家猫ACE2基因克隆、测序及生物信息学分析

Domestic Cat ACE2 Gene Cloning,Sequencing and Bioinformatic Analysis

本研究从家猫组织中扩增并鉴定血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)基因,为阐明SARS相关冠状病毒(SARS-CoV)感染家猫的机制提供理论依据.从家猫肺脏中提取总RNA,以Oligo dT-Adaptor为引物合成第一链cDNA,根据人及小鼠的ACE2基因mRNA序列设计系列引物,通过聚合酶链式反应,分段扩增出家猫ACE2基因的编码区的特异性片段并测序鉴定.家猫ACE2基因mRNA编码区共包括2 418个核苷酸,推测编码蛋白含805个氨基酸残基.其氨基酸序列与人、小鼠、大鼠ACE2氨基酸序列的同源性分别达到85%,81%和81%.利用多种生物信息学工具软件对家猫ACE2蛋白的理化及生物学性质进行分析.比较人、家猫、果子狸、小鼠、大鼠等ACE2序列中与SARS-CoV结合有关的3个功能区,发现家猫ACE2与人ACE2的同源性最高,提示家猫ACE2在SARS病毒跨种感染中可能发挥重要作用.

Angiotensin-converting enzyme 2 （ACE2） has been determined as the most efficient receptor of SARS-CoV. Domestic cats are one of the susceptible hosts for SARS-CoV. It is likely for SARS-CoV to infect cats by using the cellular ACE2 as the common receptor. Therefore this study is targeted to identify the cat ACE2. The domestic cats were sacrified and the lung was used to extract the total RNA. The cD NA was synthesized by reverse transcription. Four overlapped mRNA fragments were amplified by using the cDNA as the templates and spiced. The domestic cats ACE2 mRNA coding region consists of 2418 nu cleotides encoding putative protein of 805 amino acids. Alignment analysis demonstrated that cat ACE2 has a similarity of 85%, 81% and 81% at the amino acid level with human, mouse and rat ACE2 respectively. A series of bioinformatics analyses were performed to predict the structure and function of this protein. The results confirmed that the sequence is the coding sequence of domestic cat ACE2. Furthermore, the cat ACE2 sequences in three ACE2 domains of SARS CoV association have the highest similarity with human ACE2 suggesting cats may play an important role in SAR＆CoV interspecies infection.