Abstract：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 2 435 bp; the pcDNA3.1(+ ) -ACE2 plasmid was successfully constructed, and single enzyme digestion verified that a single band appeared at about 7 863 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 ACE2 recombinant 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.
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