血管紧张素转换酶的克隆及序列分析

Cloning and Sequence Analysis of Agiotensin Converting Enzyme

根据GenBank上公布的小鼠血管紧张素转换酶(ACE)基因序列,经多重比较设计1对特异性引物,从小鼠Balb/c肺部组织中提取总RNA,进行RT-PCR。产物经纯化回收,克隆到pMD-18T载体,转化大肠杆菌DH5α感受态细胞,检测阳性克隆、测序并进行序列分析。测序结果显示,该片段全长4 023 bp,开放阅读框(ORF)包含3 985 bp,编码1 328个氨基酸,相对分子质量为152.77 kD,等电点6.35。此序列与GenBank已登录的小鼠ACE同源性达99%,差异的5个碱基位于基因的非关键部位,说明小鼠ACE成功克隆。经同源性比较分析,达50%以上相同性的物种有17个,且符合种属之间的进化关系。因此,该序列可于研究物种亲缘关系或遗传距离的理想标记,为下一步研究其在酵母中的表达、生物活性和应用奠定了一定的基础。

One pair of primers to amplify the gene of agiotensin converting enzyme was designed and synthesized according to the published results of ACE sequence in GenBank.The production of RT-PCR was purified and cloned into pMD-18T vector,then converted to competent cell of the E.coli DH5α.Positive plasmids were selected and the sequence was analyzed.The consequence showed that the sequence was comprised a fragment of 4 023 nucleotides,including a ORF of 3 985 nucleotides,encoding 1 328 amino acids.The molecular weight was 152.77 kD and pI was 6.35.The nucleotide homology between the sequence and the published mouse sequence in GenBank reached up to 99%,only 5 nucleotides mutant in the unimportant areas,approving that the gene ACE of mouse had been cloned successfully.The homology and phylogenetic analysis of sequences revealed that there were 17 species with more than 50% identity compared with the sequence,suggesting that ACE is conserved through the evolution process.The cloning would facilitate the further research on the expression in yeast,bioactivity and application of ACE.