分子生物学
免疫球蛋白轻链
克隆(编程)
多克隆位点
生物
重组DNA
表达式向量
分子克隆
限制地点
多克隆站点
克隆载体
RNA剪接
抗体
限制性酶
肽序列
聚合酶链反应
遗传学
载体(分子生物学)
基因
限制性片段长度多态性
核糖核酸
计算机科学
程序设计语言
作者
M. Josefina Coloma,Alice Hastings,Letitia A. Wims,Sherie L. Morrison
标识
DOI:10.1016/0022-1759(92)90092-8
摘要
A new family of vectors has been produced which facilitates the cloning and expression of immunoglobulin variable regions cloned by polymerase chain reaction (PCR). The vectors are designed to express the cloned variable regions joined to human constant regions and take advantage of priming in the leader sequence so that no amino acid changes will be introduced into the mature antibody molecule. Both the heavy chain and light chain vectors utilize a murine V11 promoter provided with an EcoRV restriction site so that the amplified variable regions can be directly cloned into a functional promoter. For the heavy chain an NheI restriction site has been generated at the first two amino acids of C11l and the cloned leader and variable region are fused directly to the C11l domain of the constant region. When the leader and variable regions of the light chain were fused directly to Cκ, no expression was observed. Therefore the light chain expression vector was designed with a Sall restriction site for cloning into a splice junction 3′ of the variable region; VL then is joined to Cκ by splicing. Both vectors direct the expression of functional. fully assembled immunoglobulin molecules with the expected molecular weight. Use of redundant oligomers to prime the PCR permits the cloning and expression of recombinant antibodies without any prior information as to their sequence and makes it possible to rapidly generate recombinant antibodies from any monoclonal antibody producing cell line.
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