重组鼠源性肠激酶轻链在大肠杆菌中的表达

doi:国家自然科学基金资助课题（30070705）

重组鼠源性肠激酶轻链在大肠杆菌中的表达

孙琳¹，吴秀丽¹，王燕媚¹，于永利²，王丽颖^1*

1. 吉林大学基础医学院分子生物学教研室，吉林长春130021；2. 吉林大学基础医学院免疫学教研室，吉林长春130021

收稿日期:2005-10-25 修回日期:1900-01-01 出版日期:2006-05-28 发布日期:2006-05-28
通讯作者: 王丽颖

Expression of recombinant mouse enterokinase light chain in E.coli

SUN Lin¹, WU Xiu-li¹, WANG Yan-mei¹, YU Yong-li², WANG Li-ying^1*

1.　Department of Molecular Biology, School of Basic Medical Sciences, Jilin University, Changchun 130021，China；2. Department of Immunology, School of Basic Medical Sciences, Jilin University, Changchun 130021，China

Received:2005-10-25 Revised:1900-01-01 Online:2006-05-28 Published:2006-05-28
Contact: WANG Li-ying

摘要/Abstract

摘要： 目的：制备能在原核表达系统中表达并特异性识别(Asp)₄-Lys序列的鼠源性肠激酶，为推广应用重组肠激酶提供技术平台。方法：采用RT-PCR从C57BL/6J小鼠的十二指肠肠系膜黏膜组织中钓取肠激酶轻链的cDNA，将其克隆入pET32a原核表达载体中，并在大肠杆菌BL21(DE3)中进行表达，然后以镍亲和层析法对表达产物进行纯化。结果：所钓取的肠激酶轻链编码序列与GenBank中的序列一致，比较发现其编码的氨基酸序列在小鼠与人、牛之间的同源性大于75%。利用pET32a/BL21(DE3)表达系统成功地在大肠杆菌中表达了重组鼠源性肠激酶轻链，表达量约占大肠杆菌BL21(DE3)总蛋白的30%，但多以包涵体的形式存在。经镍亲和层析法纯化的重组肠激酶多以聚体形式存在。结论：鼠源性肠激酶轻链在原核细胞中多以包涵体形式表达，天然构象重组肠激酶的获得还需对表达系统进行优化。

关键词: 镍亲和层析, 包涵体

Abstract: Objective To establish a prokaryotic expression system for expressing mouse enterokinase which could recognize and cut the sequence of (Asp)₄-Lys in order to provide a technique platform for application of recombinant enterokinase. Methods The cDNA coding sequencing of enterokinase light chain was amplified by RT-PCR from mouse dodecadactylon mucosa and cloned into pET32a expression plasmid. The recombinant enterokinase light chain was expressed in BL21(DE3) and purified with Ni-affinity chromatography.Results The cDNA sequence amplified was identical with that in GenBank and possessed more than 75% homologous in amino acid sequence with that from human and cow. The recombinant mouse enterokinase light chain could be well expressed in Ecoli, but most of them were inclusion. Almost all recombinant enterokina se proteins were polymers after purification by Ni-affinity chromatography. Conclusion Recombinant mouse enterokinase can be expressed in Ecoli in inclusion manner so that the prokaryotic expression system for recombinant enterokinase with native conformation needs to be optimized.

Key words: Ni-affinity chromatograp hy, inclusion

中图分类号:

孙琳，吴秀丽，王燕媚，于永利，王丽颖. 重组鼠源性肠激酶轻链在大肠杆菌中的表达[J]. J4, 2006, 32(3): 360-363.

SUN Lin, WU Xiu-li, WANG Yan-mei, YU Yong-li, WANG Li-ying. Expression of recombinant mouse enterokinase light chain in E.coli[J]. J4, 2006, 32(3): 360-363.

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