Generation of a cholangiocyte-specific cDNA expression library for the identification of B and T cell autoantigens in murine biliary disease
Article first published online: 11 JAN 2012
© 2012 The Japan Society of Hepatology
Volume 42, Issue 5, pages 502–507, May 2012
How to Cite
Tucker, R. M. and Mack, C. L. (2012), Generation of a cholangiocyte-specific cDNA expression library for the identification of B and T cell autoantigens in murine biliary disease. Hepatology Research, 42: 502–507. doi: 10.1111/j.1872-034X.2011.00951.x
- Issue published online: 15 APR 2012
- Article first published online: 11 JAN 2012
- Received 25 August 2011; revision 27 October 2011; accepted 24 November 2011.
- autoimmune response;
- bile duct diseases;
- cDNA library;
- serological analysis of recombinant cDNA expression libraries
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 × 106 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.