SV40T抗原胃壁细胞特异性表达载体的构建与鉴定  

Construction and identification of a specific expression vector for SV40 large T antigen in gastric parietal cell

作　　者：陈辉[1] 侯艺芳[2] 乐晓平[3] 金辉[3] 马慧洁[3] 张钦宪[3] 

机构地区：[1]郑州大学基础医学院细胞生物学与医学遗传学教研室河南省分子医学重点实验室,河南省郑州市450052 [2]郑州大学第一附属医院妇产科,河南省郑州市450052 [3]郑州大学基础医学院组织胚胎学教研室,河南省郑州市450052

出　　处：《世界华人消化杂志》2007年第18期2004-2008,共5页World Chinese Journal of Digestology

基　　金：河南省重大科技攻关计划资助项目;No.0623031400~~

摘　　要：目的:构建在胃壁细胞中特异性表达SV40T抗原的真核表达裁体并进行鉴定.方法:采用酚-氯仿法从昆明小鼠肝细胞中提取基因组DNA,聚合酶链式反应(PCR)扩增H^+/K^+ATPaseβ亚基启动子,产物命名为HK.将PCR产物纯化回收后与pMT18-T载体相连,并将其克隆至真核表达载体pcDNA3.1(-),构建pcDNA3.1(-)/HK;从含SV40T基因片段的质粒p LITAg中酶切回收SV40T基因,与pcDNA3.1(-)/HK相连,构建胃壁细胞特异性表达载体pcDNA3.1(-)/HKSV,并测序鉴定.结果:pcDNA3.1(-)/HKSV用XbaⅠ、BarnHⅠ双酶切可得到1kb H^+/K^+ATPaseβ亚基启动子,2.7 kb SV40T基因与5.4 kb pcDNA3.1(-)载体3条DNA条带.用XbaⅠ、KpnⅠ双酶切电泳,可见到约3.7与5.4 kb的两条DNA条带;用BamHⅠ单酶切电泳,可见到2.7与6.4 kb的2条DNA条带;用EcoRⅠ单酶切,只见到约9.1 kb的1条DNA条带,酶切电泳结果均与设计一致.测序结果显示,H^+/K^+ATPaseβ亚基启动子与SV40T基因成功构建于pcDNA3.1(-)真核表达载体中.结论:构建在胃壁细胞中特异性表达SV40T基因的真核表达载体,为进一步转基因小鼠及胃癌动物模型的建立提供了稳定、可靠的分子工具.AIM： To construct and identify a eukaryotic specific expression vector of SV40 large T antigen in gastric parietal cell. METHODS： Genome DNA was extracted from liver cells of Kunming mice by a phenol-chloroform method. The H^＋/K^＋ ATPase β subunit promoter gene was amplified by polymerase chain reaction （PCR） and the product was named as HK. This was then ligated with the pMT18-T vector after purification. A cloning vector of pcDNA3.1 （-）/HK was constructed by ligating the H^＋/K^＋ATPase β subunit promoter and the eukaryotic vector pcDNA3.1 （-）. SV40 large T genes were digested by restricted enzymes from PLITAg recombinant and then inserted into the prokaryotic expression vector pcDNA3.1 （-）/ HK. Thus, an expression vector specific for pcDNA3.1 （-）/HKSV was constructed and identified in gastric parietal cell. RESULTS： Three DNA bands were seen when pcDNA3.1（-）/HKSV was digested by Xba I and BamH I, which were the 1 kb H^＋/K^＋ ATPase β subunit promoter gene, the 2.7 kb SV40T gene and the 5.4 kb pcDNA3.1（-） vector. Two DNA bands, 3.7 kb and 5.4 kb were seen when pcDNA3.1（-）/HKSV was digested by Xba I and Kpn I. Another two DNA bands, 2.7 kb and 6.4 kb, were seen when pcDNA3.1（-）/HKSV was digested by BamH I. One DNA band, 9.1 kb, was seen when pcDNA3.1（-）/HKSV was digested by EcoR I . All electrophoresis results were consistent with the design. The sequencing results showed that H^＋/K^＋ ATPase β subunit promoter and SV40 large T gene had been successfully cloned into pcDNA3.1（-） eukaryotic vector. CONCLUSION： The recombinant plasmid pcDNA3.1 （-）/HKSV which is specially expressed in gastric parietal cell, is a stable and valuable molecular tool for establishing transgenic mice and animal models of gastric carcinoma.

关 键 词：SV40病毒 T抗原 真核表达 载体构建 

分 类 号：R735.2[医药卫生—肿瘤]