人源性抗-HBc单链噬菌体抗体库的构建

目的 构建人源性单链噬菌体抗体库，为筛选人源性抗—HBc单链抗体奠定基础。方法 利用逆转录—聚合酶链反应(RT—PCR)和噬菌体表面展示技术，直接从乙肝病毒核心抗体(抗—HBc)阳性患者淋巴细胞中提取总RNA，逆转录成cDNA；合成全套人抗体可变区引物扩增抗体可变区基因，并将重、轻链可变区基因进行拼接装配成单链抗体(ScFv)基因，重组于噬菌粒载体叶pHEN1，转化抑制型大肠埃希菌E.coliTG1，以辅助噬菌体援救后，构建成人源性单链噬菌体库。结果 成功地构建了人源性抗—HBc单链噬菌体库，库容量达10^6。结论 利用RT—PCR和噬菌体表面展示技术可以成功构建人源性单链抗体库，并达到建库标准，可进一步从中筛选人源性单链抗体。

Construction of a phage display library of human single-chain Fv antibodies from peripheral blood lymphocytes of people with positive serum antibody against hepatitis B virus core protein

Objective To construcr a phage display library of human single-chain Fv antibodies from peripheral blood lymphocytes of people with positive serum antibody against hepatitis B virus core protein. Methods The heavy chain and light chain variable region gene of human immunoglobulin were amplified individually by RT-PCR from peripheral blood Lymphocytes mRNA of patients with positive serum hepatitis B virus core protein antibody and randomly combined through a DNA linker encoding the peptide (Gly 4Ser) 3 to construct single-chain variable fragments (ScFv)gene. In the presence of helper phage M13KO7, the ScFv were displayed on the surface of recombinant phages. Results Repertoire antibody library was obtained with 10 6 clonies resistant to ampicillin and kanamycin. Conclusions A typical phage display library of human single-chain Fv antibodies has been constructed Using RT-polymerase chain reaction (RT-PCR) and phage display technique, human single-chain Fv antibodies can be screened from this library.