本文選題：重組尿酸氧化酶 + 聚乙二醇修飾�。� 參考：《浙江大學》2014年碩士論文

【摘要】：目的：制備一種新型支鏈(Y型)聚乙二醇(polyethylene glycol, PEG)修飾的尿酸氧化酶,并通過大鼠體內藥效學、藥代動力學和免疫原性評估該修飾酶的體內外酶活性和穩(wěn)定性,為其臨床研究提供實驗基礎與理論依據(jù)。 方法：(1)通過不同pH、時間、投料比,考察對PEG耦合尿酸氧化酶的影響,采用離子交換色譜、超濾等手段對修飾產物進行分離純化,制備PEG修飾尿酸氧化酶耦合物；(2)運用凝膠電泳、高效液相色譜技術、TNBS法、酶法研究支鏈PEG修飾產物的理化特征、修飾度等；(3)采用酶反應—紫外分光光度法對修飾的尿酸酶生物活性及其體外穩(wěn)定性進行檢測；(4)并以大鼠為模型,研究不同聚乙二醇修飾的尿酸氧化酶在體內的藥代動力學和藥效動力學；(5)通過ELISA方法分析比較修飾產物的免疫原性。 結果：(1)在pH8.0的磷酸鹽緩沖溶液中,在室溫(25℃)條件下,以1：5的摩爾比加入尿酸氧化酶和Y型SPA-mPEG20000MW (SPA-mPEG2)反應30min,可以得到修飾度和保留活性較高的PEG化蛋白藥物；采用Q Sepharose Fast Flow層析柱和截留分子量為50kD的超濾膜堆進行分離純化,可以得到純度≥95%的PEG化尿酸氧化酶樣品。(2)Y型PEG尿酸氧化酶修飾物rUOX-mPEG2的表觀分子量比線性PEG修飾的產物rUOX-mPEG更大,PEG分子的修飾度更低而活性更高；(3)在相同溫度、pH條件下的活性穩(wěn)定性明顯高于直鏈型PEG尿酸氧化酶修飾物；(4)Y型PEG尿酸氧化酶修飾物在體內半衰期顯著延長,在體內具有更長的藥效維持時間；(5)免疫原性實驗結果表明,Y型PEG修飾能有效降低免疫原性。 結論：新型聚乙二醇修飾尿酸氧化酶的酶學性質明顯優(yōu)于重組酶和商業(yè)直鏈型修飾的尿酸氧化酶,藥效維持時間顯著高于后者,免疫原性和國外品種采用的直鏈修飾尿酸氧化酶相當,且明顯低于重組酶,在大鼠體內呈線性動力學特征。
[Abstract]:Aim: to prepare a novel branched poly (ethylene glycol) polyethylene-glycolene (peg) modified uric acid oxidase and evaluate its activity and stability in vitro and in vivo by pharmacodynamics, pharmacokinetics and immunogenicity in rats. To provide experimental and theoretical basis for its clinical research. Methods the effects of different pH, time and feed ratio on PEG coupled uric acid oxidase were investigated. The modified product was separated and purified by ion exchange chromatography and ultrafiltration. The coupling compound of PEG modified uric acid oxidase was prepared by gel electrophoresis. High performance liquid chromatography (HPLC) was used to study the physicochemical characteristics of branched PEG modified products and the degree of modification. The bioactivity and in vitro stability of modified uric acid enzymes were determined by enzyme reaction-UV spectrophotometry) and the rat model was used as a model. The pharmacokinetics and pharmacokinetics of uric acid oxidase modified by polyethylene glycol in vivo were studied. The immunogenicity of the modified product was analyzed by ELISA method. Results (1) in the phosphate buffer solution of pH8.0, at room temperature (25 鈩，

本文編號：1961198