Cloning And Expression Of Mouse Endostatin Gene

The growth of neoplasms is associated with induced angiogenesis. The new vasculature provides the needed oxygen and nutrients for the tumor cells. Without the formation of new blood vessels,tumors cannot expand beyond a few cubic millimeters, and that the rate of tumor growth correlates with the formation of new blood vessels.Neovascularization may also be a critical role of tumor metastasis since it enhances entry of tumor cells into the circulation.Consequently,highly vascular tumors may have the potential to produce metastases at a higher rate than less angiogenic tumors. So,by inhibiting the neovascularization to prevent the tumor growth and metastasis is a strategy for tumor therapy.Endostatin was isolated firstly from the conditioned media of a non-metastatic murine hemagioendothelioma cell line EOMA by O'Reilly in 1997. Endostatin is a 20KD C-terminal fragment of collagen XVH3. The generation of endostatin is catalyzed by proteolytic enzymes, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. The target of endostatin is endothelia of the blood vessel, inhibiting the endothelial cell proliferation and migration , demonstrating an intense angiogenic activity. Studies of the crystal structure of endostatin have shown a compact globular fold,with one face particularly rich in arginine residues acting as a heparin-binding epitope, this site was recently shown to be involved in the inhibition of induced angiogenesis.Experimental studies show that recombinant endostatin specifically inhibits the proliferation of endothelial cells in a dosedependent fashion.Recombinant endostatin from bacteria is largely insoluble,but still efficient in arresting tumor growth after injection into mice.Intermittent therapy with recombinant bacterially produced endostatin reduces several experimental tumors,including Lewis lung carcinoma,to a dormant state.No sign of drug induced resistance has been reported and,in the original study,the treatment dormancy appeared to persist even when therapy was discontinued.Sowe regard endostatin as a promising anti-tumor drug.The purpose of my work was to clone and express of mouse endostatin gene, We construct a temperature-sensitive expression system: pBV220-endostatin. The main work was as following:1. Total RNA was extracted from hepatic cells of mouse.A EcoRI and BamHI restriction sites were introduced into endostatin gene at specific primer F R, and endostatin was amplified by RT-PCR,this endostatin gene contained BamHI and EcoRI restriction sites at its 5' and 3' ends respectively.2. EcoRI / BamHI digested PCR products were inserted into the corresponding restriction site in the expression plasmid pBV220. Construction of non-fusion expression plasmid pBV220-endostatin.3. pBV220-endostatin was transformed into E.coli DH5a. The positive colony was screened and identified by PCR and restriction endonuclease digestion.4. The seguence of cloned endostatin gene was confirmed with the dideoxy chain-termination method and is consistent with reported sequence of mouse endostatin.5. The recombinant mouse endostatin gene was expressed in DH5a at under the condition of temperature induction.6. Detect exprssion with SDS-PAGE.7. Purification of the recombinant expressed endostatinIn this work we have successfully cloned mouse endostatin gene ,and constructed pBV220-endostatin expression plasmid That is a non-fusion expression vector. The positive pBV220-endostatin was transformed into DH5a,and expressed mouse endostatin protein successfully.This study is the basis of the endostatin gene-engineering production.