人HSP75基因重组腺病毒载体构建及在C17.2神经干细胞的表达  

Construction of Adenovirus Vector Carrying Human HSP75 Gene and Expressing in C17.2 Neural Stem Cells

作　　者：王艳[1] 陈庆状 蒋德旗[1] 李明星[1] 贾思远[3] 朱宁 王勇[1] 

机构地区：[1]南方医科大学珠江医院药剂科,广东广州市510282 [2]广州市中西医结合医院药剂科,广东广州市510800 [3]南方医科大学珠江医院烧伤科,广东广州市510282 [4]南海区第三人民医院药剂科,广东佛山市528244

出　　处：《中国康复理论与实践》2015年第3期264-268,共5页Chinese Journal of Rehabilitation Theory and Practice

基　　金：国家自然科学基金项目(No.30973162);广东省自然科学基金项目(No.S2012010008199;No.S2013010015546)

摘　　要：目的构建HSP75基因重组腺病毒载体,观察HSP75蛋白在C17.2神经干细胞中的表达。方法采用PCR方法从含人HSP75基因质粒中扩增HSP75基因,连接到腺病毒穿梭质粒的多克隆位点区域,构建重组穿梭质粒p HBAd-MCM-HSP75-GFP,在LipofiterTM转染试剂的介导下与腺病毒辅助骨架质粒p HBAd-BHGloxΔE1,3 Cre共转染HEK293细胞,整合包装成重组腺病毒,TCID50法测定病毒滴度。使用荧光显微镜、流式细胞仪和Western blotting观察重组腺病毒在C17.2细胞中的感染情况及HSP75蛋白的表达水平。结果通过酶切鉴定、测序、PCR鉴定,HSP75基因已正确插入穿梭质粒中,并与病毒骨架质粒整合包装成重组腺病毒;重组腺病毒滴度为1.0×1010PFU/ml;Western blotting检测,转染后的神经干细胞表达HSP75蛋白。结论 HSP75重组腺病毒载体成功构建,对C17.2神经干细胞具有较高的表达效力。Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid p HBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid p HBAd-MCMV-HSP75-GFP and large adenovirus helper plasmid p HBAd-BHGloxΔE1,3 Cre in mediation of Lipofiter^TM. The recombinant adenovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detected by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplification revealed the successful construction of recombinant shuttle plasmid and recombinant adenovirus. The titer of recombinant adenovirus was 1.0×10^10PFU/ml. Western blotting indicated HSP75 gene could be expressed effectively in neural stem cells after transfection. Conclusion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2neural stem cells.

关 键 词：热休克蛋白75 重组腺病毒载体 C17.2神经干细胞 

分 类 号：R741[医药卫生—神经病学与精神病学]