The expression of anti-bacterial peptide cecropin gene in COS-7 cells and the preliminary study on the activities of its gene product

To investigate the expression of anti-bacterial peptide cecropin in COS-7 cells, the open reading frame (ORF) sequence of cecropin was amplified at the C-terminus containing 6 x His-marker with two specific primer designed with cecropin gene as template. The gene sequences were recombinant with eukaryotic expression vector pcDNA 3.1(+) after being identified correctly, to construct the recombinant plasmid pcDNA 3.1(+)/cecropin-His-6. Thereafter, the liposome lipofectaminerTM2 000 was used as vector, and COS-7 cells were transfected with recombinant plasmid pcDNA 3.1(+)/cecropin-His-6 and blank vector pcDNA3.1(+). The normal cells were used as controls. Meanwhile, the supernatants were collected for purification by His-Trap HP affinity chromatograohy column and its bacteriocidal activity was detected 72 hours later. It was demonstrated that the purified product of the cell culture supernatants transfected with the recombinant plasmid pcDNA 3.1(+)/cecropin-His-6 showed only a single target band as demonstrated in the tricine-SDS-PAGE electrophoresis,and it was consistent with the expected molecular weight of the cecropin-His-6 fusion protein. The supernatants of cell cultures transfected with this recombinant plasmid exhibited bacteriocidal activity to E.coli K12D31 in comparison with that from the normal cells and cells transfected with blank vector. It is concluded that the cDNA of cecropin-Hia-6 can be successfully in COS-7 cells,thus providing the basis for further study of its eukaryotic expression.