重组蛋白sCAR-TMTP1增强腺病毒对骨肉瘤细胞基因转染效率的研究

摘要

Objective To enhance the transduction efficiency of adenovirus on coxsackievirus and adenovirus receptor (CAR) deficient human osteosarcoma cells. Methods The recombinant protein sCAR-TMTP1 was expressed and purified by Bac-to-Bac system, then identified by SDS-PAGE and Western blotting. The expression level of CAR and the infection efficiency of ADV/GFP in different cell lines (osteosarcoma cells& 293) were detected by flow cytometry (FCM), and the positive rate of FITC-TMTP1 binding to above cell lines was also measured by FCM. MG-63 cells were exposed to Ad/sCAR-ligand complexes mixtures, and the infection efficiency of ADV/GFP was measured by FCM. Results Either of expressed and purified sCAR-TMTP1 showed a specific protein band with a relative molecular mass of 31000, with which the supernatant of lysed sf9 cells infected showed specific reaction with rabbit anti-mouse sCAR monoclonal antibody. FITC-TMTP1 specifically bound to the MG-63 cells with a lower level of CAR and poor infection efficiency of ADV. ADV/GFP modified with sCAR-TMTP1 mediated an obvious enhancements of GFP expression in MG-63, compared with those cells exposed to ADV/GFP. Conclusion The recombinant fusion protein sCAR-TMTP1 has the ability to enhance the gene transfer efficiency of ADV on CAR deficient human osteosarcoma cells.%目的 提高腺病毒(adenovirus,ADV)在缺乏柯萨奇腺病毒受体(coxsackie and adenovirus receptor,CAR)的骨肉瘤细胞中的转染效率.方法 通过昆虫(Bac-to-Bac)杆状病毒表达系统表达含有CAR胞外段(sCAR)及TMTP1肽的重组蛋白(sCAR-TMTP1),并对其纯化、鉴定.流式细胞仪分别检测骨肉瘤细胞及人胚肾细胞293(HEK293)的CAR表达水平,ADV对其的感染效率及FITC-TMTP1的结合能力.重组蛋白sCAR-TMTP1与ADV共同孵育形成复合物,流式细胞仪研究其在缺乏CAR的骨肉瘤细胞(MG-63)中的转染效率.结果 表达及纯化的sCAR-TMTP1蛋白在相对分子质量约31000处可见特异蛋白条带,感染的sf9细胞裂解上清可与兔抗小鼠sCAR单克隆抗体发生特异性反应.MG-63可与FITC-TMTP1特异结合,其CAR表达水平低,ADV转染效率低.经sCAR-TMTP1修饰后的ADV/GFP对MG-63细胞的感染效率显著高于未经修饰的含报告基因GFP的ADV(ADV/GFP).结论 重组蛋白sCAR-TM-TP1可显著提升ADV对缺乏CAR的骨肉瘤细胞的转染效率.