Preparation And Application Of A Polyclonal Antibody Against Mouse NH2-terminal Domain Of Polycystin-1

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic diseases in the kidneys and is characterized by numerous fluid-filled renal cysts. ADPKD is caused by mutations in either PKD1or PKD2genes. Both genes encode two proteins which are named by polycystin-1(PC1) and polycystin-2(PC2), respectively. Recent studies indicate that PCI and PC2are able to form a protein complex via their COOH-terminal interaction and regulate several common cell signaling pathways. Loss of either polycystins can dysregulate the signal pathways and leads aberrant ionic transportation, polarity, proliferation and apoptosis in the renal epitherlial cells, eventually result in renal cyst formation. However, precisely how PC1and PC2affect in the disease at the molecular level still remains some unknown. Some studies indicate that PC1ectodomain can be cleaved by the G protein-coupled receptor (GPCR) proteolytic site (GPS) domain, and be then released to the tubular lumen.PC1entire extracellular region as a ligand which is likely to exert their biological function, and may play an important function in the process of tubule formation, and thus it is necessary to produced a rabbit polyclonal antibody, mPkdl-Np, against the extracellular portions of polycystin-1(PC1) in order to explore the functional roles of the PCI NH2-terminus. In this study, we performed hydrophobic/hydrophilic analyses and chose a cDNA fragment that encodes amino acids474E-640L of PC1. The PCR product which encoded this PC1region was then cloned into a prokaryotic expression vector pGEX-GST. With IPTG induction, the antigen mPkdl-N was produced and further purified. A Japanese white rabbit was immunized with this antigen and its antiserum was collected. The mPkdl-Np antibody was validated to be specific for PC1protein through western blotting, immunohistochemistry, and immunofluorescence approaches. In addition, We established immortalized renal collecting duct cell lines from the kidneys of Im::Pkd1-/-mice and their wildtype littermates. Using these cell lines and mPkdl-Np antibody, we found that loss of PC2downregulates PC1expression in vitro and in vivo.