胃黏膜特异表达JC病毒T抗原质粒构建及表达鉴定  

The Construction and Expression Confirmation of JC Virus T Antigen Expression Plasmid in Gastric Mucosa

作　　者：夏蒲[1] 徐小燕[2] 贾宝萍[3] 王薇[2] 关一夫[1] 高野康雄 郑华川[1] 

机构地区：[1]中国医科大学基础医学院生物化学研究室,沈阳110001 [2]中国医科大学基础医学院病理生理学教研室 [3]中国医科大学档案馆,沈阳110001 [4]日本富山大学医学部病理诊断教室

出　　处：《中国医科大学学报》2010年第1期18-21,共4页Journal of China Medical University

基　　金：沈阳人才资源开发基金项目;辽宁省百千万人才工程资助项目;教育部归国人员科研启动基金项目;人事部留学人员科技活动择优资助项目;2009年沈阳市科学技术计划资助项目(1091175-1-00)

摘　　要：目的利用鼠胃黏膜上皮细胞中高活性的角蛋白19(K19)启动子构建K19-JCVT抗原表达质粒并进行鉴定。方法利用PCR方法突变JC病毒T抗原中的NdeⅠ位点,并在两端插入BclⅠ位点,通过BamHⅠ位点将DNA片段与K19启动子连接,构建K19-JCVT抗原质粒。利用酶切和DNA测序确认其DNA序列。免疫组化筛选细胞角蛋白19阳性胃癌细胞,对细胞角蛋白19表达阳性的胃癌细胞系AGS进行K19-JCVT抗原质粒转染,用Western blot方法检测JCVT抗原的表达情况。结果K19-JCVT抗原表达质粒成功构建,AGS胃癌细胞系高表达细胞角蛋白19并用于K19-JCVT抗原表达质粒转染,转染K19-T抗原质粒的AGS细胞系有T抗原表达。结论同义突变和相似连接在质粒构建中起到关键作用,酶切位点的甲基化也是值得注意的问题。Objective To construct and confirm the JC virus（JCV）T antigen expression plasmid using mouse keratin 19（K19）promoter specific for the gastric epithelial cells. Methods The NdeI site was mutated by PCR with BclI insertion at both sides. The DNA fragment digested by BclⅠ was ligated with the plasmid containing K19 promoter via BamⅠ site. The DNA sequence was confirmed by restriction enzyme digestion and direct DNA sequencing. Cytokeratin 19 protein was examined to screen gastric carcinoma cell for transfection of K19- JCV T antigen expression plasmid by immunohistochemistry. The Western blot was employed to detect the JCV T antigen expression in the gastric carcinoma transfectant. Results K19-JCV T antigen expressing plasmid was successfully constructed. The AGS strongly expressed cytokeratin 19 protein and was selected for the transfection of K19-JCV T antigen expressing plasmid. JCV T antigen was positively expressed in the AGS transfectant. Conclusion The synonymous mutation and compatible ligation are useful in the plasmid construction. The methylation of restriction enzyme should be considered. It is meaning for the transgenic animal model of gastric carcinoma to successfully construct the JC virus T antigen expression plasmid in gastric mucosa.

关 键 词：JC病毒 T抗原 真核表达 载体构建 

分 类 号：R446.5[医药卫生—诊断学] R394[医药卫生—临床医学]