Anti - Cd28 Scfv - C_h3 - Kccysl Molecular Cloning, Expression And Activity Of Identification

In this study, the anti-CD28scFv was humanized with human IgG1CH3domain, and s-C gene was connected with oligopeptide KCCYSL, then the combinanted gene s-C-K was obtained. The fusion protein s-C-K had double functions of inducing proliferation of T cells and targeting Her2-overexpressing tumor.An anti-CD28scFv was selected using ribosome display technology in our previous study. But it will be cleared rapidly in vivo and hard to identify in the absence of Fc fragment. In this study, total RNA was isolated from mononuclearcells of human peripheral blood, then, IgG1CH3gene was amplified by reverse transcription-PCR (RT-PCR). The amplified CH3gene was connected with anti-CD28scFv by SOE PCR.Her2-targeting KCCYSL gene was joined with anti-CD28scFv-CH3by SOE PCR in order to add tumor targeting function. The fusion gene was inserted into vector pET43.1a, expressed in BL21(DE3) E.coil,and the fusion protein was purified by metal chelate affinity chromatography (Ni-NTA). We also produced C-K and s-C proteins as negative controls for accurate function determination. The fusion protein s-C-K showed high affinity to extracellular region of CD28through indirect ELISA and satisfactory proliferation effect on peripheral blood T cells (PBTC) by MTT. Otherwise, it also exhibited high affinity to SK-OV-3cells with cell ELISA. Anti-CD28scFv-CH3-KCCYSL may provide the basis for the development of tumor diagnosis and immunotherapeutic strategies with the double functions of T cell proliferation and SK-OV-3targeting.