Molecular characterization of Rab5A, and involvement in innate immunity in Yellow River Carp Cyprinus carpio

Rab5A play important roles in regulating trafficking organelles, especially in phagosome formation. In the present study, full-length cDNA sequences of Rab5A were cloned from Yellow River Carp Cyprinus carpio, which was designated as CcRab5A. The full-length cDNA of the CcRab5A cDNA sequence is 2434 bp and included an open reading frame (ORF) encoding 216 amino acids polypeptide with an estimated molecular weight of 23.47 kDa. Bioinformatics analysis showed that the CcRab5A protein was highly conserved during evolution. CcRab5A's deduced amino acid sequence showed high identity to Cyprinus carpio (99.54%) in comparison. The guanine-base binding motif (G), phosphate/magnesium-binding motif (PM), and Rab family motif (Rab F) of CcRab5A are highly conserved among various species, but the N- and C-terminal regions were hypervariable, according to the results of multiple sequence alignment and phylogenetic analysis. Additionally, 11 tissues of Yellow River Carp were examined using Real-time Fluorescence Quantitative PCR (qRT-PCR) to determine the expression levels, with the highest expression levels in head kidney and blood. Followed by heart, liver, muscle, brain, gill, skin, spleen and intestine; The expression level in body kidney was the lowest. Yellow River Carp was immunized with Aeromonas hydrophila and Spring viremia of carp virus (SVCV) respectively, and the expression changes of CcRab5A in gill, spleen, liver, intestine and skin of Yellow River Carp were detected. The results showed that the expression level of the gene was obviously up-regulated at different time points. The eukaryotic recombinant plasmids of CcRab5A, pEGFP-N3 were constructed and transfected into GCO cells for subcellular localization. The results showed that CcRab5A were mainly distributed in nuclear membrane and various endosome membranes. These results showed that CcRab5 were involved in viral and bacterial infection in the immune response of Yellow River Carp.