ABCA1胞外第四环缺失突变体的构建及其抗砷性初步研究  

Construction and Basic Study on Arsenic-Resistant Ability of a Deletion Mutant in the Fourth Extracellular Loop of ABCA1

作　　者：程建兵 谭晓华[2] 罗燕 杨磊[1,2] 

机构地区：[1]石河子大学新疆地方病与民族高发病省部共建教育部重点实验室新疆石河子大学生命科学学院,石河子832002 [2]杭州师范大学医药卫生管理学院,杭州310036

出　　处：《中国生物工程杂志》2010年第3期27-32,共6页China Biotechnology

基　　金：国家自然科学基金(30860241)

摘　　要：目的:构建ABCA1细胞外第四环第1479～1597位氨基酸残基缺失的突变体。方法:采用重叠区扩增基因拼接法构建ABCA1第1479～1597位氨基酸残基缺失的突变体基因,并将其克隆至pcDNA3.1/V5-His/ABCA1重组载体上,脂质体法转染Hela细胞,激光共聚焦观察突变体定位,Cell Counting Kit-8(CCK-8)试剂盒检测其48h急性砷中毒后生存率的变化。结果:该突变体基因经DNA序列分析表明其具有正确的序列和阅读框。激光共聚焦证实其表达的蛋白仍然能正确定位在Hela细胞的细胞膜上。CCK-8结果显示转染重组质粒和突变体质粒的细胞在各种砷浓度下生存率都较空载体组高。结论:成功构建了ABCA1胞外第四环第1479～1597位氨基酸残基缺失的突变体,且其表达的蛋白仍然定位于细胞膜上,突变体仍然具有一定的抗砷性,提示ABCA1胞外第四环可能不是关键抗砷结构域。Objective：To construct a ABCA1 mutant with the 1 479th to 1 597th amino acid codons deleted.Methods：The mutant gene was amplified by gene splicing by overlap extension,then inserted into pcDNA3.1/V5-His/ABCA1 vector and transfected into Hela cell by lipofection.The location and arsenic-resistant ability of the mutant were investigated by confocal microscopy and CCK-8 assay.Results：Sequence analysis showed that the sequence of the mutant was completely correct.The mutant could still located at the plasma membrane,and the survival rates of the mutant were higher than the control as the wild-type ABCA1.Conclusion：ABCA1 mutant with the 1 479th to 1 597th amino acid codons deleted was successfully constructed,and expressed protein could still located at the plasma membrane with some arsenic-resistant ability,which would provide a condition for further study the ABCA1.

关 键 词：ABCA1 突变体 重叠区扩增基因拼接法 

分 类 号：Q75[生物学—分子生物学]