阅读说明：本技术 一种用琼脂糖凝胶4b吸附尿液中尿激酶的方法 (Method for adsorbing urokinase in urine by using agarose gel 4B ) 是由 顾京 李争 刘艳红 郭芬 唐维 魏飘飘 于 2020-07-09 设计创作，主要内容包括：本发明公开了一种用琼脂糖凝胶4B吸附尿液中尿激酶的方法,涉及分离提取技术领域。该方法包括如下步骤：(1)将琼脂糖凝胶4B亲和包放入过滤瓶中,使尿液穿过琼脂糖凝胶4B亲和包流入过滤瓶；(2)尿液流完后取出琼脂糖凝胶4B亲和包,装入层析柱,用缓冲液洗涤,然后用解脱液解脱,待解脱液流出颜色发生变化时进行收集,即得尿激酶溶液；(3)将步骤(2)所得的尿激酶溶液冷冻干燥,得尿激酶粉末。本发明提供的吸附尿液中尿激酶的方法无需对尿液进行处理,操作简单,得到的尿激酶收率佳、纯度高。(The invention discloses a method for adsorbing urokinase in urine by using agarose gel 4B, and relates to the technical field of separation and extraction. The method comprises the following steps: (1) putting the agarose gel 4B affinity bag into a filter flask, and enabling the urine to flow into the filter flask through the agarose gel 4B affinity bag; (2) taking out the agarose gel 4B affinity bag after the urine flow is finished, filling the agarose gel 4B affinity bag into a chromatographic column, washing the chromatographic column by using a buffer solution, then desorbing the affinity bag by using a desorption solution, and collecting the affinity bag when the color of the elution solution is changed, thus obtaining a urokinase solution; (3) and (3) carrying out freeze drying on the urokinase solution obtained in the step (2) to obtain urokinase powder. The method for adsorbing urokinase in urine provided by the invention does not need to treat urine, is simple to operate, and has good yield and high purity of the obtained urokinase.)

1. A method for adsorbing urokinase in urine by using agarose gel 4B is characterized by comprising the following steps:

(1) putting the agarose gel 4B affinity bag into a filter flask, and enabling the urine to flow into the filter flask through the agarose gel 4B affinity bag;

(2) taking out the agarose gel 4B affinity bag after the urine flow is finished, filling the agarose gel 4B affinity bag into a chromatographic column, washing the chromatographic column by using a buffer solution, then desorbing the affinity bag by using a desorption solution, and collecting the affinity bag when the color of the elution solution is changed, thus obtaining a urokinase solution;

(3) and (3) carrying out freeze drying on the urokinase solution obtained in the step (2) to obtain urokinase powder.

2. The method as claimed in claim 1, wherein in step (1), the volume ratio of the urine to the agarose gel 4B is 400000-600000:15, and the flow rate of the urine passing through is 1-10 mL/min.

3. The method according to claim 1, wherein in the step (2), the buffer is at least one selected from the group consisting of a phosphate buffer, a Tris-HCl buffer, an ammonium sulfate buffer, a citrate buffer, an acetate-Tris buffer, and a glycine-Tris buffer.

4. The method according to claim 3, wherein in the step (2), the buffer is a phosphate buffer.

5. The method according to claim 1, wherein in the step (2), the buffer is used in an amount of 2 to 4 column volumes.

6. The method as claimed in claim 1, wherein in step (2), the stripping solution is a mixed solution containing glacial acetic acid and sodium chloride solution, and the volume ratio of glacial acetic acid to sodium chloride solution is 3: 400-.

7. The method according to claim 1, wherein in the step (2), the concentration of the sodium chloride solution is 0.01-0.03 g/mL.

8. The method according to claim 1, wherein in the step (2), the amount of the stripping solution is 1.5 to 2.5 column volumes.

9. The method according to claim 1, wherein in the step (2), the flow rate of the stripping solution is 1-4 mL/min.

10. The method according to claim 1, wherein in step (3), the freeze-drying specifically comprises: and (3) putting the urokinase solution obtained in the step (2) into a freeze dryer, freezing the urokinase solution for 2 to 4 hours at the temperature of minus 35 to 55 ℃, and then heating the solution to room temperature for 1.5 to 2.5 hours.

