与肝癌细胞系HepG2特异性结合单链抗体的筛选与序列分析  

Identification and sequence analysis of scFvs binding specifically to HepG2

作　　者：余冰[1] 倪明[2] 雷萍[1] 李文涵[1] 朱慧芬[1] 张悦[1] 唐珍洁 程欣[1] 沈关心[1] 

机构地区：[1]华中科技大学同济医学院免疫学系,武汉430030 [2]华中科技大学同济医院感染科,武汉430030

出　　处：《中国免疫学杂志》2005年第3期179-182,共4页Chinese Journal of Immunology

基　　金：国家重点基础研究发展规化基金资助(2002CB513109)

摘　　要：目的从人源噬菌体抗体库中筛选与肝癌细胞系HepG2特异性结合的单链抗体,为寻找肝癌细胞表面特异性标志及靶向研究奠定基础。方法以正常肝细胞系L02为阴性筛选细胞,以肝癌细胞系HepG2为阳性筛选靶细胞,从人源噬菌体抗体库(Griffin.1Library)经三轮筛选后,阳性菌质粒PCR确定其中含有单链抗体基因的克隆,通过细胞ELISA及FCM筛选特异性的单链抗体噬菌体,通过ABI3730全自动荧光测序仪测序,并通过GeneBank比对进行同源性分析,IPTG诱导可溶性单链抗体表达,并检测其与肝癌细胞株结合的特异性。结果获得了2个特异性较高的阳性噬菌体单个克隆。经DNA测序后,在Genebank中与人的免疫球蛋白库进行比对,并用IMGTVQuest软件进行分析,确定为2个插入序列不同的单链抗体片段。经细胞ELISA证明其阳性克隆菌培养上清可与肝癌细胞株特异性结合。获得的序列成功登陆Genebank,序列号为AY686498AY686499。结论从人源噬菌体抗体库中筛选到与肝癌细胞系HepG2特异性结合的具有功能活性的单链抗体,为进一步寻找肝癌细胞表面特异性抗原及靶向研究奠定了基础。Objective:To identify the specific antibodies binding to HepG2 by biopanning from a large human naive scFv phage library.To find a potential direct and broadly applicable route to identify human antibodies suitable for antitumor therapy study.Methods:A large human naive scFv phage library was used to search for specific targets by biopanning with hepatocellular carcinomas cell line, HepG2, and counter-selecting with a liver cell line, L02.After three rounds of biopanning, single clones bound selectively to HepG2 cells. PCR was carried out for identification of the positive clones which combined with scFv gene sequence. High affinity and specificity positive clones were identified by ELISA and FCM assay. DNA sequencing of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3730, and their homologous sequences were searched in GeneBank and analyzed by using IMGT/V-Quest tools. The expression of positive scFv antibody fragments were induced by IPTG and their binding activity to HepG2 were detected by ELISA.Results:Two different positive clones were chosen and were regarded as target of HepG2. The functional variable region sequences were identified by IMGT/V-Quest tools.Conclusion:The phage library biopanning combined with expression cloning permits identification of specific antibody ligand to HepG2 and affords experiment evidence to search for applicable route for antitumor therapy study. [

关 键 词：人源噬菌体抗体库 单链抗体 阴性筛选 阳性筛选 生物淘洗 

分 类 号：R733[医药卫生—肿瘤]