Experimental Studies Of Fusion Protein MCRT/vGPCR In Antitumor Immunotheraphy

Objective In order to develop an universal technique that could make CRT-coating more efficiently in the tumor cells, in this study, a mouse CRT recombinant gene with virus G-protein coupled receptor (vGPCR) was constructed and cloned into vector pcDNA3.1(+). And then the plasmids pcDNA3.1(+)-mCRT/vGPCR were stably transfected into the mouse melanoma B16-F1 cells. Proliferation assay for B16-F1 cell lines transfected with exgenous genes were performed in vivo and in vitro. mCRT/vGPCR mediated phagocytosis was also investigated. When mCRT-vGPCR coated B16-F1 cells were used as a cell-antigen to immunize mice, the specific anti-tumor immune response against the homologous tumor cells was tested. This novel approach may provide a new possibility for CRT-mediated tumor immune prevention and treatment.Methods The full length CDS of vGPCR was amplified from plasmid pSG5/vGPCR by PCR technique, and the full-length CDS of mouse calreticulin (mCRT) was amplified by RT-PCR using total RNA derived from mouse B16-F1 cells as the template. Two PCR products were ligated first and then inserted into pcDNA3.1(+). The resulted plasmid pcDNA3.1(+)-mCRT/vGPCR was transfected into B16-F1 cells by LipofectamineTM 2000 reagent. Expression and localization of mCRT/vGPCR fusion protein in the transfected cells was identified by RT-PCR, Western blotting, flow cytometry (FCM) and cell immunofluorescrence (CIF). The possible implication of mCRT/vGPCR in the phagocytosis was investigated by FCM. The different cell lines were inoculated into Bbal/C mouse to test their proliferation rate in vivo. We also assayed the proliferation rate of different cell lines by MTT in vitro. Three different apoptotic B16-F1 cells were prepared and used as the cell antigens to inoculate animals, they are BENS-treated B16-F1, BENS-treated B16-mCRT/vGPCR and mitoxantrone-treated B16-F1. And then the inhibition effects of immuno-inoculation on the growth of B16-F1 live cells inoculated latterly were observed. ELISA assay was used to detect the cytokines in the experimental mouse serum.Results 1) Eukaryotic expression plasmid pcDNA3.1(+)-mCRT/vGPCR and pcDNA3.1(+)-vGPCR were constructed succeffuly. 2) A cell line (B16-mCRT/vGPCR) with recombinant mCRT/vGPCR coated on the cell surface was obtained. 3) mCRT/vGPCR on the surface of B16-F1 cells enhanced phagocytosis in vitro. 4) Overexpression of mCRT/vGPCR can decrease proliferation of B16-F1 in vivo while increase proliferation of B16-F1 in vitro. 5) Apoptotic B16-F1 cells coated with mCRT/vGPCR induced a specific antitumor immunological effect against homogeneous tumor cells in mice.Conclusion When mCRT-vGPCR coated B16-F1 cells were used as a cell-antigen to immunize mice, the specific anti-tumor immune response against the homologous tumor cells was initiated efficiently. This novel approach may provide a new possibility for CRT-mediated tumor immune prevention and treatment.