Abstract:

Objective To establish a stable cell line expressing transmembrane form of human blood group A antigen mimotope vaccine by transfecting malignant melanoma cell line B16, and to detect the cytotoxicity of the vaccine against melanoma cells. Methods Cultured B16 cells were classified into 4 groups, i.e., P/F-M-pIRES group ［transfected with the recombinant plasmid mimotope peptide/Fas-macrophage inflammatory protein （Mip）-pIRES］, P/F-pIRES group （transfected with the recombinant plasmid mimotope peptide/Fas-pIRES）, M-pIRES group （transfected with the recombinant plasmid Mip-pIRES）, and pIRES group （transfected with the empty plasmid pIRES）. B16 cells were transfected through Lipofectamine 2000. Subsequently, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of the mimotope peptide/Fas fusion gene and Mip3β in transfected B16 cells. Cell counting kit-8 （CCK-8） was used to evaluate the vaccine-mediated complement dependent cytotoxicity （CDC） and antibody-dependent cell-mediated cytotoxicity （ADCC） against B16 cells. Results RT-PCR yielded specific DNA fragments with expected size. Western blotting revealed the anti-A antibody-binding activity of the recombinant mimotope peptide/Fas fusion protein. Factor analysis indicated significant differences in CDC （F = 244.522, P < 0.01） and ADCC （F = 71.593, P < 0.01） against B16 cells between the 4 groups. Group comparisons demonstrated more intense CDC and ADCC in P/F-M-pIRES and P/F-pIRES groups compared with M-pIRES and pIRES groups, stronger ADCC in P/F-M-pIRES group in comparison with P/F-pIRES group （F = 15.42, P < 0.05）, but no significant difference in CDC was observed between M-pIRES and pIRES group. Conclusions The transmembrane form of human blood group A antigen mimotope vaccine could be stably expressed in B16 cells, and mediate ADCC and CDC against B16 cells in vitro.

Key words: Malignant melanoma