中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (46): 8600-.doi: 10.3969/j.issn.1673-8225.2010.46.012

• 织构建与生物活性因子 • 上一篇    下一篇

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

卢卓强，晋学庆   

  1. 福建医科大学附属第一医院心血管内科，福建省福州市  350000
  • 出版日期:2010-11-12 发布日期:2010-11-12
  • 通讯作者: 晋学庆，男，主任医师，副教授，硕士生导师，福建医科大学附属第一医院心血管内科，福建省福州市 350000 jxueqing@medmail.com.cn
  • 作者简介:卢卓强★，男，1979年生，福建省龙岩市人，汉族，2009年福建医科大学毕业，硕士，主要从事心血管内科临床工作。 luzq109@126.com
  • 基金资助:

    国家人事部留学回国人员科技活动择优资助项目。

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

Lu Zhuo-qiang, Jin Xue-qing   

  1. Department of Cardiology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350000, Fujian Province, China
  • Online:2010-11-12 Published:2010-11-12
  • Contact: Jin Xue-qing, Chief physician, Associate professor, Master’s supervisor, Department of Cardiology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350000, Fujian Province, China jxueqing@medmail.com.cn
  • About author:Lu Zhuo-qiang★, Master, Department of Cardiology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350000, Fujian Province, China luzq109@126.com
  • Supported by:

    the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry*

PDF

340

被引次数

4

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。

关键词: 血管紧张素转化酶2基因, 载体, COS7细胞, 真核细胞, 基因表达, 组织构建

Abstract:

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.

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