Preparation of polyclonal antibodies to the chicken Beclin1 protein and its application in the detection of nephropathogenic infectious bronchitis virus

Abstract

Beclin1, also known as ATG6, has been shown to be closely related to coronavirus, however, the link between Beclin1 and nephropathogenic infectious bronchitis virus (NIBV) has been poorly investigated and there are no available antibodies specifically targeting the chicken Beclin1 protein. The study aimed to prepare and assay a polyclonal antibody to Beclin1, enabling a deeper understanding of the mechanism of action of Beclin1 in NIBV. In this study, we amplified the chicken Beclin1 target gene and constructed a recombinant plasmid using prokaryotic expression techniques, then obtained the recombinant target protein by induced expression. Finally, the serum is obtained by immunizing rabbits with the purified and concentrated protein. The results show that the antiserum potency of the ELISA assay was >1:204800. By western blotting and immunofluorescence, the antibodies we prepared specifically recognized the chicken Beclin1 protein, which is mainly found in the nucleus of trachea, lung, kidney, spleen and fabricant cells. NIBV infection significantly decreased the expression of Beclin1 in the trachea, but increased in others. We have successfully prepared specific rabbit anti-chicken Beclin1 polyclonal antibodies, and detected changes in tissues of diseased chickens infected with NIBV, laying the foundation for further studies on the role of Beclin1 in avian diseases.