摘要
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.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第46期8600-8603,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家人事部留学回国人员科技活动择优资助项目~~