Human APP promoter-containing Luci cell line and construction method and application thereof

A technology of APP initiation and construction method, applied in animal cells, vertebrate cells, embryonic cells, etc., can solve the problem of lack of efficient screening and inhibition of APP promoter region active drug platform and other problems

Inactive Publication Date: 2017-02-15
NORTHEASTERN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is still a lack of a platform for efficient screening of drugs that inhibit the activity of the APP promoter region in the technical field.

Method used

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  • Human APP promoter-containing Luci cell line and construction method and application thereof
  • Human APP promoter-containing Luci cell line and construction method and application thereof
  • Human APP promoter-containing Luci cell line and construction method and application thereof

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Experimental program
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Embodiment Construction

[0075] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0076] The present invention contains the human APP promoter-luci cell line, which has the amino acid sequence shown in SEQ ID NO:1.

[0077] The construction method of the present invention containing the human APP promoter-luci cell line is specifically implemented according to the following steps:

[0078] Step 1. Construct an expression vector containing human APP promoter region-luciferase chimeric gene, the expression vector is: pGL3-APPP_luc-puro plasmid, specifically constructed according to the following steps:

[0079] Step 1.1, using the pRL-SV40 vector as a template, and using primers 1 and 2, obtain the SV40 enhancer / promoter region sequence fragment through PCR amplification;

[0080] SV40 enhancer / promoter region sequence fragment can be as figure 1 Shown: PCR product analyzed by agarose gel electrophoresis; figure 1 The 7...

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Abstract

The invention discloses a construction method of a human APP promoter-containing Luci cell line, and the specific method is as follows: construction of an expression vector containing a human APP promoter region-luciferase chimeric gene; cotransfection of constructed pGL3-APPP Luc-Puro plasmids and pRL-SV40 plasmids into a human embryonic kidney cell line HEK-293T cell line; laying and planting of transfected HEK-293T cells after trypsinization into a 10cm culture dish, puromycin pressurization screening to obtain five cell lines with genomes capable of stably integrating the human APP promoter region-luciferase chimeric gene, and detection of activity of firefly luciferase and renilla luciferase in the five cell lines to obtain the cell line containing the human APP promoter region-luciferase chimeric gene. The cell line prepared by the cell line construction method can be used for screening of new anti senile dementia compounds and therapeutic drugs for inhibiting the expression of human APP.

Description

technical field [0001] The invention belongs to the technical field of cell line construction methods, and specifically relates to a human APP promoter-luci cell line, and also relates to the construction method and application of the human APP promoter-luci cell line. Background technique [0002] Alzheimer's Disease (AD), commonly known as senile dementia, is a neurodegenerative disease characterized by progressive dementia. Patients with Alzheimer's disease experience progressive decline in cognitive and memory abilities, accompanied by various neuropsychiatric symptoms and behavioral disturbances. As far as the prior art is concerned, there is no clinically effective treatment for Alzheimer's disease. [0003] In recent years, studies at home and abroad have shown that in the progression of Alzheimer's disease, the aggregation of Aβ and the formation of amyloid fibrils are the key steps in the formation of senile plaques and an important factor leading to neuronal damag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/02
CPCC12N5/0603C12N15/85C12N2503/02C12N2510/00C12N2800/107C12N2800/60G01N33/5008G01N2500/10
Inventor 赵蒲王占友
Owner NORTHEASTERN UNIV
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