幽门螺杆菌细胞毒素相关基因A绿色荧光蛋白真核表达载体的构建  

Construction of the enhanced green fluorescent protein eukaryotic expression vector carrying CagA of Helicobacter pylori

作　　者：冯百岁[1] 姚雪华[1] 赵国强[2] 

机构地区：[1]郑州大学第一附属医院消化内科 河南省高等学校临床医学重点学科开放实验室,450052 [2]郑州大学基础医学院微生物教研室

出　　处：《中华医学杂志》2007年第8期570-572,共3页National Medical Journal of China

摘　　要：目的构建含幽门螺杆菌细胞毒素相关基因 A(CagA)基因片段的绿色荧光蛋白表达载体 pEGFP-C3-CagA。方法从幽门螺杆菌细胞株中扩增出 CagA 基因片段,经回收、纯化,连接到质粒 pGEM-T 上,测序、酶切后与质粒 pEGFP-C3连接,脂质体法将 pEGFP-C3-CagA 转染入胃癌细胞株BGC823,用荧光显微镜观察转染的细胞。结果通过测序、酶切证明 CagA 插入质粒正确,构建出载体 pEGFP-C3-CagA,转染后经荧光显微镜观察到胃癌细胞株 BGC823中有绿色荧光。结论成功构建表达 CagA 的真核绿色荧光载体。Objective To construct a eukaryotic green fluorescent protein expressing vector caontaining the fragment of cytotoxin associated gene A （ CagA ） of Helicobacter pylori （ Hp ） so as to lay a foundation for the research of gene vaccine of gastric cancer, pEGFP-C3-CagA. Methods The fragment of gene CagA was amplified from Hp using PCR. The amplified product was examined by electrophoresis and sequence determination. This fragment was inserted into pGEM-T plasmid and pEGFP-C3 fluorescent expression vector. The recombined plasmid pEGFP-C3-CagA was transfected into the gastric carcinoma cells of the strain BGC823 by lipoplast method. Fluorescence microscopy was used to observe the expression of pEGFP-C3-CagA under. Results CngA was inserted in the plasmid correctly. It was verified by DNA sequencing and restriction enzyme. The enhanced green fluorescent protein eukaryotic expression vector carrying CagA of Helicobacter pylori （pEGFP-C3-CagA） was recombined correctly and transfected in gastric carcinoma cell strain BGC823. Green fluorescence was observed in transfected gastric carcinoma cell. Conclusion Construction of the enhanced green fluorescent protein eukaryotic expression vector carrying CagA was successful.

关 键 词：螺杆菌 幽门 载体蛋白质类 克隆 分子 细胞毒素相关基因A 

分 类 号：R346[医药卫生—基础医学]