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Agkistrodon acutus hemocoagulase gene and methods for preparing expression vector, host cell and recombinant protein thereof

A Agkistrodon acutus and expression carrier technology, applied in the field of biomedicine, can solve the problems of thrombosis and hemorrhagic diseases that cannot be treated, and achieve significant technological progress and highlight the effect of substantive features

Inactive Publication Date: 2012-09-12
郑颖 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Since human thrombin directly enters the blood, it is easy to cause thrombus formation, so it cannot be used for the treatment of bleeding diseases in the body except for topical application.

Method used

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  • Agkistrodon acutus hemocoagulase gene and methods for preparing expression vector, host cell and recombinant protein thereof
  • Agkistrodon acutus hemocoagulase gene and methods for preparing expression vector, host cell and recombinant protein thereof
  • Agkistrodon acutus hemocoagulase gene and methods for preparing expression vector, host cell and recombinant protein thereof

Examples

Experimental program
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Embodiment Construction

[0066] (1) Cloning of 36KDa single-chain haemocoagulase gene from Agkistrodon akistros

[0067] 1.1 The extraction of Agkistrodon venom gland total RNA comprises the following steps:

[0068] (1) The venom glands of Agkistrodon acutus were decapitated from live vipers, and the snake glands were ground in liquid nitrogen, and 1 mL of total RNA extraction buffer (Trizol solution, product of GIBCOBRL, USA) was added to each 50-100 mg of tissue, and homogenization was carried out with a homogenizer. For processing, blow and suck repeatedly with a pipette, and incubate at room temperature for 5 min.

[0069] (2) Add 0.2ml chloroform for every 1mL Trizol used, shake vigorously for 15 seconds, incubate at room temperature for 3 minutes, then centrifuge at 10000g 4°C for 15 minutes, and take the supernatant.

[0070] (3) Add 500-700 uL isopropanol to 1 mL of supernatant, and incubate at room temperature for 10 min. Centrifuge at 10,000 g at 4°C for 10 min, discard the supernatant, w...

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Abstract

An agkistrodon acutus hemocoagulase gene and methods for preparing an expression vector, a host cell and recombinant protein thereof belong to biomedicine. Hemocoagulase maturation protein is single-chain glycoprotein with the molecular weight of 36000+ / -3000Da and 6.59 isoelectric points, wherein the single-chain glycoprotein contains 236 amino acid residue, the total order of proteinogenic amino acid is SEQNO:1, and a gene for encoding the protein is formed by 783 nucleotides and has the order of SEQNO:1. The hemocoagulase has arginine esterase activity and fibrinogen hydrolytic activity and can preferentially degrade an A Alpha chain of fibrinogen. The agkistrodon acutus hemocoagulase gene can be applied to genetic engineering preparation. Methods utilizing the agkistrodon acutus hemocoagulase gene to construct the expression vector and utilizing the host cell to obtain recombinant hemocoagulase protein are provided, and a method for obtaining the agkistrodon acutus hemocoagulase gene in the molecular biology is restated.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to Agkistrodon akistrodon ( Agkistrodon acutus ) gene of hemocoagulase (haemocoagulase HCA) and its application. Background technique [0002] Among the many complex active ingredients of snake venom, one of them is a kind of serine protease, which has arginine esterase activity, which can break the Arg-Gly peptide bond at a specific part of fibrinogen, release fibrinopeptide and convert it into fibrin, and act on plasma Thrombin is very similar and is called a thrombin-like enzyme. Most venom-like thrombin can only cleave the Aα chain or Bβ chain of fibrinogen, release fibrinopeptide A (FPA) or B (FPB), and form non-crosslinked fibrin monomers. The coagulation effect of snake venom thrombin in vitro is similar to that of thrombin, and it can directly aggregate fibrinogen without passing through various coagulation factors in the body. The fibrinolytic system in the blood is degraded a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/64C12N15/10C12N15/70C12N15/81
Inventor 郑颖范泉水叶锋平沈居仁
Owner 郑颖
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