幽门螺杆菌CagA与YWHAE互作区段的确定  

Determination of the regions of Helicobacter pylori CagA mediating the interaction with YWHAE

作　　者：张晓艳 温春燕 陈豪 佘菲菲 ZHANG Xiao-yan;WEN Chun-yan;CHEN Hao;SHE Fei-fei(Key Laboratory of Ministry of Education for Gastrointestinal Cancer,Fujian Key Laboratory of Tumor Microbiology,School of Basic Medical Sciences,Fujian Medical University,Fuzhou 350122,China)

机构地区：[1]福建医科大学基础医学院,消化道恶性肿瘤教育部重点实验室,福建省肿瘤微生物学重点实验室,福州350122

出　　处：《中国人兽共患病学报》2022年第11期975-981,共7页Chinese Journal of Zoonoses

基　　金：福建省自然科学基金(No.2018J01837,No.2020J02019)。

摘　　要：目的探讨幽门螺杆菌CagA蛋白与宿主细胞YWHAE蛋白相互作用的区段。方法先在酵母细胞水平构建CagA区段蛋白[-N、-M、-(N+M)、-C]的表达载体,经“滤纸法”检测、一对一交配试验及“ONPG液相法”分析,筛选互作区段;进而在哺乳动物细胞水平,选用Cos7细胞经共转染、Co-IP方法确定互作区段,选用AGS细胞经上调/下调YWHAE表达、共转染、双荧光素酶报告基因检测等分析细胞NF-κB活性检测CagA候选区段与YWHAE互作的功能。结果成功构建CagA蛋白的N、M、(N+M)和C区段的酵母表达载体,CagA各区段蛋白“滤纸法”检测反式激活特性均为阴性,“一对一”交配后“液相法”筛选到CagA区段[-N、-(N+M)、-C]分别可与YWHAE在酵母中相互作用;Cos7细胞实验确定CagA区段[-N、-C]可分别沉淀外源性YWHAE,AGS细胞中CagA区段[-N、-C]对细胞NF-κB的激活,上调YWHAE能增强、下调YWHAE则抑制。结论幽门螺杆菌CagA蛋白N-末端与C-末端均分别与宿主细胞YWHAE蛋白相互作用,并可激活细胞NF-κB。This study was aimed at investigating the regions of Helicobacter pylori CagA mediating the interaction with YWHAE.In yeasts,plasmids for expression of CagA fragments[-N,-M,-(N+M),-C]were constructed successfully.The fragment proteins had no effect on transactivation,as tested with filter assays.ONPG liquid culture assays in mating yeast indicated that the CagA fragment[-N,-(N+M),-C]protein interacted with YWHAE.In Cos7 cells,the CagA fragment[-N,-C]protein was precipitated with exogenous YWHAE in Co-IP.In AGS cells,dual luciferase reporter assays indicated that NF-κB activation,mediated by the interaction between CagA fragments[-N or-C]and YWHAE,was enhanced with higher expression,and inhibited with lower expression,of YWHAE.The findings regarding the CagA fragments interacting with YWHAE aid in understanding of the carcinogenic mechanisms of CagA.

关 键 词：幽门螺杆菌 CAGA YWHAE 相互作用 NF-ΚB 

分 类 号：R378[医药卫生—病原生物学]