中国家鹅血管紧张素转化酶2(ACE2)基因的克隆、表达及序列解析

Gene cloning,expression and sequence information analysis of angiotensin converting enzyme 2(ACE2)gene in Chinese domestic goose

通过对中国家鹅血管紧张素转化酶2(angiotensin converting enzyme 2,ACE2)基因进行克隆、表达、序列信息分析以及对中国家鹅SARS-CoV-2易感性进行分析,旨在为鹅ACE2方面的研究提供基础资料。以中国家鹅为研究对象,检测其不同组织的ACE2基因表达,并扩增出肾组织的ACE2全基因编码区序列。将ACE2目的基因与pMD-19T重组后导入DH5α感受态细胞,随机挑取单菌落进行菌液PCR和酶切验证,并对验证为阳性的单菌落进行测序。对测序所得序列进行生物信息学分析,同时对鹅与人及其他动物ACE2结合SARS-CoV-2的氨基酸序列进行了差异性比较。结果显示,鹅体内有ACE2表达,并在肾脏中有较高的表达水平;扩增出该鹅的ACE2基因ORF序列长度为2473 bp,编码808个氨基酸,氨基酸序列与中国麻鸭的同源性最高,且处在同一进化分支中;进一步对ACE2编码蛋白质分析显示,该中国家鹅ACE2蛋白有较高亲水性,为Ⅰ型跨膜蛋白,信号肽位于第1~17氨基酸之间;此外,对鹅与人及其他动物ACE2的SARS-CoV-2结合关键氨基酸序列的比较显示,鹅与人等对SARS-CoV-2易感性较高动物的该序列差异较大,而与鸡、鸭等禽类较接近,提示鹅对SARS-CoV-2无易感性。研究获得了鹅ACE2基因与蛋白的相关资料,为鹅ACE2相关研究提供了基础理论依据和资料。

The ACE2 gene of Chinese domestic geese was cloned,expressed,sequenced and analyzed to analyze the susceptibility of Chinese domestic geese to SARS-CoV-2,in order to provide basic data for the study of ACE2 in Chinese domestic geese.Taking Chinese domestic geese as the research object,the expression of ACE2 gene in different tissues was detected,and the whole coding region of ACE2 gene in kidney tissue was amplified.The gene of ACE2 was cloned in pMD-19 T and introduced into DH5αcompetent cells.The single colonies were randomly selected for PCR and restriction endonuclease digestion,and the positive colonies were sequenced.The sequences were analyzed by bioinformatics,and the amino acid sequences of ACE2 binding SARS-CoV-2 between goose and human and other animals were compared.Studies have confirmed that ACE2 was expressed in geese and had a high expression level in the kidney.The ORF sequence of ACE2 gene of the goose was 2473 bp in length,encoding 808 amino acids.The amino acid sequence had the highest homology with that of Chinese sesame duck and was in the same evolutionary branch.Further analysis of the protein encoded by ACE2 showed that the ACE2 protein of national goose had high hydrophilicity and was a type I transmembrane protein,and the signal peptide was located between1and 17amino acids.In addition,the comparison of the key amino acid sequences of SARS-CoV-2binding of ACE2between geese and humans and other animals showed that there was a significant difference between geese and animals with high susceptibility to SARS-CoV-2,but close to that of birds such as chickens and ducks,suggesting that geese were not susceptible to SARS-CoV-2.The related data of goose ACE2gene and protein were obtained,which provided the theoretical basis and data for goose ACE2related research.