关键词: γδ TCR, 单链抗体, 原核表达, 生物学功能

Abstract: Objective To construct and express the single chain variable fragment ( Fv) of a monoclonal antibody against human γδ TCR which can stimulate proliferation of human γδ T cells．Methods The genes of the heavy (VH) and light chains (VL) of antibody were cloned by RT-PCR from a hybfidoma cell line G5-4，which secreted the monoclonal antibody (mAb) against human γδ TCR．Overlapping extension PCR was used to connect the VH and VL genes with a short linker encoding (Gly4Ser)3 to construct the anti-hγδTCR-ScFv gene G5-4ScFv. Recombinantplasmid pET22b(＋)-G5-4ScFv was constructed by inserting G5-4ScFv fragment into the prokaryotic expression vector pET22b(＋). The anti-hγδTCR-ScFv (G5-4ScFv) was expressed in E.coli TransB(DE3) by induction with IPTG and purified with Nickel-affinity chromatography column. Then G5-4ScFv was analyzed by SDS-PAGE and Western-blot assay. Its binding ability to human γδ T cells was measured by flow cytometry. The purity and biological characteristics including cytokine secretion and cytotoxicity of γδ T cells expanded by G5-4ScFv stimulation, were detected by flow cytometry and LDH methods．Results G5 -4ScFv showed a molecular weight of 30ku in SDS-PAGE. G5-4ScFv binded to γδ T cells and greatly inhibited the binding of a pan-γδTCR antibody with γδ T cells. The purity of γδ T cells expanded by G5-4ScFv stimulation was up to 90% after culture of 2 weeks. These γδ T cells exhibited IFN-γ and TNF-α secretion ability and strong cytotoxicity against Daudi cells. Conclusion Anti-hγδTCR-ScFv with biological functions has been successfully constructed and expressed in prokaryotic expression system, which provides the basis for further studying on its potential application in tumor immunotherapy.

Key words: γδ TCR, ScFv, Eukaryotic expression, biological function