Expression And Purification Of VMIP-Ⅱ N-Terminal GST Fusion Protein And Identification Of Its Bioactivity

Objective:To express and purify viral macrophage inflammatory protein-Ⅱ (vMIP-Ⅱ)N-terminal GST fusion protein, and analyze its bioactivity of inhibiting theproliferation, colony formation, adhesion and chemotaxis in breast cancer cell strainSK-BR-3by blocking CXC chemokine receptor-4(CXCR4) in vitro. Methods:According to the amino acid sequence and the nucleotide sequence of vMIP-Ⅱ(GenBank: AAB62642; GenBank： U93872.2:21495..21779), We determined thenucleotide sequence encoding the vMIP-Ⅱ N terminal21amino acid peptide as thetarget gene.Two oligonucleotide chains designed were synthesized by Sangon Biotech(Shanghai) Co., Ltd. Using Polymerase Chain Reaction instrument, two syntheticoligonucleotides were transformed into double-stranded DNA after annealing reaction.The dsDNA, the target gene was inserted into the high efficiency prokaryotic expressionvector pGEX-KG with T4DNA ligase to generate recombinant plasmidpGEX-KG-NT21MP. The sequence of recombinant plasmid pGEX-KG-NT21MP wasverified by DNA sequencing, and then transformed it into E.coli BL21(DE3).Use IPTGto induce expression the recombinant GST fusion protein GST-NT21MP, which waspurified by affinity chromatography, ultrafiltration and fast protein liquidchromatography. Through Western Blot we identified the recombinant fusion proteinGST-NT21MP. We detected its blocking effect on the proliferation of SK-BR-3in thedifferent concentrations through the method of MTT assay, the clone formation ratewas assessed by agar clone assay. We also use adhesion and tumor cell chemotaxisassays to evaluate its effect on the SK-BR-3in different steps of the metastasis. Results:The sequence of recombinant plasmid pGEX-KG-NT21MP was successfullyconstructed which was identified by DNA sequencing. The best conditions of inducingexpression of GST-NT21MP is that the concentration of IPTG was0.8mmol/L,37℃induced5hours. GST-NT21MP was successfully induced by IPTG and its form ofexpression was mainly soluble in E.coli. High purity GST-NT21MP was obtained bypurification(>99%). GST-NT21MP is able to suppress the proliferation,colonyformation, adhesion and chemotaxis in SK-BR-3cell lines in dependence on the concentrations in vitro. Conclusion: The recombinant plasmid pGEX-KG-NT21MPwas successfully constructed by using molecular biological techniques. The vMIP-ⅡNterminal GST fusion protein GST-NT1MP was successfully induced in E.coli. Highpurity GST-NT21MP was obtained by purification. The results indicate that theproliferation， colony formation， adhesion and chemotaxis inducing by stromalcell-derived factor-1α in breast cancer cell line SK-BR-3were inhibited byGST-NT21MP, and it may serve as a potential target drug in the treatment of breastcancer metastasis.