Development of rapid screening and production technique of antibody Fv for protein chip

蛋白芯片抗体Fv快速筛选及生产技术开发

基本信息

  • 批准号:
    13854003
  • 负责人:
  • 金额:
    $ 75.55万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (S)
  • 财政年份:
    2001
  • 资助国家:
    日本
  • 起止时间:
    2001 至 2005
  • 项目状态:
    已结题

项目摘要

We constructed HE and LE chimeric receptors comprising V_H and V_L of anti-hen egg lysozyme (HEL) antibody fused with extracellular D2 and transmembrane/intracellular domains of erythropoietin receptor, respectively. H_g and L_g chimeric receptors were also constructed by replacing intracellular domain with that of gp130. Furthermore, a series of chimeric receptors whose ligand recognition domain is anti-fluorescein antibody single chain F_v were constructed. After the gene transduction, genetically modified cells were successfully amplified by addition of cognate antigen in the culture medium.Next, we randomized 4 amino acids of HEL-recognition site in V_H, linked to gp130, and transduced the resultant vector into LE-expressing cells. After HEL selection, the mutant V_H showed comparable binding affinity to the wild-type V_H. Furthermore, V_H library was amplified by PCR from splenocyte of mice immunized with superoxide dismutase (SOD), and fused with gp130 intracellular domain. When cells were transduced with this V_H library, some growing clones were obtained, indicating the possibility of library selection by using the chimeric receptor.In addition, we constructed a vector in which chimeric receptor gene is flanked with two loxP sequences. By using this vector, we successfully developed a system in which a chimeric receptor-expressing cell can be sequentially converted into an antibody producer cell, which was attained by expressing Cre recombinase after antigen selection in the cell. Furthermore, we developed a technique to make 324 homogeneous spots of antibody at 150 μm diameter on a glass plate (9mm x 9mm) by an electrospray deposition method. Sandwich ELISA using this antibody microarray reproducibly detected antigen concentration at 0.1 to 1 ng/ml of detection limit.
我们构建了HE和LE嵌合受体,分别由抗鸡蛋溶菌酶(HEL)抗体的V_H和V_L与细胞外D2和红细胞生成素受体的跨膜/胞内结构域融合。用gp130取代胞内结构域构建H_g和L_g嵌合受体。构建了一系列以抗荧光素抗体单链F_v为配体识别域的嵌合受体。基因转导后,在培养基中加入同源抗原,成功扩增出基因修饰细胞。接下来,我们随机选取V_H中hell识别位点的4个氨基酸,与gp130连接,并将合成载体转导到表达le的细胞中。经过HEL选择,突变体V_H表现出与野生型V_H相当的结合亲和力。用PCR方法从超氧化物歧化酶(SOD)免疫小鼠脾细胞中扩增出V_H文库,并与gp130融合。当细胞用该V_H文库转导时,获得了一些生长的克隆,表明使用嵌合受体进行文库选择的可能性。此外,我们构建了一个载体,其中嵌合受体基因两侧有两个loxP序列。利用该载体,我们成功构建了一个嵌合受体表达细胞依次转化为抗体产生细胞的系统,该系统通过在细胞中表达抗原选择后的Cre重组酶获得抗体产生细胞。此外,我们开发了一种技术,通过电喷雾沉积法在玻璃板(9mm x 9mm)上以150 μm的直径制作324个均匀的抗体斑点。夹心ELISA采用该抗体芯片可重复性地检测抗原浓度在0.1 ~ 1 ng/ml的检出限。

项目成果

期刊论文数量(65)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Kawahara, M. et al.: "AMEGA : antigen-mediated genetically modified cell amplification"J.Immunol.Methods. 284. 187-194 (2004)
Kawahara,M.等人:“AMEGA:抗原介导的基因修饰细胞扩增”J.Immunol.Methods。
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
河原 正浩: "抗体を用いた受容体のエンジニアリング-効果的な細胞医療を目指して-"バイオインダストリー. 20. 23-33 (2003)
Masahiro Kawahara:“使用抗体进行受体工程 - 旨在实现有效的细胞治疗 -”Bioindustry 20. 23-33 (2003)。
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv.
用于产生糖基化单链 Fv 的哺乳动物细胞的抗原介导的选择系统。
Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain.
通过跨膜结构域工程改善抗体/受体嵌合体的生长反应。
Animal cell technology creation of new era (JAACT 2000)
动物细胞技术开创新时代(JAACT 2000)
  • DOI:
  • 发表时间:
    2002
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Kaawhara;M;10名
  • 通讯作者:
    10名
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NAGAMUNE Teruyuki其他文献

NAGAMUNE Teruyuki的其他文献

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{{ truncateString('NAGAMUNE Teruyuki', 18)}}的其他基金

Development of technologies for maintaining undifferentiated state and inducing growth and differentiation of human iPS cell by using chimeric receptor
开发利用嵌合受体维持未分化状态并诱导人iPS细胞生长和分化的技术
  • 批准号:
    24360337
  • 财政年份:
    2012
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Development of selective cell collection method using photo-degradable PEG-lipid
开发使用光降解 PEG-脂质的选择性细胞收集方法
  • 批准号:
    24656498
  • 财政年份:
    2012
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Challenging Exploratory Research
Development of a cell microarray chip for evaluating invasion of cancer cells by transfer printing of transfected cells
开发细胞微阵列芯片,通过转染细胞的转印来评估癌细胞的侵袭力
  • 批准号:
    22656189
  • 财政年份:
    2010
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Challenging Exploratory Research
Development of artificial receptor-based amplification system of dendritic cells for cancer therapy
开发基于人工受体的树突状细胞扩增系统用于癌症治疗
  • 批准号:
    18206083
  • 财政年份:
    2006
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
Development of mammalian cells capable of growing rapidly in serum-free media
开发能够在无血清培养基中快速生长的哺乳动物细胞
  • 批准号:
    11555216
  • 财政年份:
    1999
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Study on molecular design of artificial antibody and its application to immunosensor
人工抗体分子设计及其在免疫传感器中的应用研究
  • 批准号:
    09450301
  • 财政年份:
    1997
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Development of a homegeneous immunodiagnostics system utilizing luminescence wavelength transformation by energy transfer
利用能量转移的发光波长变换开发同质免疫诊断系统
  • 批准号:
    08555199
  • 财政年份:
    1996
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
BASIC RESEARCH AIMING CONSTRUCTION OF ON-LINE MONITORING SYSTEM FOR MAMMALIAN CELL CULTURE SYSTEM
哺乳动物细胞培养系统在线监测系统构建的基础研究
  • 批准号:
    06453105
  • 财政年份:
    1994
  • 资助金额:
    $ 75.55万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
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