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目的 构建卵巢癌抗独特型单链抗体 6 B11Sc Fv和鼠 GM- CSF融合蛋白 (6 B11m GM) ,以观察其在动物体内诱导的特异性免疫反应 ,为卵巢癌抗独特型疫苗应用于临床提供依据。 方法 用 DNA重组技术 ,将m GM- CSF连于单链抗体 6 B11Sc Fv羧基末端 ,构建重组质粒 p ET30 - 6 B11m GM,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- 1尿素溶解包涵体后直接稀释复性 ,SDS- PAGE分析蛋白纯度 ,EL ISA分析技术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。采用氧化型谷胱甘肽 (GSSG)浓度为 1mm ol.L- 1 ,还原型谷胱甘肽 (GSH)浓度为 5 m mol.L- 1 ,10℃复性 48h,复性率达 36 %。表达的融合蛋白分别能与 COC16 6 - 9单抗和大鼠抗小鼠 GM- CSF单抗特异结合 ,并能刺激 m GM- CSF依赖株 NFS- 6 0细胞增殖。 结论 以包涵体表达的融合蛋白 6 B11m GM保留了两种蛋白的活性 ,为研究融合蛋白在体内的免疫功能提供了基础
OBJECTIVE: To construct ovarian cancer anti-idiotypic scFv 6 B11Sc Fv and mouse GM-CSF fusion protein (6 B11m GM) to observe its specific immune response induced in animals, and to provide an anti-idiotypic vaccine for ovarian cancer for clinical application in accordance with. Methods Recombinant plasmid p ET30 - 6 B11m GM was constructed by ligating m GM - CSF to the carboxyl terminus of single chain antibody B11Sc Fv by DNA recombination. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The inclusion body was obtained by ultrasonication of bacterial cells. The inclusion body was solubilized with 8mol.L-1 urea and then directly diluted and refolded. SDS-PAGE was used to analyze the protein purity. Antibody and cytokine activity of the fusion protein were measured by ELISA assay and cell proliferation assay . Results renatured protein purity of 90% or more. Using glutathione oxidase (GSSG) concentration of 1mmol.L-1, reduced glutathione (GSH) concentration of 5mol mol.L-1, refolding 48h at 10 ℃, the refolding rate was 36% . The expressed fusion protein could specifically bind COC16 6-9 monoclonal antibody and rat anti-mouse GM-CSF monoclonal antibody respectively and stimulate the proliferation of mGM-CSF-dependent NFS-60 cells. Conclusion The fusion protein 6 B11m GM expressed in inclusion bodies retains the activity of the two proteins and provides a basis for studying the immunological function of the fusion protein in vivo