抗人Her2抗原羊驼纳米抗体的筛选与鉴定  

Generation of an alpaca derived nanobody recognizing human Her2 antigen

作　　者：王兰东 刘枫 成岩 李大磊 韩冰 张淑敏[1,3] 冯东晓[1,2] 

机构地区：[1]滨州医学院药学院,山东烟台264000 [2]山东绿叶制药有限公司,山东烟台264000 [3]山东国际生物科技园发展有限公司,山东烟台264000

出　　处：《军事医学》2017年第3期184-189,共6页Military Medical Sciences

摘　　要：目的获得靶向人Her2纳米抗体。方法使用重组人Her2蛋白免疫羊驼,分离免疫后羊驼的淋巴细胞提取总RNA,利用PCR技术扩增羊驼Ig G的可变区(VHH)基因片段,构建噬菌体表面展示的纳米抗体文库,采用Her2抗原对文库进行亲和筛选,将筛选到的克隆转化大肠杆菌BL21(DE3),IPTG诱导表达重组蛋白,镍离子亲和层析柱纯化获得纳米抗体并用ELISA技术和Biacore检测其与抗原Her2的亲和力,采用免疫组化方法检测Her2阳性乳腺癌组织中Her2的表达。结果第二轮筛选后得到2个抗体H3和H5,ELISA结果显示两者都具有抗原结合活性,其中H5的亲和力达8.106×10^(-10)mol/L。H5可识别乳腺癌组织中Her2抗原的表达。结论从Her2免疫的羊驼体内,通过噬菌体展示技术筛选获得了1个具有较高抗原结合活性的抗Her2纳米抗体,可用于乳腺癌的病理诊断。Objective To obtain alpaca single domain antibody targeting Her2. Methods An alpaca was immunized with human recombination Her2 protein mixed with Freund＇s adjuvant. Total RNA was extracted from the alpaca＇s blood and was used to synthesize first strand cDNA. Single domain antibody variable region （VHH） gene of the alpaca was amplified by PCR and cloned into pMES4 vector for library construction. After screening, E. coli BL21 （ DE3 ） was transformed with selected clones and was induced with IPTG for the expression of recombinant proteins. The nanobody was purified by nickel ion affinity chromatography column. The affinity of the nanobodies to Her2 was tested. Results After the second round of screening, two antibody clones were selected, H3 and H5. The affinity of H5 was 8. 106×10^-10mol/L. Histochemistry results showed that H5 could recognize Her2 antigen in breast tumor tissue. Conclusion An Her2 specific nanobody derived from alpaca is obtained through phage display library screening, which can recognize human Her2 antibody in human breast tumor tissue.

关 键 词：Her2 纳米抗体 噬菌体展示技术 乳腺肿瘤 诊断 

分 类 号：R737.9[医药卫生—肿瘤]