人免疫球蛋白重链可变区基因引物设计方法的改良

针对抗体胚系基因数据库的数据不断更新和完善,为获得人全部免疫球蛋白(Ig)重链可变区基因,改进引物设计方法,自主设计针对可变区基因高度保守的框架区1(FR1)和框架区4(FR4)的引物,提取未经免疫的健康人外周血单个核细胞,通过RT-PCR扩增重链可变区基因.其DNA序列与GenBank数据库和IMGT/V-QUEST软件比对,序列分析符合人免疫球蛋白重链基本框架结构,为胚系基因重排产生的序列.多个克隆的测序结果对比分析显示了良好的多样性.获得的重链序列为研制基因工程抗体及构建噬菌体抗体库奠定了物质基础,也为扩增其他物种Ig可变区基因的引物提供新的设计思路.

Design Method Amelioration of Human Immunoglobulin Gene Primer in Heavy Chain Variable Region

Aiming at the continuous renovation and consummation of data of databank on antibody germ line gene,in order to obtain full gene of heavy chain variable region of human immunoglobin(Ig),to ameliorate primer design method,a primer was independently designed aiming at variable gene highly conservative region framework 1(FR1) and framework 4(FR4),and extracted peripheral blood mononuclear cells(PBMCs) from healthy volunteer,and amplified its heavy chain variable region gene through RT-PCR.After comparison with databank in GenBank and IMGT/V-QUEST software and sequence analysis,the DNA sequence accorded with basic framework structure of human Ig heavy chain,and was the sequence produced by the rearrangement of germ line gene.Comparative analysis of multiple clones sequencing showed a good diversity.The obtained data of heavy chain sequence had provided the material foundation for research and development of gene engineering antibodies and phage libraries,and new design ideas for amplifying Ig variable region gene primers of other species.