天然人源性单链抗体文库的构建及鉴定  

Construction and identification of naive human single chain Fv library

作　　者：张文帅[1] 迟莹[1] 焦永军[1] 

机构地区：[1]江苏省疾病预防控制中心,南京210009

出　　处：《江苏预防医学》2017年第3期245-247,共3页Jiangsu Journal of Preventive Medicine

基　　金：国家自然科学基金(31570926);国家自然科学基金青年基金(81601732);江苏省卫生和计划生育委员会"科教强卫"工程医学重点人才(ZDRCA2016031)

摘　　要：目的构建天然人源性单链抗体(scFv)文库,并对文库质量进行鉴定。方法采集220份未经主动免疫健康人外周血,分离单个核细胞后提取总RNA,逆转录成cDNA,混匀,以此为模板PCR扩增人抗体重链可变区(VH)和轻链可变区(VL,包括Vκ和Vλ),再经过overlap-PCR法,将VH基因和VL基因随机拼接成人scFv基因文库后,插入噬菌体载体pComb3XSS中,电转化大肠杆菌XL1-Blue制备scFv文库。通过菌落数量计算库容,并随机挑选100个单菌落验证scFv基因阳性克隆率,基因测序分析scFv文库基因的多样性。结果经过1次电转化得到容量为1.8×108的scFv文库,scFv基因阳性克隆率为96%,其中78%为正确插入,其scFv基因序列均不相同。结论成功构建了一种大容量、多样性高的天然人源性scFv文库,为进一步筛选特异性中和抗体提供了基础材料。Objective To construct and identify a naive human single chain Fv（scFv）library. Methods The total RNA of mononuclear cells from peripheral blood of 220 non-immununized healthy donors was isolated and cDNA was synthesized and mixed to serve as templates to amplify genes of heavy variable chains （VH）and light variable chains （VL, including Vκ and VX） by PCR. The VH and VL genes were ligated randomly to make scFv gene library by overlap-PCR, which were cloned into vector pComb3XSS and transformed into electronic XL1-Blue competent cells to construct scFv library. The volume of constructed library was calculated, 100 colonies were selected randomly to validate scFv cloning efficiency, the diversity was evaluated by sequencing analysis. Results A scFV library with the volume of 1.8 × 10^8 was constructed after 1 time electronic transformation. The scFv cloning efficiency was 96 %, among which 78% clones with right unrepeated insertion. Conclusion A large volume naive human scFv library with high diversity was constructed successfully, which can be used to screen specific human antibodies in the future.

关 键 词：单链抗体文库 噬菌体 人源 PCR 库容量 多样性 

分 类 号：R117[医药卫生—公共卫生与预防医学]