Construction and expression analysis of recombinant eukaryotic expression plasmids of CD8α and CD207 genes in Ctenopharyngodon idella

Dendritic cells are the outposts of the immune system, which link innate immunity with adaptive immunity, and thus are considered as the most powerful antigen-presenting cells in vivo. To further investigate the functions of CD8α and CD207 in grass carp (Ctenopharyngodon idella) dendritic cells (DCs), the recombinant eukaryotic expression plasmids of CD8α and CD207 genes were constructed and successfully expressed in DCs of C. idella. The open reading frames (ORFs) of CD8α and CD207 were obtained from DCs of C. idella by RT-PCR and inserted into recombinant eukaryotic expression plasmid pcDNA3.1, respectively, to construct recombinant eukaryotic expression plasmids pcDNA3.1-CD8α and pcDNA3.1-CD207; after the sequencing was confirmed to be correct, the recombinant eukaryotic expression plasmids were transfected into DCs of C. idella using liposome method, and the expressions of CD8α and CD207 were verified by cell immunofluorescence technology and Western blot. The results showed that in the Western blot assay, the CD8α and CD207 proteins were expressed normally and significantly overexpressed after transfection of the recombinant eukaryotic expression plasmid, and the size was consistent with that of the predicted result. Cellular immunofluorescence showed that the CD8α and CD207 proteins were mainly expressed on the cell membrane of DCs. Hence, DCs of C. idella exhibit immunophenotypes and functions that are conserved in their mammalian counterparts. The recombinant eukaryotic expression plasmids of pcDNA3.1-CD8α and pcDNA3.1-CD207 were successfully constructed, which laid a foundation for further research on the action mechanism of CD8α and CD207 genes in DCs. Carrying out studies on the immunophenotype of DCs will play a positive role in analyzing the DCs-mediated immune regulation mechanism of C. idella. In the cultivation process, C. idella are easily infected by pathogens and induce inflammatory reactions. The development of DCs-based immunotherapy can provide new ideas for the prevention and control of C. idella related diseases, which has a wide application prospect in reducing diseases and enhancing the immune efficacy of vaccines for teleost fish.