抗EV71病毒人源抗体Fab段表达基因的构建及序列分析

Construction and sequence analysis of expression cassette for Fab fragments of human-origin immunoglobulin against EV71

目的从成人外周血淋巴细胞中扩增抗体可变区并构建人源抗体Fab段表达基因,为构建抗EV71病毒人源化单克隆抗体库奠定基础。方法从EV71病毒中和抗体阳性且免疫功能健全的成人全血中,分离淋巴细胞并提取总RNA,经反转录获得cDNA。用一组人IgG Fab基因特异性引物,从合成的cDNA中分别扩增抗体轻、重链可变区,应用重叠PCR技术构建Fab段表达基因,测序分析抗体基因的多样性。结果成功扩增人抗体可变区基因并组装Fab段表达盒,表达盒的长度约为1 500bp,测序分析结果表明,Fab段的多样性可达94.12%。结论成功构建人源抗体Fab段基因,为抗EV71病毒人源性单克隆抗体库的制备奠定了基础。

Humanized monoclonal antibodies against enterovirus type 71 （EV7 t） are ideal options in clinical treatment of EV71 infections. In this study, variable region genes of human-origin immunoglobulin were amplified to construct recombinant Fah fragments against EVT1. Total RNA was extracted from peripheral blood [ymphocytes of a normal adult that was positive with EV71 neutralizing antibody. With the reverse-transcribed cDNAs as templates, variable region genes of light and heavy chains were amplified by human IgG antihody primer sets, and the expression cassettes for Fab fragments were subsequently constructed by splicing by overlap extension PCR （SOE-PCR）. It was indicated by sequence analysis of obtained 1 500 bp ampl- icons that diversity in Fab fragments was 94. 12 %, suggesting successful construction of a Fab fragment expression library, which facilitated preparation of human-origin monoclonal antibody library against EV71.