CAJ | 학술논문

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-JCV T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Nde Ⅰ位点,并在两端插入Bcl Ⅰ位点,通过BamH Ⅰ位点将DNA片段与K19启动子连接,构建K19-JCV T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-JCV T抗原质粒转染,用Western blot方法检测JCV T抗原的表达情况.结果 K19-JCV T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-JCV T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.
목적 이용서위점막상피세포중고활성적각단백19(K19)계동자구건K19-JCV T항원표체질립병진행감정.방법 이용PCR방법돌변JC병독T항원중적Nde Ⅰ위점,병재량단삽입Bcl Ⅰ위점,통과BamH Ⅰ위점장DNA편단여K19계동자련접,구건K19-JCV T항원질립.이용매절화DNA측서학인기DNA서렬.면역조화사선세포각단백19양성위암세포,대세포각단백19표체양성적위암세포계AGS진행K19-JCV T항원질립전염,용Western blot방법검측JCV T항원적표체정황.결과 K19-JCV T항원표체질립성공구건,AGS위암세포계고표체세포각단백19병용우K19-JCV T항원표체질립전염,전염K19-T항원질립적AGS세포계유T항원표체.결론 동의돌변화상사련접재질립구건중기도관건작용,매절위점적갑기화야시치득주의적문제.
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 Ndel site was mutated by FCR with Bell insertion at both sides.The DNA fragment digested by Bcl Ⅰ was ligated with the plasmid containing K19 promoter via Bam Ⅰ 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 ACS 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 methy lation 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.