孙 巍, 林 珩, 花 芳, 胡卓伟. 优化宿主菌在重组系统中构建大容量天然噬菌体抗体库J. 药学学报, 2013,48(1): 66-70.
引用本文: 孙 巍, 林 珩, 花 芳, 胡卓伟. 优化宿主菌在重组系统中构建大容量天然噬菌体抗体库J. 药学学报, 2013,48(1): 66-70.
SUN Wei, LIN Heng, HUA Fang, HU Zhuo-wei. Optimizing the host bacteria to make a large naive phage antibody library in the recombination systemJ. 药学学报, 2013,48(1): 66-70.
Citation: SUN Wei, LIN Heng, HUA Fang, HU Zhuo-wei. Optimizing the host bacteria to make a large naive phage antibody library in the recombination systemJ. 药学学报, 2013,48(1): 66-70.

优化宿主菌在重组系统中构建大容量天然噬菌体抗体库

Optimizing the host bacteria to make a large naive phage antibody library in the recombination system

  • 摘要:

    制备大容量天然噬菌体抗体库。从健康成人外周血和新生儿脐带血中提取淋巴细胞总RNA, 反转录后PCR分别扩增抗体重链和轻链基因。将获得的基因片段通过overlap PCR连接为单链抗体基因 (scFv), 限制性内切酶酶切后连入pDAN5a载体中, 连接产物转化大肠杆菌XL2-blue MRF’。与XL1-blue菌株相比, 初级库库容增加了3.9倍。再将初级库噬菌体感染含有Cre重组酶的大肠杆菌BS1365, 抗体基因在重组酶作用下发生同源重组, 最终得到库容为1.7×1011的噬菌体抗体库。经检测, 该抗体库多样性良好, 利用该库可获得针对6种抗原的抗体。以上结果表明, 应用XL2-blue MRF’作为抗体基因的转化宿主, 提高了噬菌体抗体库的库容, 获得的抗体库可用于下一步抗体药物的筛选。

     

    Abstract:

    To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system.  The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood.  The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF’ with the Hte gene.  Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times.  The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations.  The library was calculated to have a diversity of 1.7×1011 and validated by the selection of antibodies against six different protein antigens.  This library provides the basis for further selection of antibody-based drugs.  It is the first time to report that XL2-blue MRF’ can be used to improve the diversity of the library in the recombination system.

     

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