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Construction and Study of ABCA1 Mutants with Arsenic-fast

Author: ZhangXiaoFei
Tutor: YangLei；HuangZuo
School: Shihezi University
Course: Biochemistry and Molecular Biology
Keywords: Arsenic resistance genes ABCA1 gene pcDNA3.1/ABCA1 plasmid Carrier Mutation
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 7
Quote: 0
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Abstract


Objective: This topic overlap extension using improved gene splicing (gene splicing by overlap extension SOE) were constructed missing the ABCA1 protein extracellular ring first 323-579,535-625 amino acid codon gene eukaryotic expression The laser scanning confocal microscopy expression of the mutant. Cell arsenic tolerance experiments, and observe changes in intracellular arsenic content and apoptosis detection, to determine whether the mutant arsenic resistance. Method: gene splicing by overlap extension principle, stitching extends, and then conduct a PCR amplification to obtain the ABCA1 missing the first 323-579, the first 535-625 amino acid codon of the two genes, cloned into pcDNA3. 1/V5-His eukaryotic expression vector. The mutant body eukaryotic expression vector was transfected into Hela cells will under the Lipofectamine2000 mediated laser confocal microscope mutants after transfection. 48h acute arsenic exposure optical density (OD) of the survival rate and median inhibitory concentration (IC50) is detected by the the thiazole basket (MTT), analysis of changes in cell resistance to arsenic. Then use atomic fluorescence spectrometry (AFS) to detect changes in the amount of intracellular arsenic accumulation. Hela cell apoptosis annexin-V/PI double staining flow cytometry mutant and comparative analysis of the full of ABCA1 and empty carrier. Results: The Construction of the first ring missing ABCA1 protein cellular bits 323-579, named extracellular-A; missing the first 535-625 to its name the extracellular-B. Amino acid codon of the gene. By laser scanning confocal microscopy cellular localization of the protein to pcDNA3.1/ABCA1Δ323-579AA, pcDNA3.1/ABCA1Δ535-625AA gene expression analysis. The plasmid to pcDNA3.1/ABCA1 control results show: the experimental group and the control group combined the mouse anti IgG/DyLight488 excited photoexcited green fluorescence, are distributed in the cell membrane. Therefore considers that the mutated gene expression basically no change, may be located in the cell membrane for further research. Hela cells transfected with the extracellular-A the extracellular-B wild pcDNA3.1/ABCA1 plasmid group pcDNA vector group given different concentrations of arsenic trioxide after 48 hours of incubation with MTT assay OD value calculated survival rate and IC50. The results showed that the wild group of cells in various concentrations survival rate is higher than the the simultaneous mutation group and empty vector group, randomized block analysis of variance wild group of various concentrations of extracellular-A, the extracellular-B, and the empty vector group difference in survival was statistically significant (P lt; 0.05); mutation group with empty vector group had no statistical significance (P gt; 0.05). The extracellular-A, cell Xi (?)-B, the pcDNA vector group empty vector and the wild group IC50 respectively for 18.31μmol / l, 18.26μmol / l, 18.52μmol / l and 29.4μmol / l of. Atomic fluorescence spectrometry results show that the experimental group within 10 hours intracellular accumulation of arsenic in volume than the control group, annexin-V/PI double staining flow cytometry results showed that early withered with sodium arsenite After 24h the cells of the experimental group mortality and late apoptosis rate, compared with the wild control group, apoptotic cell proportion increased significantly (p lt; 0.05). Conclusions: first ring ABCA1 protein extracellular deletion mutation basic loss of arsenic resistance ABCA1 may be important in arsenic resistance.

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