抗CD133纳米抗体的分离与鉴定  

Isolation and identification of a nanobody which bound with human CD133

作　　者：李玲霞[1] 翟田甜[1] 马晓玲[1] 冯亚宁[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,新疆乌鲁木齐830046

出　　处：《生物技术》2016年第1期87-92,共6页Biotechnology

基　　金：国家自然科学基金项目(“骆驼单域抗体框架区作为小分子抗体通用移植骨架的研究”,No.31370933)资助

摘　　要：[目的]获得骆驼来源的与人CD133抗原特异性结合的纳米抗体。[方法]从3峰新疆双峰驼外周血中分离淋巴细胞,采用巢式PCR扩增获得cDNA并构建VHH天然噬菌体展示文库。包被CD133-606于ELISA板,对天然文库进行3轮亲和筛选,菌落PCR和基因测序确定能结合CD133的候选克隆。挑选重复序列VHH克隆构建到pET28a并转BL21用IPTG诱导表达。IMAC方法纯化重组VHH抗体蛋白,Western Blot和ELISA检测与CD133的抗原结合性。[结果]从109淋巴细胞中构建了库容为1.4×109cfu的噬菌体展示文库,Western Blot及ELISA结果显示重复4次核苷酸序列的VHH-13的纳米抗体能与CD133-606有较好的结合活性。[结论]通过对VHH天然展示文库的亲和筛选及鉴定,成功获得了能与人重组CD133胞外区特异结合的纳米抗体。[ Objective ] It＇s to obtain a camel derived nanobody which recognized human CD133 antigen. [ Methods ] The peripheral blood lymphocytes were collected from three Bactrian camels and used for library construction. A naive VHH library was created from amplified eDNA by nested PCR. The antigen CD133 _6o6 one of CD133 ectodomain which expressed in E. coli were coated on ELISA plate for screening the VHH library. By three rounds of bio -panning,the resulted CD133 binders were identified with colony PCR and DNA sequencing. The dominant VHH clones were ligated into pET28a vector and expressed in E. coli. BL21 （ DE3 ） with IPTG induction. The expressed recombinant VHH proteins were purified by immobilized metal affinity chromatography （IMAC） and its binding activity with CD133 -606 were detected by Western Blot and ELISA. [ Results] A 1. 4 × 10^9 cfu sized phage display library was generated from 109 peripheral blood lymphocytes. The Western Blot and ELISA re- sults showed that VHH - 13 which holds four repeats in their nucleotide sequence respectively can specifically bound with CD133 [ Conclusion] The study confirmed that a nanobody capable of binding with recombinant human CD133 eetodomain can be produced through the creation and panning of naive VHH phage display library.

关 键 词：CD133 纳米抗体 天然噬菌体展示文库 亲和筛选 ELISA 

分 类 号：Q784[生物学—分子生物学]