细胞内PCR法克隆抗体可变区基因

Antibody variable genes cloning by in cell PCR

报道一种克隆免疫脾细胞中抗体可变区基因的新方法。抗原免疫小鼠的脾细胞经分散、固定、渗透处理 ,直接在细胞内进行反转录 ,再采用半巢式PCR扩增 ,获得的序列经测序证明是抗体可变区编码序列。这种不经mRNA纯化、从细胞内直接获取目的基因的方法既可用于抗体库的建立 ,亦可用于新基因的快速克隆。

A novel method in cloning antibody variable genes from immunized spleen cells was described in the report. After the antigen sensitized spleen was dispersed, the cells were treated by fixation and permeabilization. Reverse transcription and polymerase chain reaction (RT PCR) was conducted inside the cells and semi nested PCR was used to amplify the variable genes that proved to be. The method, without mRNA purification and direct cloning inside the cells, may provide a new way on construction of antibody library or fast cloning of novel genes.