Fused gene expression and Purified protein activity of Endostatin

• Main Authors: Wen-Huan Pai, 白文環
• Other Authors: Chun-Fan Chang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/57729437642077106509

碩士 === 中國文化大學 === 生物科技研究所 === 95 === Abstract Angiogenesis inhibitors, a new class of antitumor therapy associated with low toxicity and low drug resistance, suppress tumor growth by blocking the formation of new blood vessels, which provide oxygen and nutrients for growth . One such inhibitor is endostatin, a 20-kDa fragment cleaved from a collagen XVIII COOH terminus that inhibits endothelial cell proliferation, migration, invasion, and tube formation and that has shown antiangiogenesis and antitumor effects in animal models . GM-CSF ( Colony Stimulating Factor) : In the body's bone marrow (the soft, sponge-like material found inside bones) blood cells are produced. There are three major types of blood cells; white blood cells, which fight infection; red blood cells, which carry oxygen to and remove waste products from organs and tissues; and platelets, which enable the blood to clot. Cancer treatments such as chemotherapy and radiation therapy can effect these cells which put a person at risk for developing infections, anemia and bleeding problems. Colony-stimulating factors are substances that stimulate the production of blood cells and promote their ability to function. They do not directly affect tumors but through their role in stimulating blood cells they can be helpful as support of the persons immune system during cancer treatment. GM-CSF is used for : 1. Used to accelerate the recovery of white blood cells following chemotherapy. 2. Used following induction chemotherapy in Acute Myelogenous Leukemia (AML). 3. After bone marrow transplantation. 4. Before and/or after peripheral blood stem cell transplantation. 5. Sargramostim is a support medication. It does not treat cancer. It is important to procure mouse endostatin-GM-CSF fusion protein with biological activity. Now, mouse endostatin-GM-CSF fusion gene is successfully expressed in the prokaryotic expressing vector pQE31 , and mouse endostatin-GM-CSF protein can stimulative the proliferation of NFS-60 cells. This research has divided into three parts: (1)The gene structure The total RNA was extracted from fetal liver and amplified to acquire human endostatin gene by reverse transcription polymerase chain reaction (RT-PCR). Then the obtained gene was cloned into expression vector pQE31 and transformed into E. coli BL21 (DE3). (2)The protein expression and purification： Endostatin was expressed in the E. coli by IPTG inducement, and then purified by . (3) Potency confirmation assays： MTT assay was used to detect the inhibitory activity of mouse Endostatin-GM-CSF fusion protein on NFS-60 cells. Microarray analysis: was used to detect the change quantity of the specific gene group mRNA Induced. Key word: Endostatin, GM-CSF, collagen XVIII, Angiogenesis inhibitors