鸡Ii结合Rab5a和Rab7b分子的分析

doi:10.11843/j.issn.0366-6964.2021.012.025

Abstract

Abstract: This study aimed to investigate the binding domain and intracellular localization of chicken invariant chain (Ii) to the endosome transporters Rab5a and Rab7b. First, the cytoplasmic region and transmembrane region (Ii_(Cyt-Tra)), Ii CLIP (class Ⅱ-associated invariant chain peptide) with trimer region (Ii_(CLIP-TRIM)) and Ii mutants (Ii_(M81-87aa),Ii_(M91-99aa)and Ii_(M81-99aa)) obtained by PCR and gene mutation techniques were inserted into pET-32a and pEGFP-C1 to consturct prokaryotic and eukaryotic recombinant plasmids, respectively, the corresponding eukaryotic and prokaryotic recombinant expression plasmids were constructed. Secondly, the constructed recombinant plasmid containing green fluorescent protein and the recombinant plasmids containing red fluorescent Rab5a and Rab7b were co-transfected into human embryonic kidney cell line 293 T, and their co localization was observed. Finally, the binding domain of Ii with Rab5a and Rab7b was detected by pull-down method and Western blot. The results showed that the recombinant plasmids of Ii domain and mutant were successfully constructed. Both Ii _(cyt-tra) and Ii mutants could co localize with Rab5a and Rab7b, but Ii _(CLIP-TRIM) and empty vector could not. The cytoplasmic and transmembrane domains of Ii are the main domains binding Rab5a and Rab7b, not CLIP and trimer domains. In conclusion, the colocalization and binding regions of chicken Ii with Rab5a and Rab7b are cytoplasmic and transmembrane regions, not endoplasmic reticulum cavity regions. These results further suggest that Rab molecule is involved in the transport mechanism of Ii in intracellular organelles, and provide a new way for further study of the transport mechanism and function of Ii and its carrier in cells.

Key words: invariant chain, Ii, domains, Rab5a, Rab7b, co-localization, bind

CLC Number:

S852.4

CHEN Fangfang, GUI Yaping, YU Fengmei, ZHAGN Jun, TAN Yang, LI Jinchun, LIU Cuiyan, ZHA Lisha. Analysis on Ii Binding Rab5a and Rab7b Molecules[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3588-3597.

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Analysis on Ii Binding Rab5a and Rab7b Molecules

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