| 摘要: | 本項論文擬針對血管生成相關蛋白質，建立大腸桿菌系統表現融合型蛋白質( Endostatin-GMCSF ) 的實施技術平台，涵蓋基因構築系統與發酵純化確效等醫用蛋白質的量產製程任務。本研究以大腸桿菌表現系統製備高純度的醫用融合蛋白質為目標，利用融合蛋白質 6x-His標籤片段對特定金屬螯定親合特性，使用 Ni2+-NTA 親和層析篩選進行蛋白質分離與anti-Endo專一性抗體驗證，目前已經初步建立純化系統技術平台參數，融合型蛋白質( Endostatin-GMCSF )已經被純化出來並且經由anti-Endo專一性抗體驗證；由於驗證結果顯現，部分蛋白質有被Cut的現象，故發酵條件採用
本項研究涵蓋三部分：
1. 基因構築的轉形與純系：經由榮總醫院癌症中心實驗室藍老師所構建的融合基因載體(pQE31 Endostatin-GMCSF)，經轉殖技術運用轉殖技術將融合型蛋白質重組基因體質pQE31，經由DNA限制酶切電泳技術驗證載體已經成功帶入E. coli (BL21)，並選殖特定群菌株進行發酵培養。
2. 基因表現與發酵純化：融合基因於E. coli 宿主細胞內進行複製轉錄與轉譯，產生融合型蛋白質。實驗發現不經IPTG誘導菌體也能表現出融合蛋白質，所產生的蛋白質有微量的可溶性蛋白與包涵體兩種形式，而研究目標在於純化可榮幸蛋白質，故發酵條件定在溫度30℃、無IPTG誘導、發酵時間3小時。純化部份也經由Ni2+-NTA 親和層析篩選成功分離融合蛋白質，並經由anti-Endo專一性抗體驗證出來。
3. 初步確效：運用生物分析法初步定性確效。實驗部分完成大腸桿菌系統表現融合型蛋白質的培養發酵條件與抗體染色與蛋白質膠泳分析，並且也已經進行融合蛋白純化與NFS-60細胞生長曲線分析與基因微陣分析；由於融合可溶性蛋白質濃度低，故需要在近一部作深度的純化工程，已進行更深入的活性分析。
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. |
