HUANG Yu;;YU Bing;XING Wei;SHEN Xin;XIAO Daiwen;DIAO Zhijuan;LIU Jing;DAI Wei;ZHAO Xiaorong;LEI Ping;SHEN Guanxin" /> 肝癌细胞特异性结合双价抗体的构建表达与鉴定*
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  2006, Vol. 25 Issue (7): 0-0    
  疫苗及分子生物药物专栏 本期目录 | 过刊浏览 | 高级检索 |
肝癌细胞特异性结合双价抗体的构建表达与鉴定*
黄宇;余冰;邢薇;沈昕;肖代雯;
刁智娟;刘静;代维;赵晓蓉;雷萍;沈关心
1. 华中科技大学同济医学院免疫学系,武汉430030 ;2. 福建省厦门市第一医院检验科,361000
Construction,Expression and Identification of a Bivalent Single Chain Variable Fragment (BsFv)Specifically Binding to Liver Cancer Cell
HUANG Yu;;YU Bing;XING Wei;SHEN Xin;XIAO Daiwen;DIAO Zhijuan;LIU Jing;DAI Wei;ZHAO Xiaorong;LEI Ping;SHEN Guanxin
1.Department of Immunology,Tongji Medical College, Huazhong University of Science and Technology,Wuhan 430030,China;2.Department of Clinical Laboratory,the First Hospital of Xiamen City,Fujian Province 361000,China
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摘要 目的构建和表达与肝癌细胞特异结合的双价单链抗体(bivalent singlechain Fv,BsFv),并鉴定该双价抗体的生物活性。方法以与肝癌细胞特异结合的单链抗体(scFv)为模板,设计引物引入中间连接肽(G4S)及酶切位点AscI,通过聚合酶链反应(PCR)方法扩增出两个scFv片段,将其连入原核表达载体pAB1;构建的表达载体转化大肠埃希菌TG1,挑取单克隆细菌进行扩增,提取质粒酶切、测序鉴定。阳性菌经IPTG诱导表达,表达产物经SDSPAGE,Western blot鉴定,间接免疫荧光流式细胞术(FACS)测定与肝癌细胞特异结合的特性。结果经酶切鉴定以及DNA测序结果表明该BsFv构建成功,SDSPAGE、Western blot鉴定表明表达蛋白相对分子量与BsFv理论值一致,且表达产物同亲本scFv比较,与肝癌细胞特异结合活性增强。结论BsFv构建成功,BsFv较scFv具有明显优势,为其用于临床肿瘤的诊断和治疗奠定了基础。
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关键词 单链抗体肝癌细胞双价抗体    
Abstract:To produce and express a biralent siglechain Fv(BsFv) specifically binding to liver cancer cells,and to identify its biologic activity. MethodsPCR primers were designed to introduce interlinker G4S and the restriction endonuclease AscI,and two fragments were produced from the parent scFv SLH10 by PCR,and then linked into the expression vector pAB1. The cellbinding activities of the BsFv and scFv were analyzed by FACS using some cell lines. ResultsBsFv was found to bind to selected cell lines more specifically than parent scFv. ConclusionBsFv is successfully constructed with prominent superiority to scFv,laying a foundation for its use in the diagnosis and treatment in clinical tumors.
收稿日期: 1900-01-01      出版日期: 2006-07-01
:  A  
通讯作者: 沈关心   
引用本文:   
黄宇;余冰;邢薇;沈昕;肖代雯;刁智娟;刘静;代维;赵晓蓉;雷萍;沈关心 . 肝癌细胞特异性结合双价抗体的构建表达与鉴定*[J]. , 2006, 25(7): 0-0.
HUANG Yu;;YU Bing;XING Wei;SHEN Xin;XIAO Daiwen;DIAO Zhijuan;LIU Jing;DAI Wei;ZHAO Xiaorong;LEI Ping;SHEN Guanxin. Construction,Expression and Identification of a Bivalent Single Chain Variable Fragment (BsFv)Specifically Binding to Liver Cancer Cell. , 2006, 25(7): 0-0.
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