Technical Field

The invention relates to the technical field of separation and extraction, and particularly relates to a method for adsorbing urokinase in urine by using agarose gel 4B.

Background

Urokinase is a thrombolytic drug extracted from fresh human urine, is a serine protease produced by human renal tubular epithelial cells, is an alkaline protein, has an isoelectric point of about pH8.7, is white amorphous powder, and is easily soluble in water. The dilute solution is unstable in property, needs to be used fresh, and needs not to be diluted by an acid solution, and the freeze-dried state can be stable for years. Urokinase is a very specific proteolytic enzyme. The activity of the synthetic substrate is similar to that of trypsin and plasmin, and the synthetic substrate also has esterase activity, no antigenicity and no in vivo antibody production. The half-life period in vivo is (14 +/-6) min.

Urokinase activates plasminogen to active plasmin, which converts insoluble fibrin to soluble peptides, thereby dissolving the thrombus. Therefore, it is clinically used for treating thrombosis, thromboembolism and other diseases. When urokinase is combined with an anticancer agent, the urokinase can dissolve fibrin around cancer cells, so that the anticancer agent can penetrate into the cancer cells more effectively, thereby improving the capability of the anticancer agent in killing the cancer cells. Therefore, urokinase is also a good cancer adjuvant, and it has no problem of antigenicity and can be used for a long time.

As urokinase has important medical value, a plurality of special journals report methods for extracting crude urokinase, and because the urokinase content in human urine is very low, the key point of taking urokinase is how to enrich urokinase from a large amount of urine, and from the prior published documents, three methods are mainly provided: (1) and a foaming method: stirring at high speed to foam the enzyme solution, liquefying the foam, and adding ammonium sulfate to precipitate urokinase; (2) the precipitant method: adding a precipitating agent into urine to precipitate urokinase, wherein the urokinase is remained in the precipitate; (3) adsorbent, the most used in this method, and the selection of a suitable adsorbent for selective adsorption of urokinase.

Chinese patent application 201510549397.1 discloses a method for enriching urine protein by directly adsorbing urine protein in urine in a urinal or a urinal through a filter cloth bag containing modified silica gel, and transporting the filter cloth bag adsorbing urine protein to a processing point for subsequent treatment. The method utilizes the isoelectric point property of specific urine protein,the activity of the freeze-dried powder obtained by the method is 4.12 × 10, and the freeze-dried powder obtained by the method has the advantages of directly and effectively adsorbing urine proteins such as urine trypsin inhibitor, human urine kininogenase, urokinase and the like by using modified silica gel, macroporous resin, chitin, ion resin and the like, and avoiding the step of collecting urine⁵IU, which does not meet the requirements.

Chinese patent application 201510691424.1 provides a method for extracting urokinase and ulinastatin from urine, comprising: collecting urine and adjusting the pH value of the urine; adding powdered silica gel into urine and stirring to make urokinase protein in urine adsorbed by silica gel; obtaining silica gel by a filtering mode and adjusting the pH value of the filtered urine to obtain urokinase protein liquid from the silica gel; adding urokinase protein liquid into saturated water to extract urokinase, and adding ammonium sulfate into the ulinastatin protein liquid to extract ulinastatin. The invention can extract crude urokinase and crude ulinastatin from urine, thereby greatly improving the economic value of urine. However, urokinase is also eluted during the adsorption process, thereby causing a loss of urokinase, thereby decreasing the yield of urokinase.

For collecting urokinase from human urine, it is very important to improve the extraction rate of urine protein in urine, and therefore, there is a need to develop a method for improving the yield of urokinase. In view of this, in order to solve the problems in the prior art, the present invention provides a method for adsorbing urokinase in urine by using agarose gel 4B, which can significantly increase the yield of urokinase and improve the purity of urokinase.

