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Method for adsorbing urokinase in urine by using agarose gel 4B

An agarose gel, urokinase technology, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve the problems of reducing the yield of urokinase, the loss of urokinase, etc., and achieve good separation, reduced loss, and simple operation. Effect

Active Publication Date: 2020-10-09
河南省尤里卡生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, urokinase will also be washed during the adsorption process, which will cause the loss of urokinase, thereby reducing the yield of urokinase

Method used

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  • Method for adsorbing urokinase in urine by using agarose gel 4B
  • Method for adsorbing urokinase in urine by using agarose gel 4B
  • Method for adsorbing urokinase in urine by using agarose gel 4B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] (1) Put 15mL Sepharose 4B affinity pack into the filter bottle, take 500L of urine through the Sepharose 4B affinity pack and flow into the filter bottle at a flow rate of 4mL / min, the total urine test is 3.8 million iu;

[0035] (2) After the urine flow, take out the Sepharose 4B affinity bag, put it into the chromatography column (XK16 / 20 / GE) and press it tightly. The volume ratio of Sepharose 4B and the chromatography column is 0.3:1, Wash with 3 times the volume of 0.02M phosphate buffer, and then use 2 times the volume of eluent (1L 0.02g / mL sodium chloride solution + 6mL glacial acetic acid) for elution, the flow rate of the eluent is 2.5mL / min, until the color of the eluent changes from Collect when the light gray turns into dark gray to obtain urokinase solution;

[0036] (3) Put the solution collected in step (2) into a lyophilizer, freeze at minus 45 degrees for 3 hours, then warm up to room temperature for vacuum freeze-drying, keep for 2 hours, and take out ...

Embodiment 2

[0038] (1) Put 15mL Sepharose 4B affinity pack into the filter bottle, take 400L of urine through the Sepharose 4B affinity pack and flow into the filter bottle at a flow rate of 1mL / min, the total urine test is 3.02 million iu;

[0039] (2) Take out the Sepharose 4B affinity pack after the urine has finished flowing, put it into a chromatography column (GCC / Lisui Technology) and press it tightly. The volume ratio of Sepharose 4B to the chromatography column is 0.1:1, Wash with 2 times the volume of 0.02M phosphate buffer, and then use 1.5 times the volume of eluent (0.8L 0.02g / mL sodium chloride solution + 6mL glacial acetic acid) for elution. The flow rate of the eluent is 1mL / min until the color of the eluent changes from Collect when the light gray turns into dark gray to obtain urokinase solution;

[0040] (3) Put the solution collected in step (2) into a lyophilizer, freeze at minus 35 degrees for 4 hours, then warm up to room temperature for vacuum freeze-drying, keep f...

Embodiment 3

[0042] (1) Put 15mL of Sepharose 4B affinity bag into the filter bottle, take 600L of urine through the Sepharose 4B affinity bag and flow into the filter bottle at a flow rate of 10mL / min, the total urine test is 4.58 million iu;

[0043] (2) After the urine flow, take out the Sepharose 4B affinity bag, put it into the chromatography column (XK16 / 20 / GE) and press it tightly. The volume ratio of Sepharose 4B and the chromatography column is 0.5:1, Wash with 4 times the volume of 0.02M phosphate buffer, and then use 2.5 times the volume of the eluent (1.2L 0.02g / mL sodium chloride solution + 6mL glacial acetic acid) to release, the flow rate of the eluent is 4mL / min, until the color of the eluent changes from Collect when the light gray turns into dark gray to obtain urokinase solution;

[0044] (3) Put the solution collected in step (2) into a lyophilizer, freeze at minus 55 degrees for 2 hours, then warm up to room temperature for vacuum freeze-drying, keep for 2.5 hours, and...

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Abstract

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 an agarose gel 4B affinity bag into a filter bottle, so that urine passes through the agarose gel 4B affinity bag and flows into the filter bottle; (2) taking out the agarose gel 4B affinitybag after the urine completely flows out, filling the agarose gel 4B affinity bag into a chromatographic column, washing by using a buffer solution, then releasing by using a release solution, and collecting when the color of the release solution is changed, so as to obtain a urokinase solution; and (3) freeze-drying the urokinase solution obtained in the step (2) to obtain urokinase powder. According to the method for adsorbing urokinase in urine, urine does not need to be treated, operation is easy, and the obtained urokinase is good in yield and high in purity.

Description

technical field [0001] The invention relates to the technical field of separation and extraction, in particular to a method for absorbing urokinase in urine with Sepharose 4B. Background technique [0002] Urokinase is a thrombolytic drug extracted from fresh human urine. It is a serine protease produced by human renal tubular epithelial cells. It is a basic protein with an isoelectric point of about PH8.7 and is white non-crystalline. powder, easily soluble in water. The dilute solution is unstable and must be used fresh, and must not be diluted with an acidic solution. The freeze-dried state can be stable for several years. Urokinase is a highly specific proteolytic enzyme. The activity on synthetic substrates is similar to that of trypsin and plasmin, and it also has the activity of esterase. It has no antigenicity and does not cause antibodies in the body. The half-life in vivo is (14±6)min. [0003] Urokinase can activate plasminogen into active plasmin, and plasmin...

Claims

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Application Information

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IPC IPC(8): C12N9/72
CPCC12N9/6462C12Y304/21073
Inventor 顾京李争刘艳红郭芬唐维魏飘飘
Owner 河南省尤里卡生物科技有限公司