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High-activity blood coagulation factor VIII or VIII polypeptide variant Gly710Ala

A technology of coagulation factor and high activity, applied in the field of hemophilia, can solve problems such as high cost, achieve the effects of superior stability, improved drug efficacy, and good clinical application prospects

Pending Publication Date: 2022-03-18
THE FIRST AFFILIATED HOSPITAL OF GUANGXI MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The half-life of FⅧ in vivo is only less than 12 hours. In order to maintain sufficient plasma concentration to prevent bleeding, it needs to be injected 3 times a week, which is expensive

Method used

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  • High-activity blood coagulation factor VIII or VIII polypeptide variant Gly710Ala
  • High-activity blood coagulation factor VIII or VIII polypeptide variant Gly710Ala
  • High-activity blood coagulation factor VIII or VIII polypeptide variant Gly710Ala

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Embodiment 1

[0027] The mutant protein of highly active blood coagulation factor VIII or VIIIa polypeptide variant Gly710Ala has an amino acid sequence as shown in any one of SEQ ID NO: 21-29, and the amino acid at position 710 of the mutant is Ala instead of Gly of wild type VIII or VIIIa. In this embodiment, the mutein of hFVIII cDNA is taken as an example.

[0028] 1. Expression and purification of FⅧGly710Ala mutant

[0029] (1) Expression: using the QuikChange site-directed mutagenesis kit ( Ⅱ XL Site-Directed Mutagenesis Kit, Agilent, USA), using human B-region-deleted blood coagulation factor VIII (BDD-hFVIII) as a template, using PCR site-directed mutagenesis to introduce mutation sites into wild-type FVIII, replacing 710Gly with 710Ala , the amplified product is transformed after being digested with Dpn I (add 5-10 microliters of the mutant product digested by Dpn I to every 100 microliters of competent bacteria), and the transformed bacteria are coated to contain 1 / 2000 ampicil...

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Abstract

The invention relates to a polypeptide variant Gly710Ala of a blood coagulation factor VIII or VIII a with high activity. The mutant does not destroy or inhibit the blood coagulation activity of FVIII, the blood coagulation specific activity is 1.5-2 times that of a wild type, and the mutant is more excellent in stability; the mutant obviously improves the drug efficacy of the FVIII, and has a good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of hemophilia, in particular to a highly active coagulation factor VIII or VIIIa polypeptide variant Gly710Ala. Background technique [0002] Hemophilia A is a hereditary bleeding disorder caused by coagulation factor Ⅷ (FⅧ) deficiency. The incidence rate in the male population is 1 / 5000. Patients with coagulation dysfunction manifest as spontaneous muscle hematoma and joint cavity hemorrhage. FⅧ replacement therapy is currently the only effective treatment. With the development of biotechnology, genetically recombined FⅧ has gradually replaced plasma or plasma-derived FⅧ as the main preparation for replacement therapy. The half-life of FⅧ in vivo is only less than 12 hours. In order to maintain sufficient plasma concentration to prevent bleeding, it needs to be injected 3 times a week, which is expensive. The development of FⅧ preparations with higher coagulation activity can not only achieve the same therapeutic e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/755C12N15/85A61K38/37A61P7/04
CPCC07K14/755C12N15/85A61K38/37A61P7/04C12N2800/107
Inventor 韦红英
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGXI MEDICAL UNIV
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