| 鼠抗人B7-2分子单链抗体基因的构建及其在家蚕中的表达 |
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| 引用本文： | 胡静平,董海辉,邱玉华,陈永井,王文兵,黄光镁,吴小锋,朱江.鼠抗人B7-2分子单链抗体基因的构建及其在家蚕中的表达[J].蚕业科学,2010,36(2):274-281. |
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| 作者姓名： | 胡静平 董海辉 邱玉华 陈永井 王文兵 黄光镁 吴小锋 朱江 |
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| 作者单位： | 苏州大学基础医学与生物科学学院,江苏,苏州,215123;苏州大学基础医学与生物科学学院,江苏,苏州,215123;苏州大学医学生物技术研究所,江苏苏州,215007;江苏大学生命科学研究院,江苏,镇江,212013;浙江大学动物科学学院,杭州,310029 |
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| 基金项目： | 国家科技重大专项项目，江苏省自然科学基金项目 |
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| 摘 要： | B7-2蛋白为免疫球蛋白超家族(IGSF)成员之一。采用PCR法从分泌鼠抗人B7-2抗体的杂交瘤细胞株克隆出该单克隆抗体的重链(VH)和轻链(VL)可变区基因,通过重叠延伸PCR(SOE-PCR)方法,在VH和VL可变区基因间引入连接肽(Gly4Ser)3编码序列,体外构建抗人B7-2的鼠源单链抗体基因B7-2-ScFv。利用新型杂交病毒HyNPV的Bac-to-Bac表达系统,将B7-2-ScFv基因克隆到杆状病毒转移载体pFastBacHTa中,获得重组转移载体pFastBacHTa-B7-2-ScFv,转化大肠杆菌DH10Bac,获得重组杆粒Bacmid-B7-2-ScFv。将此重组杆粒的DNA转染Sf9细胞,获得重组病毒HyNPV-ScFv。感染该重组病毒的BmN细胞经SDS-PAGE和Western blotting分析表明,至感染24h时目的蛋白已有表达,96h达到最高表达水平,表达产物的分子质量约31kD。用该重组病毒感染家蚕5龄起蚕,感染后96h蚕体表现出明显的发病症状。RT-PCR结果表明,5龄起蚕感染后48h,在脂肪体内已有目的基因转录,一直持续到感染后120h;SDS-PAGE和Western blotting分析表明,5龄起蚕感染96h后方可在脂肪体中检测到目的蛋白表达。构建并在家蚕中成功表达鼠抗人B7-2单链抗体,为深入研究和开发该抗体奠定了基础。
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| 关 键 词： | B7-2蛋白 单链抗体 杂交病毒HyNPV 家蚕 |
Construction of Murine Anti-human B7-2 Molecular Single Chain Antibody Gene and its Expression in Bombyx mori |
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| HU Jing-Ping,DONG Hai-Hui,QIU Yu-Hua,CHEN Yong-Jing,WANG Wen-Bing,HUANG Guang-Mei,WU Xiao-Feng,ZHU Jiang.Construction of Murine Anti-human B7-2 Molecular Single Chain Antibody Gene and its Expression in Bombyx mori[J].Acta Sericologica Sinica,2010,36(2):274-281. |
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| Authors: | HU Jing-Ping DONG Hai-Hui QIU Yu-Hua CHEN Yong-Jing WANG Wen-Bing HUANG Guang-Mei WU Xiao-Feng ZHU Jiang |
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| Institution: | HU Jing-Ping1 DONG Hai-Hui1 QIU Yu-Hua1,2 CHEN Yong-Jing1,2 WANG Wen-Bing3 HUANG Guang-Mei1 WU Xiao-Feng4 ZHU Jiang1(1School of Basic Medicine , Biological Sciences,Soochow University,Suzhou Jiangsu 215123,China,2Medical Biotechnology Institute,Suzhou Jiangsu 215007,3Institute of Life Sciences,Jiangsu University,Zhenjiang Jiangsu 212013,4College of Animal Sciences,Zhejiang University,Hangzhou 310029,China) |
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| Abstract: | B7-2 protein is one member of the immunoglobulin superfamily.The variable domain genes encoding heavy(VH)and light(VL)chains of B7-2 antibody were cloned from a murine anti-human B7-2 hybridoma cell line.Using splicing by overlapping extension PCR(SOE-PCR)approach,coding sequence of a short linker peptide(Gly4Ser)3 was inserted between the VH and VL gene fragments to construct the anti-human B7-2 single-chain antibody gene B7-2-ScFv of murine origin in vitro.The mouse B7-2-ScFv gene was subcloned into a bac... |
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| Keywords: | B7-2 protein Single chain antibody Hybrid virus HyNPV Bombyx mori |
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