Cloning and expression of human Endostatin cDNA in HT1080 cells

Objective To construct recombination eukaryotic expression vector pcDNA3.1(-)/SEND. Methods Human Endostatin cDNA was obtained from the human liver cDNA library by PCR, and a fragment comprising human insulin signal peptide coding sequence fused to the human Endostatin cDNA was produced by PCR. After being verified by sequence, the recombination eukaryotic expression vector pcDNA3.1(-)/SEND was constructed. The plasmid pcDNA3.1(-)/SEND was transferred into HT1080 cells and positive clones were obtained after being selected by G418. The expression of Endostatin was verified by RT PCR and Western blot. Results The human Endostatin cDNA and human insulin signal peptide coding sequence fused to the human Endostatin cDNA were verified by sequence; RT PCR indicated the transcription of Endostatin in the transfected HT1080 cells and Western blot detected that the Endostatin protein, about 20kD in size, in the supernatant of HT1080 cells was transfected by pcDNA3.1(-)/SEND. Conclusion The expressed Endostatin protein was secreted into the supernatant of HT1080 cells transfected by pcDNA3.1(-)/SEND.