从外周血单个核细胞扩增多样性人免疫球蛋白基因的研究

Amplification of human diversified immunoglobulin genes from peripheral blood mononuclear cells

目的 建立成熟的体外扩增多样性人免疫球蛋白基因的实验方法。方法 设计多对具有简并性特点的人IgG、IgM扩增引物 ,从人外周血单个核细胞 (PBMCs)中提取RNA并反转录为cDNA ,以其为模板聚合酶链反应 (PCR)扩增人免疫球蛋白κ轻链基因和重链Fd段基因 ,对扩增产物进行凝胶电泳和DNA指纹分析鉴定。结果 利用不同引物进行的PCR反应均成功扩增出相应的免疫球蛋白基因 ,经鉴定所获得的基因产物具有良好的多样性 ,在天然抗角蛋白自身抗体研究和抗体库的构建中得到初步应用。结论 合理设计引物能够从PBMCs中成功扩增出多样性的人免疫球蛋白基因 。

Objective To establish a usable method for the amplification of human diversified immunoglobulin genes.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals and in which RNA was extracted and reverse-transcripted into cDNA.κ light chain genes and Fd fragments of human IgG and IgM were amplified by PCR with designed primers and identified by gel analysis and DNA fingerprinting.Results All the intent immunoglobulin genes were successfully amplified and the obtained products were identified correct and with very good diversity.Conclusion It is feasible to amplify human diversified immunoglobulin genes from PBMCs by RT-PCR using properly designed primers.This method will be useful in the studies of human antibodies,correlative immunological molecules and autoimmune diseases.