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Mechanism Of DHCR24 Protein Regulating Bovine Viral Diarrhea Virus Replication

Posted on:2021-01-16Degree:MasterType:Thesis
Country:ChinaCandidate:Y Y MaFull Text:PDF
GTID:2370330602491123Subject:Prevention of Veterinary Medicine
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Bovine viral diarrhea-mucosal disease(BVD-MD)is a febrile fever infectious disease caused by Bovine viral diarrhea virus(BVDV),which is characterized by diarrhea,inflammation,erosion and necrosis in the digestive tract mucosa,and caused infections in respiratory,leading topersistent infections and immunosuppression,influencing the performance of cattle and causing huge economic losses to the cattle industry.Therefore,it is of great significance to study the infection and pathogenic mechanism of BVDV deeply.Transcriptome and proteome integration analysis is a comprehensive analysis of expression regulation mechanism of biological gene.It can realize the complementation and integration of m RNA and protein,and then analyze the stress mechanism in the specific state of the organism comprehensively.To further understand the process of BVDV infection,in this study,we used RNA-seq technology and i TRAQ technology to analysis the changes of transcriptome sequencing and proteomic of MDBK at 48 hours(early infection)after BVDV infection.The results provided a base of excavation of key cytokines and potential regulatory mechanisms that interact with the host at the early stage of BVDV infection.Furthermore,we found that the DHCR24 protein promoted the BVDV replication by the integrated analysis of transcriptome and proteomics data.The specific research content is as follows:Firstly,we used RNA-seq technology to perform transcriptome sequencing of MDBK cells infected with BVDV,and the sequencing data was blasted with the NCBInr / Swiss Prot / Uniprot / IPI reference database.After that,we screened the genes expressed differentially through gene abundance measurement,then the GO(Gene Ontology)function annotation and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis were taken.The results showed that by using | log2 FC |> 1.5 and p-value <0.05 as the criterion,a total of 168 m RNAs including NGF,BCL2A1,BIRC3,m TOR,DHCR24 and other related genes involved in cell cycle,apoptosis,autophagy and lipid metabolism were expressed differently significantly,among them,43 m RNAs were up-regulated significantly and 125 were down-regulated significantly;the differentially expressed m RNAs were analyzed by GO function and KEGG pathway enrichment analysis,and the results showed that these m RNAs were mainly enriched in TNF signal pathway,NLRs signal pathway,NF-?B signal pathway,TLRs signal pathway,MAPK signal pathway,and lipid metabolism signal pathway,etc.Secondly,we used i TRAQ technology to analysis the changes of proteomics sequencing of MDBK cells infected with BVDV,the proteins expressed differentially were screened and analyzed by GO functional annotation and KEGG pathway enrichment analysis.The results showed that by using | log2 FC |> 1.5 and p-value <0.05 as the judgment criteria,a total of 72 proteins expressed differentially significantly were screened out,of which 16 were up-regulated significantly and 56 were down-regulated significantly;the results of GO function Annotations and KEGG pathway enrichment analysis showed that these proteins were significantly enriched in endoplasmic reticulum protein processing,steroid synthesis,endocytosis,metabolism and other pathways;compared the results of transcriptome sequencing and proteomics sequencing,we found that the DHCR24 protein has the same change level of m RNA transcription and protein.Some researchers found that DHCR24 has a variety of biological activities such as anti-apoptosis and regulating cholesterol synthesis,indicating that the DHCR24 plays an important role in the process of BVDV replication at the early stage of BVDV infection.Thirdly,we explored the role of host protein DHCR24 in promoting BVDV infection and its mechanism.We used RT-q PCR and Western blot to verify the m RNA transcription level and protein expression level of the DHCR24 in MDBK cells after BVDV infection,and the results were consistent with the results of RNA-seq and i TRAQ.The results showed that in the early stage of BVDV infection,compared with the uninfected groups,the m RNA Transcription levels and protein levels of DHCR24 up-regulated significantly;the results of overexpression of DHCR24 showed that the overexpression of DHCR24 promoted BVDV replication significantly.The intracellular cholesterol level decreased and the replication capacity of BVDV was inhibited after knocking down the expression level of DHCR24 gene,indicating that BVDV infection induced the DHCR24 to regulate the cholesterol synthesis and then promoted replication of BVDV;moreover,we added Cholesterol to the cell culture system,the replication capacity of BVDV was significantly restored on the MDBK cells which the level of DHCR24 gene were knocked down,,indicating that cholesterol has an important role in the process of BVDV replication.The replication ability of BVDV was significantly restored after the MDBK cells were treated with DHCR24 inhibitor U18666 A,and the replication ability has dose-dependent with the U18666 A.The replication ability of BVDV was significantly restored after adding Cholesterol to the cell cultures.The research results further showed that the cholesterol synthesis pathways regulated by DHCR24 plays an important role in BVDV replication.In summary,this study used RNA-seq technology and i TRAQ technology to obtain the host m RNA expression profile and protein expression profile data in the early stage of BVDV infection,and screened out m RNAs and proteins expressed differently significantly in the host,the results provided a basic data to the exploration of the interaction mechanism at the early stage of between BVDV infection with the host.At the same time,this study clarified the role and mechanism of BVDV in regulating the expression of the DHCR24 to promote the replication,and laid the foundation for further revealing the mechanism of lipid metabolism in BVDV invasion and replication.
Keywords/Search Tags:Bovine viral diarrhea virus, transcriptome, proteome, DHCR24, virus replication
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