathogen Biology

基　　金：山东省自然科学基金项目(No.ZR2018LH016);山东省医药卫生科技发展计划面上项目(No.2017WS103,2017WS104);国家自然科学基金项目(No.81702026);山东省重点研发计划(公益类专项)项目(2019GSF107054);山东省医学科学院医药卫生科技创新工程项目。

摘　　要：目的采用酵母双杂交技术筛选弓形虫免疫作图蛋白1(TgIMP1)在人类宿主胞内的相互作用因子并鉴定其相互作用。方法以刚地弓形虫RH株基因组为模板,PCR扩增TgIMP1全长编码序列,扩增产物经双酶切后插入诱饵表达质粒pGBKT7中。重组质粒转化入酵母Y2H Gold细胞中,采用酵母细胞融合方法与人胎脑cDNA酵母文库菌双杂交筛选互作因子。筛选的阳性质粒经测序和共转化验证TgIMP1体内相互作用。结果成功构建了不具备细胞毒性和自激活活性的pGBKT7-IMP1重组诱饵表达质粒,该诱饵在人胎脑cDNA文库中共计筛选得到Ringfinger protein2(RNF2)、Murine retroviru sinte grationsite1homolog(MRVI1)、Heterogeneous nuclear ribonucleoprotein A3(HNRNPA3)等6种相互作用因子。结论筛选得到6种人类宿主能够与弓形虫TgIMP1发生相互作用的蛋白因子,其中RNF2体内可与TgIMP1直接互作。Objectives To screen for interaction factors for Toxoplasma gondii immune mapped protein 1(IMP1)in a human library using ayeast two-hybrid(Y2 H)system and to identify those interactions.Method The full length of the TgIMP1 gene was amplified using PCR,with the extracted genome of the RH strain of T.gondii serving as a template.The PCR product was double digested and inserted into the bait-expressing plasmid pGBKT7 to construct the recombinant pGBKT7-IMP1,which served as the IMP1 bait for the following screening.After the bait was transformed into Y2 HGold competent cells,the interaction factors for the IMP1 bait were screened in a human fetal brain cDNA library using yeast mating.Positive colonies were identified using sequencing and co-transformation.Results The recombinant bait plasmid pGBKT7-IMP1 was successfully constructed without toxicity and with no autonomous activity.The bait plasmid was used to conduct screening according to selection procedures.Six types of novel host cell proteins,such as ring finger protein 2(RNF2),murine retrovirus integration site 1 homolog(MRVI1),and heterogeneous nuclear ribonucleoprotein A3(HNRNPA3),were identified as interaction factors with TgIMP1.Conclusion A total of 6 host interaction factors were identified with the Y2 Hsystem,and direct interaction between TgIMP1 and RNF2 in vivo was evident.

关 键 词：酵母双杂交 互作因子 免疫作图蛋白1 弓形虫 

分 类 号：R382.5[医药卫生—医学寄生虫学]