单 位：
南京农业大学农业部动物生理生化重点开放实验室
关键词：
血管紧张素转化酶2(ACE 2);真核表达质粒;转染;CHO细胞
摘 要：
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。
译 名：
Construction of eukaryotic expression plasmid pcDNA3.1(+)-ACE2 and its expressions in CHO cells
作 者：
XIAO Hang;WANG Kai;WANG Huanhuan;WANG Kun;ZHANG Yuanshu;Key Laboratory of Animal Physiology and Biochemistry,Ministry of Agriculture,Nanjing Agricultural University;
关键词：
angiotensin angiotensin converting enzyme 2(ACE 2);;eukaryotic expression plasmid;;transfection;;CHO cells
摘 要：
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.
