Cloning and expression of mouse endostatin gene and identification of its biological activity

AIM To clone and to express mouse endostatin gene and identification of in vitro activity of recombinant endostatin protein to explore new gene therapy methods for gliomas. METHODS The cDNA encoding mouse endostatin gene was amplified and cloned into vector pUC19 and was sequenced. Then the DNA fragment was cloned into expression vector pDH. The pDH Endo was induced at 42℃ to express the target protein. After the purification and refolding, the biological activity of the recombinant protein was identified by the CAM experiment. RESULTS The sequence of the amplified DNA fragment consisted with that of the known gene. After the induction, a new band corresponding to the 20 ku protein was observed in the bacteria carrying pDH Endo on SDS PAGE gel. Biological assay results indicated that the recombinant endostatin protein could suppress the new blood vessel formation in CAM in vitro . CONCLUSION The recombinant pDH Endo has been successfully constructed and highly expressed in Escherichia coli . In the CAM, the recombinant protein has high suppressive effects on the vessels in chick embryo chorioallantoic membrane.