Development and application of polyclonal antibodies against S2 of nephropathogenic infectious bronchitis virus（Original data of western blots）

This study examines the S2 protein of the renal infectious bronchitis virus (NIBV), essential for the virus's adaptation and expanded tissue and host range. Using bioinformatics, we analyzed its hydrophilicity, hydrophobicity, transmembrane regions, signal peptides, and antigenic epitopes. We also studied the secondary and tertiary structures to amplify the renal-type NIBV S2 gene via RT-PCR. The gene was cloned into the pET-32a (+) vector, forming the recombinant plasmid pET-SX9-S2, and introduced into BL21 (DE3) cells. An anti-rHis-S2 polyclonal antibody was produced using the expressed rHis-S2 as the antigen. The indirect ELISA titer of this polyclonal antibody was 1:1024,000. The rabbit polyclonal antibody against the NIBV-S2 protein developed in this study demonstrates efficacy in the detection of NIBV-S2. Detection revealed a significant increase in the expression of the NIBV-S2 protein from 3 to 9 days post-infection (dpi) with NIBV, peaking at 9 dpi. At this peak expression level, there was a notable downregulation of the ABCG2 protein, a uric acid transporter, in the kidneys of NIBV-infected chickens compared to the control group. This downregulation impaired renal excretion of uric acid, potentially leading to urate deposition. Furthermore, molecular docking and fluorescence confocal microscopy analyses demonstrated that the NIBV-S2 protein can bind to ABCG2, thereby inhibiting its transport pathway. This interaction results in significant urate deposition and induces pathological changes. In conclusion, NIBV exploits its S2 protein to interact with ABCG2, facilitating viral colonization within the kidneys and hindering uric acid clearance. This process contributes to urate accumulation and the development of the "mottled kidney" condition.