摘要
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 。
Objective To construct expression vector which expresses fusion protein of antiidiotypic single chain antibody 6B11ScFv and murine GM CSF(6B11mGM) for observing its possible immune reactions in vivo. Methods Using DNA recombinant techniques, the murine GM CSF cDNA gene was recombined to 6B11ScFv and they were cloned into expression vector pET 30a(+) to produce insoluble protein. Inclusion bodies collected after the breakage of bacteria through sonication were subjected to repeatedly washing. Inclusion bodies solubilized in the resence of 8 mol·L -1 urea were diluted with renaturation solutions so that folding process could be initiated. The purity was examined by SDS PAGE, ELISA and cell proliferation assay were used to determine the activities of fusion protein. Results 6B11mGM fusion proteins were obtained with a purity of over 90%. The optimum conditions were 1mmol·L -1 and 5 mmol·L -1 for the concentrations of GSSG and GSH, respectively, with 48 hours at 10℃ as renatured time. Fusion protein could specifically react with the primary anti ovarian carcinoma monoclonal antibody(COC166 9) and rat anti mouse GM CSF monoclonal antibody, respectively, and fusion proteins could also stimulate murine GM CSF dependent cell line NFS 60 cells to proliferate. Conclusion Fusion proteins 6B11mGM expressed as inclusion bodies can keep the activity of both the proteins and provid foundation to research the immunoreactions in vivo.
出处
《解剖学报》
CSCD
北大核心
2000年第3期226-230,共5页
Acta Anatomica Sinica
基金
教育部优秀年轻教师基金
关键词
卵巢癌
重组融合蛋白
抗独特型单链抗体
Ovarian carcinoma
Recombinant fusion proteins
Antibodies
Anti idiotypic
Granulocyte macrophage colony stimulating factor
Inclusion bodies
