Establishment of Chicken Angiotensin Converting Enzyme 2 (ACE2) Prokaryotic Expression System and Preparation of It's Polyclonal Antibody
JI Xiao-Xia1, Wu Yu-Long2, CHEN Xi-Wen1, ZHOU Xiao1, DU Xin-Yu1, ZHANG Yuan-Shu1*
1 Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China; 2 School of Food Science, Nanjing Xiaozhuang University, Nanjing 211171, China
Abstract：Angiotensin converting enzyme 2 (ACE2) can serve as a protective and regulatory agent for various pathophysiological processes, as well as a functional receptor for human coronavirus. However, there have been no reports on research related to chickens (Gallus gallus domesticus). In this study, broiler chickens were used as research subjects. Firstly, the coding region of the ACE2 gene was amplified by PCR and connected to the prokaryotic expression vector pET32a to construct a chicken ACE2 prokaryotic expression system. The expression of the ACE2 protein was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the induction conditions were optimized to determine the expression pattern. The purified recombinant chicken ACE2 protein obtained through KCl staining gel purification was identified for specificity and molecular weight using SDS-PAGE and Western blot techniques. The isoelectric point of the ACE2 protein was determined using isoelectric focusing polyacrylamide gel electrophoresis. The purified recombinant ACE2 protein was used as an immunogen to produce chicken ACE2 polyclonal antibodies in rats (Rattus norvegicus). The efficacy of the antibodies was measured, and their specificity was validated to determine the tissue distribution and cellular localization of ACE2 in chickens. The study successfully constructed a prokaryotic expression system for ACE2 in chickens using the pET32a vector, prepared chicken ACE2 polyclonal antibodies with good efficacy and specificity, and confirmed the widespread distribution of ACE2 in chickens for the first time. This study provides an experimental basis and data for further research on ACE2 in chickens.
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