阅读说明：本技术 一种粗品尿激酶的纯化方法 (Method for purifying crude urokinase ) 是由 顾京 郭芬 张茜 韩庆坤 熊心磊 丁晶 于 2020-07-10 设计创作，主要内容包括：本发明提供了一种粗品尿激酶的纯化方法,涉及生物工程及化学工程领域,该纯化方法包括以下步骤：(1)将新鲜男性尿液加入硅胶吸附,用水冲洗硅胶,氨水洗脱,收集洗脱液,向洗脱液中加入固体硫酸铵进行沉淀,过滤收集棕色的尿激酶沉淀,即为人尿激酶粗品；(2)使用平衡缓冲液平衡层析柱,待紫外、pH和电导稳定后,将步骤(1)得到的人尿激酶粗品溶解并上样,经过滤、葡聚糖凝胶层析后,使用洗脱液进行洗脱至直至UV280nm<0.1,收集洗脱液,得到尿激酶中间品；(3)使用异丙醇和NaOH溶液清洗层析柱,再用注射水冲洗至中性。从而实现了尿激酶粗品的高效回收,尿激酶的活性收率可达70％以上,满足量产经济效益要求。(The invention provides a purification method of crude urokinase, which relates to the field of biological engineering and chemical engineering, and comprises the following steps: (1) adding silica gel into fresh male urine for adsorption, washing the silica gel with water, eluting with ammonia water, collecting the eluate, adding solid ammonium sulfate into the eluate for precipitation, and filtering and collecting brown urokinase precipitate to obtain a human urokinase crude product; (2) balancing the chromatographic column by using an equilibrium buffer solution, dissolving and sampling the crude human urokinase obtained in the step (1) after ultraviolet, pH and electric conductivity are stable, eluting by using an eluent until the UV280nm is less than 0.1 after filtering and sephadex chromatography, and collecting the eluent to obtain a urokinase intermediate product; (3) the column was washed with isopropanol and NaOH solution and washed to neutrality with water for injection. Therefore, the high-efficiency recovery of the crude urokinase is realized, the activity yield of the urokinase can reach more than 70 percent, and the requirement of mass production on economic benefit is met.)

1. A method for purifying crude urokinase is characterized in that: the method comprises the following steps:

(1) adding silica gel into fresh male urine for adsorption, washing the silica gel with water, eluting with ammonia water, collecting the eluate, adding solid ammonium sulfate into the eluate for precipitation, and filtering and collecting brown urokinase precipitate to obtain a human urokinase crude product;

(2) balancing the chromatographic column by using an equilibrium buffer solution, dissolving and sampling the crude human urokinase obtained in the step (1) after ultraviolet, pH and electric conductivity are stable, eluting by using an eluent until the UV280nm is less than 0.1 after filtering and sephadex chromatography, and collecting the eluent to obtain a urokinase intermediate product;

(3) the column was washed with isopropanol and NaOH solution and washed to neutrality with water for injection.

2. The purification process according to claim 1, characterized in that: the sephadex in step (2) comprises sephadex model number CM sephadex C-25, SP sephadex C-25, CM sephadex C-50 or SPsephadex C-50 of the United states general electric company.

3. The purification method according to claim 2, characterized in that: the sephadex is a sephadex model CM sephadex C-50 from american general company.

4. The purification process according to claim 1, characterized in that: the temperature of the precipitate in the step (1) is 0-10 ℃.

5. The purification process according to claim 1, characterized in that: the eluent in the step (2) comprises an acetate buffer solution, a citrate buffer solution, a phosphate buffer solution or a Tris-HCl buffer solution containing sodium chloride.

6. The purification method according to claim 5, characterized in that: the eluent is acetate buffer solution containing sodium chloride.

7. The purification process according to claim 1, characterized in that: the electric conductivity of the eluent in the step (2) is 15-90ms/cm, and the pH value is 6.5-9.0.

8. The purification process according to claim 1, characterized in that: the equilibration buffer solution in the step (2) comprises one or more of acetate buffer solution, citrate buffer solution, phosphate buffer solution and Tris-HCl buffer solution.

9. The purification process according to claim 1, characterized in that: the balance state in the step (2) is that the upper and lower deviation of an ultraviolet horizontal line is less than 0.1Mau, the conductance value is 1-25ms/cm, and the pH value is 6.5-9.0.

10. The purification process according to claim 1, characterized in that: the step (3) is to wash the chromatographic column by using 1 to 30 mass percent of isopropanol and 0.1 to 1M of NaOH solution, and the washing time is 0.5 to 2 hours.