[Preparation and characterization of a polyclonal antibody against human Fibrocystin-L].

Aim: PKHDL1 (the gene for Polycystic Kidney and Hepatic Disease Like-1) had been recently identified, but characteristics of the gene product, Fibrocystin-L (FPC-L), still remain unknown. We therefore produced a rabbit polyclonal antibody hFL-Np to explore the cellular characteristics of this novel protein.

Methods: Based on the hydrophobic/hydrophilic analyses, chose a cDNA fragment which encodes 633L-768K amino acids of the FPC-L and amplified it by RT-PCR. The PCR product was then cloned into a prokaryotic expression vector pGEX-GST. With IPTG induction, the antigen hFL-N was produced and further purified. A rabbit was immunized with the antigen and its antiserum was collected. Applied Western blot with the polyclonal antiserum hFL-Np and validated the antibody specific for FPC-L protein. In addition, also used immunofluorescence staining with hFL-Np to detect the subcellular distribution in cultured HEK293 cells.

Results: The prokaryotic expression vector pGEX-hFL-N was successfully constructed and a hFL-N antigen was produced in E.coli Rossetta cells. Using the antigen, a polyclonal antibody hFL-Np was produced and the specificity for FPC-L was also proved by biochemistry and cellular assays. Using the antibody, the cellular staining reveals that FPC-L was a cytosolic protein.

Conclusion: We produced an anti-FPC-L polyclonal antibody hFL-Np. By biochemistry and cellular characterization, proved that the polyclonal antibody hFL-Np is specific for FPC-L and demonstrated FPC-L is a cytosolic protein. The finding provides a platform for further dissecting FPC-L functions in mammalian development.