Knock-Out Of TLR7 Coding Sequence In DF-1 Cells And Its Differential Analysis Of The Innate Immune Signal Pathway Response To FAdV Infection

Fowl adenovirus(FAdV)is a non-enveloped DNA virus,which is a kind of non-enveloped particle with a diameter of 70-90 nm,and its capsid contains the double-stranded linear DNA molecules,which have the inverted repeat sequences with approximately 100 bp on both ends respectively.Adenovirus infection can probably result in the corresponding symptoms or death when the host is under a stress or an impairment of immunity.Fowl adenovirus serotype-4 posts a predominant situation in poultry industry,and the acute infection is the common characteristic of this disease.The susceptible chickens aged from 3 to 5 weeks,and the short duration of disease and high mortality wrought great losses to the breeding industry.The current circumstance is that there is no effective therapeutic and vaccines toward the assorted diseases caused by fowl adenovirus,and the prevention can merely depend on strengthening the feeding management and the sanitary and anti-epidemic affairs.Denoting the congenital normal physiological defense function of the organism,innate immunity is a significant constituent of the immunologic defense of organism.It can make corresponding immune responses to the invasion of varied pathogenic microorganisms and foreign matters,and it has the function to eliminate pathogens and activate acquired immunity.Innate immunity originated in the early phase of system development and in the preliminary stage of anti-infection responses of the hosts,innate immunity recognizes and eliminates various pathogens in an antigen-non-specific manner,and performs the collective defense mechanism of immune function.Firstly,the key factors of innate immune signal transduction in FAdV infected DF-1 cells were detected,and the genes with obvious changes in transcription level were studied.sgRNA and a suitable CRISPR/Cas9 gene editing system were constructed to knock out the genes involved in the innate immune signal pathway of DF-1 cells.The antagonistic mechanism of innate immune system against FADV infection and the difference of virus proliferation efficiency between pre-modified and post-modified DF-1 cells were studied.1.qRT-PCR was used to detect the transcription level of innate immunity-related genes in DF-1 cells in response to FAdV infection.During the 24-hour infection period,84 expressed genes were measured in 5 periods.Among them,some principal genes of the innate immune reaction and inflammation reaction made responses to FAdV infection and increased significantly in DF-1 cells.2.Finally,six genes including IFNB,TLR7,IL6,IL8,FASLG and STAT4 were selected to construct Cas9 expression vector and sgRNA expression vector,which were co-transfected into DF-1 cells.Single cell clones were sorted and cultured according to the GFP positive characteristics.After PCR verification,four cell pools and four single cell clones were obtained.Virus titer was detected in the specific gene knock-out cells.At the same time,the standard curve of FAdV conservative sequence was constructed.DF-1-TLR7-KO#3 single cell line was selected and named as DF-1-TLR7-KO cell.This cell line showed the best potence of virus proliferation.The titer of progeny FAdV derived from DF-1 cells was 7.7 lgTCID50/mL constantly,and that from DF-1-TLR7-KO cells was 8.50?8.93 lgTCID50/mL.Cell growth of DF-1-TLR7-KO cells was compared with DF-1 cells via CCK-8 kit.It was concluded that there was no significant difference in proliferative ability between DF-1-TLR7-KO cells and DF-1 cells.3.The transcription level of innate immune-related genes was detected by qRT-PCR in DF-1-TLR7-KO cells in response to FAdV infection.The expression of 84 genes in DF-1-TLR7-KO cells were measured at 5 intervals during the first 24-hour infection period.The expression of related genes in DF-1-TLR7-KO cells decreased more than that of the DF-1 cells,indicating that the innate immune response to FAdV infection was weakened in knock-out cells.The TLR7 agonist,Imiquimod,was used to interfere with both DF-1 cells and knock-out cells.Imiquimod treatment inhibited the proliferation of FAdV in DF-1 cells significantly,which indicated that Imiquimod was beneficial to the anti-virus effect in DF-1 cells.Imiquimod treatment had no significant effect on the proliferation of FAdV in DF-1-TLR7-KO cells,and the number of viral copies was much higher than that in DF-1 cells.It might be inferred that knock-out of TLR7 gene is beneficial to virus proliferation.Traditional chicken embryo is easy to proliferate virus,but it needs to consume a large number of specific pathogen free(SPF)chicken embryos.The production cycle is long and the yield is unstable.It is difficult to deal with the outbreak of large-scale epidemic,and it is also difficult to carry out large-scale production,and the sufficient vaccine products cannot be provided during a short period.The purpose of this study was to make cells amplify the virus stably,reduce the use of chicken embryo,reduce the cost,shorten the production cycle,increase the virus titer,and provide good raw materials for vaccine production.In addition,the mechanism of the interaction between FAdV infection and the innate immune system of avian cells was explored in this study,and the specific gene knock-out was successfully tried to enhance the virus proliferation ability in cells.It provides a basis for studying the response mechanism of host to virus invasion,constructing,and screening high-quality avian cells and increasing vaccine production.