中国麻鸭血管紧张素转化酶2(ACE2)的基因克隆及基因结构的序列解析

doi:10.11843/j.issn.0366-6964.2020.07.012

摘要/Abstract

摘要： 血管紧张素转化酶2（ACE2）被发现以来，其病理生理功能，特别是作为SARS、COVID-19等冠状病毒侵入细胞的功能性受体，显示了巨大的潜能，明确其在不同物种的基因序列及结构可为冠状病毒感染机制研究提供依据。本研究以中国麻鸭为研究对象，利用RT-PCR和Western blot检测ACE2在中国麻鸭各组织中的表达，然后用PCR技术，扩增出中国麻鸭ACE2基因的完整ORF序列，再TA克隆至pMD-19T载体中进行测序，对所得序列进行生物信息学解析。结果证实，ACE2在中国麻鸭心、肝、肺、肾等组织中均有表达。基因克隆结果显示，该中国麻鸭ACE2基因的CDS序列全长为2 435 bp，编码805个氨基酸残基，与人ACE2核苷酸序列和氨基酸序列同源性分别为66.2%和66.4%，且处在不同进化树分支上。通过对与SARS病毒S蛋白结合有关的18个关键氨基酸残基分析发现，中国麻鸭除第330和353位氨基酸外，其余均与人不相同。结构分析发现，中国麻鸭ACE2为Ⅰ型跨膜蛋白，有多处N-糖基化位点。研究首次获得中国麻鸭ACE2基因的完整ORF序列及相关基础资料，所得序列已上传GenBank并被成功收录，研究结果为开展ACE2在鸭上的功能研究提供了理论依据。

关键词: 中国麻鸭, 血管紧张素转化酶2, 基因克隆, 基因序列解析

Abstract: Since the discovery of angiotensin-converting enzyme 2 (ACE2),its pathophysiological functions,especially as functional receptors for coronaviruses such as SARS and COVID-19,have shown great potential.To clarify the gene sequence and structure of different species can provide a basis for the study of the mechanism of coronavirus infection. In this experiment,RT-PCR and Western blot were firstly used to detect the presence of ACE2 in different tissues of China Sheldrake duck.Then the homologous cloning and PCR technology were used to amplify the complete ORF sequence of the China Sheldrake duck ACE2 gene,and then TA cloned into pMD-19T.The vector was sequenced,and the obtained sequence was analyzed by bioinformatics. The expression of ACE2 gene and protein in heart,liver,lung,kidney and other tissues was confirmed.Gene cloning results showed that the full-length CDS sequence of the China Sheldrake duck ACE2 gene was 2 435 bp,encoding 805 amino acid residues,and its nucleotide sequence and amino acid sequence homology with human ACE2 were 66.2% and 66.4%,respectively,and on different branches of the evolutionary tree.Analysis of the 18 key amino acid residues related to the binding of the SARS virus S protein in humans found that except for the 330th and 353th amino acids, the rest were different from humans. Structural analysis revealed that the duck ACE2 was a type I transmembrane protein with multiple N-glycosylation sites.The study obtained the complete ORF sequence and related basic data of the ACE2 gene of China Sheldrake duck for the first time. The obtained sequence had been uploaded to GenBank and successfully included.The results provided a theoretical basis for the functional study of ACE2 on ducks.

Key words: China Sheldrake duck, ACE2, gene clone, gene sequence analysis

中图分类号:

S852.2

李帅, 纪晓霞, 杨博, 许濛, 张源淑. 中国麻鸭血管紧张素转化酶2(ACE2)的基因克隆及基因结构的序列解析[J]. 畜牧兽医学报, 2020, 51(7): 1597-1606.

LI Shuai, JI Xiaoxia, YANG Bo, XU Meng, ZHANG Yuanshu. Gene Cloning and Sequence Analysis of Angiotensin Converting Enzyme 2 (ACE2) in China Sheldrake Duck[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1597-1606.

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