论文部分内容阅读
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。
Objective: To construct a recombinant adenovirus vector targeting hepatocellular carcinoma targeting Staphylococcal enterotoxin A (SEA) and CD80 gene. Methods: Firstly, the shuttle plasmid pShuttle and pShuttleCMV were used to construct a new shuttle plasmid with polyA tailed signal without CMV enhancer / promoter, named as pShuttle2. The AFP enhancer, promoter, SEA and CD80 genes were respectively subcloned into pShuttle2 from the constructed pKSEP vector and pMD18TBIS vector, and co-transformed into E. coli BJ5183 with the adenovirus backbone plasmid pAdEasy1. A recombinant adenovirus was prepared after transfection of HEK293 cells with the obtained recombinants and then infected with Hepa16 hepatoma cell line highly expressing AFP and B16 melanoma cell line not expressing AFP and NIH3T3 fibroblast cell line. The expression of SEA and CD80 on the cell membrane surface was detected by indirect immunofluorescence, confocal laser scanning microscopy and flow cytometry. The membrane-expressed SEA-induced lymphocyte proliferation activity was examined by 3H incorporation assay. Results: After infected with the prepared recombinant adenovirus, SEA and CD80 could be co-expressed on the membrane of Hepa16 cell which expressed AFP, but not on the cell membrane of B16 and NIH3T3 which did not express AFP. CONCLUSION: The successful construction of recombinant adenovirus vector targeting hepatocellular carcinoma-associated SEA and CD80 gene provides a basis for further study on the combined application of SEA and CD80 in targeted gene therapy of hepatocellular carcinoma and its anti-tumor immune mechanism.