Department of Medicine, University of Colorado, Denver Department of Pediatric Gastroenterology, Hepatology and Nutrition, Children's Hospital Colorado, Aurora, Colorado, USA.
Hepatol Res. 2012 May;42(5):502-7. doi: 10.1111/j.1872-034X.2011.00951.x. Epub 2012 Jan 11.
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.
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).
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.
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.
存在几种炎症性胆管病的小鼠模型，包括胆道闭锁、原发性胆汁性肝硬化、自身免疫性肝炎和原发性硬化性胆管炎。为了努力鉴定浸润性自身反应性 T 细胞和血清自身抗体的靶抗原，我们旨在生成能够表达多种蛋白的胆管细胞衍生 cDNA 文库。
从正常小鼠胆管细胞系中分离 mRNA，并将其反转录成 cDNA。在将 cDNA 最初克隆到转移载体（pDONR222）后，整个文库被转移到大肠杆菌表达载体（pDEST160）中。
文库包含 2.3×10^6 个独立克隆，表达的蛋白分子量高达 100kD。使用多种技术，包括 Western blot 分析、单个克隆的质谱分析和质粒的直接 DNA 测序，已在文库中鉴定出多种广泛表达和胆管细胞特异性蛋白（例如细胞角蛋白 19）。
已生成全面的小鼠胆管细胞 cDNA 表达文库，可作为免疫研究中多种胆管细胞特异性抗原的来源。该文库可用于筛选 T 细胞系或杂交瘤的特异性。此外，该文库在自身抗体反应性 SEREX 分析中具有潜在用途。胆管细胞特异性 cDNA 文库是鉴定炎症性胆管病中靶抗原的有力工具，可作为共享资源。
