Subcloning, Expressing, Preparing Polycolonal Antibody Of HES And Primary Study Of It's Anti-Angiogenic Activity

Cancers threaten severely to man' heath. The growth and metastasis of cancers depend on angiogenesis, which means the development of new blood vessels from preexisting ones. Inhibition of angiogenesis can suppress tumor growth. Endostatin , an endogenous inhibitor of angiogenesis and tumor growth had been discovered by Folkman and O'Reilly in 1997, which is a fragment of the C-terminal of collagen XVIII. The protein of endostatin is consist of 184 amino acid residues, composing 16 acidic amino acids, 29 alkaline amino acids and 4 cysteines, hydrophobe amino acids acount for 42 percent too. The pI of it is 9.3 . Endostatin is an endogenous and effective inhibitor to form capillaries, and especially regress the proliferation of endothelial cells.It suppesses tumors from varieties of tissues to growand metastasize without drug-resistance.Our task was to subclone endostatin gene which was cloned from human embryo liver and express endostatin protein in prokaryote cells in fusion protein with genetic engineering technique, detect the anti-angiogenesic biological activity of recombinant human endostatin, and prepare high titer and especial polycolonal antibody of endostatin . We have obtained human endostatin gene from recombinant clonal vector pT-hES and constructed fusion expressional vector pGEX-hES by subcloning technique, transformed the recombinant expressional plasmid vector into the Escherichia coli BL 21 with the routinal CaCl2 methods, induced recombinant bacteria to express human endostatin by IPTG. Inclusion body of fusion protein GST-hES crudely purified was used to immune rabbit to generate polyclonal antibody, by which detected the expression of endostatin in varieties of tissues of murine using Western-blotting and identified the especialty of the polyclonal antibody to endogenous endostatin. We renatured endostatin inclusion body by density and pH gradual dialysis, and used affinity chromatographic column of Glutahione Sepharose 4B beads to purify fusion protein GST-hES, GST-hES was digested by thrombin to produce t endostatin. The anti-angiogenesis activity of the protein was tested in vitro and in vivo byendothelial cells(ECV304)proliferation inhibitory assay and chick embryo chorio-allantoic membrane (CAM)assay respectively. The fusion protein GST-hES expressed in Escherichia coli BL 21 was insoluble inclusion body, the mass of GST-hES in total protein of the recombinant bacteria accounted for 48.5 percent. Soluble fusion protein was obtained by renaturing and the purity was up to 90.5 percent. Recombinant endostatin was collected after fusion protein GST-hES was digested by thrombin and the moleculor weight was about 20kDa. Biological activity tests showed that recombinant endostatin suppessed endothelial cells growth dramatically in vitro, the cells numember was reduced obviously when the dose of endostatin was over 100μg/ml. Endostatin regressed neovascularisation of chick embryo chorio-allantoic membrane stimulated by VEGF to 30 percent, all of the above indicated recombinant endostatin had activities to suppess endothelial cells growth and anti-angiogenesis in vitro and in vivo respectively. The antibody we prepared had detected different expression in different tissues of murine, which showed the antibody recognized endogenous endostatin especially.The results of our research showed that the recombinant endostatin has functions to suppress the proliferation of endothelial cells and neovascularisation in vitro and in vivo, and indicated it's potent in treat ofsolid tumors and diseases associated with angiogenesis. The successful expression of human endostatin and the preparation of antibody lay the foundation for the anti-angiogenesis therapy and diagnosis of solid tumors.