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卵巢癌抗独特型单链抗体6B11ScFv mGM |
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刘蓓 崔恒 冯捷 叶雪 李艺 曹善津 葛华 付天云 姚煜 钱和年
【摘要】 目的 构建卵巢癌抗独特型单链抗体6B11ScFv和鼠GM-CSF融合蛋白(6B11mGM)，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mGM-CSF连于单链抗体6B11ScFv羧基末端，构建重组质粒pET30-6B11mGM，转化大肠杆菌BL21(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，ELISA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1mmol.L-1，还原型谷胱甘肽(GSH)浓度为5mmol.L-1，10℃复性48h，复性率达36%。表达的融合蛋白分别能与COC166-9单抗和大鼠抗小鼠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 以包涵体表达的融合蛋白6B11mGM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。
【关键词】 卵巢癌；重组融合蛋白；抗体；抗独特型；粒细胞-巨噬细胞集落刺激因子；包涵体
【中图分类号】 R392.11 【文献标识码】 A 【文章编号】 0529-1356(2000)03-226
THE CONSTRUCTION， EXPRESSION AND ACTIVITY EXAMINATIONS
OF 6B11ScFv／MURINE GM-CSF FUSION PROTEIN
LIU Bei,CUI Heng, FENG Jie, YE Xue, LI YI, CAO Shan-jin, GE Hua, FU Tian-yun, YAO Yu, QIAN He-nian
(Gynecologic Oncology Center, People s Hospital, Beijing Medical University, Beijing 100044, China)
【Abstract】 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 renat[1] [2] [3] 下一页
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