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山东大学学报(医学版)

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EGFPKallikrein重组真核表达载体的构建及其在兔内皮祖细胞中的表达

姜虹,李大庆,张运,刘春喜,冯进波,王荣   

  1. 山东大学齐鲁医院心内科暨心血管重构与功能教育部重点实验室山东 济南 250012
  • 收稿日期:2006-03-29 修回日期:1900-01-01 出版日期:2006-06-24 发布日期:2006-06-24
  • 通讯作者: 姜虹

Construction of pEGFPKallikrein expression vectorand its expression in endothelial progenitor cells in rabbit

JIANG Hong,LI Da-qing.ZHANG Yun,LIU Chun-xi,FENG Jin-bo,WANG Rong   

  1. Department of Cardiology, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China
  • Received:2006-03-29 Revised:1900-01-01 Online:2006-06-24 Published:2006-06-24
  • Contact: JIANG Hong

摘要: 目的:构建携带人组织激肽释放酶(tHK)基因,以绿色荧光蛋白(GFP)为报告基因的重组真核表达载体,并导入内皮祖细胞(EPCs)中表达,验证tHK基因修饰EPCs用于血管病治疗的可行性。方法:PCR扩增tHK目的基因,将该全长基因定向克隆至真核表达载体pEGFPN3上,构建重组质粒载体。并利用脂质体转染体外培养的EPCs,在活细胞状态下用荧光显微镜直接观察tHK EGFP融合蛋白在细胞中的表达;用RTPCR 方法验证tHK在mRNA水平的表达;用ELISA方法验证tHK蛋白水平的表达。结果:酶切和测序证实pEGFPKallikrein重组质粒构建正确,重组质粒载体在EPCs 中获得了表达,表达的融合蛋白具有tHK 和EGFP 的双重活性。结论:通过基因克隆方法成功构建了pEGFPKallikrein重组质粒载体,并且在EPCs 中稳定表达,为进一步研究tHK基因修饰EPCs双重治疗血管病的技术策略奠定了基础。

Abstract: To construct pEGFPKallikrein eukaryotic expression vector by using green fluorescence protein as reportor gene and transfecting rabbit endothelial progenitor cells. Methods: The human tissue kallikrein(tHK) genes were amplified with PCR technique and inserted into pEGFPN3 vector. The rabbit endothelial progenitor cells (EPCs) were transfected with the formed plasmid by means of lipidosome. The KallikreinEGFP fused protein was viewed directly with fluorensce microscope, and the expression of tHK was detected by RTPCR(reverse transcriptionPCR) and ELISA. Results: Plasmids was formed correctly and was detected by PCR meanwhile it being sequenced and expressed in the rabbit EPCs. The fused protein has activites of both kallikrein and EGFP. Conclusion: tHK gene modified EPCs could play a role in the treatment of artery injury and endothelial dysfunction.

Key words: Kallireins, Endothelium, Stem Cells, Gene Transfer Techniques

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