Method for preparing restrictive thrombin

A thrombin and restrictive technology, applied in the field of bioengineering, can solve the problems of insufficient thrombin purity and difficult industrial production, and achieve the effects of reducing equipment investment, simple operation, and reducing environmental pollution.

Active Publication Date: 2016-06-01
FUJIAN HUAGENE BIOSCI CO LTD
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Problems solved by technology

This patent application involves the use of gel chromatography, which is not easy to achieve industrial prod

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  • Method for preparing restrictive thrombin
  • Method for preparing restrictive thrombin
  • Method for preparing restrictive thrombin

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preparation example Construction

[0019] Please refer to figure 1 , the preparation method of restriction thrombin, comprises the steps:

[0020] Step 1. Add anticoagulant to fresh pig blood, centrifuge, and take supernatant anticoagulant plasma;

[0021] Step 2, the supernatant anticoagulated plasma is subjected to anion-exchange column chromatography, and then the anion-exchange column is eluted with eluent I, and the obtained eluent is the prothrombin solution;

[0022] Step 3, activating the prothrombin in the prothrombin solution at normal temperature and pressure to obtain a crude thrombin solution;

[0023] Step 4, performing ultrafiltration and concentration on the thick thrombin solution to obtain a thrombin concentrate;

[0024] Step 5, subjecting the obtained thrombin concentrate to cation-exchange column chromatography, and then eluting the cation-exchange column with eluent II to collect the thrombin protein peak to obtain a preliminary purified thrombin solution;

[0025] Step 6, dialyzing the...

Embodiment 1

[0039] Please refer to figure 1 Take 9L of fresh pig blood, add 1L of 3.8% trisodium citrate solution anticoagulant, and centrifuge. Take 5L of supernatant anticoagulated plasma. The anticoagulant plasma was loaded onto the pre-balanced DEAESepharose FastFlow anion exchange column, and the pH was adjusted to 7, and the concentration was 0.02mol / L. Trisodium citrate eluent was eluted to obtain 2200 mL of prothrombin eluent. The eluate is desalted by ultrafiltration to obtain a prothrombin solution. Add 1mol / L CaCl 2 Solution 165mL, so that Ca in the prothrombin solution 2+ The final concentration of prothrombin was 0.07 mol / L, and prothrombin was activated for 3 hours at normal temperature and pressure to obtain 2365 mL of thrombin solution. Concentrate the thrombin solution by an ultrafiltration machine about 11 times to obtain 200 mL of a crude thrombin solution, which is marked as A1.

[0040] Load 200mL of crude thrombin solution A1 onto the equilibrated DEAESephrose C...

Embodiment 2

[0044] Thrombin detection

[0045] 1. Detection of thrombin titer and protein concentration

[0046] According to the relevant standard detection method of thrombin potency on page 1561-1562 of the second part of the 2015 edition of the Pharmacopoeia of the People's Republic of China, make a standard curve, and carry out the titer of the thrombin solution A1, A2, A3, A4 in the above-mentioned implementation case 1. Determination. In addition, the protein concentration of the thrombin solution A1, A2, A3, and A4 was detected, and the determination method used the "Second method forin phenol method (Lowry method)" in the "Chinese Pharmacopoeia" 2015 edition four general rules 0731 protein content determination method. ", the thrombin titer and protein concentration detection results are shown in Table 1.

[0047] According to the thrombin titer detection results and the corresponding protein concentration detection results, the specific activity and yield of thrombin were calc...

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Abstract

The invention provides a method for preparing restrictive thrombin which is high in purity and specific activity. The method includes the steps that an anion exchange chromatography medium is utilized to adsorb prothrombin from pig plasma, an anionic chromatography column is subjected to elution, the prothrombin is activated, and a crude solution of thrombin is obtained; ultrafiltration and concentration are conducted, and a concentrated solution of the thrombin is obtained; then a cation exchange chromatography medium is utilized to adsorb thrombin, a cationic chromatography column is subjected to elution, a thrombin solution is obtained, ultra-filtration desalination concentration is conducted, finally freeze-drying is conducted, and a finished product of the restrictive thrombin is obtained. The method has the advantages that pig blood is utilized to prepare the restrictive thrombin, and it is beneficial to fully utilize pig blood resources and reduce environmental pollution; the restrictive thrombin can be prepared at a normal temperature and under normal pressure, and equipment investment is reduced; the prepared restrictive thrombin is high in purity, can reach electrophoretic purity and can be used for conducting restriction enzyme digestion on GST fusion protein; operation is simple, only steps of centrifugation, fed sample chromatography, ultrafiltration, freeze-drying and the like are needed, and industrial production is achieved easily.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a preparation method of restricted thrombin. Background technique [0002] Thrombin (Thrombin, EC3.4.21.5) is a highly specific serine proteolytic enzyme in the plasma coagulation system, which can be used as a local hemostatic drug and is the main raw material of thrombin freeze-dried powder. Thrombin can hydrolyze fibrinogen to form fibrin monomer, and promote the secretion and aggregation of platelets. Therefore, thrombin plays an important role in the process of blood coagulation and hemostasis. Clinically, thrombin is widely used in local hemostasis, and can be used for hemostasis of small blood vessels that are not easy to ligate during surgery, gastrointestinal bleeding and traumatic bleeding. Thrombin can recognize the amino acid sequence of LeuValProArg↓GlySer, so thrombin has a unique role in the subsequent processing of fusion protein products obtained in genet...

Claims

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Application Information

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IPC IPC(8): C12N9/74
CPCC12N9/6429C12Y304/21005
Inventor 黄宏宝黄发灿郑登忠郭晓乾章丽丽
Owner FUJIAN HUAGENE BIOSCI CO LTD
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