Construction of Wif1-IgG1/Fc Recombinant Vector and Characterization of Its Expression in CHO Cells.

Abstract

Wnt signalling dysregulation has been implicated in cancers, including lymphoma. The Initiation of Wnt signalling modulated by soluble Wnt ant agonists, including soluble frizzled related proteins, dickkopf proteins, and Wnt inhibitory factor-1 (Wif1). These natural inhibitors can be used directly as theraputic reagents for cancers. But the recombinant antagonist only give very low blood levels, are cleared rapidly from the body (only several hours) and must be administered daily via subcutaneous injection. Therefore, development of new forms of Wnts inhibitors with prolonged circulating half-life is of significant interest. Human IgG immunoglobulins have longer circulating half-life (21 days in humans). When fused with a Wnt inhibitor, its dimeric structure further increases the effective size and circulating half-life. Thus administration of vector plasmid may be every 2~3 week. Moreover, IgG Fc can interact with FcR of CTL, NK, MΦ, and complements and the target cells can be killed through ADCC and CDCC. We constructed a Wif1-IgG1/Fc expressing vector and expressed it in Chinese hamster ovum cells in order to seek effective methods to inhibit lymphoma growth. Wif1 and IgG/Fc cDNAs were amplified by RT-PCR from human lymphocyte and cloned into the eukaryotic expression vector pcDNA3.0 by direct cloning and the resultant recombinant plasmid pcDNA Wif1-IgG1/Fc was transfected into 293 cells with lipofectmin. The constitutively expressing cells were obtained by G418 screening. Expression of the fusion protein was confirmed by Western blot, the fusion protein was purified with affinity chromatography, and its' biological activity was examined by flow cytometry, MTT colorimetry and ELISA. Our results show that the fusion protein significantly upregulated Wif1 density and promoted molt3 cell apoptosis, which was time dependent. The molt3 cell apoptosis induced by the fusion protein increased in the presence of NK cells or complements. Thus, we have successfully constructed and functionally expressed Wif1-IgG1/Fc fusion protein and shown that the purified fusion protein could kill molt3 cells through apoptosis and by ADCC and CDCC.