幽门螺杆菌CagⅤ蛋白的克隆表达及初步分析.pdfVIP

中国人兽共患病学报 2013 , 29 (1 0) Chinese J ournal of Zoonoses 955 DOI: 10. 3969/cjz. j. issn. 1002 - 2694.2013. 10.004 幽门螺忏菌CagV 蛋白的克隆表达及初步分析 王艳冬，宫雅楠，肖 边，张建中 摘 要:目的 构建幽门螺杆菌C Helicobacter 户ylori) IV 型分泌系统 cagVChp0530) 基因的原核表达系统，表达并纯化 CagV 蛋白，初步分析其结构和抗原性，为进一步研究幽门螺杆菌的致病机制和治疗药物的筛选奠定基础。方法 以 H. py- lori ATCC70039 2 株基因组为模板，采用聚合酶链反应CPCR) 扩增获得目的片段，将其插入表达载体 pET28a 后转化大肠杆菌 BL21CDE3) ，表达纯化后利用蛋白质印迹CWestern blot) 分析 CagV 蛋白的抗原性。结果 双酶切鉴定结果证实 cagV 基因重 组表达载体构建成功;重组表达蛋白经飞行质谱鉴定为幽门螺杆菌 CagV 蛋白;Western blot 检测结果显示 CagV 蛋白具有特 异抗原性。结论 所克隆表达的 CagV 蛋白具有较好的抗原性，对后续的的 H. pylori 致病性和检测、分析研究有比较重要的 意义。 关键词:幽门螺杆菌;蛋白;纯化;抗原性 中图分类号:R378.2 文献标识码:A 文章编号: 1002-2694(2013) 10 一 0955-04 Cloning and analysis of CagV in Helicobacter pylori WANG Yan-dong ,GONG Ya-nan ,XIAO Di ,ZHANG Jian-zhong (National 1nstitute for Communicable Disease Control and Prevention , China CDC , Beij ing 102206 , China) ABSTRACT: The aim is to clone , express and purify the CagV protein of Helicobacter pylori (H. pylori) type lV secre- tion system , and conduct preliminarily analysis on its structure and antigenicity for the further study of pathogenic mechanism and treatment of Helícobacter pylorí. The cagV gene was amplified from H. pylori ATCC700392 genomic DNA and inserted into expression vector pET28a , and then transformed into E . coli BL21 CDE3) for the expression. The expression product was identified by SD5and MALDI-TOF-MS and its antigenicity was tested by Western blot. Results indicated that the re combinant protein was identified as Helicobacter 户ylori CagV protein by MALDI-TOF-MS. Western blot test result showed CagV protein had specific antigenicity for Helicobacter 户ylori. The CagV protein with good