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Alzheimer disease pathogenic gene and application thereof

A technology for Alzheimer's disease and pathogenic genes, which can be applied in the field of molecular pathology and can solve problems such as increased levels

Inactive Publication Date: 2012-07-25
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most mutations in the FAD gene alter APP protein processing, resulting in increased Aβ42 or increased relative Aβ42 / 40 levels

Method used

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  • Alzheimer disease pathogenic gene and application thereof
  • Alzheimer disease pathogenic gene and application thereof
  • Alzheimer disease pathogenic gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Verification of the relationship between Alzheimer's disease-causing genes and AD pathogenesis

[0042] The inventor collected samples from patients with AD and FTD and their relatives, see Table 1 for details.

[0043] Table 1 Collection of AD samples

[0044]

[0045]

[0046] The Alzheimer's disease-causing genes in the genomes of the above 12 normal people, 14 AD patients, 2 FTD patients and 9 FAD patients were detected, and the Alzheimer's disease genes were obtained by sequencing after PCR amplification. The sequence of exons 16 and 17 of the pathogenic gene was used to verify the correlation between the pathogenic gene of Alzheimer's disease and AD according to the sequence determination results combined with clinical data.

[0047] Specific steps are as follows:

[0048] 1) Collection of AD peripheral blood samples

[0049] According to the American NINCDS-ADRDA diagnostic criteria, patients with probable AD or confirmed AD were diagnosed. Pe...

Embodiment 2

[0079] Example 2 Construction of APP695K724M eukaryotic expression vector and establishment of stable expression cell line

[0080] 1. A small amount of plasmid pcDNA3.1(-) (Invitrogen) DNA was extracted.

[0081] 2. Preparation of APP695K724M gene mutant:

[0082] K724M site-directed mutation was introduced by Megaprimer-PCR method.

[0083] The primers used in PCR with overlapping primers are shown in Table 2:

[0084] Table 2 Primers used in Megaprimer-PCR

[0085]

[0086]

[0087] (1) The first PCR

[0088] Using APP695 cDNA (commercially available or synthesized according to the GenBank sequence) as a template, the mutant primer MP (K724M) was introduced as the upstream primer, APP-3' was used as the downstream primer, and PrimeSTARTM HS DNA Polymerase was used to amplify PP.

[0089] The PCR reaction system is (50μL):

[0090]

[0091] PCR reaction conditions:

[0092] Pre-denaturation at 94°C for 5 minutes;

[0093]Denaturation at 98°C for 10s, annealin...

Embodiment 3

[0131] Example 3 Construction of adenoviral expression vector for Alzheimer's disease-causing gene

[0132] To study the pathogenic mechanism of FAD APP mutants or to screen anti-AD drugs, it is usually necessary to establish primary neuronal cell or animal models. Common transfection methods are difficult to transfect primary neurons, and it is necessary to establish viral vectors that express APP efficiently, such as adenovirus, lentivirus, retrovirus, baculovirus, etc. This embodiment describes the construction process of recombinant adenovirus vector, and the specific steps are as follows:

[0133] 1. Construction of shuttle carrier

[0134] 1) Small amount extraction of shuttle plasmid pShuttleCMV

[0135] Take 1 μL shuttle plasmid pShuttleCMV (Stratagene) to transform Top10 competent cells, screen on Kan-resistant LB agar plate (containing 50 μg / mL kanamycin), pick a single colony and inoculate it in 5 mL LB medium (containing 50 μg / mL kanamycin prime), 37°C, 250rpm s...

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Abstract

The invention discloses an Alzheimer disease (AD) pathogenic gene and an application thereof, which belong to the technical field of molecular pathology. The cDNA nucleotide sequence of the gene is represented by SEQ ID NO:1. The invention also relates to a recombinant vector containing the Alzheimer disease pathogenic gene, a recombinant cell containing the recombinant vector, a coding protein of the Alzheimer disease pathogenic gene, and an exon of the Alzheimer disease pathogenic gene. The detection of the gene with the Alzheimer disease pathogenic gene exon can be used in gene molecular diagnosis of an AD patient, or in determining pathogenic risk prediction of AD patient relatives. The invention assists in providing an important clue for the AD pathogenic mechanism. An exon vector containing the Alzheimer disease pathogenic gene can be used for preparing an AD cell or an animal model.

Description

technical field [0001] The invention belongs to the technical field of molecular pathology, and specifically relates to an Alzheimer's disease-causing gene and its application. Background technique [0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that has become the main cause of dementia. The clinical symptoms of AD are mainly manifested as progressive memory loss, cognitive decline, and abnormal mental behavior, which eventually lead to extremely high mortality and disability rates. The pathological features of AD are: extracellular deposition of β-amyloid peptide (Aβ) in the cerebral cortex and hippocampus to form senile plaques (SP), and neurofibrils formed by abnormal aggregation of tau protein in brain nerve cells. Tangle (neurofibrillary tangle, NFT), neuronal synaptic dysfunction and loss of pyramidal nerve cells. [0003] AD can be divided into familial AD (familial Alzheimer's disease, FAD) and sporadic AD (sporadic Alzheimer's disease...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/47C12N15/63C12N15/85C12N15/861C12N5/10C12Q1/68A01K67/027
Inventor 彭向雷管小亭何金生郑妍鹏张莹周淑杰
Owner BEIJING JIAOTONG UNIV
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