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目的 :深入研究血管紧张素转化酶 2 (angiotensin- converting enzyme2 ,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 c DNA序列 ,TA克隆到p GEM- T easy载体 ,亚克隆到 pc DNA3.1+载体 ,构建重组真核表达质粒 pm ACE2 ,测序鉴定。采用脂质体法将pm ACE2转染 Cos7细胞 ,分别用 RT- PCR和 SDS- PAGE检测 ACE2的转录和表达。 CLUSTAL X 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 pm ACE2测序结果表明 ,克隆片段存在 A70 1G、T110 2 C和 T1330 C3处变异 ,其序列与以往报道不一致 ,c DNA全长为 2 397bp,而与 NCBI Refseq数据库 (XM- 136 130 )中 c DNA全长190 2 bp不符 ;2 SDS- PAGE显示 pm ACE2表达产物的相对分子量约 80 k D;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- 76 5位残氨 )组成 ;4小
OBJECTIVE: To study the role of angiotensin-converting enzyme 2 (ACE2) in the pathogenesis of cardiovascular diseases and its application in the gene therapy of hypertension. To clone and express mouse ACE2 gene in vitro and to perform nucleotide sequencing And amino acid sequence analysis and tissue distribution characteristics. METHODS: The full-length cDNA of ACE2 gene was amplified from mouse kidney tissue by RT-PCR. TA cloned into p GEM-T easy vector and subcloned into pcDNA3.1 + vector to construct recombinant eukaryotic expression vector Plasmid pm ACE2, sequencing identification. Pm ACE2 was transfected into Cos7 cells by liposome method, and the transcription and expression of ACE2 were detected by RT-PCR and SDS-PAGE respectively. CLUSTAL X 1.8 Biological Software Analysis of multiple sequence comparisons of mouse and rat and human ACE2 proteins. The specificity of mouse ACE2 tissue distribution was studied by RT-PCR. Results: The amplified fragment of 1RT-PCR was about 2.6 kb. The sequencing results of the recombinant eukaryotic expression plasmid pm ACE2 showed that the cloned fragment was mutated at A70 1G, T110 2 C and T1330 C3. The sequence was different from previous reports. Length of 2 397bp, which was inconsistent with the 1902 bp of c DNA in NCBI Refseq database (XM-136 130). 2 SDS-PAGE showed that the relative molecular mass of pm ACE2 expression product was about 80 kD; 3 amino acid sequence analysis showed that small The murine ACE2 consists essentially of the N-terminal signal peptide sequence (residues 1 to 18 residues), the catalytic and transmembrane domains containing the zinc binding site conserved sequence HHEMGHIQ (residues 373-380) (region 738- 76 5 residue ammonia) composition; 4 small