人酸性成纤维细胞生长因子在大肠杆菌的高效表达

doi:国家973重大基础科研基金资助课题

吉林大学学报(医学版) ›› 2004, Vol. 30 ›› Issue (1): 1-5.doi: 国家973重大基础科研基金资助课题

• 基础研究 • 下一篇

人酸性成纤维细胞生长因子在大肠杆菌的高效表达

刘青¹，万敏²，卫红飞²，吴秀丽²，张培因²，王燕媚²，杨翰仪³，王丽颖^2*

1. 吉林大学第一医院内分泌科，吉林长春 130021；2. 吉林大学基础医学院分子生物学教研室，吉林长春 130021；3. 吉林大学基础医学院生物化学教研室，吉林长春 130021

收稿日期:2003-09-04 修回日期:1900-01-01 出版日期:2004-01-28 发布日期:2004-01-28
通讯作者: 王丽颖

Overexpression of human acidic fibroblast growthfactor in Escherichia coli

LIU Qing¹, WAN Min², WEI Hong-fei², WU Xiu-li², ZHANG Pei-yin²,WANG Yan-mei², YANG Han-yi³, WANG Li-ying^2*

1. Department of Endocrinology, First Hospital, Jilin University, Changchun 130021, China;2. Department of Molecular Biology, School of Basic Medical Sciences, Jilin University,Changchun 130021,China;3. Department of Biochemistry, School of Basic Medical Sciences, Jilin University, Changchun 130021,China

Received:2003-09-04 Revised:1900-01-01 Online:2004-01-28 Published:2004-01-28
Contact: WANG Li-ying

摘要/Abstract

摘要： 目的：探讨人酸性成纤维细胞生长因子（acidic fibroblast growth factor, aFGF）在大肠杆菌中高效表达的方法及重组人aFGF的生物学活性。方法：①通过PCR法扩增人aFGF引入点突变，对其密码子进行改造；②构建aFGF-pET-28a重组质粒并在大肠杆菌BL21(DE3)中表达；③重组蛋白的纯化及生物学活性研究。结果：通过PCR扩增，将设计的核酸水平突变引入到aFGF DNA中，使其自起始密码ATG后的前50个碱基均为原核细胞偏爱密码子、并保持原氨基酸序列不变，成功地实现了重组人aFGF在大肠杆菌中的高效表达，并通过降低培养温度使大肠杆菌中可溶性目的蛋白的含量大幅增加，原核表达的aFGF亦具有天然生物学活性。结论：通过对aFGF DNA碱基进行改造，可大幅度提高aFGF在大肠杆菌中的表达量，且目的蛋白具有良好的生物学活性。

关键词: 成纤维细胞生长因子1/分析,大肠杆菌,重组, 遗传,聚合酶链反应

Abstract: Objective To overexpress human acidic fibroblast growth factor (aFGF) in Escherichia coli (E.coli) and study the biological activities of recombinant aFGF. Methods ①The codon of aFGF was altered by amplifying aFGF DNA and introducing point mutation; ②Human aFGF was recombined with pET-28a and expressed in E.coli; ③Recombinant protein was purified and the biological activities were studied. Results The projected point mutation was introduced into aFGF DNA fragment and the first 50 bp after ATG start codon of aFGF were altered to the partiality codon of E.coli by PCR, and the original sequence of amino acid was kept. So the overexpression of recombinant aFGF in E.coli was successfully accomplished. The expression volume of soluble aFGF in E.coli was greatly increased by lowering culture temperature. Recombinant aFGF protein had biological activities of nature aFGF. Conclusion By altering the bases of aFGF DNA, the expression volume of aFGF in E.coli is increased greatly, and the recombinant protein has nature biological activities.

Key words: fibroblast growth factor 1/analysis, Escherichia coli, recombination,genetic, polymerase chain reaction

中图分类号:

Q784

刘青, 万敏, 卫红飞, 吴秀丽, 张培因, 王燕媚, 杨翰仪, 王丽颖. 人酸性成纤维细胞生长因子在大肠杆菌的高效表达[J]. 吉林大学学报(医学版), 2004, 30(1): 1-5.

LIU Qing, WAN Min, WEI Hong-fei, WU Xiu-li, ZHANG Pei-yin, WANG Yan-mei, YANG Han-yi, WANG Li-ying. Overexpression of human acidic fibroblast growthfactor in Escherichia coli[J]. Journal of Jilin University(Medicine Edition), 2004, 30(1): 1-5.

人酸性成纤维细胞生长因子在大肠杆菌的高效表达

Overexpression of human acidic fibroblast growthfactor in Escherichia coli

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