| Crucian carp is a common freshwater fish,is one of the important economic fish in China.Cyprinid Herpesvirus 2(Cy HV-2)belongs to double-stranded DNA virus,which mainly causes Herpes Viral Haematopoietic Necrosis(HVHN).The mortality rate after infection was as high as 90-100%,which brought serious economic losses to the crucian carp breeding industry.There is a special protein in Cy HV-2,called thymidine kinase(TK),which corresponds to the ORF55 gene.Previous studies have proved that TK gene was the main virulence gene in Cy HV-2.By interfering with TK gene,it could significantly reduce the titer of Cy HV-2 and have an impact on the virulence of the virus.However,the biological function of TK gene in Cy HV-2 is still unknown.Therefore,this study mainly focuses on the TK proteins and its interacting peptides and proteins.The main results are as follows:1 Phage display technology was used to screen TK protein-interacting polypeptides.At the same time,the preparation of TK protein polyclonal antibody was completed.In order to investigate the biological function played by the TK gene in the process of Cy HV-2 infection,we cloned TK gene into p MAL-c5 X prokaryotic expression vector.After TK protein was obtained by purification,a random phage 12 peptide library was used to screen TK protein-interacting polypeptides.With three rounds of panning,the phage was significantly enriched.The amplified phage was coated on the LB plate containing tetracycline,fifty plaques were randomly selected for sequencing.The results showed that 38 polypeptides were identical,and the sequence was LSPGANSHVSRH.In addition,the purified recombinant TK protein was used to obtain polyclonal antibody serum,with a titer of 1:512K,laying a foundation for further research on the mechanism of TK.2 The interaction between TK protein and polypeptide was confirmed by Dot Blot overlay experiment and bioinformatics analysis.TK protein-interacting proteins were identified using subcellular colocalization and immunoprecipitation.In order to explore the function of TK protein,the interaction betweem TK protein and polypeptide were first confirmed by Dot-Blot overlay experiments,and the specific interaction sites were predicted by bioinformatics analysis.Four potential interacting proteins were further predicted by NCBI: gastrula zinc finger protein Xl CGF8.2DB-like,WD repeat-containing protein 7 isoform X1,actin-binding Rho-activating protein and zinc finger protein 516-like Isoform X1.The relative expression of these four genes was detected after the virus infected cells,and two of them were screened for subsequent interaction identification.The results of subcellular co-location showed that TK protein and two potential interaction proteins were co-located in the nucleus,while the immune co-precipitation test identified that TK protein and actin-binding Rho-activating protein had interaction,which provided theoretical basis for further study of the function of TK gene.3 TK protein-interacting polypeptides were added to the Cy HV-2 infected cells,and it was found that this polypeptide had a significant promoting effect on virus-infected cells.Transcriptome analysis identified 59 up-regulated genes and 202down-regulated genes between the virus group and the virus plus polypeptide group.In the KEGG enrichment analysis,Regulation of action cycloskeleton and Focal adhesion pathways were chosen,and eight genes were verified by RT-q PCR.The results showed that the validation results were consistent with the sequencing results.In order to reveal the effect of TK protein-interacting polypeptide on virus infection,the effect of interaction peptides on virus infection was observed by adding peptides into the cells infected with the virus.The results showed that when the concentration of polypeptides was greater than 0.1 mg/m L,it could significantly promote the replication of the virus in host cells.In view of the above phenomenon,the transcriptome sequencing technology was used to analyze the cells infected with virus and the cells infected with virus plus polypeptide.This transcriptome analysis identified261 differentially expressed genes(DEGs).The enrichment analysis of KEGG pathway showed that 181 DEGs were annotated into 73 KEGG pathways.Through KEGG enrichment analysis,Regulation of actin cytoskeleton and Focal adhesion pathway were screened,and RT-q PCR validation of 8 DEGs showed that the expression of all genes was significantly down-regulated,which was consistent with the results of sequencing.Through preliminary analysis of this signal pathway,it can be seen that during the process of virus infected cells,the Rho A gene、MLC gene and other eight genes were all significantly down-regulated,which might affect the cell proliferation,mechanical stretch,cell adhesion and other aspects,thus accelerating cell apoptosis.To sum up,this study successfully screened an interactive polypeptide of TK protein,and this polypeptide could significantly promote the replication of virus in host cells.This process would significantly reduce the Focal adhesion pathway,and its specific mechanism remained further exploration.Meanwhile,four potential interacting proteins were successfully predicted by bioinformatics analysis,and one of the actin-binding Rho-activating protein could interact with TK protein,which laid a foundation for further exploring the biological function of the TK gene in the process of viral infection. |
