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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 ,为下一步用其强化肝癌的免疫治疗奠定了基础
OBJECTIVE: To construct a superantigen expression vector regulated by AFP cis-acting elements and to express SEA (D2 2 7A) specifically on the surface of AFP positive hepatocellular carcinoma cell membrane. Methods: AFP gene promoter, enhancer, linker CD80tm and SEA (D2 2 7A) were amplified by PCR. The above fragment was inserted into the multi-cloning site of the retroviral vector pLXSN to construct a hepatoma-specific attenuated superantigen expression vector (pLXSNSEA (D2 2 7A) linker CD80tm) regulated by the cis-acting element of the AFP gene. Tumor cell lines expressing or not expressing AFP were transfected with the expression vector by liposomes, and the expression of SEA was detected by RT PCR and indirect immunofluorescence staining. Results: The AFP gene promoter, enhancer, linker CD80tm and SEA (D2 2 7A) were successfully cloned into the multi-cloning site of retroviral vector pLXSN. The restriction enzyme digestion and DNA sequence analysis were correct. RT PCR and indirect immunization Fluorescence detection confirmed that SEA (D2 2 7A) can be specifically expressed in AFP-positive liver cancer cell membrane. CONCLUSION: The construction of AFP cis-acting superantigen-modified superantigen expression vector lays the foundation for further immunotherapy of hepatocellular carcinoma