鸭血管紧张素转换酶2(ACE2)真核表达载体的构建和稳定细胞株的筛选  

Construction of Duck(Anas platyrhynchos)Angiotensin-converting Enzyme 2(ACE2)Eukaryotic Expression Vector and Screening of Stable Cell Line

作　　者：纪晓霞 李帅 曹西月 张崇昊 张源淑[1] JI Xiao-Xia;LI Shuai;CAO Xi-Yue;ZHANG Chong-Hao;ZHANG Yuan-Shu(Key Laboratory of Animal Physiology and Biochemistry,Ministry of Agriculture,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学农业部动物生理生化重点开放实验室,南京210095

出　　处：《农业生物技术学报》2022年第7期1421-1431,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972640)。

摘　　要：血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)是肾素血管紧张素系统(renin angiotensin system,RAS)的负调节分子和SARS-coV-2等冠状病毒的功能性受体。为丰富禽类鸭(Anas platyrhynchos)ACE2蛋白的研究内容,探寻该蛋白与禽冠状病毒的关系,本研究利用PCR扩增鸭ACE2基因编码区全长,通过同源重组与pcDNA3.1(+)载体连接,构建真核表达体系,确定中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞的新霉素(geneticin,G418)最佳筛选浓度,pcDNA3.1(+)-ACE2转染细胞后通过G418加压筛选单克隆细胞,采用Western blot和免疫荧光法对获得的细胞株进行鉴定,建立稳定表达鸭ACE2蛋白的细胞株,并测定其ACE2重组蛋白的酶活性。结果显示,成功克隆了鸭ACE2基因,其编码区全长为2435 bp;成功构建pcDNA3.1(+)-ACE2真核表达质粒,单酶切验证7863 bp左右出现单一条带;脂质体法将重组质粒转染入CHO细胞,可表达外源ACE2重组蛋白;获得3株单克隆细胞株,分别命名为pcDNA3.1(+)-ACE2-1、pcDNA3.1(+)-ACE2-2、pcDNA3.1(+)-ACE2-3;单克隆细胞株中提取的ACE2重组蛋白具有较高的酶活性。本研究构建的鸭ACE2真核表达体系以及获得的可以稳定表达ACE2重组蛋白细胞株,为鸭ACE2蛋白与冠状病毒等生物学功能的研究提供了基础,丰富了该蛋白的相关理论。Angiotensin-converting enzyme 2(ACE2)is a negative regulator of the renin angiotensinogen system(RAS)and a functional receptor of coronavirus such as SARS-CoV-2.To enrich the research content of ACE2 in ducks(Anas platyrhynchos)and explore the relationship between the ACE2 and avian coronavirus,the full-length coding sequence of the duck ACE2 gene was amplified by PCR,and the eukaryotic expression system was constructed by connecting homologous recombination with pcDNA3.1(+)vector.The optimal screening concentration of geneticin(G418)was determined for Chinese hamster ovary(CHO)cells,after transfecting of cells,selected monoclonal cell lines for G418.Western blot and immunofluorescence were used to identify the obtained cell lines.The cell line for expressing duck ACE2 protein was established,determined the enzyme activity of ACE2 recombinant protein.The results showed that the duck ACE2 gene was successfully cloned,and its coding region was 2435 bp;the pcDNA3.1(+)-ACE2 plasmid was successfully constructed,and single enzyme digestion verified that a single band appeared at about 7863 bp.The recombinant plasmid was successfully transfected into CHO cells by Lipofectamine,and exogenous ACE2 recombinant protein was expressed.Three monoclonal cell lines named pcDNA3.1(+)-ACE2-1,pcDNA3.1(+)-ACE2-2,pcDNA3.1(+)-ACE2-3 were successfully obtained,respectively.The recombinant ACE2 protein extracted from the monoclonal cell line showed high enzymatic activity.This study successfully constructed a duck ACE2 eukaryotic expression system and obtained 3 cell lines that can stably express ACE2recombinant protein.This study provides a basis for the study of duck ACE2 protein and coronavirus and other biological functions and enriches the related theories of the protein.

关 键 词：真核表达 脂质体转染 血管紧张素转化酶2(ACE2) 细胞株 鸭 

分 类 号：S852.23[农业科学—基础兽医学]