Construction of Chicken (Gallus gallus domesticus) Angiotensin-converting Enzyme 2 (ACE2) Eukaryotic Expression System and Screening of Stable Cell Lines
JI Xiao-Xia1,2, LIU Ben-Fu1, WANG Huan-Huan2, CHEN Xi-Wen2, ZHANG Yuan-Shu2,*
1 College of Rural Revitalization, Linyi Vocational College, Linyi 276023, China; 2 Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
Abstract：Angiotensin-converting enzyme 2 (ACE2) serves as a negative regulator of the renin-angiotensin system (RAS) and also acts as a key functional receptor for multiple coronaviruses, including Severe acute respiratory syndrome coronavirus (SARS-CoV), facilitating viral entry into host cells. To explore the relationship between chicken (Gallus gallus domesticus) ACE2 protein and avian coronaviruses including Infectious bronchitis virus (IBV), this study amplified the complete coding sequence of the chicken ACE2 gene by RT-PCR and cloned it into the pcDNA3.1(+) eukaryotic expression vector via homologous recombination. After verification by colony PCR, restriction enzyme digestion, and nucleotide sequencing, the recombinant plasmid was transfected into Chinese hamster ovary (CHO) cells using liposome-mediated transfection. Stable cell lines constitutively expressing chicken ACE2 were then established through geneticin (G418) selection combined with monoclonal expansion. The expression of ACE2 in CHO cells was assessed by RT-PCR, Western blot, and immunofluorescence staining, while the enzymatic activity of the recombinant ACE2 protein was quantitatively measured using a double-antibody sandwich assay. The results demonstrated that the recombinant eukaryotic plasmid pcDNA3.1(+)-ACE2 was successfully constructed in vitro, yielding a single restriction fragment of approximately 7 872 bp, and sequencing data matched the predicted sequence perfectly. After efficient transfection into CHO cells, RT-PCR confirmed abundant transcription of the target gene, immunofluorescence revealed uniform distribution of the recombinant protein on the cell membrane with high transfection efficiency, and Western blot further confirmed robust expression of the recombinant protein with an apparent molecular weight of about 105 kD. Following G418 selection and monoclonal culture, 4 stable cell lines were obtained, designated pcDNA3.1(+)-ACE2-A/B/C/D. The double-antibody sandwich assay showed that the ACE2 enzymatic activity reached 601.77 U/L, indicating high catalytic activity. In summary, this study successfully established a eukaryotic expression system for chicken ACE2 and generated stable cell lines producing the recombinant protein, thereby providing a solid foundation for further investigations into the biological functions of chicken ACE2, particularly its involvement with coronaviruses.
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