Construction, Fusion Expression, And Biological Characterization Of Calreticulin And Apo-2L

Angiogenesis, the development of new capillaries from preexisting blood vessels, is an important process both in physiologiocal and pathophysiological situations. Neovascularization is a physiological process involved in embryonic development, female reproduction, and wound healing. It is hypothesized that the interactions between the angiogenic and antiangiogenic factors regulate the neovascularization. The angiogenesis is a complex process, which involves in a series of on and off regulatory switches. Disturbance of these factors results in abnormal angiogenesis. Abnormal angiogenesis plays an important role in a wide spectrum of diseases, including tumor growth, metastasis, inflammation, 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. Studies have shown that tumor growth is dependent on angiogenesis. This provides the rationale for antiangiogenic therapy in cancer.Calreticulin (CRT), a 47 kD highly conserved and ubiquitous protein, is a multifunctional Ca2+-binding molecular chaperone in the endoplasmic reticulum. It was found firstly from skeletal muscle sarcoplasmic reticulum by Ostwald and Maclennan in 1974. CRT plays an important role in nascent protein folding, antigen-presentation, angiogenesis, apoptosis, excepting of calcium balance regulation. Studies have shown that CRT is highly related to occurrence, development, treatment and prognosis of tumor. Vasostatin is a novel inhibitor for endothelial cell proliferation which purified from the supernatant of an Epstein-Barr virus-immortalized cell line and identified as the N-terminal domain of calreticulin, it was the part of calreticulin at its amino acid 1-180 . Vasostatin is a specific inhibitor for 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-related apoptosis-inducing ligand (TRAIL) was identified in 1995 by screening the expressed sequence tag (EST) databases using a conserved sequence contained in TNF family members. TRAIL is a typeâ…¡membrane protein and induces apoptosis via death receptors in a wide variety of tumor cells but not in normal cells.MMPs are an important ECM degrading enzyme. They play an essential role in tumor invasion and metastasis by degrading ECM. MMPs are a family consisiting twenties members and Zn-depending endopeptidase. Gelatinase is one of the member of this family. A large number of research showed enhanceed MMP-2 or MMP-9 expression in a variety of cancer, cultivated tumor cells and cancer gene transformed cells. Invasion assay in vivo and vitro showed that the tumor hyperinvasibility was highly related to the over expression of gelatinase.There are two treatment strategies against solid tumors. One is directly killing tumor cells, the other is anti-angiogenesis. This dissertation described the expression of a bifunctional fusion protein of vasostatin with TRAIL by SOE-ing PCR with the PLGLWA linker. The main works include: (1) cloning, expression and bioassay of vasostatin120-180 in expressed by E. coli. (2)cloning, expression and biological characterization of the bifunctional fusion protein Vasostatin120-180-TRAIL114-281. (3)enzymatic digestion characters of fusion protein both in vitro and in vivo.Part one:Cloning, Expression and Bioassay of Human Vasostatin120-180 in E.coliVasostatin inhibits tumor growth by inhibiting endothelial cell proliferation, migration and angiogenesis. The encoding gene of vasostatin120-180 was amplified by PCR from human liver cDNA. E. coli BL21 was used as host strain for cloning and expression experiments. Vasostatin120-180 was expressed with pMAL-C2 expression vector and purified with Amylose Resin column. Western Boltting showed a 47 kD protein band. The biological activity of vasostatin120-180 was detected by endothelial cell proliferation test and tube formation inhibition assay in vitro. The result showed MBP-vasostatin120-180 had an obvious dose-dependent effect on inhibiting endothelial cell proliferation and tube formation.Part two:Fusion expression and bioassay of TRAIL114-281 -Vasostatin120-180 with the PLGLWA linkerThe aim of this study is to design a fusion protein of antiagiogenic inhibitor Vasostatin120-180 and cytotoxic TRAIL in order to improve its anti-tumor activity. The novel fusion protein is predicated to kill tumor cells as well as to inhibit angiogenesis near the tumor site. A restriction sequence of PLGLWA of MMPs was introduced in order to cleave the fusion protein in tumor tissues. Fusion gene of Vasostatin120-180 and TRAIL114-281 were amplified by SOE-ing PCR and cloned into pMAL-C2 expression vector. Recombinant vectors were transformed into E. coli BL21 and expressed under IPTG induction. We obtain the purified MBP-VT and MBP-TV fusion proteins by Amylose Resin affinity purification column. The results of endothelial cell proliferation and tumor cells apoptosis showed that MBP-VT had an obvious apoptotic effect on tumor cells as well as an inhibitory effect on endothelial cell proliferation. While, MBP-TV had almost no apoptotic effect against tumor cells. Flow cytometric detection and DNA ladder experiment showed the same results.Part three: Enzymatic digestion of fusion proteins in vitro and in vivoMBP-TV was tested its apoptotic effect against SW1990 for 24 h and 72 h, respectively. The results showed that the apoptotic effect improved obviously at 72 h. The reason is that MBP-TV is digested by secreted MMPs of incubated SW1990 at 72 h and the active site of TRAIL is expressed. MBP-VT was tested for MMP-2 digestion by SDS-PAGE and Western Blot. SDS-PAGE indicated there were three bands and in accordance with the sizes of MBP-VT, MBP-Vs and TRAIL114-281, respectively. Western Blot showed the same result which suggested that the MBP-VT could be cleaveged correctly. The antitumor activities of MBP-TV and MBP-VT were demonstrated in hepatoma-bearing nude mice model. The size and the weigh of the tumors were similar after daily injection (i.p.) of MBP-TV and MBP-VT (5 mg/kg/day) for 12 days, suggesting that MBP-TV may be digested and to exhibite their anti-tumor activity.