| Herpes simplex virus 1(HSV-1)belongs to the family Herpesviridae alpha herpesvirus.It is a DNA virus with complex transcription mechanism and genome structure.It infects more than 80%of the world's population.Previous studies have shown that the virus can reshape the gene expression of the host cells,change the host genes and proteins,cell metabolism,antiviral immunity and other related signal pathways to promote the replication of the virus itself.However,the transcriptome changes caused by HSV-1 and infected cells and their potential biological significance are not clear.The purpose of this study is to detect the transcriptome expression characteristics of human fibroblasts KMB-17 after HSV-1 infection by using the deep sequencing technology,and to screen the differentially expressed genes and long-chain non coding RNA(IncRNA)caused by HSV-1 infection,and to analyze the biological functions of differentially expressed genes,lncRNA and related signal pathways,so as to provide new regulatory genes for further study of the interaction between HSV-1 and cells And IncRNA.The results of transcriptome analysis showed that there were 3885 protein coding genes(2-fold difference and FDR<0.05),including 3885 up-regulated genes and 1377 down regulated genes.Furthermore,go functional annotation and KEGG metabolic pathway analysis showed that the differentially expressed genes were mainly enriched in JAK-STAT signaling pathway,pattern recognition receptor signaling pathway and cytokine cytokine receptor interaction pathway.By integrating differential expression genes and functional enrichment analysis,10 immune related genes were obtained,including IL24,C2CD4A,CXCL5,CXCL2,CSF2,IL6,CCL20,CCL20,CSF3 and ADCY8.The analysis of differential IncRNA expression showed that 891 IncRNAs were significantly differentially expressed after HSV-1 infection,of which 515 were up-regulated and 376 were down regulated.Go functional annotation and KEGG metabolic pathway analysis of differentially expressed IncRNA target genes showed that the target genes of lncRNA were mainly enriched in JAK-STAT signaling pathway and pattern recognition receptor signaling pathway,suggesting that differentially expressed IncRNA mainly played an antiviral role by regulating immune related genes.About 50 million people are infected with dengue virus(DENV)every year in the world,resulting in tens of thousands of deaths.However,there is still a lack of effective vaccines and drugs to prevent and treat DENV infection.Studies have shown that neutralizing antibody has not only preventive effect,but also therapeutic effect on DENV infection.Based on the current situation that there is no effective prevention and treatment method for DENV infection in clinic,we propose:since the broad-spectrum neutralizing antibodies against four types of viruses can not be effectively induced by vaccines,we can deliver the antibody gene through adeno-associated virus(AAV),directly give instructions to the body,and generate a new dengue prevention and treatment strategy for specific broad-spectrum neutralizing antibodies against four types of dengue viruses.Adeno-associated virus(AAV)vector is a powerful technology for delivering monoclonal antibodies,which has no pathogenicity,low immunogenicity,can transduce divided and non divided cells and can stably express foreign genes with little side effects.The purpose of this study is to construct a recombinant adenovirus system with broad-spectrum neutralizing antibody against dengue virus,which can stably express active antibodies,and to determine and optimize their biological activity in vitro,and to evaluate the expression of broad-spectrum monoclonal antibody gene delivered(expressed)by recombinant adenovirus.Firstly,the expression system of DV82.1 plasmid was constructed and transfected into 293 cells to express DV82.1 in vitro and verify whether the antibody can bind to DENV,then the AAV expression system that can stably express DV82.1 was expressed and the expression effect of DV82.1 antibody gene was verified in 293 cells.The results showed that pcDNA3.1-DV82.1 could express the heavy chain and light chain of DV82.1 antibody in 293 cells with a concentration of 0.22ug/ul.The results of ELISA showed that DV82.1 antibody could react specifically with 4 serotypes of dengue virus.Further results showed that the constructed AAV-DV82.1 expression system overexpressed DV82.1 antibody in 293 cells,and the overexpression multiple reached 4.9×105 times,which indicated that AAV-DV82.1 delivery system had been successfully constructed,which laid the foundation for the next step to evaluate the protective effect of AAV-DV82.1 on dengue infection in mice. |
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