人癌胚抗原单链抗体基因的构建和筛选
DOI:
CSTR:
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:


Construction and Screening of the Anti-CEA Single Chain Antibody Gene
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    从分泌抗癌胚抗原(carcinoembryoni antigen, CEA)单抗的杂交瘤细胞株C50中提取总RNA, 逆转录成cDNA, PCR扩增分别得到抗体轻、重链可变区基因, 再利用两对PCR引物合成和扩增得到全单链抗体基因. 将含轻、重链可变区序列的DNA片段克隆于含噬菌体基因Ⅲ的噬菌粒pCANTAB5. 重组克隆在噬菌体表面表达基因Ⅲ与单链抗体的融合蛋白. 表达具抗原结合活性的单链抗体的重组噬菌体可以通过亲和筛选的方法筛选得到并富集. 利用该方法我们可以从许多分泌不同抗体的杂交瘤细胞RNA中快速克隆和筛选功能性抗体可变区基因.

    Abstract:

    A strategy based on polymerase chain reaction (PCR) and a phagedisplay system for the rapid method for constructing and screening functional single chain antibody genes was reported. Total RNA from a hybridoma (C50) producing an antibody that reacts with carcinoembryonic antigen (CEA) was used as a template to make the first strand of a cDNA, lightand heavy-chain variable-region genes were separately amplified by PCR. Then whole single chain antibody gene was synthesized and amplified by using two sets of DNA primers that(1) hybridized to the ends of the light- and heavy-chain variable regions, ( 2 ) encoded a Linker peptide, and(3)contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into pCANTAB 5 phagemid containing gene Ⅲ of the phage. Clones encoding single-chain antibody was express on the surface of the phage as a fusion with gene Ⅲ protein. Recombinant phage expressing anti-CEA single chain antibody which retained its antigen-binding capability was screened by affinity selection and enriched. By using this approach it should be possible to rapidly clone and screen the functional variable region sequences of many different antibodies from hybridoma RNA.

    参考文献
    相似文献
    引证文献
引用本文

章美云,孔健.人癌胚抗原单链抗体基因的构建和筛选[J].生物化学与生物物理进展,1996,23(5):470-474

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:1995-12-04
  • 最后修改日期:1996-02-15
  • 接受日期:
  • 在线发布日期:
  • 出版日期: