《中国康复理论与实践》

《中国康复理论与实践》 ›› 2015, Vol. 21 ›› Issue (03): 264-268.

• 基础研究 • 上一篇    下一篇

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

王艳1a，陈庆状2，蒋德旗1a，李明星1a，贾思远1b，朱宁3，王勇1a   

  1. 1.南方医科大学珠江医院，a.药剂科；b.烧伤科，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.南海区第三人民医院药剂科，广东佛山市528244。
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2015-03-25 发布日期:2015-03-25

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

WANG Yan, CHEN Qing-zhuang, JIANG De-qi, LI Ming-xing, JIA Si-yuan, ZHU Ning, WANG Yong   

  1. 1. a. Department of Pharmacy; b. Department of Burns, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, China; 2. Department of Pharmacy, Guangzhou Hospital of Integrated Traditional and West Medicine, Guangzhou, Guangdong 510800, China; 3. Department of Pharmacy, the Third People's Hospital of Nanhai, Foshan, Guangdong 528244, China
  • Received:1900-01-01 Revised:1900-01-01 Published:2015-03-25 Online:2015-03-25

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被引次数

1

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pHBAd-MCM-HSP75-GFP，在LipofiterTM 转染试剂的介导下与腺病毒辅助骨架质粒pHBAd-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1.0×1010 PFU/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。

关键词: 热休克蛋白75, 重组腺病毒载体, C17.2 神经干细胞

Abstract: Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2 neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid pHBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pHBAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. 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 1.0×1010 PFU/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 C17.2 neural stem cells.

Key words: heat shock protein 75, recombinant adenovirus vector, C17.2 neural stem cells