Gene Cloning, Expression And Activity Study Of Human Angiopoietin-1

Angiopoietin-1 (Ang-1) is a secreted glycoprotein, which functions as a cell growth factor by binding to the tyrosine kinase receptor-Tie2, leading to the phosphorylation of Tie2, and thus induces a series of biochemical reactions. hAng-1 plays an important role in many physiological and pathological processes, such as blood vessel formation, wound healing and tumor growth. The full length protein of hAng-1 consists of 498 amino acids, with a signal peptide at the N-terminal. The C-terminal region is a fibrinogen-like domain (FD), which is a determinant part of its biological activity. The coiled-coil region between the N and C terminals can polymerize Ang-1 molecule.In this research, the hAng-1 gene was amplified from marrow of a normal individual by RT-PCR, and then inserted into pMD18-T Vector. Sequence analysis showed the cloned hAng-1 gene was identical to the published one. In order to achieve prokaryotic and eukaryotic expression of hAng-1, the expression plasmids pQE31 Ang-1 and B7Ang-1 were constructed respectively.In prokaryotic expression system, the hAng-1 was expressed in form of inclusion body. The recombinant protein was purified by means of affinity chromatography on Ni-NTA columns. After refolding procedures, the recombinant hAng-1 displayed biological activity in the test of CAM blood vessel formation,2.5ug of recombinant protein could effectively stimulate the formation of CAM blood vessel.We prepared a polyclonal antibody specific to hAng-1 by immunizing normal rabbits with the purified E.coli-expressed hAng-1 protein. The polyclonal antibody was measured by ELISA assay, and the titer of antiserum was 1: 1 X 106.In order to select high-level expression of recombinant hAng-1 in CHO cells, the polyclonal antibody was immobilized to the microplate and a sandwich ELISA was established for detecting the expression of hAng-1 in CHO cells.In eukaryotic expression system, the CHO cell lines that constantly express hAng-1 were established by selection with MTX. After precipitated with saturationsolution, the hAng-1 was recovered from cell culture supernatant. In the CAM blood vessel formation test, 500ng of CHO-expressed hAng-1 could stimulate the formation of CAM blood vessel, its biological activity was better than that of the hAng-1 expressed inE.coli.As a conclusion, the hAng-1 gene was cloned, expressed in both prokaryotic and mammalian cells. Both the recombinant hAng-1 showed CAM blood vessel formation stimulating activity. Our work thus lays a foundation for further study of hAng-1.