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Protein matter with coagulation activity

A blood coagulation and substance technology, which is applied in the field of protein substances and their preparation, can solve the problems that there are no successful blood coagulation drugs of Agkistrodon acutus venom, no detailed reports on pharmacological effects, difficulties in separation and purification, etc., and achieve strong process operability , good blood coagulation activity, and easy operation

Inactive Publication Date: 2012-06-20
JIANGSU QINGJIANG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because snake venom contains a variety of proteins and polypeptides with in vitro blood coagulation activity, it is difficult to separate and purify. At present, the only domestic snake venom hemostatic products are Bangting (for injection) produced by Liaoning Jinzhou Aohong Pharmaceutical Co., Ltd. The SDS-polyacrylamide gel electrophoresis of the snake venom thrombin in this product contains three bands of 54kDa, 34kDa and 15kDa, and the thrombin-like enzyme of the three electrophoresis bands is currently on the market. Pharmacological effects have not been reported in detail
The distribution areas of Agkistrodon in my country include Anhui (southern), Chongqing, Jiangxi, Zhejiang, Fujian (northern), Hunan, Hubei, Guangxi (northern), Guizhou, Guangdong (northern) and Taiwan Province, etc., with a wide distribution range. Many domestic institutions have conducted research on the blood coagulation active components in Agkistrodon venom, and have achieved certain results, but so far, no blood coagulation drug from Agkistrodon venom has been successfully marketed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation of Substance A

[0021] Take 6 g of freeze-dried crystals of Agkistrodon venom and dissolve it in Tris-HCl buffer solution with a concentration of 20 mmol / L and pH 7.0, centrifuge at 1000 rpm at 4°C for 30 min, and take the supernatant for purification by SOURCE30Q anion column chromatography, using NaCl The method of solution linear gradient elution, first wash 5 column volumes with 20% NaCl solution, and then elute 10 column volumes with 40% NaCl solution, collect and combine active component peaks, use ultrafiltration membrane ultrafiltration with a molecular weight cut-off of 10kDa After filtration and concentration, perform Sephacryl S200 gel column chromatography purification, collect and combine active component peaks, and then perform SOURCE30Q anion column chromatography purification, using NaCl isocratic elution method, first elute with 20% NaCl solution for 3 column volumes Then it was eluted with 40% NaCl solution, and the peak of the a...

Embodiment 2

[0022] Example 2 Preparation of Substance A

[0023] Take 8 g of freeze-dried crystals of Agkistrodon venom and dissolve them in Tris-HCl buffer solution with a concentration of 20 mmol / L and pH 7.0, centrifuge at 1000 rpm at 4°C for 30 min, and take the supernatant for purification by SOURCE30Q anion column chromatography. The method of linear gradient elution of NaCl solution, first wash 3 column volumes with 20% NaCl solution, then elute 12 column volumes with 30% NaCl solution, collect and combine active component peaks, and use ultrafiltration with a molecular weight cut-off of 5kDa After concentration by membrane ultrafiltration, carry out Sephacryl S200 gel column chromatography purification, collect and combine active component peaks, and then carry out Hitrap Q XL anion column chromatography purification, adopt the method of NaCl solution isocratic elution, first wash with 10% NaCl solution After removing 3 column volumes, elute with 30% NaCl solution, collect and com...

Embodiment 3

[0024] Example 3 Determination of specific activity of coagulation of substance A in vitro

[0025] 2.1 Materials

[0026] Substance A 1, 2, 3 batches of samples were prepared by the method of Example 1 or 2

[0027] Baquting was purchased from Penglai Nuokang Pharmaceutical Co., Ltd.

[0028] 2.2 Method

[0029] The coagulation specific activity index of the substance A in vitro of the present invention is investigated by using the coagulation time of the coagulation active substance per unit protein amount to human plasma. The specific operation method is: take a standard human plasma, add 1ml of water for injection to dissolve, accurately measure 0.2ml, put it in a small test tube, keep it in a 37°C water bath for 3 minutes, and accurately add 0.2ml of the preheated test solution , mix immediately, and count the time. Use a clean long needle to pick up the mixed solution in the water bath and keep warm. At the same time, observe the solution until the long needle picks t...

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PUM

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Abstract

The invention relates to single chain protein matter with coagulation activity. The gel electrophoresis of the reducing SDS-polyacrylamide of the protein matter is performed to form a single strip under the molecular weight of 31+ / -2kDa, the molecular weight is measured to be 30500+ / -1000Da by a MALDI-TOF method, the isoelectric point is 4.4-4.8, the area normalization method is utilized to measure the purity which is more than 98%, and fifteen-amino acid sequence of N terminal end measured by the Edman degradation method is VIGGNECDTNEHRFL. The protein matter has stronger coagulation activity on human fibrinogen in vitro, and the specific activity thereof is 500-1500U / mg. Verified by in vitro coagulation activity experiment tests, the single chain protein matter with coagulation activityprovided by the invention has better coagulation activity; the preparation method thereof utilizes frequently-used separation and purification measures in the extraction separation process of currentbiochemical filed, thereby having strong process maneuverability, simple and easy operation and stable volume of production.

Description

technical field [0001] The invention relates to a protein substance with blood coagulation activity and a preparation method thereof, in particular to a protein substance with blood coagulation activity and a method for extracting the substance from snake venom. Background technique [0002] Snake venom contains a variety of proteins and polypeptides, most of which are special proteases and active polypeptides. These substances may be involved in digestion as supporting substances and are also the main cause of snake venom hemorrhagic and neurotoxic. In 1767, Fontana described the blood coagulation effect of snake venom for the first time. After more than 200 years, the research on the application of snake venom to the blood system has continuously made achievements. Since the 1970s, many snake venom drugs, such as Ancrod, Batroxobin, Crotalase and Reptilase, have been developed into clinical drugs, and they play an important role in the treatment of blood and cardiovascula...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/34C07K1/18C07K1/16A61K38/17A61P7/04
Inventor 赵伟徐宏江曹宇虹宋伟付辉张喜全
Owner JIANGSU QINGJIANG PHARMA
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