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IDE and NEP fusion gene containing CBD (Collagen Binding Domain), cell line, liquid ECM (Extracellular Matrix) and application

A technology of CBD-NEP and gene fusion, applied in the field of genetic engineering, to achieve the effect of reducing amyloid precipitation

Active Publication Date: 2019-09-17
SHANGHAI UNIV +1
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  • 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

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  • IDE and NEP fusion gene containing CBD (Collagen Binding Domain), cell line, liquid ECM (Extracellular Matrix) and application
  • IDE and NEP fusion gene containing CBD (Collagen Binding Domain), cell line, liquid ECM (Extracellular Matrix) and application
  • IDE and NEP fusion gene containing CBD (Collagen Binding Domain), cell line, liquid ECM (Extracellular Matrix) and application

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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 t...

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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 of IDE and NEP containing CBD (Collagen Binding Domain, an abbreviation of collagen binding domain, a peptide segment that can bind to collagen I), a plasmid, a cell line, a liquid ECM and its application belong 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 amyloidbata 42 (abbreviated as Aβ42), which is secreted to the extracellular deposits to form amyloid plaques and hyperphosphorylation of tau protein, which eventually leads to Neurons adhere to each other, hinder nerve conduction, and lead to memory loss (AssociationA.Alzheimer'...

Claims

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Application Information

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Patent Type & Authority Applications(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
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