论文部分内容阅读
目的构建带有血凝素(HA)标签的小鼠组蛋白变异体macroH2A1(mH2A1)的真核表达载体,并观察其在人胚肾293T细胞中的表达定位情况。方法提取内毒素休克的BALB/c小鼠肝脏组织的总RNA,通过逆转录-聚合酶链反应获得内毒素休克小鼠肝脏组织的cDNA,以cDNA为模板使用PCR方法扩增得到mH2A1编码序列,并将其酶切后连接至带有HA标记的载体pcDNA3-HA上;对阳性克隆进行酶切、PCR和测序鉴定。随后将重组质粒瞬时转染293T细胞,利用荧光显微镜观察。结果 PCR、双酶切和测序鉴定表明pcDNA3-mH2A1-HA真核表达质粒构建正确;经转染实验发现,该质粒能够在293T细胞中表达,表达产物主要定位在细胞核中。结论 mH2A1真核表达载体pcDNA3-mH2A1-HA的成功构建及明确其在哺乳动物细胞中的具体核定位,为进一步研究mH2A1作用细胞的信号通路提供了一个重要的工具。
Objective To construct the eukaryotic expression vector of hemagglutinin (HA) -tagged mouse histone variant macroH2A1 (mH2A1) and observe its expression in human embryonic kidney 293T cells. Methods The total RNA of BALB / c mouse livers subjected to endotoxic shock was extracted and the cDNA of liver tissues of endotoxic shock mice was obtained by reverse transcription - polymerase chain reaction. The mH2A1 coding sequence was amplified by PCR using cDNA as a template. And then digested and ligated into pcDNA3-HA with HA tag. The positive clones were digested by restriction endonucleases, PCR and sequencing. The recombinant plasmids were transiently transfected into 293T cells and observed by fluorescence microscopy. Results PCR, double enzyme digestion and sequencing showed that the pcDNA3-mH2A1-HA eukaryotic expression plasmid was constructed correctly. The transfection experiments showed that the plasmid was able to express in 293T cells and the expression product was mainly located in the nucleus. Conclusion The successful construction of mH2A1 eukaryotic expression vector pcDNA3-mH2A1-HA and its specific nuclear localization in mammalian cells provide an important tool for further study of mH2A1 signaling pathway.