山羊血管紧张素转换酶2基因的克隆表达与生物信息学分析

Clone expression and bioinformatic analysis of angiotensin converting enzyme 2(ACE2) gene from Capra hircus

[目的]血管紧张素转换酶2（ACE2）是RAS系统的关键调控酶,在动物上的研究较少,本试验研究了山羊ACE2的相关理化性质和功能。[方法]利用同源克隆及RT-PCR技术,根据牛的ACE2基因mRNA序列设计引物,从山羊肾脏组织中扩增出ACE2基因编码区的特异性片段,TA克隆到p MD19-T载体并转化至大肠杆菌DH5α感受态细胞,对生长出的单菌落进行验证后测序分析。根据测序分析结果设计引物,经PCR扩增和酶切连接,构建原核表达载体p ET-28a-ACE2,经过验证后转化大肠杆菌BL21（DE3）并用IPTG诱导表达ACE2蛋白。[结果]山羊ACE2基因c DNA编码区共包括2 415个核苷酸,编码804个氨基酸残基。同源性比对发现,山羊ACE2基因核苷酸序列同源性与绵羊ACE2最高,为99%,与斑马鱼最低,仅为60%。遗传进化分析也表明该ACE2基因与绵羊的遗传距离最近,与斑马鱼处在两个不同的分支上。蛋白结构及理化性质分析预测该山羊ACE2蛋白属于稳定型蛋白,蛋白信号肽序列位于第1氨基酸（aa）~18氨基酸（aa）之间,蛋白跨膜螺旋预测为Ⅰ型跨膜蛋白。成功构建山羊ACE2蛋白胞外部分（19~738 aa）表达载体,表达出的蛋白相对分子质量约为90×10^3,主要以包涵体的形式在BL21（DE3）中表达。[结论]本试验首次成功克隆了山羊ACE2基因编码区（基因序列号：KF921008.1）,并对其蛋白结构及理化性质进行了初步预测,同时在大肠杆菌中高效表达了其胞外区蛋白。

[Objectives]ACE2 is the key regulatory enzyme of RAS system,to determine ACE2 gene sequence. [Methods]We used homogeneous clone and RT-PCR method. First,primers were designed on the basis of the mRNA sequence of ACE2 in cattle.Second,specific fragments of ACE＇s coding sequence were amplified from the kidney tissues of goats. These specific fragments were then cloned into p MD19-T carrier using TA method and transferred to DH5α competent cells. In the end,the ACE2 gene sequence was determined after the colonies of DH5α were validated. To construct prokaryotic expression vector p ET-28a-ACE2 and make it express efficiently,the ACE2 gene was amplified and inserted into p ET-28 a by restriction enzyme digestion. After identified by sequencing,the ACE2 gene was induced with IPTG. [Results]The result showed the c DNA coding sequence of ACE2 in goats covered 2 415 nucleotides and coded 804 amino acid（ aa） residues. Sequence homology analysis showed that the sequence in goats shared the highest homology（ 99%） with that in sheep and the lowest homology（ 60%） with that in zebra fishes. Genetic evolution analysis showed that this gene in goats has the shortest genetic distance with such gene in sheep,but resides in a totally different branch from the ACE2 gene in zebra fishes,which accords with the general genetic evolution rules. Analysis of protein structure and physicochemical properties predicted that ACE2 protein is a stable typeⅠ transmembrane protein with its signal peptide sequences locating between 1aa and 18 aa. The combinant plasmid p ET-28a-ACE2 was constructed successfully,and with relative molecular mass of the protein of about 95×10^3,mainly in the form of inclusion body in the BL21（ DE3） for expression. [Conclusions]In this experiment,the full length of ACE2 gene in goats was first successfully cloned,and its protein structure and physicochemical properties were predicted. The sequence was uploaded to the Gen Bank,and its Gen Bank accession No. was KF921008.1. ACE2 protein was expressed in BL21（ DE3） largely.