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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 ovarian cancer anti-idiotype single chain antibody 3D5ScFv and mouse GM-CSF fusion protein (3D5mGM), and to identify its activity. Methods The recombinant plasmid pET30a-3D5mGM was transformed into E.coli BL21 (DE3) and induced by IPTG by DNA recombination technology. The 3D5ScFv gene was fused with murine granulocyte colony-stimulating factor (mGM-CSF) gene and inserted into pET30a , And expressed in the form of inclusion bodies. The inclusion bodies were obtained by ultrasonication of bacterial cells. The inclusion body was dissolved with 8 mol / L urea and then diluted directly. The purity of the protein was analyzed by SDS-PAGE. Antibodies and cells of the fusion protein were measured by ELISA and cell proliferation assay Factor activity. Results renatured protein purity of 90% or more. The expressed fusion protein is capable of binding OC125 mAb and also specifically to rat anti-mouse GM-CSF SF. And can stimulate the proliferation of mGM-CSF-dependent NFS-6 cells. Conclusion The expressed fusion protein 3D5mGM retains the activity of the two proteins and provides a basis for further study on the possibility of the fusion protein in the treatment of ovarian cancer.