卵巢癌抗独特型单链抗体3D5ScFv/鼠GM-CSF融合蛋白(3D5mGM)的构建、表达

【摘要】目的构建卵巢癌抗独特型单链抗体3D5ScFv和鼠GM—CSF融合蛋白(3D5mGM)，并对其活性进行鉴定。方法用DNA重组技术，将3D5ScFv基因与鼠粒细胞集落刺激因子(mGM—CSF)基因融合，插入到pET30a( )中，构建重组质粒pET30a一3D5mGM，转化大肠杆菌B121(DE3)，IPrG诱导，以包涵体形式获高效表达，超声破碎细菌细胞获得包涵体，用8mol／L尿素溶解包涵体后直接稀释复性，SDS-PAGE分析蛋白纯度，ELISA和细胞增殖实验测定融合蛋白的抗体和细胞因子活性。结果复性蛋白纯度达90％以上。表达的融合蛋白能够与OC125单抗结合，也能与大鼠抗小鼠GM-CSF单抗特异结合。并能刺激mGM-CSF依赖株NFS-60细胞增殖。结论表达的融合蛋白3D5mGM保留了两种蛋白的活性，为进一步研究该融合蛋白治疗卵巢癌的可能性提供了基础。

Construction and expression of the fusion protein 3D5ScFV/murine GM-CSF

Objective To construct a novel plasmid expressing the fusion protein (3D5mGM ) of anti-idiotypic single-chain antibody 3D5ScFv and murine GM-CSF,and to observe its expression and specific immunoreactions. Methods Using DNA recombination techniques,3D5ScFv gene was fused with murine GM-CSF cDNA,then cloned into the expression vector pET30a( ).The recombinant plasmid pET30a-3D5mGM was used to transform E.coli BL21(DE3).High expression of 3D5mGM was induced by IPTG in the form of inclusion bodies.Inclusion bodies were obtained by sonication and dissolved with 8 mol/L carbamide,renatured by direct dilution.The purity was determined by SDS-PAGE,and activities of antibodies of the fusion protein and cell factors was evaluated by ELISA and cell proliferation assay. Results The purity of renatured 3D5mGM fusion protein is over 90%.Expressed fusion protein specifically interacted with the monoclonal antibody OC-125 and the rat-anti-mouse GM-CSF monoclonal antibody,and stimulated the proliferation of murine GM-CSF-dependent cell line NFS-60. Conclusions Expressed fusion protein 3D5mGM maintains activities of the two proteins,and provides a basis for studying immunologic functions of fusion proteins in the treatment of ovarian carcinoma.