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

Construction of recombinant eukaryotic expression plasmid containing mouse angiotensin-converting enzyme 2 gene and its in vitro expression

背景:通过转基因疗法使血管紧张素转化酶2(angiotensin-converting enzyme,ACE2)过度表达来治疗心血管病是当前ACE2研究的趋势,而构建含ACE2基因的载体则能为该研究方法提供工具。目的:构建含小鼠血管紧张素转化酶2基因的真核表达载体,并验证其蛋白的表达。方法:采用RT-PCR的方法从C57小鼠肾脏扩增出ACE2基因全长cDNA序列,并通过酶切连接和转化等分子生物学方法将其定向克隆至载体pcDNA3.1/Hygro(+)上,构建小鼠ACE2基因的真核表达载体pm-ACE2,并通过酶切及测序鉴定。通过脂质体转染法将所构建的pm-ACE2转染COS7细胞,并利用Western blot检测COS7细胞中ACE2蛋白的表达。结果与结论:通过测序并与GeneBank上的小鼠血管紧张素转化酶2序列(NM_027286.3)比对,证实实验成功克隆小鼠ACE2基因全长cDNA至载体pcDNA3.1/Hygro(+)上,测序结果提示存在3处同义突变;所构建的pm-ACE2在真核细胞中具有ACE2蛋白的表达。结果提示实验成功构建了小鼠ACE2基因真核表达载体,此载体具有表达ACE2蛋白的功能。

BACKGROUND：Overexpression of angiotensin-converting enzyme 2（ACE2） by transgene treatment is a tendency on curing angiocardiopathy.The construction of ACE2 gene expression plasmid can provide tools for those relative researches.OBJECTIVE：To construct eukaryotic expression vector containing mice ACE2 gene and to verify its protein expression.METHODS：The full-length cDNA of mice ACE2 was amplified from mouse kidney by RT-PCR,and cloned into pcDNA3.1/Hygro（＋） to construct eukaryotic expression vector pm-ACE2 containing mouse ACE2 gene,and identified by enzyme digestion and sequencing.The constructed pm-ACE2 was transfected into COS7 cell by Lipofectamine 2000,and its expression in eukaryotic cell of pm-ACE2 was detected by Western-Blot.RESULTS AND CONCLUSION：Mice ACE2 gene was successfully cloned and linked to vehicle pcDNA3.1/Hygro（＋）.Sequencing result showed that there were three spots of samesense mutation.The pm-ACE2 expressed ACE2 protein in eucaryon.All findings demonstrated that a mouse ACE2 eukaryotic expression vector is constructed successfully,which can express ACE2 protein.