Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (40): 7491-7495.doi: 10.3969/j.issn.2095-4344.2012.40.016

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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

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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