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Affinity medium for human urinary trypsin inhibitor as well as synthesis and application for same

A trypsin inhibition and urine trypsin technology is applied to the human urine trypsin inhibitor affinity medium and its synthesis and application fields, which can solve the problems of long purification time, low recovery rate of protein and activity, etc. The effect of large economic benefits and large industrial application potential

Active Publication Date: 2013-10-02
宁夏妙朗生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Too many purification steps lead to longer purification time and lower recovery of protein and activity

Method used

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  • Affinity medium for human urinary trypsin inhibitor as well as synthesis and application for same
  • Affinity medium for human urinary trypsin inhibitor as well as synthesis and application for same
  • Affinity medium for human urinary trypsin inhibitor as well as synthesis and application for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Synthesis and application of Sepharose-ASP-GLY

[0030] Sepharose CL 4B (100g) was washed with 10 times the volume of deionized water, and dried into a wet cake; suspended in 50ml activation buffer (0.8M NaOH, 20% dimethylsulfoxide, 10% epichlorohydrin, 0.5mg / ml sodium borohydride) shaken at 40°C for 2.5h, then poured into a glass frosted funnel, and washed with 10 times the volume of distilled water each time under suction filtration until the pH of the washing solution was neutral, and then drained to form a wet cake shape. The activated Sepharose CL 4B medium was suspended in 500ml of 0.1M NaOH solution, 20ml of ethylenediamine solution was added, and the gel was kept at 30°C for 12h under stirring (200rpm). Rinse with deionized water. NH 2 - Suspend Sepharose CL 4B in 350ml 50% (v / v) ice-bathed acetone solution, then dissolve 4g triazoxide in 80ml-20°C pre-cooled acetone, quickly add the medium suspension, check the pH value in real time, use satur...

Embodiment 2

[0038] Embodiment 2: Synthesis and application of Sepharose-SER-GLY

[0039] Sepharose CL 4B (100g) was washed with 10 times the volume of deionized water, and dried into a wet cake; suspended in 50ml activation buffer (0.8M NaOH, 20% dimethylsulfoxide, 10% epichlorohydrin, 0.5mg / ml sodium borohydride) shaken at 40°C for 2.5h, then poured into a glass frosted funnel, and washed with 10 times the volume of distilled water each time under suction filtration until the pH of the washing solution was neutral, and then drained to form a wet cake shape. The activated Sepharose CL 4B medium was suspended in 500ml of 0.1M NaOH solution, 20ml of ethylenediamine solution was added, and the gel was kept at 30°C for 12h under stirring (200rpm). Rinse with deionized water. NH 2 - Suspend Sepharose CL 4B in 350ml 50% (v / v) ice-bathed acetone solution, then dissolve 4g triazoxide in 80ml-20°C pre-cooled acetone, quickly add the medium suspension, check the pH value in real time, use satur...

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PUM

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Abstract

The invention relates to an affinity medium for human urinary trypsin inhibitor, formed by connecting an affinity ligand for human urinary trypsin inhibitor with a chromatography medium. Preferably, the affinity ligand for human urinary trypsin inhibitor is an analogue of the site S1 of serine protease, the chromatography medium is Sepharose, chitosan, silica gel and ceramic particles, and the affinity ligand for human urinary trypsin inhibitor is connected with the chromatography medium by using cyanuric chloride as a spacer arm. A synthesis method for the affinity medium for human urinary trypsin inhibitor, and a method for purifying human urinary trypsin inhibitor in a large scale by using the affinity medium for human urinary trypsin inhibitor are further provided. Human urinary trypsin inhibitor can be rapidly separated and purified in a large scale by using the affinity medium for human urinary trypsin inhibitor, and human urinary trypsin inhibitor pure product can be obtained in case of only one-step affinity chromatography; and the human urinary trypsin inhibitor pure product is short in the consumed time of whole purification step, high in the recovery rates of protein and activity, and suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the technical field of enzyme production for medical treatment and diagnosis, in particular to a human urinary trypsin inhibitor affinity medium and its synthesis and application. Background technique [0002] Human urinary trypsin inhibitor is a Kunitz-type broad-spectrum protease inhibitor that can effectively inhibit the activity of trypsin, chymotrypsin, lactase, lipase, hyaluronidase and other pancreatic enzymes. In addition, it has the functions of stabilizing lysosomal membrane, inhibiting the release of lysosomal enzymes, inhibiting the production of myocardial inhibitory factor (MDF), scavenging oxygen free radicals and inhibiting the release of inflammatory mediators. Human urinary trypsin inhibitors can also improve immune function decline, abnormal protein metabolism and renal function reduction caused by surgical stimulation, prevent damage to internal organs ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C07K1/22C07K17/14C07K17/10C07K17/08
Inventor 辛瑜杨海麟张玲张玉然陈亦仝艳军
Owner 宁夏妙朗生物科技有限公司
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