S-layer Protein Of Lactobacillus Acidophilus Antagonizes H9N2 Avian Influenza Virus Invading Dendritic Cells

Avian influenza(AI)is caused by avian influenza virus(AIV),which is a kind of poultry infectious diseases.It was divided into Category A disease by the International Bureau of OIE.The virus also can infect human and cause death.So that avian influenza virus has become a serious threat to healthy development of China's poultry industry and human safety.It has been shown that avian influenza virus can combine with DC-SIGN to promote viral replication in human dendritic cells,further studies have found that S-layer protein of Lactobacillus also can combine with DC-SIGN receptor,regulating dendritic cells and preventing the virus infect cells which expressing DC-SIGN.In order to investigate whether S-layer protein can bind to DC-SIGN and block the invasion of dendritic cells in the mucosal region,we constructed recombinant E.coli expressing S-layer protein of L.acidophilus ATCC 4356,and the recombinant S-layer protein was extracted and purified.The biological activity of S-layer protein and its stimulating effect on dendritic cells were explored.In addition,the invasion of dendritic cells by H9N2 avian influenza virus was detected by flow cytometry and qRT-PCR,and whether the virus was able to replicate effectively in dendritic cells and cause dendritic cell responses were detected.Finally,the antagonistic effect of S-layer protein on the invasion of dendritic cells of H9N2 avian influenza virus and the response of cells to the antagonism were investigated.The contents of this study are divided into the following four parts:1?The expression and extraction of S-layer proteinIn this study,the recombinant plasmid pGEX-4t-2-S was constructed by amplifying the S-layer protein gene with the genome of L.acidophilus ATCC 4356 as template.The recombinant plasmid pGEX-4t-2-S was transformed into E.coli Rosetta-gami2(DE3).Recombinant S-layer protein was purified by affinity chromatography.The results of enzymatic digestion showed that the pGEX-4t-2-S plasmid was successfully constructed and the sequencing results were correct.The results of gel electrophoresis showed that the constructed plasmid was successfully transferred into E.coli Rosetta-gami2(DE3)and successfully expressed.The results of gel electrophoresis showed that there was a single band at 70 KD.The results of grayscale analysis showed that the recombinant S-layer protein was obtained by affinity chromatography with purity of 95%and 11.9 mg/1111CFU.2?S-Iayer protein activates dendritic cellsIn this study,we examined the effect of S-layer protein on dendritic cells.The cultured dendritic cells were treated with S-layer protein,and the cells were harvested 24 hours post infection.The expression level of the costimulatory molecules on the cell surface was detected by flow cytometry.In addition,RNA was extracted from the collected cells to detect the effects of S-layer protein treatment on the secretion of antiinflammatory cytokines IL-10 and proinflammatory cytokines TNF-a in dendritic cells.The results showed that the expression of CD86 and CD80 on the surface of dendritic cells was significantly up-regulated by S-layer protein,and the expression of anti-inflammatory factor IL-10 was also been promoted.Therefore,we can conclude that S-layer protein can stimulate the expression of costimulatory molecules on the surface of dendritic cells,and also induce dendritic cells to secrete anti-inflammatory factor IL-10 and promote the maturation of dendritic cells.3?H9N2 virus invade into dendritic cellsIn this study,H9N2 virus was used to infect dendritic cells.Virus infection for 1 h,flow cytometry and immunofluorescence technique were used to detect whether the virus can infect dendritic cells.The relative level of HA and NA genes of H9N2 avian influenza virus were detected by qRT-PCR at 1 h,6 h,12h and 24 h after infection.The content of NP protein in 1 h and 24 h was observed by confocal microscopy in dendritic cells.The levels of Mx1,Isgl5 and Ddx58,and the levels of inflammatory cytokines IL-10 and TNF-a were detected by qRT-PCR after 24 hours of viral infection.Experimental results show that H9N2 virus can infect dendritic cells,and can replicate effectively in dendritic cells.Post 24 h of virus infection,the expression level of costimulatory molecules on the cell surface of the virus infection group was significantly up-regulated compared with the control group.In addition,the levels of ISGs Mxl,Isgl5 and Ddx58 were also increased with the replication of the virus in the cells,and the levels of inflammatory cytokines IL-10 and TNF-a secreted by dendritic cells also increased.All of these results show that H9N2 virus can infect mouse dendritic cells and replicate effectively in the cells,following a strong response of the dendritic cells.4?S-layer protein inhibit H9N2 virus invade into dendritic cellsIn the virus antagonistic experiment,dendritic cells was treated with S-layer protein before infected with H9N2 virus.The level of H9N2 virus in dendritic cells was detected by flow cytometry and qRT-PCR.The relative level of H9N2 virus was detected by qRT-PCR at different time points post infection.The NP protein of H9N2 virus was observed 24 h after virus infection.Flow cytometry was used to detect changes in cell phenotype during antagonism.qRT-PCR was used to detect the levels of HA,NA and ISGs Mxl,Isg15 and Ddx5 8,and the relative levels of inflammatory cytokines IL-10 and TNF-?.All of the results showed that S-layer protein could antagonize the invasion of dendritic cells by H9N2 virus within 24 h of virus infection,and the antagonistic effect was gradient-dependent.In addition,the treatment of S-layer protein on dendritic cells before infection of H9N2 virus can stimulate the expression of dendritic cell phenotype,improve the level of interferon-stimulating gene,which are conducive to cell antagonism of the virus.Compared with the single virus group,the level of anti-inflammatory factor IL-10 mRNA was higher in the S-layer protein treatment group,while the trend of proinflammatory cytokine TNF-a was opposite.This is conducive to reducing cell inflammatory response,prevent the virus from escaping.