小鼠ATP酶β亚单位真核表达载体的构建和细胞内定位研究

Construction and intracellular localization of eukaryotic expression vector of mice ATPaseβ

目的构建小鼠ATP酶β亚单位(ATPaseβ)真核表达载体,并观察其在哺乳动物细胞中的表达定位情况。方法取BALB/c小鼠肝脏组织的总RNA,反转录为cDNA,以cDNA为模板PCR扩增得到ATPaseβ编码序列,将其连接到pMD18-T载体上,然后以pMD18-ATPaseβ为模板扩增得到ATPaseβ编码序列,再将其克隆到真核表达载体pcDNA3/HA上。采用脂质体转染法转染NIH 3T3细胞及293细胞,在荧光显微镜下观察结果。结果 PCR、双酶切和测序结果表明,重组质粒pcDNA3/HA-ATPaseβ构建正确;转染实验发现,该质粒能够在NIH 3T3细胞中表达,表达产物主要定位于细胞质,细胞膜上也有表达,而在293细胞中只表达于细胞质。结论成功构建了带有HA标签的小鼠ATPaseβ真核表达载体,该载体能够在哺乳动物NIH 3T3及293细胞中有效表达并正确定位,为进一步研究ATPaseβ的细胞内生物学功能提供了一个重要的工具。

Objective To construct the eukaryotic expression plasmid of mice ATPaseβ and determine the expression and intracellular localization in mammalian cells.Methods The total RNA was extracted from the liver of BALB/c mice.The ATPaseβ gene fragment was synthesized and amplified from the total RNA by RT-PCR.The resulted products were cloned into pMD18-T vector and then sequenced.The open reading fragments of ATPaseβ were successfully inserted into pcDNA3/HA vector to construct eukaryotic expression plasmid pcDNA3/ATPaseβ with HA tag.For detecting the intracellular localization,NIH3T3 cells and 293A cells were transfected with the mouse ATPaseβ eukaryotic plasmid.The expression of ATPaseβ protein was analyzed by immunofluorescence assay.Results（1） The extracted total RNA was isolated and the concentration was 1.6g/L.（2） The ATPaseβ sequences were specifically amplified through RT-PCR.（3） The purified RT-PCR product was cloned into pMD18-T vector and then sequenced,and it was verified as the same as that of ATPaseβ gene in GenBank（GenBank accession no.AF030559）.（4） The expression vector pcDNA3-ATPaseβ was constructed and verified by polymerase chain reaction（PCR）,the 5400bp and 1590bp fragments were produced by EcoR I and Kpn I double digestion,and were identified as ATPaseβ by DNA sequencing.（5） With fluorescence microscopy,it was found that the HA-ATPaseβ protein was expressed in NIH3T3 cells and 293 cells with a cytoplasmic distribution.It was also expressed in cytolemma of NIH3T3 cells.However,the same detection in pcDNA3/HA cells gave negative results.Conclusion The eukaryotic expression plasmid of HA-ATPaseβ fusion protein has been successfully constructed and effectively expressed in mammalian cells,and it provides an important tool for the future study of the intracellular biological functions of ATPaseβ.