中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (40): 7491-7495.doi: 10.3969/j.issn.2095-4344.2012.40.016

• 移植与免疫 transplantation and Immunology • 上一篇    下一篇

小鼠次级淋巴组织趋化因子的原核表达及纯化

张力丹1，罗彦彦1，林 青2，任向荣2，苏绍波2，林有坤1   

  1. 1广西医科大学第一附属医院皮肤性病科，广西壮族自治区南宁市 530021
    2中山大学中山眼科中心，眼科学国家重点实验室，广东省广州市 510060
  • 收稿日期:2012-01-26 修回日期:2012-03-28 出版日期:2012-09-30 发布日期:2012-09-30
  • 通讯作者: 林有坤，主任医师，广西医科大学第一附属医院皮肤性病科，广西壮族自治区南宁市 530021 linyoukun7@ yahoo.com.cn
  • 作者简介:张力丹，女，1987年生，贵州省遵义市人，汉族，2010年广西医科大学毕业，主要从事结缔组织病的研究。 lidan311@ yahoo.com.cn

Prokaryotic expression and purification of mouse secondary lymphoid-tissue chemokine

Zhang Li-dan1, Luo Yan-yan1, Lin Qing2, Ren Xiang-rong2, Su Shao-bo2, Lin You-kun1   

  1. 1Department of Dermatology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
    2State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China
  • Received:2012-01-26 Revised:2012-03-28 Online:2012-09-30 Published:2012-09-30
  • Contact: Lin You-kun, Chief physician, Department of Dermatology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China linyoukun7@yahoo. com.cn
  • About author:Zhang Li-dan, Department of Dermatology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China lidan311@yahoo. com.cn

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摘要：

背景：次级淋巴组织趋化因子(secondary lymphoid-tissue chemokine，SLC/CCL21)是近年来发现的，具有免疫调节作用及抗肿瘤活性。
目的：构建小鼠次级淋巴组织趋化因子原核表达载体，并在大肠杆菌中高效表达重组蛋白。
方法：取C57BL/6小鼠淋巴结细胞，体外用Poly(I:C)刺激后提取RNA并反转录，以此cDNA为模板，通过PCR技术扩增出CCL21成熟蛋白编码序列。克隆入原核表达载体pBEn-SBP-SET1a，构建融合表达载体pBEn-CCL21。将表达载体转化大肠杆菌BL21(DE3),经异丙基-β-D-硫代吡喃半乳糖苷诱导后TRICINE-SDS-PAGE电泳分析和鉴定重组蛋白的表达。CCL21融合蛋白经链霉亲和柱层析纯化。
结果与结论：实验成功构建了CCL21基因的原核表达载体pBEn-CCL21，并获得相应的融合蛋白。结果显示CCL21可在大肠杆菌中高效表达，经链霉亲和柱层析纯化即可获得CCL21重组目的蛋白。

关键词: 原核表达, CCL21, 蛋白纯化, 大肠杆菌, 小鼠

Abstract:

BACKGROUND: Secondary lymphoid-tissue chemokin (CCL21) is a recently discovered chemotactic factor with a broad range of immunoregulatory activities and anti-tumor effects.
OBJECTIVE: To establish the prokaryotic expression vector of mouse CCL21, and highly express the recombinant protein in Escherichia coli.
METHODS: Lymph node cells in C57BL/6 mice were collected and subsequently stimulated using Poly(I:C) in vitro, then its extracted RNA was reverse-transcripted. This cDNA was used as template in polymerase chain reaction amplification to collect CCL21 coding sequence. The gene was cloned into the prokaryotic expression vector pBEn-CCL21 and was expressed in Escherichia coli BL21 on the induction of isopropy-β-D-thiogalactoside. Then the recombinant protein expression were analyzed by TRICINE-SDS-PAGE electrophoresis and purified by streptevidin affinity chromatography.
RESULTS AND CONCLUSION: The prokaryotic expression vector pBEn-CCL21 of mouse CCL21 gene was successfully constructed, and the fusion protein was obtained. The experiment proves that CCL21 can be highly expressed in Escherichia coli BL21, and the CCL21 target protein can be obtained by streptevidin affinity chromatography.

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