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| 抗鼠CTLA-4单链抗体的构建与表达及其免疫学活性的鉴定 | |
| 引用本文： | 彭国平,周承,吴炜,孙雯,田锋,贺纪凯,陈智. 抗鼠CTLA-4单链抗体的构建与表达及其免疫学活性的鉴定[J]. 华中科技大学学报(医学版), 2008, 37(3). DOI: 10.3870/j.issn.1672-0741.2008.03.003 |
| 作者姓名： | 彭国平 周承 吴炜 孙雯 田锋 贺纪凯 陈智 |
| 作者单位： | 1. 浙江大学医学院附属第一医院传染病研究所,传染病诊治国家重点实验室,杭州,310031 2. 浙江大学医学院附属口腔医院,杭州,310003 3. 浙江大学医学院临床医学系,杭州,310023 |
| 基金项目： | 国家自然科学基金 , 省部共建基金 |
| 摘 要： | 目的 构建具有免疫学活性的抗小鼠细胞毒性T淋巴细胞相关抗原4(CTLA-4)单链抗体ScFv (single chain fragment of variety region),为进一步将其应用到动物体内奠定基础.方法 采用RT-PCR技术,从分泌抗小鼠CTLA-4 单克隆抗体(mAb) 的杂交瘤细胞中扩增mAb 的VH、VL 基因,并进一步将其组装成VH-Linker-VL 型的ScFv 片段.将ScFv 片段亚克隆至pGMET 载体,测序正确后将其克隆至真核分泌型表达载体pSect2-GFP,将pSect2-GFP-ScFv 纯化质粒转染中国仓鼠卵巢细胞(CHO)并进行表达,同时经Zeocin筛选稳定表达株.用Western blot方法检测ScFv 融合蛋白的表达,采用ELISA检测 ScFv 的亲和活性.结果 经Zeocin 筛选2周后,CHO细胞株可稳定表达GFP-ScFv 蛋白.Western blot法证实GFP-ScFv 蛋白在细胞上清和细胞裂解液中均有表达,大小约55 kD.ELISA检测表明,CHO 培养上清中的GFP-ScFv 蛋白经浓缩初纯后能与重组小鼠CTLA-4 纯化抗原结合,并具有抑制亲本单抗与其结合的能力.结论 成功构建了抗小鼠CTLA-4 的ScFv 真核表达载体,并能有效表达出具有免疫学活性的ScFv 融合蛋白. |
| 关 键 词： | 细胞毒性T淋巴细胞相关抗原4 单链抗体 真核表达 免疫学活性 单链抗体 构建与表达 免疫学 活性 Construction ScFv Murine Activity 有效表达 真核表达载体 能力 抗原结合 单抗 亲本 小鼠 重组 培养上清 大小 细胞裂解液 法证 |
Construction,Expression and Immunologic Activity of Murine Anti-CTLA-4 ScFv |
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| Peng Guoping,Zhou Cheng,Wu Wei et al Institute of Infectious Diseases,National Key Laboratory of Infectious Diseases,The First Affiliated Hospital,College of Medicine,Zhejiang University,Hangzhou. Construction,Expression and Immunologic Activity of Murine Anti-CTLA-4 ScFv[J]. Journal of Huazhong University of Science and Technology(Health Sciences), 2008, 37(3). DOI: 10.3870/j.issn.1672-0741.2008.03.003 | |
| Authors: | Peng Guoping Zhou Cheng Wu Wei et al Institute of Infectious Diseases National Key Laboratory of Infectious Diseases The First Affiliated Hospital College of Medicine Zhejiang University Hangzhou |
| Affiliation: | Peng Guoping,Zhou Cheng,Wu Wei et al Institute of Infectious Diseases,National Key Laboratory of Infectious Diseases,The First Affiliated Hospital,College of Medicine,Zhejiang University,Hangzhou 310003 |
| Abstract: | Objective To construct and express the single chain fragment of variety region (ScFv) of monoclonal antibody against murine CTLA-4 and analyze the immunologic activity of recombinant ScFv protein. Methods The VL and VH genes were cloned by RT-PCR from anti-murine CTLA-4 mAb excreted by hybridoma cells. VH-linker-VL fragment (ScFv) was constructed and subcloned into vector pGMET and then cloned into vector pSect2-GFP. The purified pSect2-GFP-ScFv plasmid was transfected into CHO cells and selected by Zeocin ... |
| Keywords: | cytotoxic T lymphocyte related antigen 4 ScFv eukaryotic expression immunologic activity |
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