肝癌靶向性SEA基因重组腺病毒载体的构建及体外生物学活性鉴定

Construction of Hepatoma-targeting Recombinant Adenovirus Vector of Staphylococcal Enterotoxin A (SEA) and Identification of Its Biological Activity

[目的]构建肝癌靶向性葡萄球菌肠毒素A(SEA)基因重组腺病毒载体。[方法]首先利用现有的腺病毒穿梭质粒pShuttle和pShuttle-CMV,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、启动子及SEA基因分别从已构建的pKS-EP载体和pMD18-T-SEA载体上,亚克隆至pShuttle2中,再与腺病毒骨架质粒pAdEasy-1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Hepa1-6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,经荧光显微镜观察和流式细胞术检测SEA在细胞膜表面的表达。采用3H掺入法检测膜表达的SEA体外诱导淋巴细胞增殖的活性。[结果]SEA能够靶向性地表达在高表达AFP的Hepa1-6细胞膜上,在不表达AFP的B16、NIH3T3细胞膜上不表达,并且体外能够诱导淋巴细胞增殖。[结论]成功地构建了肝癌靶向性SEA基因重组腺病毒载体,为进一步研究SEA在肝癌靶向基因治疗中的应用及其抗肿瘤免疫机制奠定了基础。

[Purpose ]To construct hepatoma-targeting recombinant adenovirus vector of staphylococcal enterotoxin A （SEA） gene. [Methods] A new transfer plasmid pShuttle2 was construeted with polyA signal sequence without CMV enhancer/promoter using the existing adenovirus transfer plasmids pShuttle and pShuttle-CMV. AFP enhancer, promoter and SEA gene was subcloned into pShuttle2 from the vectors pKS-EP or pMDI8-T-SEA respectively. Then the recombinant plasmid was co-transformed into E.coli BJ5183 with backbone vector pAdEasy-1 to ohtain recombinant adenovirus DNA. The recombinant adenovirus DNA was transfected into HEK293 cells to prepare adenovirus. After AFP-producing cell line Hepal-6 and AFP-nonprodueing cell lines B16 and NIH3T3 were infected by recombinant adenovirus, the expression of SEA on the surface of cell was detected using indirect immunofluorescent staining by fluorescent microscope and flow cytometry （FCM）. The in vitro activity of membrane SEA to stimulate proliferation of lymphocytes was detected by 3H-TDR. [Results] SEA targeting expresion on the surface of AFP-produeing Hepa1-6 cells could stimulate proliferation of lymphocytes in vitro hut not on AFP-nonproducing B16 and NIH3T3 cells. [Conclusion] Hepatoma-targeting recombinant adenovirus vector of SEA gene is successfully constructed, which lays the foundation for further research on application of SEA in targeted genetherapy and immunological mechanisms for hepatoma.