Construction And Expression Of DNA Vaccine Vector PVAX1-CAIX-GM Of Renal Cell Carcinoma

Objective:The present study aims to construct a DNA vaccine vector containing carbonic anhydrose IX (CAIX) gene and human granulocyte-macrophage colony stimulating factor(hGM-CSF), detect the protein expression in vitro by these expression plasmids in eukaryotic cells. These studies will pave the way for further study on novel DNA vaccine against renal cell carcinoma.Methods:The total RNA were extracted from renal cell carcinoma tissue, and gene segments encoding the CAIX protein were amplified using RT-PCR. We used plasmid pORF-hGM-CSF as template to amplify hGM-CSF using routine PCR. Both PCR products were cloned into eukaryotic vector pVAX1 respectively to construct a recombinant plasmid pVAX1-CAIX-GM by molecular-biological methods. The recombinant plasmids were analyzed by sequencing and the one containing the correct fragment was transfected into HEK293 cells. The protein translated from recombinant plasmids in eukaryotic cells were detected by immunocytochemistry methods, atomic force microscopy and Western Blotting.Results:The results of PCR, restriction endonuc lease cutting and DNA sequencing analysis confirmed that recombinant plasmid pVAX1-CAIX-GM were successfully constructed. CAIX positive-expression cells were observed by immunocytochemistry and atomic force microscopy. The protein which can bind with CAIX antibody specially were detected after recombinant plasmid was transfected into HEK293 cells.Conclusion:Successfully construct DNA vaccine vector pVAX1-CAIX-GM of renal cell carcinoma, and these recombinant vector have normal function of translation in eukaryotic cells in vitro, lays the foundation for the further research on immune response characteristics and protective immunity of such DNA vaccine.