Generation of a cholangiocyte-specific cDNA expression library for the identification of B and T cell autoantigens in murine biliary disease.
Generation of a cholangiocyte-specific cDNA expression library for the identification of B and T cell autoantigens in murine biliary disease.
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DOI：
10.1111/j.1872-034x.2011.00951.x
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发表时间：
2012-05
期刊：
影响因子：
--
通讯作者：
Mack CL
中科院分区：
文献类型：
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作者：
Tucker RM;Mack CL
存在几种炎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。在一个正在进行的努力，以确定浸润性自身反应性T细胞和血清自身抗体的靶抗原，我们的目标是产生一个胆管细胞衍生的cDNA文库，能够表达各种各样的蛋白质。从正常小鼠胆管细胞系中分离mRNA并逆转录成cDNA。在将cDNA初始克隆到转移载体（pDONR 222）中后，将整个文库穿梭到大肠杆菌表达载体（pDEST 160）中。该库包含2.3 × 10个独立克隆，表达分子量高达100 kD的蛋白质。使用各种技术，包括蛋白质印迹分析，单个克隆的质谱分析，和质粒的直接DNA测序，已经在其中鉴定了许多普遍表达的和胆管细胞特异性的蛋白质（例如细胞角蛋白19）。一个全面的小鼠胆管细胞cDNA表达文库已经产生，并可用作免疫学研究的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的SEREX分析中具有潜在的用途。胆管细胞特异性cDNA文库是一个强有力的工具，用于识别小鼠炎症性胆管病的靶抗原，并可作为共享资源。
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 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.