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- 3D5mGM ,转化大肠杆菌BL2 1(DE3) ,IPTG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OC12 5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 - 6 0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。
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.
出处
《中国妇产科临床杂志》
2004年第5期366-369,394,共5页
Chinese Journal of Clinical Obstetrics and Gynecology
