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A highly active coagulation factor xi mutant ala570thr

A blood coagulation factor, high activity technology, applied in the direction of blood diseases, genetic engineering, plant gene improvement, etc., can solve the problems of limitation and low efficiency, achieve strong catalytic ability, enhance blood coagulation activity, and good prospects for alternative treatment

Active Publication Date: 2022-01-18
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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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  • A highly active coagulation factor xi mutant ala570thr
  • A highly active coagulation factor xi mutant ala570thr
  • A highly active coagulation factor xi mutant ala570thr

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The amino acid sequence of mutant proteins of highly active coagulation factor XI mutant ALA570THR is shown in SEQ ID NO: 5.

[0037] Method for preparing mutant proteins of highly active coagulation factor XI mutant ALA570THR, including the following steps:

[0038](1) Costling factor XI encoding genes of human wild-shaped or coagulation factor XI mutant Ala570 THR is entered into the vector, resulting in a recombinant carrier; Image 6 )

[0039] (2) Transform the above recombinant carrier to obtain recombinant expression cytocytes;

[0040] (3) Culture of the above cell clones in serum-free medium, expressing the expression of macrosurbs of highly active coagulation factor XI mutant ALA570THR;

[0041] Serum-free medium for "SAFC Biosciences EX-Cell TM 302 "(commercial reagents). 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...

Embodiment 2

[0054] Detect thrombus force map (see Figure 9)

[0055] ThrombolaStogram (TEG): It is a comprehensive test for monitoring all blood coagulation processes in whole blood. It does not require blood specimens, with a small number of whole blood monitoring of coagulation factors, platelets, fibrinogen, fibrin, fibrin, and its cellular components, accurately providing patient coagulation profiles. The anticoagulation blood is added to the activation monitoring reagent bottle first, and then the volume is added to the special cylindrical cup (add CaCl in advance. 2 . The cup is rotated at an angle of 4 ° 45 'and 1 weeks / 9S, and a needle is monitored by a spirally suspended needle in the blood, and the coagulation speed and intensity curve is drawn by a computer. The coagulation process is mainly evaluated by the following curve parameters: (1) The reaction time R value, that is, the detection start to the curve amplitude to rise to 2 mm, refers to the time required to detect the fibr...

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PUM

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Abstract

The present invention relates to a highly active coagulation factor XI mutant Ala570Thr (A570T), the nucleotide sequence is shown in SEQ ID NO: 1-4, and the amino acid sequence is shown in SEQ ID NO: 5. After being activated from the zymogen state to become an active enzyme, the present invention is resistant to its physiological inhibitor, so it has high blood coagulation activity and stronger catalytic ability to non-physiological substrates, and is applied to the treatment of hemorrhagic diseases , has 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 more particularly to a highly active coagulation factor XI mutant ALA570THR. Background technique [0002] Coagulation factor defects or other human coagulation dysfunction can result in bleeding disease, in which hemorrhagic diseases caused by coagulation factors VIII / IX (FVII / FIX) defects are hemophilia (A- / B) The coagulation factor VIII / IX activity of severe patients is often less than 1% of normal, often spontaneous bleeding results in muscle hematoma or articulation. Pressing factor VIII / IX preparation (currently typically recombinted expression of thromboplane VIII / IX protein) supplementary patients The prostate factor VIII / IX level is the only effective treatment, but requires frequent administration. Genetic therapy is currently the treatment of clinical trials, and introduces normal coagulation factor VIII / IX gene into patients, thereby achieving improving coagulation fact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64C12N15/57A61K38/48A61P7/04
CPCC12N9/6443A61P7/04C12Y304/21027A61K38/00C12N15/52A61K38/39A61K38/40A61K38/4846A61K38/57A61K48/0066
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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