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Investigation On The Mechanisms Of Tight Junction Protein Occludin Affect Bovine Viral Diarrhea Virus Infection

Posted on:2021-08-19Degree:MasterType:Thesis
Country:ChinaCandidate:X Y HuFull Text:PDF
GTID:2480306602979489Subject:Veterinary science
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Bovine viral diarrhea virus(BVDV)is a contagious disease of cattle,sheep,pigs,deer and other animals.BVDV infection can cause high fever,diarrhea,leucopenia,immunosuppression,mucosal congestion,reproductive dysfunction and other clinical symptoms in infected animals,and can also cause the decrease of milk production,abortion of pregnant cattle,stillbirth and persistent infection of cattle.Seriously,BVDV infection causes diarrhea/mucosal disease(BVD/MD)in young animals,with a mortality rate of 100%.In addition,BVDV is also a common source of pollution for bovine biological products(serum,frozen semen,embryo,vaccine).Occludin,or OCLN for short,is one of the most representative proteins in tight junction(TJ),which directly participates in the barrier function and barrier function of TJS.A large number of studies have reported that OCLN is an important host factor for hepatitis C virus(HCV)to infect target cells.It plays a role of virus receptor,mediates HCV to adsorb target cells,and thus starts virus replication.However,BVDV and HCV belong to Flaviviridae,and their genome structure and biological characteristics are similar,but the mechanism of BVDV and TJ has not been reported.Therefore,this study explored whether OCLN played a key role in BVDV infection?What are the key steps of OCLN in BVDV infection?What is the mechanism?The details are as follows:1.Expression of OCLN gene in MDBK cells infected by BVDVThe results showed that the mRNA and protein levels of OCLN increased significantly 36 h after BVDV infection of MDBK cells,and compared with the differential gene expression profile of BVDV infection target cells established by transcriptome sequencing RNA-Seq in previous studies,BVDV infection of target cells resulted in a significant up regulation of OCLN gene mRNA levels.2.The effect of RNA interference on the expression of OCLN on BVDV replicationThe results showed that small interference RNA(siRNA)targeting OCLN gene significantly reduced the mRNA and protein levels of OCLN 48 h after transfection;qRT-PCR was used to detect the replication level of BVDV in OCLN knockdown cells infected by BVDV,and the untranslated 5 'region of BVDV was found The content of region,UTR mRNA decreased significantly;and the double strand RNA of OCLN knockdown cells infected by BVDV was detected by immunofluorescence staining compared with the negative control group,the content of red fluorescent labeled dsRNA in OCLN knockdown cells was significantly reduced;the detection of virus titer showed that OCLN knockdown significantly affected the formation and release of BVDV particles in offspring and reduced the virus titer.3.CRISPR/Cas9 knockout of OCLN gene and its effect on BVDV replicationWestern Blot analysis of CRISPR/Cas9 showed that the protein expression level of OCLN was significantly reduced after knocking out OCLN;gene sequencing showed that the knock out efficiency of OCLN was up to 73.9%;qRT-PCR was used to detect the replication in OCLN knockout cells infected with BVDV compared with the control group,it was found that the content of 5'UTR mRNA in BVDV decreased significantly;the content of red fluorescent labeled BVDV dsRNA in OCLN knockout cells was significantly reduced;12,24 and 48 h after OCLN knockout cells were infected with BVDV;the titer of virus decreased significantly;CPE caused by BVDV infected with OCLN knockout cells decreased significantly;BVDV binding and internalization efficiency decreased significantly after OCLN knockout through BVDV binding and internalization test.4.Mechanism of tight junction protein OCLN inhibiting BVDV replicationStrep-tag(?) system detection found that OCLN bound to BVDV E2 protein,but failed to bind to Erns and El protein;Western Blot test found OCLN-TurboID stable expression cell line with high OCLN expression;Western Blot test found the optimal concentration of biotin It was 50 ?g/mL,and the content of biotinylated protein was the highest when treated for 18 h.bioinformatics analysis showed that GO functional enrichment mainly included actin filament-based process,cell junction organization and protein localization to plasma membrane.KEGG analysis found a large number of signaling pathways related to endocytosis,in which EGFR,APs,Clathrin,Dynamin,Rab5 and EHD3 proteins constitute a protein-protein interaction network.This study found that BVDV infection of MDBK cells caused significant upregulation of OCLN genes and proteins,which was consistent with the results of differential transcriptome expression profiles of BVDV infection target cells constructed by previous RNA-Seq;OCLN gene knockdown and knockout significantly prevented BVDV mRNA and dsRNA content accumulation,reduced BVDV titer,and weaken BVDV infection CPE;at the same time,after OCLN knockout,it significantly prevented BVDV binding and internalization and other infection processes;OCLN as an important cytokine,interacted with BVDV envelope protein E2 and mediates BVDV Infection;using the adjacent protein biotin labeling technology TurboID and the biotin-avidin system to screen for proteins that interact with OCLN.It was found that proteins that interact with OCLN were mainly enriched in pathways such as endocytosis,which is mediated by OCLN for subsequent research.The molecular mechanism of BVDV infection provideed a theoretical basis.
Keywords/Search Tags:bovine viral diarrhea virus, OCLN, RNAi, CRISPR/Cas9, envelope protein, TurboID
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