Construction of eukaryotic expression vectors containing nucleosome Th epitope and CTLA4Ig genes

Objective To study the feasibility of CTLA4Ig as eukaryotic expression vector by fusing nucleosome Th epitope in CTLA4Ig cDNA. Methods Touchdown PCR was used to amplify CTLA4Ig cDNA and introduce nucleosome Th epitope. The PCR product was ligated into the multiple clone site of eukaryotic expression vector pcDNA3.1(+) by gene recombination technique. Then the recombinated plasmid pcDNA3.1(+)-CTLA4Ig-H2B was transfected into COS-7 cells using DOTAP. The expression of fusion protein in the supernatant of the cell disruption was detected with Western blotting. After the attenuated Salmonella Typhimurium SL7207 containing the recombinant expression plasmids pcDNA3.1(+)-CTLA4Ig-H2B immunized BALBc mice orally,spleens were removed to identify the recombinant proteins expression by immunohistochemistry. Results Restriction analysis and DNA sequence analysis showed that the nucleosome Th epitope and CTLA4Ig cDNA had been successfully inserted into pcDNA3.1(+) eukaryotic expression vector. The fusion protein could be detected in the supernatant of cell disruption 48 h after the transfection of pcDNA3.1(+)-CTLA4Ig-H2B. This protein specifically bound with human CTLA-4 monoclonal antibody. The fusion protein could be detected in the the cytoplasm of spleen cells. Conclusion The eukaryotic expression vector containing nucleosome Th epitope and CTLA4Ig gene is successfully constructed.