Method for separating and purifying recombined hirudin suitable for industrial production

A technology for separation and purification of recombinant hirudin, applied in chemical instruments and methods, from leech inhibitors, protease inhibitors, etc., can solve the problems of unsuitable mass industrial production, high pigment content, poor operability, etc., and improve the reliability Operability, reduction of pigment content, and cost reduction effects

Inactive Publication Date: 2017-06-13
程度胜 +1
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Problems solved by technology

[0003] Since hirudin is currently separated and purified by heating a large amount of fermented liquid and putting it into a fermenter, it is not suitable for large-scale industrial production, and when removing impurity proteins and lipids, low-temperature centrifugation is used for mass production. The operability is poor, and the protein purity is low, the pigment content is high, and the cost is high

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  • Method for separating and purifying recombined hirudin suitable for industrial production
  • Method for separating and purifying recombined hirudin suitable for industrial production

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[0023] In order to enable those skilled in the art to better understand the solution of the present invention, the method for separating and purifying recombinant hirudin suitable for industrial production provided by the present invention will be further described in detail below in conjunction with specific embodiments.

[0024] Hirudin (HV 3 ) Protein expression engineering bacteria, see "New Secretion Expression Vector and Its Application in Recombinant Hirudin Expression Technology" applied by Tan Shuhua and Wu Wutong of China Pharmaceutical University, No. 24, Tongjia Lane, Central Road, Nanjing, Jiangsu Province, patent application number: 01113526.3, authorized announcement date: 20031119.

[0025] Step 1: Activation of strains

[0026] Hirudin (HV 3 ) A protein expression engineering strain was thawed at room temperature, inoculated with an inoculation loop, streaked on the LBA solid medium, placed upside down in a constant temperature incubator, and kept at a const...

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Abstract

The invention discloses a method for separating and purifying recombined hirudin suitable for industrial production and belongs to the field of biotechnological pharmaceutics. The method comprises the following steps: taking HV3 glycerinum strain for culturing and fermenting, thereby acquiring fermentation liquor; centrifuging the fermentation liquor, thereby acquiring fermentation liquor supernate; adjusting pH of the fermentation liquor supernate with 12M hydrochloric acid; and putting into a fermentation tank and heating, thereby effectively removing impure protein, greatly reducing the difficulty of the subsequent purifying process and increasing the maneuverability of the large-scale production process. The fermentation liquor is taken out from the tank and is placed at 2-8 DEG C, and then a method for filtering through a filter is adopted, so that the impure protein and lipid matters are removed and the maneuverability of the large-scale production process is promoted. The separating and purifying process designed according to the structural characteristics of hirudin provided by the invention is capable of effectively increasing the protein purity and greatly reducing the pigment content; the process is simple and convenient in operation; and the cost is lower.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for separating and purifying recombinant hirudin suitable for industrial production. Background technique [0002] At present, hirudin is an acidic polypeptide secreted by the salivary glands of leeches, containing 65-66 amino acid residues, mainly including HV 1 、HV 2 、HV 3 These three isomers have high structural homology among them. Hirudin is the strongest thrombin-specific inhibitor found so far, hirudin (HV 3 ) The activity of the protein-expressing engineered bacteria depends on the activity intensity of antithrombin, which forms a stable complex with non-covalent bonds in an equimolar ratio with thrombin. Pharmacological and clinical studies have shown that hirudin can be used for deep vein thrombosis, type II heparin-related thrombocytopenia and disseminated intravascular coagulation, and prevent thrombosis during hemodialysis and extr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K1/36C07K1/34C07K1/20C07K1/18
CPCC07K14/815
Inventor 程度胜桑建彬薛霞韩明娣龙应国
Owner 程度胜
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