肝癌靶向性超抗原表达载体的构建与鉴定  

Design and construction of hepatocellular carcinoma specific superantigen expres sion vector

作　　者：禄韶英[1] 隋延仿[2] 李增山[2] 潘承恩[1] 武文[2] 叶菁[2] 

机构地区：[1]西安交通大学第一医院普外科,陕西西安710061 [2]第四军医大学基础部病理学教研室,陕西西安710032

出　　处：《免疫学杂志》2003年第2期101-104,共4页Immunological Journal

基　　金：国家自然科学基金资助项目 (39770 82 7)

摘　　要：目的　设计并构建受AFP基因顺式作用元件调控 ,并经减毒处理的肝癌特异性超抗原表达载体。方法　用新设计的引物作PCR扩增截短的AFP基因启动子和增强子、linker CD80tm和SEA(D2 2 7A)。将上述片断与逆转录病毒载体pLXSN的多克隆位点连接 ,构建成为AFP基因顺式作用元件调控的肝癌特异性减毒超抗原表达载体 [pLXSNSEA(D2 2 7A) Linker CD80tm],并用软件对其开放读框进行分析。结果　成功克隆了截短的AFP基因启动子、增强子、linker CD80tm和SEA(D2 2 7A)。将上述序列连接到逆转录病毒载体pLXSN的多克隆位点 ,酶切鉴定和DNA序列分析无误。结论　AFP基因转录启动元件修饰的SEA表达载体 ,在基因转录水平定量、定向、特异性调控强有力的细胞和体液免疫活化剂 (SEA) 。Objective To design and construct attenuated hepatocellul ar carcinoma (HCC) specific superantigen expression vector re gulated by th e cis acting element of AFP.Methods The abbreviated cDNA of AFP enh ancer and promoter was synthesized through PCR from genome DNA of HepG2 cells. S EA (D227A) and linker CD80tm were also amplified from the plasmid that contain the cDNA of SEA and CD80 through PCR. The four gene fragments were subcloned int o pbluescript ⅡKS(+) and identified by sequence analysis. All the gene fragment s were then cloned into the multiple cloning site of retroviral vector pLXSN. The new recombinant plasmid [pLXSN SEA(D227A) Linker CD80tm] was purified and i dentified by restriction endonucleases.Results By the methods o f restriction digestion and sequence analysis we confirmed that the length, posi tion and orientation of inserted fusion genes were all correct. The transcriptio n of SEA (D227A) Linker CD80tm is under the control of AFP cis acting element . Conclusion The new recombinant expression vector is specific for HCC,which will be used as gene vaccine for HCC. [

关 键 词：肝癌 超抗原 表达载体 构建 鉴定 甲胎蛋白 

分 类 号：R735.7[医药卫生—肿瘤]