人源性抗HBc单链噬菌体抗体库的构建

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

利用 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,反转录成 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 pHEN1,转化抑制型大肠杆菌 E.coliTG1,以辅助噬菌体援救后,构建成人源性单链噬菌体库。库容量达106。

Using RT-polymerase chain reaction(RT-PCR)and phage display technique we constructed a phage display library of human single-chain Fv antibodies.The heavy chain and light chain variable region gene of human immunoglobulin were amplified individu- ally 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 encod- ing the peptide(Gly4Ser)3 to construct single-chaiu variable fragments(ScFv)gene. These ScFv gene degested with restriction enzyme Sfi Ⅰ and Not Ⅰ were cloned into pHENl vector and transformed into E.coil TGl.In the presence of helper phage M13KO7,the ScFv were displayed on the surface of recombinant phages.Repertoire antibody library was obtained with 10~7 clonies resist to ampicillin and kanamycin.