The Construction And Identification Of Molecular Adjuvants GM-CSF, MIP-1α And Melanoma DNA Vaccine

ObjectiveMalignant melanoma is a kind of malignant tumor,which is derived from the malignant melanocytes.Due to the discovery of melanoma-associated antigens including TRP2 and the phenomenon that spontaneous tumor regression occurs in some melanoma patients suggesting clearance of tumor cells by immune system,melanoma has become the model tumor for immunotherapy.However,the therapeutic effect of tumor vaccine is weak owing to the low immunogenicity of melanoma-associated antigens such as TRP2.The present study aims to construct the molecular adjuvants based on GM-CSF and MIP-1αeukaryotic expression vectors and melanoma DNA vaccine based on TRP2.All the constructs are verified in different systems.These studies will pave the way for further investigation of immunotherapy of melanoma and related tumors.MethodsThe cDNA of mouse GM-CSF and MIP-1αwere amplified by RT-PCR from the total RNA of spleen,respectively.The eukaryotic expression vectors for them were constructed by inserting the DNA fragments into pVAX1 plasmid.Meanwhile,the cDNA of TRP2 was amplified from the total RNA of B16 melanoma cells and ligated with pEGFP-N1 to construct the eukaryotic expression vector.All the cloned genes were confirmed by DNA sequencing. The expression of these plasmids was verified by transfection HeLa cells.GM-CSF expression was detected by ELISA and its biological activity was tested by bone marrow cell colony formation assay.The expression of TRP2 in HeLa cells was detected by Westem Blotting and its distribution was observed by laser scanning confocal microscopy.Results:1.The cDNAs encoding mouse GM-CSF,MIP-1αand TRP2 were cloned and the eukaryotic expression vectors were constructed successfully.DNA sequencing showed that both GM-CSF and MIP-1αhad identical sequence to that in the GenBank.The cDNA for TRP2 had a mutation at 1441 from T to C,which is corresponding to amino acid mutation at 481 from F to L,a conserved mutation that does not affect the dominant T cell epitope TRP2_180-188 (SVYDFFVWL).The peptide sequence from 1-23 was the potential signal peptide as identified by SignalIP while a potential transmembrane region from 473-493 was predicted by TMpred, which contains the F481L mutation.2.The GM-CSF eukaryotic vector was expressed in HeLa cells.The GM-CSF concentration in the supematant 24 h and 48 h after transfection was 6.71 ng/mL and 42.19 ng/mL respectively.The GM-CSF in the supernatant could markedly stimulate the proliferation and colony formation of bone marrow cells.Additionally,GM-CSF and MIP-1αvector caused the expansion of immune cells locally in vivo in the mice.3.After transfection of HeLa cells with TRP2 expression vector,Westem Blotting showed TRP2 expression in the cells.Confocal microscopy revealed that strong green fluorescence in the transfected cells and TRP2-EGFP appeared to be localized in the inner membrane system with focusing on some region,which was different from the evenly distribution of EGFP protein. Conclusion:The eukaryotic expression vectors for GM-CSF,MIP-1αand TRP2 are constructed successfully and can be expressed in HeLa cells.The expressed GM-CSF has significant biological activity while GM-CSF vector can enhance the expansion of immune cells in vivo. The MIP-1αvector can enhance the expansion of immune cells in vivo too.These results suggest that the expression vectors for GM-CSF and MIP-1αshould be adopted as molecular adjuvants in vivo,and TRP2 expression vector could be used as DNA vaccine for treating melanoma to do deep researches.