肝癌特异性超抗原SEA(D227A)基因表达载体的构建与表达  

The construction and expression of a hepatocellular carcinoma-specific superantigen SEA (D227A) expression vector

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作  者:禄韶英[1] 隋延仿[2] 李增山[2] 潘承恩[1] 叶菁[2] 张秀敏[2] 王文勇[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:《细胞与分子免疫学杂志》2003年第4期353-356,共4页Chinese Journal of Cellular and Molecular Immunology

基  金:国家自然科学基金资助项目 (No .39770 82 7)

摘  要:目的 :构建受AFP顺式作用元件调控的超抗原表达载体 ,将SEA(D2 2 7A)特异性的表达于AFP阳性肝癌细胞膜表面。方法 :PCR扩增AFP基因启动子、增强子、linker CD80tm和SEA(D2 2 7A)。将上述片断插入逆转录病毒载体 pLXSN的多克隆位点 ,构建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)。通过脂质体介导 ,以表达载体转染表达或不表达AFP的肿瘤细胞系 ,用RT PCR和间接免疫荧光染色 ,检测SEA的表达。结果 :成功地将AFP基因的启动子、增强子、linker CD80tm和SEA(D2 2 7A)克隆到逆转录病毒载体 pLXSN的多克隆位点 ,酶切鉴定和DNA序列分析无误 ,RT PCR和间接免疫荧光法检测证实 ,SEA(D2 2 7A)能在AFP阳性的肝癌细胞膜特异性表达。结论 :AFP顺式作用元件修饰的超抗原表达载体的构建 。AIM: To construct the superantigen SEA (D227A) expression vector modulated by cis acting element of AFP and express it specifically on AFP + hepatocellular carcinoma (HCC) cells. METHODS: The enhancer and promoter of AFP gene, SEA (D227A) cDNA and linker CD80tm were amplified by PCR. The gene segments mentioned above were cloned into the multiple cloning sites of retrovirus vector pLXSN to construct an attenuated superantigen expression vector pLXSN SEA (D227A) linker CD80tm modulated by cis acting element of AFP gene. AFP + or AFP - HCCs were transfected by the superantigen expression vector through lipofectamine mediation. SEA expression was detected by RT PCR and indirect immunofluorescence staining. RESULTS: Promoter, enhancer, SEA (D227A) and linker CD80tm had been successfully cloned into the multiple cloning site of retrovirus vector pLXSN, RT PCR and indirect immunofluorescence assay were used to prove that SEA (D227A) could specifically express in the AFP + HCCs. CONCLUSION: The successful construction and expression of hepatocellular carcinoma specific superantigen SEA (D227A) gene expression vector lay the foundation for enhancing immunotherapy of hepatocellular carcinoma.

关 键 词:超抗原 逆转录病毒载体 AFP顺式作用元件 肝细胞癌 

分 类 号:R392.11[医药卫生—免疫学] R735.7[医药卫生—基础医学]

 

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