Recombinant CREG protein and application thereof

A recombinant protein and protein technology, applied in the field of recombinant CREG protein and its application, can solve the problem of low CREG protein production

Active Publication Date: 2009-09-02
GENERAL HOSPITAL OF THE NORTHERN WAR ZONE OF THE CHINESE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glycosylated CREG proteins can maximize their biological functions; at the same time, deglycosylated CREG proteins with biological functions can also be prepared using prokaryotic expression vectors to meet the requirements of mass production, thereby overcoming glycosylated CREG proteins Must use eukaryotic expression vector preparation and low yield

Method used

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  • Recombinant CREG protein and application thereof
  • Recombinant CREG protein and application thereof

Examples

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Embodiment Construction

[0022] The following examples are provided to further explain and illustrate the present invention. Example:

[0023] 1. Construction and identification of eukaryotic expression vector pcDNA3.1myc-His / hCREG (including hCREG and hCREG with glycosylation site mutation)

[0024] According to the open reading frame in the hCREG cDNA sequence registered in GenBank (NM003851), hCREG RT-PCR primers with stop codon mutations were designed and synthesized.

[0025] The upstream primer is: 5'-aa ggatcc atggccgggctatcccgc-3'

[0026] Basic Tm=66°C, salt (50mM) adjusted Tm=74°C;

[0027] The downstream primer is: 5'-gc gaattc Gcactgaactgtgacatttaatattcttctgg-3'

[0028] Basic Tm=64°C, salt (50mM) adjusted Tm=74°C;

[0029] The uppercase letters represent the stop codons of C / G mutations.

[0030] Extract mRNA from VSMCs cultured in vitro and reverse transcribe into cDNA (cDNA first-strand synthesis kit from TakaRa Company. Amplification conditions are: 25°C, 5 minutes; 42°C, 50 minut...

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Abstract

The invention relates to a eukaryotic expression vector carrying human cellular repressor gene of E1A-stimulated gene (hCREG) and a eukaryotic expression vector carrying glycosylation sites mutation hCREG. The invention also relates to preparation of recombinant hCREG protein and glycosylation sites mutation hCREG protein with the eukaryotic expression vector, the actions of the two types of proteins on restricting the proliferation of vascular smooth muscle cells (VSMCs), the preparation of the medicine preventing restenosis after percutaneous coronary intervention (PCI) with the proteins and the applications thereof.

Description

technical field [0001] The present invention relates to a eukaryotic expression vector carrying human E1A activating gene repressor (hCREG) gene and a eukaryotic expression vector carrying hCREG gene with glycosylation site mutation, and the present invention also relates to the eukaryotic expression vector It is used to prepare recombinant hCREG protein and hCREG protein with glycosylation site mutation, the effect of the two proteins on inhibiting the proliferation of vascular smooth muscle cells (vascular smoothmuscle cells, VSMCs) and the preparation of drugs with inhibitory effect on post-PCI restenosis and its use. Background technique [0002] Protein glycosylation is an important way of post-translational modification of eukaryotic proteins. About 50% of proteins in organisms are glycosylated proteins, including enzymes, carrier proteins, hormones, toxins, various lectins and structural proteins. The protein in the glycosylated protein is the main bearer of its phys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/62C07K14/47C07K19/00A61K38/17A61P9/00
Inventor 韩雅玲孙鸣宇栾波康健张效林闫承慧
Owner GENERAL HOSPITAL OF THE NORTHERN WAR ZONE OF THE CHINESE PEOPLES LIBERATION ARMY
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