正常人天然IgG抗体噬菌体呈现库的构建

Construction of a naive IgG phage display library from a normal human individual

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.