摘要
目的:构建肝癌靶向性葡萄球菌肠毒素A(SEA)和CD80基因共表达重组腺病毒载体。方法:首先利用现有的腺病毒穿梭质粒pShuttle和pShuttleCMV,构建新的不带CMV增强子/启动子而带有polyA加尾信号穿梭质粒,命名为pShuttle2。将AFP增强子、启动子、SEA及CD80基因分别从已构建的pKSEP载体和pMD18TBIS载体上,分别亚克隆至pShuttle2中,再与腺病毒骨架质粒pAdEasy1共转化E.coliBJ5183。以获得的重组子转染HEK293细胞后制备重组腺病毒,然后感染高表达AFP的肝癌细胞系Hepa16和不表达AFP的黑色素瘤细胞系B16、成纤维细胞系NIH3T3。采用间接免疫荧光法,激光共聚焦显微镜观察和流式细胞术检测SEA和CD80在细胞膜表面的表达。采用3H掺入法检测膜表达的SEA诱导淋巴细胞增殖的活性。结果:以制备的重组腺病毒感染肿瘤细胞后,SEA和CD80能够靶向性地共表达在高表达AFP的Hepa16细胞膜上,而在不表达AFP的B16、NIH3T3细胞膜上不表达。结论:成功地构建肝癌靶向性SEA和CD80基因共表达重组腺病毒载体,为进一步研究SEA和CD80在肝癌靶向基因治疗中的联合应用及其抗肿瘤免疫机制奠定了基础。
AIM： To construct hepatoma-targeting recombinant co-expression adenovirus vector of Staphylococcal enterotoxin A （SEA） and CDSO gene. METHODS： Using the adenovirus transfer plasmids pShuttle and pShuttle- CMV, we constructed a new transfer plasmid pShuttle2 with polyA signal sequence instead of CMV enhancer/promoter. AFP enhancer, promoter, SEA or CDSO gene was subcloned into pShuttle2 from the vectors pKS-EP or pMDIS-T- BIS respectively, and then the constructed plasmid pShuttle2-BIS containing AFP enhancer, promoter, SEA or CDSO gene was cotransformed into E, coil BJ5183 with backbone vector pAdEasy-1 to obtain recombinant adenovirus DNA. The recombinant adenovirus DNA was transfected into 293 cells to prepare adenovirus. After AFP-producing cell line Hepal.6 and AFP-nonproducing cell lines BI6 and NIH3T3 were infected by recombinant adenovirus, the expression of SEA and CDSO on the surface of cells was detected by indirect immunofluorescent staining, laser confocal microscope and flow cytometry （FCM）. RESULTS： SEA and CDSO was specifically co-expressed on the surface of infected Hepal-6 cells but not on BI6 and NIH3T3 cells. CONCLUSION： Hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CDSO gene was sucessfully constructed, which lays the foundation for further research on application of SEA and CDSO in targeted genetherapy for hepatoma and the underlying immunological mechanisms.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第5期613-616,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30271464)
全军医药卫生科研基金重点项目(01Z084)
