The Cloning, Expression And Biological Characterization Of Human Vasostatin And TRAIL-Vasostatin

Angiogenesis, the developmet of new capillaries from pre-existing blood vessels, is an important process in physiological and pathophysiological situations. Neovascularization is a physiological process involved in embryonic development, female reproduction, and wound healing, etc. It is hypothesized that the interplay of angiogenic and antiangiogenic factors operates to regulate neovascularization. The regulation of angiogenesis is a complex process, which involves a series of on and off regulatory switches. Disturbance of the balance of these factors results in abnormal angiogenesis. Abnormal angiogenesis plays an important role in a wide spectrum of diseases, including tumor growth, metastasis, inflammatory conditions, ischemic diseases, and degenerative disorders. Tumor growth and metastasis require angiogenesis, and this process is pivotal to the survival and subsequent growth of solid tumors beyond a few mm3 in size. Expansion of tumor mass occurs not only by perfusion of blood through the tumor but also by paracrine stimulation of tumor cells by several growth factors and matrix proteins produced by the new capillary endothelium. Studies have shown that tumor growth is dependent on angiogenesis. This provides the rationale for antiangiogenic therapy in cancer. Vasostatin is a novel inhibitor of endothelial cell proliferation which was purified from the supernatant of an Epstein-Barr virus-transformed cell line and was identified as the N-terminal domain of calreticulin, which includes N-terminal with amino acid l~180 of calreticulin. Vasostatin is a specific inhibitor of basic fibroblast growth factor (bFGF)-induced endothelial cell proliferation in vitro and a suppressor of bFGF-induced angiogenesis in vivo. When inoculated into athymic mice, Vasostatin prevented or significantly reduced experimental tumor growth.Tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) was identified in 1995 by screening the expressed sequence tag (EST) databases using a conserved sequence in comparison to certain TNF family members. TRAIL is a typeII membrane protein and induces apoptosis via death receptors in a wide variety oftumor cells but not in normal cells.Part One: Cloning, Expression of Human Vasostatin in E.coli and its BioassayIn the present study, cDNA of Vasostatin was amplified by RT-PCR from the total RNA of liver cancer cell SMMC-7721. We constructed the recombinant plasmid of pMAL -Vasostatin 1-180 and pMAL -Vasostatin 120-180, respectively. Vasostatin was expressed in BL21 with pMAL-c2 expression vector and purified with amylose resin column. The biological activity of Vasostatin was detected with the inhibition of endothelial cell proliferation, endothelial cell migration and in vitro tube formation. The expression rate of recombinant MBP-Vasostatin 1-180 and MBP-Vasostatin120-180 fusion protein in E.coli was 20% in total proteins of E.coli. The purity of fusion protein reached electrophoresis purity. MBP-Vasostatin1-180 and MBP-Vasostatin 120-180 showed a significant inhibitory effect on endothelial cell proliferation and migration in a dose dependent manner, and the exhibited the inhibition of tube formation as well. The results suggest that MBP-Vasostatin1-180and MBP-Vasostatin120-180 are effective inhibitors to endothelial cells and has potential therapeutic usage on cancer treatment. Furthermore, both fusion proteins displayed a similar inhibition on the cultivated endothelial cell proliferation and neovascularization, indicate that the antiangiogenic activity of calreticulin resides in a domain within amino acids 120-180.Part Two: Fusion expression of TRAIL-Vasostatin120-180 and its bioassayThe aim of this study is try to create a fusion protein combining the antiagiogenic inhibitor Vasostatin120-180 and TRAIL. The novel fusion protein is designed to kill tumor cells via a cytotoxic factor of TRAIL as well as to inhibit the angiogenesis in tumor mass via an angiogenic inhibitor of Vasostatin. A procaryotic expression system was used to produce TRAIL-Vasostatin 120-180 fusion protein which maintained both Vasostatin 120-180 and TRAIL activities as demonstrated by cell-based assays. In the present study, cDNA of Vasostatin120-180 and TRAIL were amplified by PCR from the pMAL-Vasostatin1-180 and pBV-TRAIL. The coding sequances for...