Disclosure of Invention

The invention aims to provide a method for adsorbing urokinase in urine by using agarose gel 4B, which can obviously improve the yield of urokinase and the purity of urokinase.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for adsorbing urokinase in urine by using agarose gel 4B comprises the following steps:

(1) putting the agarose gel 4B affinity bag into a filter flask, and enabling the urine to flow into the filter flask through the agarose gel 4B affinity bag;

(2) taking out the agarose gel 4B affinity bag after the urine flow is finished, filling the agarose gel 4B affinity bag into a chromatographic column, washing the chromatographic column by using a buffer solution, then desorbing the affinity bag by using a desorption solution, and collecting the affinity bag when the color of the elution solution is changed, thus obtaining a urokinase solution;

(3) and (3) carrying out freeze drying on the urokinase solution obtained in the step (2) to obtain urokinase powder.

Preferably, in the step (1), the volume ratio of the urine to the agarose gel 4B is 400000-600000:15, and more preferably 500000: 15.

Preferably, in step (1), the flow rate of urine passing through is 1-10mL/min, more preferably 4 mL/min.

Preferably, in the step (2), the chromatographic column is selected from any one of BPG/GE, Chromaflow/GE, Axichrom/GE, QuickScale/Merck, SAC-BIO/Lisui technology, ABH-BIO/Lisui technology, EAC-BIO/Lisui technology, XK16/20/GE and GCC/Lisui technology, and is further preferably XK 16/20/GE.

Preferably, in the step (2), the volume ratio of the sepharose 4B to the chromatography column is 0.1-0.5: 1, more preferably 0.3: 1.

Preferably, in the step (2), the buffer is at least one selected from the group consisting of a phosphate buffer, a Tris-HCl buffer, an ammonium sulfate buffer, a citrate buffer, an acetate-Tris buffer, and a glycine-Tris buffer, preferably a phosphate buffer, and more preferably a 0.02M phosphate buffer.

Preferably, in step (2), the buffer is used in an amount of 2 to 4 column volumes, preferably 3 column volumes.

Preferably, in the step (2), the desorption solution is a mixed solution containing glacial acetic acid and a sodium chloride solution; the volume ratio of the glacial acetic acid to the sodium chloride solution is 3:400-600, and is further preferably 3: 500; the concentration of the sodium chloride solution is 0.01 to 0.03g/mL, and more preferably 0.02 g/mL.

Preferably, in the step (2), the amount of the stripping solution is 1.5 to 2.5 column volumes, and more preferably 2 column volumes.

Preferably, in the step (2), the flow rate of the stripping solution is 1 to 4mL/min, and more preferably 2.5 mL/min.

Preferably, in the step (2), the color change means that the color changes from light gray to dark gray or from light yellow to dark yellow.

Preferably, in the step (3), the freeze-drying specifically means: putting the urokinase solution obtained in the step (2) into a freeze dryer, freezing for 2-4 hours at the temperature of 35-55 ℃, then heating to room temperature, and keeping for 1.5-2.5 hours; further preferably, the freeze-drying specifically means: and (3) putting the urokinase solution obtained in the step (2) into a freeze dryer, freezing the urokinase solution for 3 hours at minus 45 ℃, and then heating the solution to room temperature for 2 hours.

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the method for adsorbing urokinase in urine, urine does not need to be processed, collected urine directly penetrates through the urine and the agarose gel 4B, then the urine and the agarose gel 4B are filled into a chromatographic column, and urokinase can be obtained through elution, so that the operation is simple, and the loss of urokinase is well reduced;

(2) the method of the invention can better separate urokinase, reduce the loss of urokinase, and obtain the urokinase with good yield and high purity.

Drawings

FIG. 1 is a map of a urokinase solution obtained in example 1, in which 02 is a target peak;

FIG. 2 is a urokinase powder pattern obtained in example 1, in which 02 is the target peak.

Detailed Description

The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, Sepharose 4B affinity pack is an affinity chromatography gel prepared by Sepharose 4B of this company, Sepharose 4B being purchased from GE, Cat. No. 17-0120-05.