Eukaryotic Expression of Goose(Anser cygnoides domesticus)-derived Angiotensin-converting Enzyme 2(ACE2) and Screening for Stable Cell Lines
XU Jia-Jing, WANG Huan-Huan, CHEN Xi-Wen, WANG Gong-Min, ZHANG Yuan-Shu*
Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture and Rural Affairs, Nanjing Agricultural University, Nanjing 210095, China
Abstract：Angiotensin-converting enzyme 2(ACE2), a key regulatory enzyme in the renin-angiotensin system(RAS) and the functional receptor for multiple coronaviruses, has been extensively studied in mammals. However, the molecular characteristics of waterfowl ACE2, particularly goose(Anser cygnoides domesticus)-derived ACE2, remain largely unexplored.The aim of this study was to clone the goose-derived ACE2 gene, construct its eukaryotic expression vector, and establish Chinese hamster(Cricetulus griseus) ovary(CHO) cell lines stably expressing the protein. The full-length coding sequence of ACE2 was amplified by PCR using jejunum cDNA from the Sanhua goose as the template, and the recombinant plasmid pcDNA3.1(+)-ACE2 was constructed to determine the optimal screening concentration of neomycin(G418), and then the recombinant plasmid was transfected into CHO cells by liposome assay, and the resistant monoclonal cell lines were obtained by G418 screening, the expression and activity of ACE2 were systematically evaluated using Western blot, qPCR, immunofluorescence staining, and protease activity detection. The results showed that the coding region sequence of goose ACE2 gene was successfully cloned, with a total length of 2 427 bp, and the recombinant eukaryotic expression plasmid pcDNA3.1(+)-ACE2 was constructed, and the size of the bands verified by double enzyme digestion was in accordance with the expectation(5428 bp of vector, 2427 bp of ACE2). The optimal screening concentration of G418 was determined to be 800 μg/mL, and 2 monoclonal cell lines expressing ACE2 protein were obtained after screening, named pcDNA3.1(+)-ACE2-1 and pcDNA3.1(+)-ACE2-2, respectively. Compared with the pcDNA3.1(+) control group, ACE2 mRNA levels in both pcDNA3.1(+)-ACE2-1 and pcDNA3.1(+)-ACE2-2 cells were significantly elevated(P<0.01). Immunofluorescence staining revealed distinct ACE2-positive signals in the 2 cell lines, confirming successful protein expression.The enzyme activity assay showed that the ACE2 protease activities expressed by the 2 cell lines were(37.25±2.95) and(246.87±15.94) U/mg, respectively, which were extremely significantly higher than those of the pcDNA3.1(+) group(P<0.01). In addition, ACE2 mRNA expression and enzymatic activity remained stable across multiple passages, indicating that the established cell lines possessed good long-term expression stability. In this study, the CHO cell lines stably expressing highly active ACE2 were successfully established, which provides a reliable platform for the study of the molecular structure and function of goose ACE2.
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