| The gibel carp has a very important position in freshwater fisheries because of its good economic traits.However,in recent years,sputum hemorrhagic disease caused by infection with herpes simplex virus type ?(CyHV-2)has seriously threatened the breeding industry of gibel carp.And often occur at 15-25?,more than 25? or below 15? often can be poisoned to survive.To carry out research on the pathogenesis of hemorrhagic disease of silver carp,and to find out the drugs or methods that can effectively inhibit the proliferation of the virus,can effectively alleviate the harm caused by sputum hemorrhage.In this paper,the transcriptome sequencing of healthy gibel carp and the cytotoxic silver carp infected with CyHV-2 was carried out.The bioinformatics method was used to analyze the function of the obtained Unigene,and the metabolic pathways were analyzed.Comparative Study.Based on the identification of a series of immune-related genes,the full-length cDNA of Prx gene of gibel carp was cloned by PCR and RACE.The Prx gene was artificially infected with CyHV-2.The expression of sputum in vivo;at the same time,through the manual intervention of HSP70 promoter and inhibitor in the gibel carp,combined with CyHV-2 artificial infection,the body's physiological and biochemical level was detected,and the virus was detected under different artificial interventions.Proliferative copy number;Finally,through the artificial intervention of the vaccination against gibel carp,the serum biochemical indicators,serum and tissue immune indexes and immunoglobulin light chain genes and weight of the gibel vaccination group and the control group were detected.The amount of expression of the chain gene.Through the above research,it is necessary to trace the cause of hemorrhagic disease of gibel carp,find out the method that can artificially inhibit the proliferation of virus,and evaluate the effectiveness of the implementation of the soil vaccine.The results showed that transcriptome sequencing was performed on healthy gibel carp and inoculated intestinal mucosa infected with CyHV-2.According to bioinformatics analysis,511,179 transcripts were obtained,and 314,941 Unigenes,including 139,178 Unigene was successfully annotated.The results of KEGG annotation indicated that there were 2464,1098,788 and 443 Unigene involved in the disease infection pathway,cancer pathway,signal transduction pathway and immune system pathway,respectively.Compared with healthy gibel carp,there were 7770 genes in the intestinal mucosal transcriptome showing significant differential expression,of which 4435 genes were down-regulated and 3335 genes were up-regulated.Among them,1431,388 and 2618 differentially expressed genes were aligned into biological processes(BP),cellular components(CC)and molecular function(MF)databases.A total of 8727 differentially expressed genes were sequenced in the KEGG database,and 309 pathways were significantly enriched,mainly for the small RNA pathway associated with cancer,the inflammatory mediator signaling pathway of the ITRP pathway,and oxidative phosphorylation pathways.After analyzing the function of gene annotation,it was found that there were 28 significant up-regulations in genes related to immune function,and 20 were significantly down-regulated.Increased expression of heat shock protein 70,peroxide reductase,interleukin-6 receptor ? subunit,uridine diphosphate glucuronyltransferase and interleukin-6 after infection with CyHV-2 The expression levels of extracellular superoxide dismutase,copper-zinc superoxide dismutase and glutathione S-transferase decreased.Next,the full-length cDNA of the peroxidase reductase(Prx)gene was cloned from the liver of the gibel carp by RT-PCR and rapid amplification of cDNA ends(RACE).The cDNA of peroxidase(CaPrx)gene of gibel carp is 1 035 bp in length and 594 bp in open reading frame,encoding 197 amino acids.There is a conserved Cys residue at the N-terminus and C-terminus of the amino acid sequence,belonging to the 2-cysteine-like Prxs family.The multi-sequence alignment and phylogenetic tree analysis showed that the Prx gene of gibel carp has high homology with fish such as carp,blue carp,zebrafish rhinoceros carp and carp.The results of real-time PCR showed that CaPrx was widely expressed in gibel carp,and the expression level was the highest in blood cells and liver,and less in sputum and head kidney.In individuals infected with CyHV-2,the expression of CaPrx gene in liver and spleen decreased significantly,indicating that its antioxidant effect was partially damaged by viral infection.In view of the temperature-related morbidity characteristics of sputum hemorrhagic disease,artificial intervention of heat shock proteins in vivo was carried out on HSP70 inhibitors and promoters.The study found that the expression of HSP70 gene in the liver of the gibel carp with the typical symptoms of sputum hemorrhage in the culture pond was significantly higher than that of the healthy gibel carp(P<0.01),artificial infection of CyHV-2.After the virus,the expression of HSP70 in the liver increased significantly after 24h(P<0.05).When the fish showed symptoms,the expression of HSP70 increased significantly.In the control group,after artificial infection with CyHV-2,serum aspartate aminotransferase was significantly increased in the pathogenesis of fish(P<0.05),and there was a significant difference between the injection and the injection accelerator and inhibitor group.In the HSP70 promoter group,the serum aspartate aminotransferase and alanine aminotransferase were significantly increased at 12 o'clock after artificial infection of CyHV-2,while alkaline phosphatase and albumin did not change significantly.The number of viruses in the gibel carp with HSP70 inhibitor reached the highest point 12 h after infection,which was significantly higher than that of the control group and the HSP70 promoter group(P<0.01).In the control group and the HSP70 promoter group,there was no significant difference in the proliferation trend and the number of viruses.Finally,this paper evaluates the physiological response of the gibel carp and the expression of immunoglobulin heavy and light chain genes in vivo in the production of girap injection of gibel carp.Through the comparative study of the vaccination group and the control group,the immune effects of vaccination were identified from the aspects of growth index,serum biochemical index and immune index.The catalase(CAT),total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH)in the serum and liver homogenate of the vaccinated group and the control group were determined.-PX),total antioxidant capacity(T-AOC)and lysozyme(LZM),the results showed that the T-SOD in the serum and liver of the vaccinated group was higher than that of the control group(P<0.05).The T-AOC in the serum and liver of the vaccination group was higher than that of the control group,and there was significant difference in serum(P<0.05).In the vaccination group,the immunoglobulin light chain gene Ig? was not significantly changed in the intestinal mucosa,but was higher in the liver,spleen and head kidney than in the control group,and there was a significant difference in the liver(P<0.01).).In the vaccination group,the expression level of the immunoglobulin heavy chain gene Ighm of the gibel carp was higher in the liver and head kidney than in the control group,but there was no significant difference.The albumin content in the serum of the vaccination group was significantly higher than that of the control group(P<0.05).The fatness,liver index,liver index and liver index of the vaccine group were significantly higher than those of the control group(P<0.01)The results of this study showed that the infection of CyHV-2 in gibel carp can significantly differentially express the intestinal mucosa gene;when the heat shock protein in the gibel carp is artificially inhibited,the resistance of the fish to CyHV-2 virus is greatly reduced.HSP70 has a positive effect in resisting the proliferation of viruses. |
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