Generation of a cholangiocyte-specific cDNA expression library for the identification of B and T cell autoantigens in murine biliary disease.

AIM

Several mouse models of inflammatory cholangiopathies exist, including biliary atresia, primary biliary cirrhosis, autoimmune hepatitis, and primary sclerosing cholangitis. In an ongoing effort to identify the target antigens of both infiltrating autoreactive T cells and serum autoantibodies, we aimed to generate a cholangiocyte-derived cDNA library capable of expressing a wide variety of proteins.

METHODS

mRNA was isolated from a normal mouse cholangiocyte cell line and reverse transcribed into cDNA. After initial cloning of the cDNA into a transfer vector (pDONR222), the entire library was shuttled into an Escherichia coli expression vector (pDEST160).

RESULTS

The library contains 2.3 × 10(6) independent clones and expresses proteins up to 100 kD in molecular weight. Using a variety of techniques, including western blot analysis, mass spectrometry of individual clones, and direct DNA sequencing of plasmids, a number of both ubiquitously expressed and cholangiocyte-specific proteins (e.g. cytokeratin 19) have been identified within.

CONCLUSION

A comprehensive mouse cholangiocyte cDNA expression library has been generated and is available for use as a source of multiple cholangiocyte-specific antigens for immunological studies. The library can be used to screen for specificity of T cell lines or hybridomas. Furthermore, this library has potential uses in SEREX analysis of autoantibody reactivity. The cholangiocyte-specific cDNA library is a powerful tool for the identification of target antigens in murine inflammatory cholangiopathies and is available as a shared resource.