真核表达质粒pcDNA.3.1(+)-ACE2的构建及其在CHO细胞中的表达

Construction of eukaryotic expression plasmid pcDNA3.1(+)-ACE2 and its expressions in CHO cells

为了构建pcDNA.3.1（＋）-血管紧张素转化酶2（ACE2）真核表达质粒并检测在中国仓鼠卵巢（CHO）细胞中的表达,从羊肾脏中提取总RNA,经RT-PCR扩增出ACE2基因,后将其与pcDNA3.1载体进行连接重组,构建pcDNA3.1（＋）-ACE2真核表达质粒,经HindⅢ、XhoⅠ限制性内切酶双酶切及DNA序列测序分析等方法验证后,通过Lipofectamine 3000脂质体介导转染至CHO细胞,Western blot法检验ACE2基因在蛋白质水平上的表达。结果显示：RT-PCR扩增出大小约为2 200 bp特异ACE2基因片段,连接获得的pcDNA3.1（＋）-ACE2重组体经HindⅢ、XhoⅠ酶切后分别出现约2 200 bp和5 000 bp片段,测序分析与Gen Bank上公布的结果完全一致,表明成功克隆了重组pcDNA3.1（＋）-ACE2真核表达质粒,Western blot检测显示该pcDNA3.1（＋）-ACE2能在CHO细胞中表达。本研究成功构建了pcDNA3.1（＋）-ACE2真核表达质粒,并证实其能在CHO中表达,为后续探究ACE2蛋白活性及其作用的研究奠定了基础。

To construct eukaryotic expression plasmid pcDNA3.1（＋）-ACE2 and observe its expression in CHO,total RNA was extracted from kidney of goat and then ACE2 gene was amplified by reverse transcription polymerase chain reaction（RT-PCR）.The product was cloned into pcDNA3.1（＋） vector,and they were identified by PCR and double restrictive edonuclease digestion and sequence analysis.Then the recombinant expression plasmid pcDNA3.1（＋）-ACE2 was transfected into CHO cells by lipofectamine 3 000,and the ACE2 protein expression was identified by Western blot.The results showed that RT-PCR product was around 2 200 bp.Analysis by restricting enzyme digestion and DNA sequencing showed the pcDNA3.1（＋）-ACE2 eukaryotic expression plasmid was successfully established,and the expression of pcDNA3.1（＋）-ACE2 could be detected in CHO cells by western blotting.The pcDNA3.1-CSRP2-HA eukaryotic expression plasmid was constructed successfully,and it could be expressed in CHO cells.This study lays the foundation for the research of the activity and immunological functions of ACE2 protein.