摘要
目的构建载脂蛋白B mRNA编辑酶催化亚基3A(APOBEC3A)表达载体,真核细胞表达APOBEC3A并鉴定其胞嘧啶脱氨酶活性。方法构建APOBEC3A真核表达载体pc DNA3.0-APOBEC3A,转染HEK293T细胞和Hep G2细胞。Western blot法鉴定APOBEC3A蛋白表达,免疫荧光细胞化学染色确定APOBEC3A在HEK293T细胞和Hep G2细胞的定位,利用组蛋白(His)标签蛋白纯化方法富集纯化APOBEC3A蛋白,荧光偏振技术检测APOBEC3A脱氨酶活性。结果 DNA测序证实pc DNA3.0-APOBEC3A插入长600 bp的APOBEC3A基因序列;该质粒能在HEK293T细胞和Hep G2细胞表达APOBEC3A,APOBEC3A在HEK293T细胞中主要分布于胞质,在Hep G2细胞中胞质和胞核均匀分布;纯化获得的APOBEC3A对单链DNA中TTCA序列具有胞嘧啶脱氨基活性。结论成功构建APOBEC3A真核表达载体,APOBEC3A在HEK293T细胞和Hep G2细胞定位不同,表达的APOBEC3A具有胞嘧啶脱氨基活性。
Objective To construct the expression vector of apolipoprotein B mRNA editing enzyme catalytic subunit 3A（ APOBEC3A）,express APOBEC3 A in eukaryotic cells and identify its cytosine deaminase activity. Methods The APOBEC3 A gene was obtained by PCR and inserted into the eukaryotic expression vector pc DNA3. 0（ ＋）. The recombinant vector pc DNA3. 0-APOBEC3 A was then transfected into HEK293 T and Hep G2 cells after confirmed by DNA sequencing. The recombinant protein was purified by Ni-NTA His Bind affinity column. Western blot analysis was used to detect the expression of APOBEC3 A protein. The localization of APOBEC3 A protein in HEK293 T and Hep G2 cel s was identified by immunofluorescence cytochemistry. The deaminase activity of APOBEC3 A protein was characterized by fluorescence polarization. Results DNA sequencing confirmed that APOBEC3 A gene（ 600 bp） was inserted into pc DNA3. 0-APOBEC3 A,which was expressed in HEK293 T and Hep G2 cells successfully. APOBEC3 A protein was mainly expressed in cytoplasm of HEK293 T cells and cytoplasm and nuclei of Hep G2 cells. APOBEC3 A protein showed cytosine deaminase activity on the TTCA sequence in single-stranded DNA. Conclusion The study constructed successfully APOBEC3 A eukaryotic expression vector,identified the differential expression of APOBEC3 A protein in HEK293 T and Hep G2 cells,and confirmed that the APOBEC3 A protein had cytosine deaminase activity.
作者
成珊
曹丽妍
杜娟
王文勇
郭晏海
CHENG Shan CAO Liyan DU Juan WANG Wenyong GUO Yanhai(Department of Pharmacy, Fourth Military Medical University, Xi ＇ an 710032 Department of Pathology, Fourth Military Medical University, Xi＇an 710032, China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第2期179-184,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31570168)
