丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

Human In-Cell scFv Library from Infiltrating B Cell

互联网

957
The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not totally adapted to the study of the antibody repertoire formed in vivo during, for example, autoimmune diseases.
We describe here, the use of the in-cell PCR together with Cre-recombination applied to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the Cre–loxP system, the 800-bp band corresponding to scFv was cloned and human scFv fragments selected.
This in-cell amplification, association, and scFv selection procedure is a potentially useful tool for the study of antibody repertoire and the VH/VL pairing that occurs during the diseases’ process.
提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序