Preparation and application of a highly active blood coagulation factor xi mutant and its gene therapy/editing vector, recombinant/fusion protein

一种凝血因子、突变蛋白的技术，应用在基因治疗、肽/蛋白质成分、载体等方向，能够解决限制、效率低下等问题，达到强催化能力、增强凝血活性、高凝血活性的效果

Active Publication Date: 2021-02-26
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE +3
View PDF2 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation and application of a highly active blood coagulation factor xi mutant and its gene therapy/editing vector, recombinant/fusion protein
  • Preparation and application of a highly active blood coagulation factor xi mutant and its gene therapy/editing vector, recombinant/fusion protein
  • Preparation and application of a highly active blood coagulation factor xi mutant and its gene therapy/editing vector, recombinant/fusion protein

Examples

Experimental program
Comparison scheme
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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention relates to the preparation and application of a highly active blood coagulation factor XI mutant and its gene therapy / editing vector, recombinant / fusion protein. The nucleotide sequence is such as SEQ ID NO: 1-6, and the amino acid sequence is such as SEQ ID NO: 7 shown. The invention has high blood coagulation activity, can efficiently activate blood coagulation reaction, improve the overall blood coagulation function of the body, is applied to the treatment of hemorrhagic diseases, and has good prospects for gene therapy, gene editing and recombinant protein replacement therapy.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64C12N15/57C12N15/85A61K38/48A61K48/00A61P7/04
CPCA61K48/005C12N9/6443C12N15/85C12Y304/21027A61K38/00C12N2800/107A61P7/04
Inventor 王学锋武文漫丁秋兰
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap