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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 APOBEC3A expression vector of apolipoprotein B mRNA and to express APOBEC3A in eukaryotic cells and identify its cytosine deaminase activity. Methods APOBEC3A eukaryotic expression vector pcDNA3.0-APOBEC3A was constructed and transfected into HEK293T cells and Hep G2 cells. APOBEC3A protein was identified by Western blot. The localization of APOBEC3A in HEK293T cells and Hep G2 cells was confirmed by immunofluorescence cytochemistry. Purified APOBEC3A protein was purified by using His tagged protein purification method. APOBEC3A deaminase activity was detected by fluorescence polarization . Results DNA sequencing confirmed that pcDNA3.0-APOBEC3A was inserted into APOBEC3A gene sequence of 600 bp in length. APOBEC3A was expressed in HEK293T cells and Hep G2 cells. APOBEC3A was mainly distributed in the cytoplasm in HEK293T cells and in cytoplasm of Hep G2 cells And uniformly distributed in the nucleus. The purified APOBEC3A has cytosine deamination activity on the TTCA sequence in single-stranded DNA. Conclusion APOBEC3A eukaryotic expression vector was successfully constructed. APOBEC3A was located in different locations of HEK293T cells and Hep G2 cells. APOBEC3A expressed cytosine deaminase.