野生型和A294G突变型血管紧张素转换酶2基因真核表达质粒构建与鉴定  

Construction of eukaryotic expression system of wild type and A294G mutant human angiotensin-converting enzyme 2

作　　者：雷和平[1] 姚轻舟[1] 刘居理[1] 林秋雄[1] 邓春玉[1] 朱杰宁[1] 余细勇[1] 

机构地区：[1]广东省医学科学院广东省人民医院医学研究中心广东省心血管病研究所,广州510080

出　　处：《岭南心血管病杂志》2011年第5期402-405,共4页South China Journal of Cardiovascular Diseases

基　　金：中国博士后科学基金资助项目(项目编号:No.20100470893);国家自然科学基金资助项目(项目编号:No.81070103)

摘　　要：目的构建人野生型和突变型(A294G)血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)基因真核表达载体。方法采用逆转录聚合酶链反应(RT-PCR)方法扩增人ACE2基因,与pcDNA3.1真核表达载体连接,构建pcDNA3.1-hACE2重组质粒,通过酶切和测序进行鉴定;以此野生型ACE2真核表达载体为模板,在pyrobest DNA高保真聚合酶的作用下利用体外定点突变法构建ACE2基因突变型重组质粒,用Dpn I限制性内切酶处理聚合酶链反应产物,直接转化DH5α感受态细胞,挑取克隆并提取相应质粒,测序加以验证。结果测序结果证实,野生型和突变型ACE2基因分别插入到pcDNA3.1+中,野生型ACE2基因序列与NCBI网站人ACE2基因全长编码序列(NCBI参考序列:NM_021804.2)相应片段一致,突变型(A294G)ACE2基因除第294位碱基A被G替代以外,其余序列与野生型一致。结论成功构建了人野生型和突变型ACE2的真核表达质粒,为下一步研究突变型ACE2的生物学功能以及ACE2在疾病发病机制中的作用奠定了基础。Objectives To construct the eukaryotic expression system of human wild type and mutant A294G angiotensin-converting enzyme 2 （ACE2）. Methods Human ACE2 gene was amplified by reverse transcfiptase polymerase chain reaction （RT-PCR）, linkaged with pcDNA3.1 eukaryotic expression vector to construct recombinant pcDNA3.1-hACE2, and then identified the recombinant plasmid by enzyme digestion and sequencing. Using the wild-type ACE2 expression vector as a templet, the mutant recombinant plasmids was amplified by PCR with pyrobest DNA polymerase. The template DNA was removed by digestion with Dpn I, and the amplified DNA was directly transformed into E.coli DH5a. After plasmid DNA was recovered from a colony, the sequence was confirmed. Results The wild-type and mutant ACE2 genes were subcloned into eukaryotic expression vector pcDNA3.1 ＋, and the recombinant plasmids were validated by sequencing. The sequence of the wild-type ACE2 showed the same sequence as that in the NCBI （RefSeq： NM_021804.2）, and the sequence of mutant ACE2 was concordant with that of the wild-type ACE2 except that the number 294 base A was replaced by G. Conclusions The eukaryotic expression plasmids containing human wild and mutative ACE2 were successfully constructed, which will enable us to explore the biological function of the mutative ACE2 gene and the role of ACE2 in the pathogenesis of disease futher.

关 键 词：基因 血管紧张素转换酶2 点突变 质粒 

分 类 号：Q517[生物学—生物化学]