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| Study on the Binding Characteristics of Rab5a with Invariant Chain of Chicken (Gallus gallus) and Their Co-localization in Eukaryotic Cells |
| TAN Hong-Li, GUI Ya-Ping, LI Mei-Zhen, HUANG Xin, QIN WEN-Juan, ZHA Li-Sha, CHEN Fang-Fang* |
| Animal Molecule and Applied Immune Innovation Team of Anhui Agricultural University, Hefei 230036, China |
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Abstract Rab5a molecule is an intracellular transporter, while the invariant chain (Ii) is an immune molecule that helps MHC (major histocompatibility complex) to assemble, mature and present antigen peptide. For a long time, the intracellular pathway mechanism of Ii is not clear. The purpose of this study was to explore the structural characteristics of Rab5a in chicken (Gallus gallus) and its role in the intracellular transport of Ii. First, chicken Rab5a gene was cloned with self-designed primer, and the eukaryotic expression recombinant vectors containing chicken cIi and its mutants (IiD81-87aa and IiD91-99aa) were constructed. Secondly, the protein structure of Rab5a in chicken and mouse (Mus musculus) were compared by software. Finally, Rab5a and Ii were transfected into mouse dendritic cell line DC2.4 cells, respectively. The localization and co-localization of chicken Rab5a and Ii, cIiD81-87aa and cIiD91-99aa in eukaryotic cells were observed by immunofluorescence and laser confocal microscope. The results showed that the Rab5a gene was 648 bp, which was consistent with the expected size. The molecular structural analysis showed that Rab5a of chicken was highly similar to that of mouse, and the homology was as high as 95.4%. The results of homologous modeling analysis showed that Rab5a of chicken and mouse contained 5 alpha helixes and 6 beta helixes, indicating that Rab5a of mammal and poultry were homologous. Laser confocal microscope observation showed that chicken Rab5a and Ii could be localized in the early endocytosis of cells, but the mutants IiD81-87aa and IiD91-99aa could not be localized in the early endocytosis. The results of GST pull-down and Western blot showed that Rab5a could bind Ii molecule. In conclusion, the results of co-localization and binding of Rab5a and Ii in the early endocytosis suggest that Rab5a is involved in the transport of Ii in cells, which provides a new basis for further study of its mechanism.
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Received: 23 November 2019
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Corresponding Authors:
*, fang7828887@126.com
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