vMIP-ⅡN端重组肽的表达纯化及活性鉴定

doi:Q816

中国生物工程杂志

2010, Vol. 30

Issue (09): 80-86 DOI: Q816

研究简报

vMIP-ⅡN端重组肽的表达纯化及活性鉴定

杨清玲¹,杨志峰²,高艳军²,陈昌杰^1**

1. 蚌埠医学院生化与分子生物学教研室蚌埠 233030
2.蚌埠医学院临床检验诊断学实验中心蚌埠 233030

Expression and Purification of vMIP-Ⅱ N-Terminal GST Fusion Protein and Identification of Its Bioactivity

YANG Qing-ling¹,YANG Zhi-feng²,GAO Yan-jun²,CHEN Chang-jie¹

1.Department of Biochemistry & Molecular Biology,Beng Bu Medical College,Bengbu 233030,China
2.Clinical Testing and Diagnose Experimental Center of Bangbu Medical College, Bengbu 233000,China

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摘要：

病毒巨噬细胞炎症蛋白-Ⅱ (viral macrophage inflammatory protein-Ⅱ,vMIP-Ⅱ)由卡波西肉瘤疱疹病毒编码，前期研究证明了vMIP-II N端21肽(NT21MP)选择性的阻断趋化因子CXCR4，从而抑制乳腺癌细胞趋化的活性。通过化学合成法获得编码vMIP-ⅡN末端的基因序列，与pGEX-KG（克隆位点的N端有谷胱甘肽转移酶GST标签序列）连接构建原核表达载体，重组质粒在大肠杆菌E.coli BL21(DE3)中获得表达，免疫印迹显示重组蛋白GST-NT21MP主要在细菌裂解液上清中表达，可溶性部分经亲和层析、超滤、快速蛋白液相色谱（fast protein liquid chromatography,FPLC）纯化获得高纯度的GST-NT21MP蛋白。利用Transwell趋化试验测定GST-NT21MP的活性。结果显示，重组蛋白GST-NT21MP能够抑制乳腺癌细胞SK-BR-3的趋化活性，可作为治疗乳腺癌转移的潜在性靶向药物。

关键词： vMIP-Ⅱ; CXCR4; SDF-1; 纯化

Abstract:

The viral macrophage inflammatory protein-II (vMIP-II) is encoded by Kaposi's sarcoma-associated herpesvirus and the residues 1~21 of the N-terminal of vMIPII(NT21MP) was shown to strongly and selectively bind CXCR4 to inhibit the chemotaxis of SK-BR-3 cells.The N-terminal of vMIP-ⅡcDNA was derived by chemical sythesis method. DNA fragment encoding the NT21MP peptide was inserted into pGEX-KG vector and the recombinant plasmid was expressed in E.coli BL21(DE3). NT21MP peptide, which was recombinated with a glutathione S-transferase (GST) at the N-terminal, was expressed in E.coli cells.SDS-PAGE and Western Blot analysis demonstrated that the recombinant protein existed mainly in the supernatant of E.coli lysates. The supernatant was further purified by affinity chromatography, ultrafiltration and fast protein liquid chromatography. The bioactivity of GSTNT21MP was determined by Transwell chemotaxis on CXCR4-expressing breast cancer cell line SK-BR-3 in vitro. The results indicate that The chemotaxis of SK-BR-3 cells toward stromal cell-derived factor-1(SDF-1) was reduced by GST-NT21MP and may serve as a potential target drug for the treatment of breast cancer metastasis.

Key words: vMIP-Ⅱ CXCR4 SDF-1Purification

收稿日期: 2010-05-31 出版日期: 2010-08-25

基金资助:

安徽省教育厅自然科学研究重点项目（KJ2010A240）资助项目

通讯作者: 陈昌杰 E-mail: bbmcccj@sohu.com

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引用本文:

杨清玲杨志峰高艳军陈昌杰. vMIP-ⅡN端重组肽的表达纯化及活性鉴定[J]. 中国生物工程杂志, 2010, 30(09): 80-86.

YANG Qing-Ling, YANG Zhi-Feng, GAO Yan-Jun, CHEN Chang-Jie. Expression and Purification of vMIP-Ⅱ N-Terminal GST Fusion Protein and Identification of Its Bioactivity. China Biotechnology, 2010, 30(09): 80-86.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q816 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I09/80

［1］ Arvanitakis L, GerasRaaka E, Varma A, et al. Human herpesvirus KSHV encodes a constitutively active Gproteincoupled receptor linked to cell proliferation. Nature, 1997,385(6614): 347350.
［2］叶石敦,莫雪梅,张光,等.vMIPII各受体结合活性位点分析.中国现代医学杂志,2006,16(23):35463552. Ye S D, Mo X M, Zhang G, et al. China Journal of Modern Medicine, 2006,16(23):35463552.
［3］ Julius J, Linda B, Ken B. Chemokines and their receptors as therapeutic targets:the role of the SDF1/CXCR4 Axis. Current Pharmaceutical Design, 2004,10(11): 12451259.
［4］ Magda K, Kacper J, Ryan R, et al. CXCR4SDF1 signalling, locomotion, chemotaxis and adhesion. J Mol Histol, 2004,35(3):233245.
［5］杨清玲,李成华,丁勇兴,等.CXCR4 抑制性多肽对乳腺癌细胞株转移作用的研究.癌变.畸变.突变,2008，20(2):8992. Yang Q L, Li C H, Ding Y X, et al. Carcinogenesis, Teratogenesis & Mutagenesis, 2008,20(2):8992.
［6］陈昌杰, 章菊, 杨清玲,等. CXCR4的抑制性多肽对乳腺癌细胞CXCR4和HER2表达及HERCEPTIN药物敏感性的影响. 中国组织化学与细胞化学杂志, 2008, 17(5):442448. Chen C J, Zhang J, Yang Q L, et al. Journal of Histochemistry and Cytochemistry, 2008,17(5):442448.
［7］ Yang Q L, Ding Y X, Chen C G,et al. Suppression of murine breast cancer metastasis by selective inhibition of CXCR4 by synthetic polypeptide derived from viral macrophage inflammatory protein II. Chinese Science Bulletin, 2010, 55(20):21002107.
［8］ Gupta S K, Pillarisetti K, Thomas R A, et al. Pharmacological evidence for complex and multiple site interaction of CXCR4 with SDF1α: implications for development of selective CXCR4 antagonists.Immunol Lett, 2001, 78(1):2934.
［9］ Liang Z, Brooks J, Willard M, et al.CXCR4/CXCL12 axis promotes VEGFmediated tumor angiogenesis through Akt signaling pathway. Biochem Biophys Res Commun,2007 ,359(3):716722.
［10］ Zhou N M, Luo Z W, Luo J S, et al. Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV1 coreceptor by mutagenesis and molecular modeling studies. J Biol Chem , 2001, 276(46): 4282642833.
［11］ Huang X Q, Shen J H, Cui M, et al. Molecular dynamics simulations on SDF1a:binding with CXCR4 receptor. Biophys J, 2003, 84(1):171184.
［12］杨清玲, 丁勇兴. vMIP对细胞膜上趋化因子受体亲和力的结合特性研究. 上海交通大学学报（医学版）,2006;26（4）:389392. Yang Q L, Ding Y X. Journal of Shanghai Jiaotong University (Medical Science),2006,26(4):389392.
［13］ Cai S H,Tan Y,Ren X D, et al. Loss of Cterminal αhelix decreased SDF1αmediated signaling and chemotaxis without influencing CXCR4 internalization. Acta Phamacol Sin, 2004; 25(2):152160.

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