LAUSR

Objective To investigate the intracellular localization and binding of duck invariant chain (Ii) to major histocompatibility complex (MHC) class Iα or MHC class IIβ molecules. Methods First, the genes of duck Ii, MHC Iα, and MHC IIβ were amplified by PCR, cloned into expression plasmids, and then sequenced to compare homology between ducks and selected agricultural species. Second, Rosetta or 293T cells were transfected with plasmids encoding Ii, MHC Iα, and MHC IIβ either alone or in combination to interrogate binding following expression. The fluorescent reporter molecules used to tag/image transfected cells and expressed protein were identified by western blot. Additionally, the binding interaction of the expressed products was observed by affinity purification using pull-down assay and coimmunoprecipitation. Finally, intracellular colocalization was observed by laser confocal microscopy. Results The results of western blots demonstrated that all constructed plasmids successfully expressed proteins. Pull down and coimmunoprecipitation revealed that Ii could bind Iα or IIβ, forming Ii-Iα or Ii-IIβ complexes, and they dissociated into single molecules after SDS treatment. In 293T cells, Ii interacts with MHC Iα or MHC IIβ chains and colocalizes intracellularly. Confocal fluorescence microscopy assay indicated that MHC Iα and IIβ were located in the endoplasmic reticulum and endosome. Conclusions Both MHC and Ii proteins exhibit direct interactions and can colocalize in the endoplasmic reticulum and endosomes. Clinical Relevance This study investigates the relationship between duck Ii and MHC proteins, offering a theoretical foundation for exploring the role of Ii in immune responses and advancing the development of Ii-vectored avian vaccines.