RUI: Assembly and Expression MHC Class II Chains in the Channel Catfish

这项研究项目的长期目标是表征和定义硬骨鱼物种中MHC II类分子在细胞表面表达所必需的特定加工事件。该项目的直接目标是确定正确折叠、组装和细胞表面表达第二类异源二聚体所需的分子事件。特别是，这项工作试图确定主要组织相容性复合体(MHC)II类异源二聚体在内质网伴侣Calnexin和不变链的帮助下在硬骨鱼中的组装。通过将先前确定的编码基因导入鲶鱼II类阴性成纤维细胞系中，将测试II类α链和β链的优先配对。第二类基因和蛋白的RNA和细胞表面表达将被检测。在其他实验中，将使用免疫沉淀、免疫印迹、流式细胞术和免疫荧光显微镜来测试钙粘蛋白、钙网蛋白和不变链在处理和运输II类分子中的作用。斑点叉尾鱼的淋巴、巨噬细胞和成纤维细胞系的可获得性，以及先前表达的两个MHC II类A和B类基因座的特征，使斑点叉尾鱼成为硬骨鱼中研究MHC II类组装和细胞表面表达的独特系统。

The long-term goal of this research project is to characterize and define the specific processing events necessary for cell surface expression of MHC class II molecules in a teleost species. The immediate goal of this project is to determine the molecular events necessary for proper folding, assembly, and cell surface expression of class II heterodimers. In particular, the work seeks to determine the assembly of major histocompatibility complex (MHC) class II heterodimers in teleosts proceeds with assistance from the endoplasmic reticulum chaperone calnexin and the invariant chain. Preferential pairing of class II alpha and beta chains will be tested with transfection of the previously characterized encoding genes into catfish class II negative fibroblast cell lines. RNA and cell surface expression of class II genes and proteins will be examined. In other experiments, immunoprecipitations, immunoblots, flow cytometry, and immunofluorescence microscopy will be used to test the role of calnexin, calreticulin, and invariant chain in the processing and trafficking of class II molecules. The availability of catfish lymphoid, macrophage, and fibroblast cell lines, along with the previous characterization of two expressed MHC class II A and class II B loci, make the channel catfish a unique system among teleosts for the study of MHC class II assembly and cell surface expression.