Construction of recombinant plasmid with human endostatin cDNA in high-biosafety

Objective: To construct a high-biosafety eukaryotic expression vector carrying human endostatin cDNA and exploit the potential of gene therapy. Methods: pVAX1, which was authorized by FDA to be used in clinical trial, was taken as the expression vector. Endostatin gene with signal sequence of human IgGγ chain was amplified by PCR. The PCR products digested by EcoRI and KpnI were cloned into pVAX1 to construct a recombinant plasmid, named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI, PCR and DNA sequencing. Then pVAX-sEN was transfected into HepG2 cells by Lipofectamine 2000. The expression of Endostatin in HepG2 was detected by RT-PCR and ELISA. Results: Endostatin gene together with signal sequence was amplified by PCR. EcoRI/KpnI, PCR and DNA sequencing indicated the recombinant plasmid was correctly cloned. The recombinant plasmid was transfected into HepG2 cells. Seventy-two hours after the transfection the HepG2 cells and cell culture medium were collected. RT-PCR showed the Endostatin mRNA was expressed. The highest concentration of endostatin in culture medium was 172.34ng/ml. Conclusion: The high-biosafety recombinant plasmid pVAX-sEN was successfully constructed which lays a foundation for further research on gene therapy for tumor using anti-angiogenesis agents.