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Screening Gene Mutation of Familial Alzheimer’s Disease and Construction of APP Expression Vectors

Author: XuShaoHua
Tutor: ZhengZuoPeng；PengXiangLei
School: Beijing Jiaotong University
Course: Biochemistry and Molecular Biology
Keywords: Alzheimer's disease Gene mutation Amyloid precursor protein
CLC: R749.16
Type: Master's thesis
Year: 2011
Downloads: 50
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Abstract


Objective: Alzheimer's disease (Alzheimer's disease, AD) is a progressive neurodegenerative disease common in the elderly population. Clinical and experimental studies have determined three lead to early onset familial AD (FAD) of the gene, amyloid former body protein (amyloid of precursor protein, APP), as early as the old prime -1 (presenilin 1, PS-1), and as early as the old prime - 2 (presenilin 2, PS-2). Most of the existing AD pedigrees gene mutation data from domestic and abroad, little has been reported. In order to understand the situation of domestic FAD pathogenic gene mutation, the study on the part of the Chinese population in the three gene mutation hotspot detection analysis; Novo Mutations preliminary molecular mechanisms. Methods: AD peripheral blood samples of the patients and their relatives, genomic DNA was extracted exons 16, 17 primers were designed for the PS-1 and PS-2 genes coding exons and APP outside, PCR amplification of exon sequencing screening gene mutation. Detected mutation Megaprimer-PCR method to construct the corresponding gene cDNA mutants and eukaryotic expression plasmid vector transfected HEK293 cells liposomes, G418 screening stable cell lines; for efficient nerve cells pathogenic mechanism of expression and research FAD mutant, we constructed a recombinant adenovirus shuttle plasmid, competent cells transformed AdEasier (BJ5183) containing plasmid pAdEasy-1 recombinant adenovirus plasmid Pme I linearized by Pac I The digested ethanol precipitated liposomes law transfected HEK293 cells packaging recombinant adenovirus, were identified by Western Blot. Results: the AD clinical samples causative gene sequencing results, this filter to a PS-1 gene mutation (R278I) and a new mutations of the APP gene (K724X). Build APP695 wild-type and mutant eukaryotic expression plasmid pcDNA3.1 (-)-APP695WT pcDNA3.1 (-)-APP695K724X and pcDNA3.1 (-)-APP695K724N after broth PCR and sequencing of the corresponding The HEK293 stable cell lines. The the recombinant adenovirus plasmid pAd-APP695WT was build the pAd-APP695K724X and pAd-APP695K724N, after the broth PCR and sequencing, Western Blot identification results show the success of the recombinant adenovirus packaging. Conclusion: This study screened to a PS-1 gene the mutation (R278I) and a new lead the FAD APP gene mutation (K724X); build APP695 eukaryotic expression vector, and the establishment of a stable expressing HEK293 cell lines; build and successful packaging of the recombinant adenovirus, to lay the foundation for in-depth study of FAD pathogenesis and drug screening.

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CLC: > Medicine, health > Neurology and psychiatry > Psychiatry > Cerebral organic mental disorder > Elderly as early as possible the old disorder
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