肝癌特异性超抗原SEA(D227A)基因表达载体的构建与表达

目的：构建受AFP顺式作用元件调控的超抗原表达载体，将SEA(D227A)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(D227A)。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(pLXSN SEA(D227A)—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(D227A)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(D227A)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。

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

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.