Identification Of Thrombocytes In Grass Carp (Ctenopharyngodon Idella) And Their Immunomodulatory Functions In Viral And Bacterial Infections

Thrombocytes are an important component of peripheral blood cells that function similarly to mammalian non-nucleated platelets and play an important role in hemostasis and coagulation in the body,as well as a key role in immune regulation.However,because of its nucleated character,it has certain limitations when it comes to isolation.CD41 is one of the biomarkers of thrombocytes to identify grass carp(Ctenopharyngodon idella)thrombocytes,and its m RNA transcript level can indicate the number of thrombocytes.In this study,we prepared a polyclonal antibody to CD41to sort out grass carp thrombocytes and investigate the immunomodulatory role of grass carp thrombocytes in the face of GCRV-II challenge and A.hydrophila infection.The main findings were as follows.1.Preparation and identification of anti-grass carp CD41 polyclonal antibodyIn this study,we first compared the CD41 sequences of several species by multiple sequence alignment and phylogenetic tree,and found that the zebrafish CD41 sequence had high homology with grass carp.Then we compared the grass carp gene library with zebrafish CD41 by means of sequence alignment,identified the CDs region of grass carp CD41,and intercepted a part of it for PCR amplification,the sequence length was1077 bp,and successfully constructed p GEX-4T-1-CD41 recombinant plasmid by enzyme digestion reaction,ligation reaction and transformation,and grass carp(Ctenopharyngodon idella)CD41 protein was expressed in E.coli and purified the inclusion body protein.Subsequently,New Zealand rabbits were immunized by subcutaneous injection of the protein.The antibody potency was 1:12800 by ELISA.Rabbit polyclonal antibody was purified by CNBr coupled antibody method at a concentration of 1.9 mg/m L.Its specificity was identified by SDS-PAGE,western blot,flow cytometry and indirect immunofluorescence assay.The purified antibody was used to screen grass carp thrombocytes with 14.13%of CD41~+cells.2.Identification of thrombocytes morphology and immunomodulatory effects after pathogenic stimulation in grass carpWe found that grass carp thrombocytes were morphologically similar to other fish thrombocytes by Giemsa staining and transmission electron microscopy,with shuttle-shaped or long rod-shaped cells containing granules,microtubule systems,filamentous pseudopods and granules.It was also found that grass carp thrombocytes could express IL-1β,lysozyme and MCH II when their specific genes were verified by RT-PCR.This led us to make a preliminary investigation on the function of grass carp thrombocytes in immunity.Indirect immunofluorescence assays showed that grass carp virus(GCRV-II)could invade grass carp leukocytes but not thrombocytes;m RNA expression of CD41 in thrombocytes was not affected by viral or bacterial challenge in vitro,whereas CD41 transcripts were significantly induced after pathogen infection in vivo,which was caused by a combination of immature hematopoietic stem cells and thrombocytes,suggesting that the organism,upon pathogen stimulation,the number of thrombocytes in vivo increases.Meanwhile the m RNA expression of some representative innate immune genes(IFN1,IL-1β,TNFαand Mx2)was significantly upregulated after GCRV-II challenge.Transcripts of some innate immune genes(IL-6 and TNFα)were rapidly increased after bacterial infection.In summary,this study systematically started from the selection to the cloned gene,and successfully prepared anti-grass carp CD41 polyclonal antibody with high specificity,and confirmed that it could effectively bind to CD41 protein on the surface of grass carp thrombocytes by a series of experimental methods.It was demonstrated by indirect immunofluorescence,viral challenge and bacterial infection experiments that although GCRV-Ⅱcould not invade grass carp thrombocytes,the number of grass carp thrombocytes and the m RNA transcript levels of some representative cytokines were up-regulated when the organism was stimulated by pathogens.This suggests that grass carp thrombocytes play a crucial immunomodulatory role in the pathogen invasion process.