[Preparation and characterization of a polyclonal antibody against mouse NH2;-terminal domain of polycystin-1].

OBJECTIVE

To produce a rabbit polyclonal antibody, mPkd1-Np, against the extracellular portions of polycystin-1 (PC1) in order to explore the functional roles of the PC1 NH2;-terminus.

METHODS

Based on hydrophobic/hydrophilic analyses, we chose a cDNA fragment that encodes amino acids 474E-640L on PC1 and amplified it via RT-PCR. The PCR product was then cloned into a prokaryotic expression vector pGEX-GST. After IPTG induction, the antigen mPkd1-N was produced and further purified. A rabbit was immunized with this antigen and its antiserum was collected. The mPkd1-Np antibody was validated to be specific for PC1 protein through Western blotting, immunohistochemistry, and immunofluorescence methods.

RESULTS

The prokaryotic expression vector pGEX-mPkd1-N was successfully constructed and mPkd1-N antigen was induced to express in E.coli Rossetta cells. Using this antigen, the polyclonal antibody mPkd1-Np was produced and its specificity for PC1 was proved through biochemistry and cellular assays.

CONCLUSION

We successfully produced an anti-PC1 NH2;-terminal polyclonal antibody named mPkd1-Np. The polyclonal antibody provides a platform for further research into PC1 NH2;-terminal function, specifically renal tubulogenesis and its maintenance.