天然人源IgG Fab噬菌体抗体库的构建及多样性分析

Construction and diversity analysis of a nave human phage-displayed Fab library

目的:构建天然人源IgGFab噬菌体抗体库并分析来源于健康人外周血B淋巴细胞的抗体基因可变区的多样性及优势选择。方法:以DNA重组技术从300名健康志愿者的外周血淋巴细胞中扩增出全套人抗体轻链及重链Fd基因,分别插入噬菌体载体pFabICN相应位置,构建天然人源免疫球蛋白G基因文库;进一步从抗体库中随机挑选克隆,获得重轻链基因进行核苷酸序列测定并利用IgBLAST数据库分析抗体基因可变区的同源家族。结果:从4×108库容的天然人源IgGFab噬菌体抗体库中随机挑选克隆,对其中32个轻链及重链Fd基因插入正确的克隆进行核苷酸序列测定和氨基酸序列的推算,获得29条彼此完全不同的Fd重链基因及轻链基因。以IgBLAST数据库比对分析显示29条重链基因的V区分别属于VH3(72.4%),VH1(10.3%),VH4(6.9%),VH5(6.9%)和VH7(3.4%)基因家族。29条轻链基因分别属于Vκ1(44.8%),Vκ2(15.4%),Vκ3(11.5%),Vκ4(23.1%)基因家族和Vλ1(11.5%)基因家族。结论:以300份健康人外周血淋巴细胞的抗体基因构建的天然人源IgGFab噬菌体抗体库基因多样性良好,重链VH3基因家族存在优势表达。

Objective：To construct phage-displayed Fab library and to analyze its diversity and the usage of VH gene families of a nave human antibody from healthy volunteers.Methods：Total genes encoding the light chains and Fd region of the heavy chains of the human immunoglobulin G were amplified from the peripheral blood lymphocytes of 300 healthy volunteers by recombinant DNA technology.A nave human phage-displayed Fab library was constructed by inserting the light and heavy chain genes into the vector pFabICN.Variable regions of the light and heavy chain genes randomly selecting from the library were analyzed by comparing the sequences with known database using IgBLAST.Results：Several clones were randomly picking out from a phage-displayed Fab library with a titer of 4×108 and thirty-two plasmid DNA clones coding light chain and heavy chain were sequenced respectively.DNA sequence analysis revealed that 29 light chain genes and 29 Fd heavy chain genes could correctly translate into amino acids.These antibody genes were totally different from each other.Comparison of the variable regions of the heavy chain genes by IgBLAST revealed that biased expression of VH families occurs,with VH3 family the most frequently used（72.4%）,followed by VH1（10.3%）,VH4（6.9%）,VH5（6.9%）and VH7（3.4%）.For the light chain genes,Vκ genes were more frequently used with Vκ1（44.8%）,Vκ2（15.4%）,Vκ3（11.5%）,Vκ4（23.1%）and Vλ genes were less frequently used with Vλ1（11.5%）.Conclusion：A nave human phage-displayed Fab library with high diversity is constructed from the peripheral blood lymphocytes of 300 healthy volunteers and a VH3 biased expression of VH gene families is observed.