摘要
目的:构建一个正常人天然IgG抗体噬菌体呈现库.方法:从一献血员取50mL新鲜血液,分离淋巴细胞,提取RNA,逆转录合成cDNA,PCR扩增重链Fd和轻链cDNA,依次将PCR产物插入载体pComb3H相应位点.以点印迹检测噬菌体表面Fab的表达.结果:所有4个IgG亚类的重链Fd片段、部分κ轻链和λ轻链cDNA得到扩增.先期插入的重链Fd的实际库容达1.1×106,插入率20%.后期插入的轻链库容达1.35×107,插入率接近100%,Fab表达库容达2.70×106.噬菌体悬液的点印迹显示有Fab表达.结论:构建了一个正常人IgG的Fab噬菌体呈现库.
AIM: To construct a naive human IgG phage display library. METHODS: Peripheral blood lymphocytes were isolated from 50 mL blood, which was obtained from a healthy blood donor. The heavy chain Fd and light chain cDNAs synthesized from the total lymphocyte RNA were PCR amplified and the amplification products were inserted successively into the vector pComb3H. Dot immunoblotting was used to monitor Fab expression. RESULTS: The insertion rate of the heavy chain Fd, which was inserted first, was 20% with an actual library volume of 1.1106. Pooled chain and chain PCR products were subsequently inserted, resulting in an insertion rate close to 100% and a library volume of 1.35107. Fab expression library volume reached 2.70106. Dot immunoblotting showed that there was Fab expression on the phages of the library. CONCLUSION: A human IgG Fab phage display library from a normal unimmunized individual is constructed.
出处
《第四军医大学学报》
1999年第6期464-467,共4页
Journal of the Fourth Military Medical University
关键词
抗体库
噬菌体
抗体工程
antibody library
bacterial phage
antibody engineering