| 罗丹枫,龚静吉,程腾,等.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.骨科,2016,7(6): 442-447. |
| 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 |
| The recombinant fusion protein sCAR-TMTP1 enhances the gene transfer efficiency of ADV on human osteosarcoma cells |
| 投稿时间：2016-06-25 |
| DOI：10.3969/j.issn.1674-8573.2016.06.013 |
| 中文关键词: 骨肉瘤 腺病毒 TMTP1肽 柯萨奇腺病毒受体 重组蛋白 基因治疗 肿瘤细胞 |
| 英文关键词: Osteosarcoma Adenovirus TMTP1 Coxsackie and adenovirus receptor Recombinant protein Gene therapy Tumer cell |
| 基金项目:国家自然科学基金资助项目（81202061） |
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| 中文摘要: |
| 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31 000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-TMTP1可显著提升ADV对缺乏CAR的骨肉瘤细胞的转染效率。 |
| 英文摘要: |
| 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 31 000, 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. |
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