重组截短型人角质细胞生长因子-1的表达及纯化

J4 ›› 2010, Vol. 36 ›› Issue (4): 629-633.

重组截短型人角质细胞生长因子-1的表达及纯化

邓林^1,2,刘孝菊^1,3,4,龚守良³,王会岩^1,2,田海山¹,王晓杰¹,马吉胜^1,2,李校堃^1,3,4

1.吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春 |130118;2.吉林农业大学生命科学学院,吉林长春 130118；3.吉林大学公共卫生学院卫生部放射生物学重点实验室|吉林长春 130021;4.温州医学院药学院生物技术制药工程重点实验室,浙江温州 |325035

收稿日期:2009-12-30 出版日期:2010-07-28 发布日期:2010-07-28
通讯作者: 李校堃（Tel:0431-84533348，E-mail: xiaokunli@163.com） E-mail:xiaokunli@163.com
作者简介:邓林（1983－）|男|吉林省公主岭市人|农学硕士|主要从事生物反应器与基因工程药物的研究。
基金资助:
1.吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春 130118;2.吉林农业大学生命科学学院,吉林长春 130118；3.吉林大学公共卫生学院卫生部放射生物学重点实验室，吉林长春 130021;4.温州医学院药学院生物技术制药工程重点实验室,浙江温州 325035

Expression and purification of recombinant truncated human keratinocyte growth factor-1

DENG Lin^1,2, LIU Xiao-Ju ^1,3,4, GONG Shou-Liang³, WANG Hui-Yan^1,2, TIAN Hai-Shan¹, WANG Xiao-Jie¹, MA Ji-Sheng^1,2, LI Xao-Kun^1,3,4

1. Engineering Research Center of Bioreactor and Pharmaceutical Development, |Ministry of Education,Jilin |Agricultural University, |Changchun 130118,China;2.School of Life Science, Jilin Agricultural University,Changchun 130118,China;3. Key Laboratory of Radiobiology,Ministry of Health,School of Public Health,Jilin University,Changchun 130021,China;4. Key Laboratory of Biotechnology Pharmaceutical Engineering,School of Pharmacy,Wenzhou Medical College, Wenzhou |325035,China

Received:2009-12-30 Online:2010-07-28 Published:2010-07-28
Supported by:
1. Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education,Jilin Agricultural University, Changchun 130118,China;2.School of Life Science, Jilin Agricultural University,Changchun 130118,China;3. Key Laboratory of Radiobiology,Ministry of Health,School of Public Health,Jilin University,Changchun 130021,China;4. Key Laboratory of Biotechnology Pharmaceutical Engineering,School of Pharmacy,Wenzhou Medical College, Wenzhou 325035,China

摘要/Abstract

摘要：

目的：构建高效表达N端缺失的23个氨基酸重组人角质细胞生长因子1( rhKGF1_dest23)的基因工程菌，为治疗放化疗后口腔黏膜炎的新药开发提供实验数据。方法：利用PCR方法分别以pET3c-hKGF1及sumo-EGF为模板合成N端缺失的23个氨基酸rhKGF1_dest23及sumo基因片段，构建4种原核表达载体pET22b-rhKGF1_dest23﹑pET22b-sumo-rhKGF1_dest23﹑pET3c-rhKGF1_dest23和pET3c-sumo-rhKGF1_dest23，分别转化原核表达宿主菌Rosetta(DE3) plysS、BL21(DE3) 、BL21(DE3)Star plysS、origima(DE3) 和BL21AI，筛选rhKGF1_dest23蛋白表达的最佳质粒与宿主菌组合。CM离子交换和肝素亲和层析法纯化rhKGF1_dest23蛋白，Western blotting法鉴定rhKGF1_dest23蛋白。结果： pET22b-rhKGF1_dest23质粒与BL21AI 宿主菌为最佳组合表达，经IPTG与阿拉伯糖诱导， SDS-PAGE电泳后表明目的蛋白大部分以可溶性形式存在，约占菌体总蛋白的10％，经CM和肝素两步纯化后rhKGF1_dest23蛋白纯度为95%以上，Western blotting法鉴定为rhKGF1_dest23蛋白。结论：成功构建表达人KGF1_dest23重组蛋白的基因工程菌，经IPTG与阿拉伯糖诱导，CM弱阳离子与肝素亲合层析后，得到了纯化的rhKGF1_dest23蛋白。

关键词: 高效表达;重组截短人角质细胞生长因子-1;纯化

Abstract:

Objective To construct the genetic engineering bacteria highly expressing 23 amino acids human keratinocyte growth factor -1 (rhKGF1_dest23) missing N terminal,and provide experimental data for development of new drug for treatment of oral mucositis after radiotherapy and chemotherapy.Methods PCR was used to synthese 23 amino acids rhKGF1_dest23 missing N terminal and sumo gene fragments,and construct four kinds of recombinant prokaryotic expression vectors: pET22b-rhKGF1_dest23,pET22b-sumo-rhKGF1_dest23,pET3c-rhKGF1_dest23 and pET3c-sumo-rhKGF1_dest23,then they were transformed into prokaryotic expression host bacteria: Rosetta(DE3) plysS,BL21(DE3),BL21(DE3)Star plysS,origima(DE3) and BL21AI,the best expression combination of plasmid and host strain of rhKGF1_dest23 protein was screened and purified by CM ion-exchange and heparin affinity chromatography and identified with Western blotting.Results pET22b-rhKGF1_dest23 plasmid and the BL21AI host bacteria was the best combination of expression,after induced by IPTG and arabinose,the majority of recombinant protein was expressed in soluble form,accounting for about 12% of the total bacterial proteins.Its purity reached to more than 95% of the protein after two steps chromatography,then conformed with Western blotting.Conclusion Human genetic engineering bacteria of KGF1_dest23 is successfully constructed and induced by IPTG and arabinose,then after CM weak cation exchange and heparin affinity chromatography,the purified rhKGF1_dest23 protein is obtained

Key words: high expression;recombinant truncated human keratinocyte growth factor -1;purification

中图分类号:

邓林, 刘孝菊, 龚守良, 王会岩, 田海山, 王晓杰, 马吉胜, 李校堃. 重组截短型人角质细胞生长因子-1的表达及纯化[J]. J4, 2010, 36(4): 629-633.

DENG Lin, LIU Xiao-Ju, GONG Shou-Liang, WANG Hui-Yan, TIAN Hai-Shan, WANG Xiao-Jie, MA Ji-Sheng, LI Xao-Kun. Expression and purification of recombinant truncated human keratinocyte growth factor-1[J]. J4, 2010, 36(4): 629-633.

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