生物学杂志

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重组人KGF-1蛋白的表达、纯化及活性研究

  

  1. 1、安徽大学生命科学学院,合肥230039;2.安徽安科生物股份有限公司,合肥 230088
  • 出版日期:2016-02-18 发布日期:2016-02-18
  • 通讯作者: 宋礼华,博士生导师,基因工程药物研究方向,E-mail:Songlh@ankebio.com
  • 作者简介:周昭,硕士研究生,主要研究方向:生物化学与分子生物学,E-mail:truefire@sina.com;
  • 基金资助:
     
    安科生物股份有限公司自拟

Expression、purification and bioactivity of recombinant human keratinocyte growth factor-1

  1.  
    1.School of Life Science,Anhui University,230039 Hefei;

    2.Anhui Anke Biotechnology (Group) Co.Ltd,230088 Hefei,China

  • Online:2016-02-18 Published:2016-02-18

摘要:  

目的:在大肠杆菌中表达可溶性的重组人rhKGF-1蛋白;研究其生物学活性,为rhKGF-1蛋白进一步制药提供基础。方法:合成ΔN23 rhKGF-1(N端缺失23个氨基酸残基的rhKGF-1形式)的基因序列,应用重叠PCR技术,将其与poly His-sumo标签基因序列拼接。构建重组表达质粒pET30a-6His-sumo-rhKGF1,转入E.coli BL21(DE3)中,表达融合蛋白。融合蛋白经Ni-NTA纯化后,切除SUMO标签,再经Ni-NTA纯化获得ΔN23 rhKGF-1蛋白。CCK-8法测定其对人永生化表皮细胞(HaCat细胞)的促增殖作用。结果:成功构建pET30a-6His-sumo-rhKGF1表达质粒,测序结果与预期一致。实现了融合蛋白在E.coli BL21(DE3)中的可溶性表达,经纯化、酶切获得了ΔN23 rhKGF-1蛋白。制备的ΔN23 rhKGF-1蛋白具有促进HaCat细胞增殖的作用,EC50=6.11ng/ml。

关键词: 重组人角质细胞生长因子-1, SUMO蛋白, 原核表达

Abstract:  

Objectives:To express soluble recombinant human keratinocyte growth factor-1(rhKGF-1) in Escherichia coli and determine the bioactivity of rhKGF-1 to provide the basis for developing new drugs containing rhKGF-1. Methods:The sequence encoding ΔN23 rhKGF-1(the form of KGF-1 missing 23amino acids in N terminal) was synthesized and fused with SUMO through overlap PCR to obtain 6His-sumo-rhKGF-1 fusion gene,which was the subcloned into expression plasmid pET30a.The recombinant plasmid pET30a-6His-sumo-rhKGF-1 was then transfected to E.coli BL21(DE3) for expressing the fusion protein. Firstly ,the fusion protein was purified by Ni-NTA.Then,it was digested by Sumo Protease,and purified by Ni-NTA again to obtain purified ΔN23 rhKGF1. The ΔN23 rhKGF1 was used to promote proliferation of HaCat cells,then assay its bioactivity by CCK-8 methods.Results:Recombiant expression plasmid pET30a-6His-sumo-rhKGF1 was constructed correctly,the result of sequencing was the same as the expected.The fusion protein was expressed in E.coli BL21(DE3) in soluble form.After purification and SUMO Protease digestion,purified ΔN23 rhKGF-1 had been obtained.It could promote HaCat cells proliferation,the value of EC50 was 6.11ng/mL.

Key words: recombinant human keratinocyte growth factor-1, SUMO protein, prokaryotic expression