Proteomics Analysis Of Channa Argus Spleen Infected With Nocardia Seriolae And Study On Immune Effect Of NCCRP1

Channa argus is an important economic fish for aquaculture in China.Nocardia seriolae is a class of Gram-positive and parthenogenic intracellular parasitic bacterium that are widely found in the aquatic environment and can infect a variety of farmed fish,including C.argus,which can result in seriously affecting the healthy development of C.argus aquaculture.In this study,we analyzed the histopathological changes of C.argus infected by N.seriolae,screened and identified immune-related genes in C.argus in response to N.seriolae infection based on proteomics and bioinformatics analysis techniques,explored the immune-related pathways after infection,and performed recombinant protein expression in prokaryotic form for the differentially expressed gene nonspecific cytotoxic cell receptor(NCCRP1)and prepared polyclonal antibodies to label nonspecific cytotoxic cell(NCC)to identify the NCC cell population in C.argus.The main studies and results were as follows:(1)The analysis of the pathological sections showed that hepatocytes appear vacuolated,with consolidation and disappearance of intrahepatic nuclei and a high level of sinusoidal stasis after 96 h of infection with N.seriolae,and kidney tissues with scattered tubular arrangement and the cells were necrotic and exfoliative;the spleen cells in the spleen tissues were reduced,and there were large nodules,the boundary of the nodules is clear,and there were a large number of necrotic cells in the nodules.N.seriolae caused varying degrees of acute damage to the histocytes of the C.argus,of which nodules were present in the spleen,showed more severe damage.(2)Proteome sequencing of C.argus spleen tissue 96 h after N.seriolae infection.The results showed that a total of 70,671 precursors,63,305 peptides,7,688 protein groups and7,825 proteins were identified.For protein annotation,GO annotation showed that 7,253 proteins were annotated in biological processes,cellular components and molecular functions;KOG annotation showed that a total of 6,252 proteins were annotated in 25 subcategories including translation,ribosomal structure and biogenesis,RNA processing and modification;KEGG annotated 3,691 proteins in six major categories,of which 619 proteins were annotated by the immune system,ranked first in the organismal systems.A total of 700 differentially expressed proteins(DEPs)were obtained,among them 353 were up-regulated and 347 were down-regulated after the infection.The DEPs were mainly concentrated in some important immune signal pathways such as complement and coagulation cascades,chemokine signaling pathway,natural killer cell mediated cytotoxicity,protein network interaction analysis further identifies some proteins that may play an important role in the resistance of C.argus to N.seriolae infection,The combined analysis of proteomic and transcriptomic data revealed that92.9% of the differentially expressed proteins showed a positive correlation with the protein levels and m RNA levels of the differentially expressed genes,including NCCRP1,STAT3,TFRC,KRT18,etc.(3)In this study,the CDS region of NCCRP1 was cloned,the expression plasmid was constructed and expressed in prokaryotic form,and the recombinant protein of NCCRP1 was purified for polyclonal antibody preparation.The expression patterns of NCCRP1 at the m RNA and protein levels were analyzed using q RT-PCR and Western-blot techniques,and the results showed that NCCRP1 was mainly expressed in the liver,spleen and kidney tissues of the snakehead.After N.seriolae infection,both m RNA and protein levels of NCCRP1 were upregulated,with sustained up-regulation in spleen tissues,rapid up-regulation of m RNA levels in kidney and liver tissues at 1 and 2 d after infection,and up-regulation of protein levels at 4d after infection.(4)Based on the verification of the specificity of the antibodies,the anti-NCCRP1 antibody was able to identify NCC by blood,spleen tissue and flow cytometry,and the cell morphology analysis showed that the NCC of the C.argus was a class of neutrophil population.In summary,N.seriolae infection in C.argus can lead to spleen,liver and kidney tissues damage.N.seriolae activate or inhibit the differential expression of a large number of immunerelated proteins,anti-NCCRP1 antibody can be used to identify NCC,identified NCC as a class of neutrophils,explored their proliferation pattern after infection,and preliminarily demonstrated that NCCRP1 played an important role in the resistance of C.argus to N.seriolae infection,which laid the foundation for the in-depth study of the molecular mechanism of resistance of C.argus to N.seriolae infection.