High-activity blood coagulation factor XI mutant Ala570Thr

A technology of coagulation factor and high activity, applied in blood diseases, genetic engineering, plant genetic improvement, etc., can solve problems such as low efficiency and limitations, achieve strong catalytic ability, improve overall coagulation function, and high coagulation activity.

Active Publication Date: 2020-12-25
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE +3
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  • Application Information

AI Technical Summary

Problems solved by technology

However, wild-type factor XI catalyzes the cleavage of bypass coagulation substrates including FX, FV, or TFPI very inefficiently, limiting its ability to promote coagulation through bypass

Method used

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  • High-activity blood coagulation factor XI mutant Ala570Thr
  • High-activity blood coagulation factor XI mutant Ala570Thr
  • High-activity blood coagulation factor XI mutant Ala570Thr

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The amino acid sequence of the mutant protein of highly active blood coagulation factor XI mutant Ala570Thr is shown in SEQ ID NO:5.

[0037] The preparation method of the mutant protein of highly active coagulation factor XI mutant Ala570Thr comprises the following steps:

[0038](1) The human blood coagulation factor XI coding gene of human wild type or blood coagulation factor XI mutant Ala570Thr is connected in the vector to obtain the recombinant vector; (see Figure 6 )

[0039] (2) Transforming the above-mentioned recombinant vector into a host cell to obtain recombinant expression cell clones;

[0040] (3) Cultivating the above-mentioned cell clones in serum-free medium, expressing the expression of the mutant protein of highly active blood coagulation factor XI mutant Ala570Thr;

[0041] The serum-free medium is "SAFC Biosciences EX-CELL TM 302” (commercialized reagent). In order to ensure product safety and prevent blood-derived preparations from spreading...

Embodiment 2

[0054] Detection of thromboelastogram (see Figure 9)

[0055] Thromboelastogram (TEG): is a comprehensive test used to monitor the overall coagulation process in whole blood. It does not require blood sample processing, and uses a small amount of whole blood to monitor the interaction between coagulation factors, platelets, fibrinogen, fibrinolytic system and other cellular components, and accurately provide the patient's coagulation profile. When testing, first add anticoagulant blood to the activation monitoring reagent bottle, then suck out a certain volume and add it to a special cylindrical cup (add CaCl in advance 2 ). The cup rotates at an angle of 4°45' at a constant speed of 1 cycle / 9s. The coagulation state of the blood is monitored through a needle immersed in the blood and suspended by a helical wire, and the coagulation speed and intensity curve is drawn by the computer. The blood coagulation process is mainly evaluated through the following curve parameters: (1...

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Abstract

The present invention relates to a high-activity blood coagulation factor XI mutant Ala570Thr (A570T), nucleotide sequences are shown as SEQ ID NO: 1-4 and an amino acid sequence is shown as SEQ ID NO: 5. A zymogen state is activated into an enzyme with activity to produce resistance of a physiological inhibitor, thus the high-activity blood coagulation factor XI mutant Ala570Thr has very high coagulation activity and stronger catalytic capability on a non-physiological substrate, is applied to treatments of hemorrhagic diseases, and has very good prospects for gene treatment, gene editing andrecombinant protein replacement.

Description

technical field [0001] The invention belongs to the field of hemorrhagic disease treatment, and particularly relates to a highly active coagulation factor XI mutant Ala570Thr. Background technique [0002] Blood coagulation factor deficiency or other human coagulation disorders can lead to bleeding disorders, among which bleeding disorders caused by coagulation factor VIII / IX (FVIII / FIX) deficiency are called hemophilia (type A / B) In severe patients, the coagulation factor VIII / IX activity is often lower than 1% of normal, and spontaneous bleeding often occurs leading to muscle hematoma or joint deformity. Infusion of factor VIII / IX preparations (currently usually recombinantly expressed coagulation factor VIII / IX proteins in vitro) to supplement the patient's blood coagulation factor VIII / IX levels is currently the only effective treatment, but requires frequent administration. Gene therapy is a treatment method currently undergoing clinical trials, which introduces the no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/57A61K38/48A61P7/04
CPCC12N9/6443A61P7/04C12Y304/21027A61K38/00C12N15/52
Inventor 武文漫王学锋丁秋兰
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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