噬菌体表面展示技术筛选HCBP6人源单链可变区抗体  

Screen for human single chain variable region in antibody against human hepatitis C virus core protein binding protein 6

作　　者：钟彦伟[1] 成军[1] 张忠东[1] 孙敏[1] 李强[1] 李克[1] 王琳[1] 李莉[1] 张玲霞[1] 陈菊梅[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心

出　　处：《世界华人消化杂志》2003年第4期389-393,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金;No.C39970674;No.C03011402

摘　　要：目的:制备丙型肝炎病毒(HCV)核心蛋白的肝细胞结合蛋白6(HCBP6)的特异性人源化单链可变区抗体(scFv)。方法:采用噬菌体表面展示技术,将生物合成的应用酵母双杂交技术筛选得到的HCV核心蛋白的肝细胞结合蛋白6(HCBP6)16肽固相包被于Nunc板,从噬菌体单链可变区抗体库中经过5轮“黏附-洗脱-扩增”筛选过程,随机挑选出48个克隆,利用酶联免疫黏附法(ELISA)、交叉反应和竞争抑制实验,对其进行免疫学检测,获得与HCBP6结合活性较强的单链可变区抗体片段的阳性克隆,并对HCBP6特异性scFv的编码序列进行序列测定分析。结果:对噬菌体单链可变区抗体库经过5轮“黏附-洗脱-扩增”的筛选后,结合到包被平皿的噬菌体与第一轮相比,富集了162倍。用酶联免疫黏附法测定第5轮筛选后上清液中含有的噬菌体抗体与HCBP6结合活性。其中有30株克隆ELISA的吸光度(A450 nm)值较高(P8 nm 0.77,P240.714,P26 0.728,P29 0.723,P38 0.803,P39 0.762,P43 0.747等)。对这些噬菌体抗体进行与牛血清白蛋白(BSA)的交叉反应后,确定其中有7株交叉反应较弱(P80.145,P24 0.119,P26 0.17,P29 0.186,P38 0.118,P39 0.138,P43 0.178),结合2次ELISA重复实验的/4值及竞争抑制实验结果,最后确定1株(P24)阳性克隆。提取质粒,SfiI/NotI双酶切后,将片段亚克隆到pCANTABSE载体,进行DNA序列测定,DNA大小为771 bp,符合人源化单链可变区抗体典型的骨架区(FR)及互补决定区(CDR)的序列结构。结论:用噬菌体抗体库技术能够成功地获得HCBP6的scFv。AIM:TO screen human single chain variable fragment (scFv) in antibody against hepatitis C virus (HCV) core proteinbinding protein 6 (HCBP6) by phage display technique to determine HCBP6 in human liver tissue. METHODS:The semisynthetic phage antibody library was panned by HCBP6 peptide which was coated on a microtiter plate, after five rounds of bio-panning, 48 clones specific for HCBP6 were determined with the enzyme-linked immunosorbent assay (ELISA). The specificity of HCBP6 scFv was identified by ELISA, cross reaction with bovine serum albumin (BSA) and competition inhibition assay. The DNA sequence of the positive clone was determined. RESULTS:Specific phage antibody against HCBP6 was enriched by 162 times after five rounds of panning with HCBP6 peptide. Binding activities of 48 clones with HCBP6 peptide were determined by ELISA, and the results indicated that 30 clones could bind to HCBP6 peptide, 7 clones had low absorbance value at A450 nm; Direct and competition inhibition ELISA showed that one clone named P24, exhibited specific binding to HCBP6 peptide. Data of DNA sequence showed that the scFv gene encoded 771 bp. CONCLUSION:The scFv fragments to HCBP6 can be successfully selected by phage display technique, which paves a way for study of distribution of HCBP6 in human liver tissues.

关 键 词：丙型肝炎病毒 核心蛋白 肝细胞结合蛋白6 特异性人源化单链可变区抗体 噬菌体表面展示技术 酵母双杂交技术 

分 类 号：R392[医药卫生—免疫学]