Construction And Study Of Abca1 Mutants With Arsenic-fast

Objective:The purpose of this research is to construct deletion mutant of ABCA1 with the first extracellular loop of with the 323th to 579th,535th to 625th amino acid codons deleted by gene splicing by overlap extension. Eukaryotic expression and observe by Laser Scanning Confocal Microscope. At the same time carry on the cell arsenic tolerance experiments, and to observe changes in intracellular levels of arsenic and apoptosis rate detection in order to determine mutant whether to have the arsenic resistance function.Methods:In line with the mechanism of gene splicing by overlap extension (SOE), an additional PCR was used to get the ABCAlmutant in which the 323th to 579th,535th to 625th amino acid codons were deleted. Then the mutated gene was cloned into pcDNA3.1/V5-His mammalian expression vector. Eukaryotic expression mutant was transfected into Hela cells by Lipofectaonmine2000. Laser confocal microscopy detect transfection of mutant expression. After exposed in arsenite for 48 hours, cell survival rate and IC50 were detected by MTT assay. We also examined the arsenic accumulation in Hela cell by atomic fluorescence spectrophotometry ehich reflected the effct of arsenic-resistance. At the same time, annexin-V/PI double staining flow cytometry technique was used to examine mutant influence which perishes weakly to the Hela cell.Results:Mutants with 323th to 579th(named extracellular-A) and 535th to 625th(named extracellular-B) amino acid codons deleted was constructed successfully. pcDNA3.1/ABCA1Δ323-579AA, pcDNA3.1/ABCA1Δ535-625AA proteins cellular localization was analysised by laser scanning confocal microscope. According to plasmid pcDNA3.1/ABCA1 the result showed that:The experimental group and control group after the combination of mouse anti-IgG/DyLight488 excited by the excitation light yellowish green fluorescence and distributed in the cell membrane. Therefore the basic expression of mutant gene was considered may be located at the cell membrane, further studies could be carryed. Hela cells transfected with mutantl group and mutant2 group and wild pcDNA3.1/ABCA1 plasmid group, empty vector group given different concentrations of arsenic, incubation with the MTT method 48 hours and then calculate survival rate and measured OD values of IC50. Results showed that survival rate of those cells wild group were higher than that of control under all concentration of arsenic. Randomized block design ANOVA showed that:The wild group Compared with the mutation group land mutation group 2 and the empty vector survival rate was significantly (P<0.05); mutation group and the empty vector was no statistical significance; mutation group and the empty vector was no statistically significant(P>0.05). IC50 of mutation groupl, Mutation group2, empty vector group and the wild group of IC50 was 18.31μmol/l,18.26μmol/l,18.52μmol/l and 29.4μmol/l. the results of atomic fluorescence spectrophotometry showed that arsenic accumulation of HeLa cells in experimental groups was lower than that of control groups after incubation with 10μmol/l NaAsO2 for up to 10 h,and arsenic accumulation of experimental groups reached a steady state in 4 h and did not increase significantly thereafter,In contrast,arsenic accumulation of control groups increased throughout the 10h incubation period. Annexin-V/PI double staining flow cytometry results showed that, NaAsO2 role in the experimental group after 24h cells in early apoptosis and late apoptosis rate, and the wild control group, the proportion of apoptosis cells increased significantly (p<0.05).Conclusion:ABCA1 protein in the first extracellular loop deletion mutants are basically the loss of arsenic resistance capabilities may be related to arsenic resistance function of ABCA1 has an important relationship.