小鼠ACE2基因的克隆和体外表达以及序列分析与组织分布

目的:深入研究血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)在心血管疾病发病机理中的作用及在高血压基因治疗中的应用,克隆和体外表达小鼠ACE2基因,并进行核苷酸和氨基酸序列分析和组织分布特征研究.方法:采用RT-PCR方法,从小鼠肾组织中扩增出ACE2基因的全长cDNA序列,TA克隆到pGEM-T easy载体,亚克隆到pcDNA3.1 载体,构建重组真核表达质粒pmACE2,测序鉴定.采用脂质体法将pmACE2转染Cos7细胞,分别用RT-PCR和SDS-PAGE检测ACE2的转录和表达.CLUSTALX 1.8生物软件分析小鼠和大鼠及人ACE2蛋白的多序列比较.采用RT-PCR技术研究小鼠ACE2组织分布的特异性.结果:①RT-PCR扩增片段约2.6 kb,重组真核表达质粒pmACE2测序结果表明,克隆片段存在A701G、T1102C和T1330C 3处变异,其序列与以往报道不一致,cDNA全长为2 397 bp,而与NCBI Refseq数据库(XM-136130)中cDNA全长1 902 bp不符;②SDS-PAGE显示pmACE2表达产物的相对分子量约80 kD;③氨基酸序列分析表明,小鼠ACE2主要由N末端的信号肽序列(第1-18位残氨)、含有锌结合位点保守序列HHEMGHIQ(第373-380位残氨)的催化结构域和跨膜结构域(第738-765位残氨)组成;④小鼠ACE2与人有84%的相似性,与大鼠有90%的相似性,三者同源;⑤ACE2在肺、心和肾组织中大量表达,在睾丸和肝组织中也有表达.结论:成功克隆并体外表达了小鼠ACE2基因,不同物种ACE2组织分布的特异性不尽相同.

Cloning, expression and sequence analysis and tissue distribution of angiotensin-converting enzyme 2 (ACE2) gene in adult mice

Objective: To clone angiotensin converting enzyme 2(ACE2) gene, to analyze its amino acids and nucleotides sequence and to investigate tissue distribution of ACE2 in adult mice. Methods: WTBZ The full length ACE2 encoding sequence was amplified from the RNA of mice kidney tissue by RT PCR technique, cloned into plasmid pGEM T easy, then subcloned into plasmid pcDNA3.1+. After identification of DNA sequence, the recombinant plasmid pmACE2 was transfected into Cos7 cells with lipofectin reagent. The transient expression of ACE2 molecule was detected by SDS PAGE. Sequence analysis was conducted with CLUSTALX program. Tissue distribution of ACE2 in mice was detected by RT PCR. Results: A fragment about 2.6 kb was amplified and the recombinant plasmid pmACE2 was confirmed by two enzyme digesting and DNA sequencing. The cloned DNA sequence was consistent with that previously reported, except for 3 variations: A701G, T1102C and T1330C. SDS PAGE proved that expression of a soluble, truncated products form of ACE2 was a glycoprotein of approximately 80 kD in Cos7 cells. The predicted mice ACE2 sequence contained an N terminal signal sequence(amino acid residues 1-18), a single HHEMGHIQ zinc binding domain (amino acid residues 373-380) and C terminal membrane anchor (amino acid residues 738-765). Mice ACE2 showed 84% identity with that of human, and 90% identity with that of rat. Expression of ACE2 was the greatest in lungs, hearts and kidneys, and moderate levels were also detected in testes and livers. Conclusion: Mice ACE2 gene has been cloned and successfully expressed in vitro . The tissue specific expression of ACE2 in different species is not identical.