人类新细胞因子CKLFSF2羧基端蛋白在大肠杆菌中的表达纯化、抗体制备与鉴定

Expression, Purification and Identification of A Novel Human Cytokine C-terminal 51 Amino Protein of CKLFSF2 in E. coli

哺乳动物细胞表达的人类新细胞因子——趋化素样因子超家族成员-2(CKLFSF2)存在分泌形式,位于CKLFSF2分子的羧基端,具有细胞趋化作用.为进一步研究CKLFSF2羧基端蛋白的结构和生物学功能及抗体制备,构建了GST-CKLFSF2C51原核表达质粒,经原核表达、亲和层析、凝胶过滤,获得GST-CKLFSF2C51融合蛋白和CKLFSF2羧基端蛋白(CKLFSF2C51),纯度可达到95%以上.GST-CKLFSF2C51融合蛋白用于制备多克隆抗体,ELISA方法检测抗体效价阳性,蛋白质印迹检测CKLFSF2哺乳动物细胞超表达细胞裂解液,获得特异性条带与预期大小一致.CKLFSF2C51经N端测序,质谱鉴定与预期结果一致,该蛋白质具有对PC-3细胞趋化的活性,并且该活性可被制备的多克隆抗体中和.上述结果表明,原核CKLFSF2羧基端蛋白具有与CKLFSF2真核表达蛋白类似的细胞趋化活性,原核CKLFSF2羧基端蛋白制备的多克隆抗体可用于免疫组织化学、蛋白质印迹检测,并能中和CKLFSF2蛋白的趋化活性作用.

Overexpressed CKLFSF2 （chemokine-like super family member 2） can be secreted into the supernatant of cultured cells, which exhibits the effects on cell-chemotaxis. To study the structural and functional characteristics of CKLFSF2 and make the polycolonal antibody, the recombinant plasmid pGEX-4T-3-CKLFSF C51 was constructed, following the expression in E. coli with high efficiency, purification using Glutathione Sepharose 4B and Sephacyl S-200, the recombinant CKLFSF2 C51 protein （typically 〉95% pure） was obtained. The N-terminal animo acid sequencing of CKLFSF2 C51 was performed and relative molecular mass is 5871.48 measured by ASIMA-CFRTM plus MALDI-TOF. ELISA assay detected the reactivity of the polycolonal antibody and Western blot show that CKLFSF2 was overexpressed in mammalian cells and the specific band was corresponding to the expectation. The recombinant CKLFSF2 C51 protein exhibits significantly chemotactic effect in PC-3 cells, which can be neutralised by anti-CKLFSF2 C51 polyantibody. The anti-CKLFSF2 C51 antibody can also be used for immunohistochemistry. Taken together, the recombinant CKLFSF2 C51 from E.coli has similar bioactivity as that from eukaryocytic expression system. The polycolonal antibodies of recombinant CKLFSF2 C51 can be used in immunohistochemistry, Western blot and to neutralize the chemotactic effect.