Application of a kind of prothrombin mutant protein and its coding nucleic acid

A prothrombin mutation and encoding nucleic acid technology, which is applied to peptide/protein components, medical preparations containing active ingredients, enzymes, etc., can solve problems such as limiting the hemostatic effect, weakening the hemostatic effect of prothrombin, and unsatisfactory effects , to achieve the effect of improving blood coagulation function, good treatment prospect and high promoting effect

Active Publication Date: 2021-05-11
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE +3
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Prothrombin complex (including prothrombin, coagulation factor IX, coagulation factor X, coagulation factor VII) is also used in the treatment of hemophilia, especially in the presence of inhibitors, but the effect is not ideal
After prothrombin is used in patients with hemophilia, in addition to being activated to promote blood coagulation, it can also activate protein C, inhibit blood coagulation, and weaken the hemostatic effect of prothrombin
At the same time, since thrombin is rapidly inactivated by antithrombin, it also limits the hemostatic effect of prothrombin in hemophiliacs

Method used

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  • Application of a kind of prothrombin mutant protein and its coding nucleic acid
  • Application of a kind of prothrombin mutant protein and its coding nucleic acid
  • Application of a kind of prothrombin mutant protein and its coding nucleic acid

Examples

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Effect test

Embodiment 1

[0035]This example provides a coagulation selective high-active thrombus protrusion protein amino acid sequence as shown in SEQ ID NO: 2.

[0036]The preparation method includes the following steps:

[0037](1) A recombinant carrier is obtained in the carrier in the carrier in the carrier (see) the human thrombin generated coding gene in the human wild-shaped or thrombin protoplast R541W.figure 1 )

[0038](2) Transform the above recombinant carrier to obtain recombinant expression cytocytosis;

[0039](3) The above cell clones were cultured in a serum-free medium, expressing the expression of mutant proteins of highly active coagulation factor XI;

[0040]Serum-free medium for "SAFC Biosciences EX-CellTM 302 "(commercial reagent). To ensure product safety, prevent blood source preparations from propagating infectious diseases, so serum-free medium is used for mammalian cell culture, protein expression, and cellular vectors have grown steady state, The cell density was maintained within the target...

Embodiment 2

[0056]Thrombin protoplast R541W to treat hemophilia A mice

[0057](1) The purified wild-type thrombin or mutant R541W was injected with a hemophilia mouse with a 1.5 times quantity (150 ug / ml). The 4-8 weeks of bloody mice were selected, and 3 mice were injected with 2 ml of physiological saline by tail veins. PBS injection of 3 mice were a negative control.

[0058](2) After anesthesia mice, the tail at a diameter of 2 mm is placed in a cage to observe its survival rate. byFigure 4 It can be seen that coagulation active selective thrombin protoplast R541W can correct coagulation defects caused by coagulation factor VIII defects in vivo, reduce bleeding, and improve the survival rate.

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Abstract

The invention relates to the application of a prothrombin mutant protein and its coding nucleic acid for preparing gene therapy medicine, including linking it with a promoter and / or termination sequence and constructing an expression plasmid. The mutant protein and its encoding nucleic acid of the present invention have high blood coagulation activity, can efficiently promote blood coagulation, improve the overall blood coagulation function of the body, and have good prospects for gene therapy, gene editing and recombinant protein replacement therapy.

Description

Technical field[0001]The invention belongs to the field of bleeding treatment, and in particular, the present invention relates to a prothrombin mutant protein and its encoding nucleic acid.Background technique[0002]Coagulation factor defects or other human coagulation dysfunction can result in hemorrhagic diseases, in which hemorrhagic diseases (A -1 / B) due to blood-bleeding disease caused by coagulation factor VIII / IX (FVII / FIX) defects , Severe patients' coagulation factor VIII / IX activity is often less than 1% of normal, often spontaneous bleeding results in muscle hematoma or articulation. Infusion factor VIII / IX preparation (currently usually a thirty-expression of thromboplane VIII / IX protein) supplemented with the patient's body is the only effective treatment, but frequent administration is required. Genetic therapy is currently in the treatment of clinical trials, and introduces normal coagulation factor VIII / IX gene into patients, thereby achieving improving...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/48A61P7/04
CPCA61K38/4833A61P7/04C12Y304/21005
Inventor 王学锋武文漫丁秋兰
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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