抗乙型肝炎病毒表面抗原人单链可变区片段抗体的筛选与鉴定

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen.

作者信息

Zhang Jian-Lin, Gou Jian-Jin, Zhang Zi-Yan, Jing Yi-Xin, Zhang Lin, Guo Rui, Yan Ping, Cheng Niu-Liang, Niu Bo, Xie Jun

机构信息

Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001,China.

出版信息

Hepatobiliary Pancreat Dis Int. 2006 May;5(2):237-41.

PMID:16698583

Abstract

BACKGROUND

Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable (ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator.

METHODS

mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg, the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay (ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE.

RESULTS

The variable heavy (VH) and variable light (VL) and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 x 10(6) and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa.

CONCLUSION

The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

摘要

背景

噬菌体展示技术已成为旨在鉴定与特定靶标结合分子的研究中的重要工具。它能够快速生成并筛选出针对基本上任何适合抗体治疗的疾病靶标的高亲和力、全人源抗体候选产品。在本研究中，我们通过噬菌体展示技术制备了针对乙型肝炎病毒表面抗原（HBsAg）的重组单链可变片段（ScFv）抗体，以获得一种病毒靶向介质。

方法

从一名健康志愿者的B淋巴细胞中分离mRNA并转化为cDNA。分别扩增重链和轻链的可变片段，并通过聚合酶链反应与特殊构建的DNA接头组装成ScFv DNA。将ScFv DNA连接到噬菌粒载体pCANTAB5E中，连接后的样品转化到感受态大肠杆菌TG1中。用M13K07辅助噬菌体感染转化细胞以形成人重组噬菌体抗体库。通过菌落计数和限制性分析评估文库的库容和重组率。用HBsAg进行两轮淘选后，通过酶联免疫吸附测定（ELISA）从富集的噬菌体克隆中筛选出展示抗体ScFv的噬菌体克隆。通过竞争ELISA检测阳性克隆的抗原结合亲和力。用竞争ELISA证实的阳性噬菌体克隆转染HB2151大肠杆菌以产生可溶性形式的抗HBsAg ScFv。用ELISA检测可溶性抗HBsAg ScFv的抗原结合亲和力。最后，通过SDS-PAGE测定可溶性抗HBsAg ScFv的相对分子质量。

结果

重链可变区（VH）、轻链可变区（VL）和ScFv DNA分别约为340 bp、320 bp和750 bp。文库库容高达2×10⁶，10个随机克隆中有8个是重组体。两个噬菌体克隆能与原始HBsAb强烈竞争结合HBsAg。在两个强阳性噬菌体克隆中，发现来自一个克隆的可溶性抗HBsAg ScFv具有与HBsAg的结合活性。SDS-PAGE显示可溶性抗HBsAg ScFv的相对分子质量为32 kDa。