Construction And Expression Of Mouse-human Chimeric Anti-human Melanoma Antibody

Malignant melanoma (MM) originate mainly from epithelial melanocytes, seen mostly on the skin, its episode is occupied the third among the cutis malignant tumor .MM is still on the increase worldwide. Recent years, immunotherapy and genetherapy for MM gain some elementary success. Effects of conventional treatment such as surgical removal,radiotherapy,and chemotherapy are far from satisfactory, Survival rate is very low. So,researchers pay close attention to early diagnosis and treatment of MM.Recent developments in genetic antibodies have made it possible to use engineered antibodies in immunotherapy. Some mouse mAbs had been used in diognose and immunotherapy of malignant cancer. But the immunogenicity of these antibodies greatly limited their clinical applications. In recent years, several kinds of engineered antibodies, especially the chimeric antibodies, reshaped antibody,single chain Fv(ScFv), were constructed in order to reduce the immunogenicity and to facilitate clinical applications. The studies about genetically engineered antibodies improve targeted therapy of MM,therefore genetically engineering antibodies show a good future.Here we report a study on constructing eukaryotic expression vector for anti-human melanoma chimeric antibody, and analysing the expression of the constructed gene in eukaryotic cell.A set of oligonucleotide primers were designed and used to amplify the Variable regions (V_H and V_L gene) of the murine antibody from the anti-human melanoma hybridoma cell line HB8759, HB8760, Mel3 by RT-PCR. The products were cloned into T-easy vector . The six genes were sequenced and compared with those published genes in EMBL genbank. Then they were inserted into the chimeric antibody eukaryotic expression vector pMH-CA. With the constructed vector transfected transiently into COS-7 cell line to express respectively. The expression of chimeric antibody was detected by RT-PCR, ELISA, and Western blot. Then X63 cell lines were established to express chimeric antibody stably.The results showed that the coding sequences of V_h, V_l were correct. They were homologous with the published mouse antibody variable region gene sequences. The V_h and V_l genes were 360bp and 330bp respectively and both of them were capable of encoding 120 and 110 amino acids. The deduced ammo acid sequences contained four FRs,three CDRs and two cysteine residues necessary for the maintenance of the antibody structure,indicating that they were functionally rearranged. Then, restriction enzyme digestion analysis confirmed that eukaryotic expression vectors of the chimeric antibody against human melanoma were successfully constructed. The expression of chimeric genes were detectable by RT-PCR, ELISA and Western blot after being transfected transiently in COS-7 cell line. The secretive expression of chimeric antibodies were confirmed by SDS-PAGE and Western Blot analysis in cell condensed supernatant. After being screened by G418, X63 cell lines were established to express chimeric antibody stably, as well as the binding specificity of the chimeric antibody with antigen of melanoma was confirmed by indirect immunofluorescence .