VWF propeptide expression vector as well as preparation method and application thereof

A technology for expressing vectors and propeptides, which is applied in the field of VWF propeptide expression vectors and its preparation, and can solve the problems of impossibility and difficulty in packaging and preparing viral vectors.

Pending Publication Date: 2021-12-31
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Usually, the method of gene therapy is to inject the complete normal VWF gene into the human body through a viral vector to correct the genetic defect. However, because the VWF molecule is particularly large and is one of the largest genes in the human body, the preparation of the viral vector packaging is very difficult and almost impossible.

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  • VWF propeptide expression vector as well as preparation method and application thereof
  • VWF propeptide expression vector as well as preparation method and application thereof

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Embodiment 1

[0014] The c.875-5T>G mutation was found in a patient with von Willebrand disease type 2A, which can lead to the deletion of exon 8 of the VWF gene (VWF E8del). Exon 8 of VWF gene encodes amino acids 293-333 of VWF propeptide D1 region. Patients showed a mild decrease in plasma von Willebrand factor antigen levels and a marked decrease in VWF high molecular weight multimers.

[0015] The full-length wild-type human VWF cDNA was cloned into the expression vector pCIneo, and the VWF expression vector pCIneoVWFWT was constructed; the coding sequence of VWF signal peptide and VWF domain D1D2 was amplified based on the sequence of pCIneoVWFWT, and a stop codon was inserted behind the 763 amino acid, The VWF propeptide expression vector pCIneoVWFpp was obtained and confirmed by direct sequencing. VWF propeptide mutants and VWF full-length plasmid mutants (including VWF E8del mutation and Y87S mutation) were constructed by site-directed mutagenesis. The study on the structure model...

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Abstract

The invention relates to a VWF propeptide expression vector as well as a preparation method and application thereof. The VWF propeptide expression vector comprises a VWF signal peptide, a encoding sequence of a VWF structural domain D1D2 region and a termination codon. Based on the action mechanism that VWF propeptide participates in VWF polymer assembly, the VWF propeptide expression vector disclosed by the invention can restore part of congenital VWF polymer formation obstacles only by introducing normal VWF propeptide, and paves a way for a gene therapy strategy for correcting congenital defects on the protein level through 'protein structure editing'.

Description

technical field [0001] The invention belongs to the field of von Willebrand disease, and in particular relates to a VWF propeptide expression vector and its preparation method and application. Background technique [0002] Von Willebrand Factor (VWF) is a very important high-molecular-weight glycoprotein in the blood coagulation system. Its main function is to mediate the adhesion of platelets to the exposed collagen under the damaged endothelium, and to protect coagulation factor VIII from being absorbed too quickly. degradation. These functions depend on the binding sites for many ligands in the VWF subunit multidomain structure: D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK . In addition, VWF monomers can be connected end-to-end to form multimers, and only high molecular weight VWF multimers have corresponding physiological functions. Defective VWF multimer formation leads to type 2A von Willebrand disease. VWF gene mutations located in the VWF propeptide region (D1D2 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/867C12N15/861C12N15/864C12N15/12A61K48/00A61K38/37A61P7/04
CPCC12N15/86C07K14/755A61K48/0008A61K48/005A61K38/37A61P7/04C12N2740/15043C12N2710/10043C12N2750/14143
Inventor 王学锋周爱武梁茜武文漫
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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