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High-activity blood coagulation factor XI mutant and preparation and application of gene therapy/editing vector and recombinant/fusion protein thereof

A technology for coagulation factors and mutant proteins, applied in gene therapy, peptide/protein components, applications, etc., can solve problems such as limitations and low efficiency, and achieve the effect of strong catalytic ability, enhanced coagulation activity, and improved coagulation function.

Active Publication Date: 2018-06-29
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE +3
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  • Abstract
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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 and preparation and application of gene therapy/editing vector and recombinant/fusion protein thereof
  • High-activity blood coagulation factor XI mutant and preparation and application of gene therapy/editing vector and recombinant/fusion protein thereof
  • High-activity blood coagulation factor XI mutant and preparation and application of gene therapy/editing vector and recombinant/fusion protein thereof

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Experimental program
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Effect test

Embodiment 1

[0046] The amino acid sequence of the mutant protein of highly active blood coagulation factor XI is shown in SEQ ID NO:7.

[0047] A method for preparing a mutant protein of highly active blood coagulation factor XI, comprising the steps of:

[0048] (1) The human blood coagulation factor XI coding gene of human wild type or blood coagulation factor XI mutant Gly397Ser is connected in the vector to obtain the recombinant vector; (see Figure 8 )

[0049] (2) Transforming the above-mentioned recombinant vector into a host cell to obtain recombinant expression cell clones;

[0050] (3) Cultivate the above-mentioned cell clones in a serum-free medium to express the expression of a mutant protein of highly active blood coagulation factor XI;

[0051] 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 infectious diseases, serum-free medium is used for mamma...

Embodiment 2

[0064] The preparation method of the gene therapy plasmid vector of highly active blood coagulation factor XI comprises the following steps:

[0065] (1) Construction of the vector plasmid The cDNA encoding FXI Gly397Ser was ligated into the pcDNA3.1 plasmid containing the CMV promoter.

[0066] (2) Inject the purified vector into type A hemophilia mice. Hemophilia mice aged 4-8 weeks were selected, and 150 μg of the plasmid vector dissolved in 2 mL of normal saline was injected into 6-7 mice respectively through the tail vein. Six mice were injected with PBS as negative control.

[0067] (3) aPTT detects coagulation factor XI activity. 48 hours after injection, orbital blood was collected to detect coagulation factor XI activity and antigen, wherein the activity was the sum of the original mouse coagulation factor XI activity and the activity of injected human wild-type or mutant coagulation factor XI in the mouse, while the antigenic principle was only is the level of pou...

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Abstract

The invention relates to a high-activity blood coagulation factor XI mutant and preparation and application of a gene therapy / editing vector and recombinant / fusion protein thereof. A nucleotide sequence is as shown in SEQ ID NO:1-6, and an amino acid sequence is as shown in SEQ ID NO:7. The mutant provided by the invention has very high blood coagulation activity, can efficiently activate blood coagulation reaction and improve the whole blood coagulation function of the organism, is applied to therapy of hemorrhagic diseases, and has very good gene therapy, gene editing and recombinant proteinreplacement therapy prospects.

Description

technical field [0001] The invention belongs to the field of hemorrhagic disease treatment, and in particular relates to the preparation and application of a highly active blood coagulation factor XI mutant, a gene therapy / editing carrier, and a recombinant / fusion protein. 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 protein in vitro) to replenish the level of coagulation factor VIII / IX in patients is currently the only effective treatment, but frequent administration is required. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/57C12N15/85A61K38/48A61K48/00A61P7/04
CPCA61K38/00A61K48/005C12N9/6443C12N15/85C12N2800/107C12Y304/21027A61P7/04
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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