Generation and expression of a recombinant singleª²chain Fv antibody fragment against hepatocellular carcinoma

    CHEN Wei, LI Xu, WANG Ying, ZHANG Liª²Ping

    Center for Laboratory Medicine, the First Affiliated Hospital, Medical College, Xi­¸an JiaoTong University, Xi¬ðan 710061, China

    ¡¾Abstract¡¿ AIM: To generate and express a singleª²chain Fv (ScFv) fragment from the HCD78 hybridoma, which produced a monoclonal antibody against hepatocellular carcinoma (HCC). METHODS: VH and VL gene segments were generated from the HCD78 hybridoma by reverse transcribedª²polymerase chain reaction (RTª²PCR). The ScFv was arranged in VH¨CVL orientation, being joined together with a 15ª²aminoª²acid (Gly4Ser)3 linker. The ScFv encoding sequence was amplified and cloned into pMD18ª²T vector. Amplification of the resultant ScFv gene was performed before it was introduced into E.coli TG1 and HB 2151 via a phagemid vector pCANTAB5E.1. Sodium dodecyl sulfateª²polyacrylamide gel electrophoresis (SDSª²PAGE) and immunohistochemistry analysis were used to determine the characteristics of the soluble ScFv . Results DNA Sequencing proved that the HCD78 ScFv gene was correctly cloned into expression vector. SDSª²PAGE analysis of the recombinant antibody revealed that a protein with apparent molecular weight of approximately 30,000 was successfully expressed in E.coli HB2151. The ScFv antibody showed a specific reaction with HCª²108, HepGª²2 and SMMCª²7721 hepatocellular carcinoma cells by immunohistochemical assay. Conclusions The HCD78 ScFv is successfully constructed and expressed, which has the potential to be used as an agent in the research and clinical management of hepatocellular carcinoma. ......