A fusion gene containing cbd, cell line, liquid ECM and application

A technology of fusion gene and CBD-NEP, applied in the field of genetic engineering

Active Publication Date: 2020-10-02
SHANGHAI UNIV +1
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of this method is that ECM is simply prepared without adding any growth factors. It only studies the effect of ECM protein on cell behaviors such as stem cell proliferation, migration and differentiation, and does not use it as a drug-loaded substance. , for the treatment of specific diseases

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
  • A fusion gene containing cbd, cell line, liquid ECM and application
  • A fusion gene containing cbd, cell line, liquid ECM and application
  • A fusion gene containing cbd, cell line, liquid ECM and application

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0051] Another aspect of the embodiments of the present invention also provides the aforementioned method for preparing the liquid ECM, which includes:

[0052] (1) Use primers to amplify the cDNA of APP, NEP gene and IDE gene by polymerase chain reaction, and then carry out enzyme digestion on the amplified gene and the original vector backbone at the same time according to the restriction site selected by the primers, Afterwards, the enzyme-digested fragments were recovered, and the enzyme-digested fragments and the carrier enzyme-digested fragments were subjected to an enzyme-linked reaction under the action of DNA ligase. After the enzyme-linked reaction was completed, the enzyme-linked products were transformed into competent cells and the plasmid was amplified, and then carried out PCR amplification, construction of recombinant plasmids or recombinant expression vectors;

[0053] (2) Integrate the recombinant plasmid or recombinant expression vector into the cell genome ...

Embodiment 1

[0060] The main technical process of the preparation method of a liquid extracellular matrix ECM enriched with enkephalinase and insulin-degrading enzyme provided in this example can be divided into four steps: 1. Carrier construction; 2. Cell construction; 3. Preparation of ECM ; 4, ECM performance testing.

[0061] Each step is described in detail below:

[0062] 1. Carrier Construction

[0063] (1) Select the appropriate carrier skeleton. The present invention selects the lentiviral vector pLVX-IRES-Puro (hereinafter referred to as PLVX) and the retroviral vector pQCXIH (hereinafter referred to as PQH) as the basic skeleton of the constructed recombinant vector.

[0064] (2) The National Center of Biotechnology Information (NCBI) website (https: / / www.ncbi.nlm.nih.gov / ) found the coding sequence (codingsequence, CDS) sequence of APP, NEP and IDE .

[0065] (3) Use the Primer5 primer design software to design the primers required for APP, NEP and IDE, and then submit them...

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 invention discloses an IDE and NEP fusion gene containing a CBD (Collagen Binding Domain), a cell line, a liquid ECM (Extracellular Matrix) and an application. The IDE and NEP fusion gene containing the CBD (Collagen Binding Domain) comprises a CBD-IDE fusion gene and a CBD-NEP fusion gene, wherein the sequence of the CBD-IDE fusion gene is as shown in SEQID NO:1, and the sequence of the CBD-NEP fusion gene is as shown in SEQID NO:2. The cell line of the IDE and NEP fusion gene containing the CBD (Collagen Binding Domain) can stably express and secrete growth factors IDE and NEP and is combined to the extracellular matrix. The liquid ECM is synthesized by the cell line and is secreted outside cells. Through a gene technique, the IDE and NEP fusion gene carrying the CBD is constructed, then the expressed IDE protein and the expressed NEP protein are combined to collagen I of the ECM, the purpose of performing enriching on the ECM is achieved, and the purpose of treating Alzheimer 's Disease is realized.

Description

technical field [0001] The present invention relates to a preparation method of liquid ECM, in particular to a fusion gene, plasmid, cell line, liquid ECM and its The invention belongs to the technical field of genetic engineering. Background technique [0002] Alzheimer's disease (Alzheimer's Disease, hereinafter referred to as AD) is one of the most common and serious systemic degenerative diseases in the elderly. The pathological features of AD are mainly that the amyloid precursor protein in the cell is cleaved incorrectly to produce toxic amyloid bata 42 (abbreviated as Aβ42), which is secreted to the extracellular deposits to form amyloid plaques and hyperphosphorylated tau protein. It leads to mutual adhesion between neurons, hinders nerve conduction, and leads to memory loss (Association A.Alzheimer's disease facts and figures.Alzheimers Dement.,2012,8(2):131-168.). At present, there are three main therapeutic strategies for the pathogenesis of AD: ① reduce Aβ and ...

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): C12N15/62C12N15/867A61K38/48A61P25/28
CPCA61K38/00A61P25/28C07K2319/036C12N9/6489C12N9/6494C12N15/62C12N15/86C12N2740/10043C12N2740/15043C12N2800/107C12Y304/24011C12Y304/24056
Inventor 索广力张书忙乔勇肖同乾
Owner SHANGHAI UNIV
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