南方医科大学学报

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (04): 416-418.

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人内皮抑素基因的克隆、表达、纯化及活性测定

杨芳1, 何援利1, 姜孝玉2, 刘芸2, 彭冬先1, 宗利丽1   

  1. 1. 南方医科大学珠江医院妇产科, 广东, 广州, 510282;
    2. 中山大学生命科学院医药分子生物学实验室, 广东, 广州, 510275
  • 出版日期:2005-04-20 发布日期:2005-04-20
  • 基金资助:
    收稿日期:2004-10-5。
    基金项目:广东省科技厅科技计划项目(2003B30502)
    作者简介:杨芳(1975-),女,南方医科大学在读硕士研究生,主治医师,电话:020-61643360

Cloning, expression, purification and characterization of human endostatin gene

YANG Fang1, HE Yuan-li1, JIANG Xiao-yu2, LIU Yun2, PENG Dong-xian1, ZONG Li-li1   

  1. 1. 南方医科大学珠江医院妇产科, 广东, 广州, 510282;
    2. 中山大学生命科学院医药分子生物学实验室, 广东, 广州, 510275
  • Online:2005-04-20 Published:2005-04-20

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摘要： 目的 获得有生物学活性的人内皮抑素蛋白。方法 用反转录多聚酶链反应(RT-PCR)得到人内皮抑素编码区基因,连接PGEM-T载体,测序确认,定向克隆入pBV220表达载体,转化大肠杆菌DH5α进行表达、纯化及活性测定。结果 经测序分析证实,所获得的552bp基因片段序列属于人内皮抑素基因,构建表达载体在大肠杆菌中表达,经SDS-PAGE分析,出现特异性蛋白质条带,并具有生物学活性。结论 人内皮抑素基因在大肠杆菌中获得非融合表达,表达蛋白能够抑制人脐静脉血管内皮细胞(ECV304)的增殖,为应用内皮抑素进行抗血管生成治疗奠定了基础。

Abstract: Objective To procure biologically active human endostatin. Methods Human endostatin gene was acquired by means of reverse transcriptase (RT)-PCR and cloned into PGEM-T vector with subsequent sequence identification. The gene fragment was inserted into the prokaryotic expression vector pBV220 and transformed into E. coli DH5α strain. Endostatin expression in the E. coli was identified and the inclusion body isolated, purified and its activity analyzed. Results The obtained gene fragment 552 bp in length was identified as the functional section of human endostatin gene by sequence analysis, and SDS-PAGE analysis showed that the expressed product was the target protein with biological activity. Conclusion Human endostatin gene was expressed in E. coli and the protein obtained can inhibit the proliferation of ECV 304 cells.

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