vMIP-ⅡN端重组肽的表达纯化及活性鉴定

Expression and Purification of vMIP-Ⅱ N-terminal GST Fusion Protein and Identification of its Bioactivity

病毒巨噬细胞炎症蛋白-Ⅱ(viralmacrophageinflammatoryprotein-Ⅱ,vMIP-Ⅱ)由卡波西肉瘤疱疹病毒编码,前期研究证明了vMIP-IIN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.coliBL21(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(fastproteinliquidchromatography,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。

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.