Hirulog as well as preparation method and application thereof

A technology of leech peptide and recombinant hirudin, which is applied to the preparation method of peptides, from leech inhibitors, chemical instruments and methods, etc., can solve the problems of small immunogenicity, affecting use, haptenism, etc., and achieve immunogenicity The effect of reducing, good development prospects and clinical application value

Inactive Publication Date: 2013-05-29
大连市食品药品检验所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The smaller the molecular weight, the smaller the immunogenicity. Because hirudin and recombinant hirudin have a large molecular weight, they are hapten, which affects their clinical use.

Method used

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  • Hirulog as well as preparation method and application thereof
  • Hirulog as well as preparation method and application thereof
  • Hirulog as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0025] (1) 100 rats were injected with recombinant hirudin into the tail vein at a dose of 6 mg / kg. Collect urine 0.5h after administration for the separation and extraction of metabolites;

[0026] (2) Preliminary separation of blank and sample urine by gel chromatography. Chromatographic conditions: gel column, TSK-GELG2000SW 7.5mm×30.0cm, injection volume: 20μl, mobile phase: 0.02mol / l PBS solution, pH7.0, flow rate 0.8ml / min, detection wavelength: 215nm. Collect the effluent, collecting 1 tube per minute. Take the collected solution at 16, 17, 20, and 21 minutes, freeze at -70°C, freeze-dry with a freeze dryer the next day, and set aside;

[0027] (3) The 16-21 min collected liquid collected by gel chromatography was separated again by C8 reverse phase chromatography. Chromatographic conditions: C8 column, injection volume 20μl, gradient elution, acetonitrile: 0.1% TFA from 15:85 to 45:55 within 25min, flow rate 0.5ml / min, detection wavelength 215nm. Inject samples rep...

Embodiment 3

[0029] Determination of molecular weight

[0030] The above-mentioned leech peptide rH 1-51 and rH 1-52 , the molecular weight of the extracted metabolites was determined by time-of-flight mass spectrometry (MALDI-TOF / TOF). Detection conditions: N2 laser source, wavelength 337nm; detection method: positive ion, linear method (flight tube length 1.5m, acceleration voltage 20kV); matrix: CCA, leech peptide rH 1-51 and rH 1-52 The molecular weight was determined to be 5162 by time-of-flight mass spectrometry.

Embodiment 4

[0032] Determination of antithrombin activity

[0033] This method uses tosyl-aminoacetyl-prolyl-arginyl-p-nitroaniline (Tosyl-glycyl-polyl-arginine-para-nitroniline; trade name: Chromozym TH) as the substrate, thrombin It can hydrolyze Chromozym TH to release p-nitroaniline (para-nitroniline, pNA), and pNA has specific absorption at 405nm. rH can be quantitatively combined with thrombin, so that thrombin loses the activity of hydrolyzing the substrate. By measuring the amount of thrombin not inhibited by rH in the reaction system, the content of rH can be indirectly measured. In this experiment, rH was used as a standard to prepare a standard curve, and the rH 1-51 and rH 1-52 Antithrombin activity.

[0034] 1. Preparation of standard curve

[0035] Recombinant hirudin is a specific inhibitor of thrombin. Thrombin can hydrolyze the chromogenic substrate (Chromozym TH, CT) to release p-nitroaniline, which has absorption at 405nm wavelength, within a certain range , the am...

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Abstract

The invention provides a hirulog as well as a preparation method and application thereof and belongs to the field of polypeptides and preparation methods thereof. A metabolic product of a recombined hirulog provided by the invention in urine of a rat has stronger antithrombin activity, which is found in the process that the recombined hirulog metabolizes in the body of the rat. Through extraction, separation and structural evaluation, the metabolic product is found to be a hirulog which is formed by retaining the N ends of a group of recombined hirulogs and removing 17-12 amino acids on the C ends of the group of recombined hirulogs, namely a hirulog rH1-51, a hirulog rH1-52 and a mixture of the hirulog rH1-51 and the hirulog rH1-52. The preparation method mainly comprises the following steps of: carrying out intravenous administration on the rat, collecting urine, extracting and purifying by using a gel filtration high performance liquid chromatography (GF-HPLC) and combining a reverse phase high performance liquid chromatography (RP-HPLC) to obtain the hirulog. The immunogenicity of the hirulog provided by the invention is remarkably reduced, and therefore, the hirulog is expected to become a new-generation antithrombus drug and is favorable in development prospect and high in clinical application value.

Description

technical field [0001] The invention belongs to the field of polypeptide and its preparation, in particular to a leech peptide and its preparation method Background technique [0002] According to the Statistical Yearbook of the World Health Organization, cardiovascular diseases, malignant tumors, respiratory diseases, traffic accidents and suicides are the top five causes of death in the world. The annual death toll of cardiovascular diseases in my country reaches one million. With the improvement of people's living standards, cardiovascular diseases will become more and more serious. [0003] Coronary heart disease is an important type of cardiovascular disease. Its pathogenesis is related to arterial and venous thrombosis and thromboembolism. It is mainly due to abnormal blood lipid metabolism, which causes inflammatory immune response and platelet aggregation in the intima of blood vessels. Lipid substances accumulate to form white plaques, blood vessels are narrowed to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/815C07K19/00C07K1/20C07K1/16A61K38/56A61P7/02
Inventor 姜素云
Owner 大连市食品药品检验所
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