真核表达质粒pcDNA.3.1(+)-ACE2的构建及其在CHO细胞中的表达
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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。
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.
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.
引文
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[5]杨维维,许媛媛,荣超,等.山羊血管紧张素转换酶2基因的克隆表达与生物信息学分析[J].南京农业大学学报,2015,38(1):120-126.
[6]高云.真核表达系统的研究进展[J].中华男科学,2002:292-294.
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[10]钟久昌,余细勇,于汇民,等.血管紧张素转换酶2基因转染对人血管内皮细胞中氧化应激水平的影响[J].中华高血压杂志,2008,16(6):499-502.
[11]吕瑾,刘增长,张全军,等.ACE2过表达对大鼠心肌梗死后心室重构的影响[J].第三军医大学学报,2013,35(16):1680-1684.
[12]Li W,Moore M J,Vasilieva N,et al.Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus[J].Nature,2003,426(6965):450-454.
[13]杨国良,费小战,陈焕春,等.SARS病毒受体ACE2的克隆,原核表达及其功能区鉴定[J].病毒学报,2006,22(2):118-122.
[14]卢卓强,晋学庆.小鼠血管紧张素转化酶2基因表达型载体的构建及体外表达[J].中国组织工程研究,2001,14(46):8600.
[15]王冲,谭亚娣,郭爱珍,等.家猫ACE 2基因克隆、测序及生物信息学分析[J].武汉大学学报(理学版),2005,51(6):739-743.
[16]Harhay G P,Sonstegard T S,Keele J W,et al.Characterization of954 bovine full-CDS c DNA sequences[J].BMC Genomics,2005,6(1):166.
[17]先俊,邱娴,张安兵,等.真核表达质粒pc DNA3.1-CSRP2-HA的构建及鉴定[J].现代生物医学进展,2014,21:4014-4017.
[18]余鹰,周鸣,曹利,等.pc DNA3.1表达载体转染对细胞生长的影响[J].生物技术,2001,11(1):1-3.
[2]Eriksson U,Danilczyk U,Penninger J M.Just the beginning:novel functions for angiotensin-converting enzymes[J].Curr Biol,2002,12(21):R745-R752.
[3]Lambert D W,Clarke N E,Turner A J.Not just angiotensinases:new roles for the angiotensin-converting enzymes[J].Cell Mol Life Sci,2010,67(1):89-98.
[4]王珊珊.血管紧张素转化酶2(ACE 2)在断奶仔猪体内的表达分布及在胃酸分泌中的作用[D].南京:南京农业大学,2014.
[5]杨维维,许媛媛,荣超,等.山羊血管紧张素转换酶2基因的克隆表达与生物信息学分析[J].南京农业大学学报,2015,38(1):120-126.
[6]高云.真核表达系统的研究进展[J].中华男科学,2002:292-294.
[7]郑光宇.真核生物表达系统研究进展[J].喀什师范学院学报,2004:33-36.
[8]Tipnis S R,Hooper N M,Hyde R,et al.A human homolog of angiotensin-converting enzyme.Cloning and functional expression as a captopril-insensitive carboxypeptidase[J].J Biol Chem,2000,275(43):33238-33243.
[9]Donoghue M,Hsieh F,Baronas E,et al.A novel angiotensin-converting enzyme-related carboxypeptidase(ACE 2)converts angiotensin I to angiotensin 1-9[J].Circ Res,2000,87(5):E1-E9.
[10]钟久昌,余细勇,于汇民,等.血管紧张素转换酶2基因转染对人血管内皮细胞中氧化应激水平的影响[J].中华高血压杂志,2008,16(6):499-502.
[11]吕瑾,刘增长,张全军,等.ACE2过表达对大鼠心肌梗死后心室重构的影响[J].第三军医大学学报,2013,35(16):1680-1684.
[12]Li W,Moore M J,Vasilieva N,et al.Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus[J].Nature,2003,426(6965):450-454.
[13]杨国良,费小战,陈焕春,等.SARS病毒受体ACE2的克隆,原核表达及其功能区鉴定[J].病毒学报,2006,22(2):118-122.
[14]卢卓强,晋学庆.小鼠血管紧张素转化酶2基因表达型载体的构建及体外表达[J].中国组织工程研究,2001,14(46):8600.
[15]王冲,谭亚娣,郭爱珍,等.家猫ACE 2基因克隆、测序及生物信息学分析[J].武汉大学学报(理学版),2005,51(6):739-743.
[16]Harhay G P,Sonstegard T S,Keele J W,et al.Characterization of954 bovine full-CDS c DNA sequences[J].BMC Genomics,2005,6(1):166.
[17]先俊,邱娴,张安兵,等.真核表达质粒pc DNA3.1-CSRP2-HA的构建及鉴定[J].现代生物医学进展,2014,21:4014-4017.
[18]余鹰,周鸣,曹利,等.pc DNA3.1表达载体转染对细胞生长的影响[J].生物技术,2001,11(1):1-3.