Prokaryotic Expression Of IFIT5 And Its Effect On ALV-J Replication

Avian leukemia is a class of infectious tumor diseases brought about by avian leukemia viruses.J subgroup avian leukemia virus infection will damage the immune organs of chickens,mainly causing immunosuppression in chickens,which in turn causes tumors,which are the most pathogenic and infectious,the global poultry industry has caused huge economic losses.However,to date,there is not any effective vaccine or therapeutic to prevent ALV-J infection,and the pathogenesis of ALV-J and the exact molecular basis for tumorigenesis remain incompletely understood.In response to the failure of ALV-J vaccine development,exploring effective anti-immunosuppressive drugs is critical to preventing and controlling such viruses.The host’s innate immune system is the principal line of defense against the invasion of foreign pathogens,in which interferon-stimulating genes play an important role.Among the many ISGs,IFITs play a major role in regulating the immune response and maintaining immune homeostasis.IFITs are mainly induced to expression by IFNs,viruses and LPS.IFITs are mainly used in viral replication,translation initiation,cell proliferation and migration,and feedback regulation of type I interferons.The IFIT family consists of four principal members:IFIT1,IFIT2,IFIT3 and IFIT5,and birds have only one member,so the chicken IFIT5 gene is selected to further study the antiviral mechanism.To study the effect of in vivo and in vitro infection of ALV-J on IFIT5 expression,we extracted RNA from ALV-J-infected chicken spleen tissue cells and reverse transcription into cDNA,and cloned the chicken IFIT5 gene as a template,with a total CDS region of 1440 bp and encoding 479 amino acids.Bioinformatics analysis showed that it has 7 typical TPR motifs.Evolutionary analysis showed that chicken IFIT5 and turkey IFIT5 have the highest amino acid homology and the furthest relationship with fish.To study the effect of in vivo and in vitro infection of ALV-J on IFIT5 expression,we extracted RNA from ALV-J-J-infected chicken spleen tissue cells and reverse transcription into cDNA,as a template,using PCR to clone the chicken IFIT5 gene,the total length of the CDS region is 1440 bp,encoding 479 amino acids.Bioinformatics analysis showed that it has 7 typical TPR motifs.Evolutionary analysis shows that the highest amino acid homology with chicken IFIT5 is turkey,and the lowest homology is fish.The obtained IFIT5 gene was attached to the p ET-28a vector,the recombinant plasmid p ET-28a-IFIT5 was constructed,dh5αand BL21 were converted sequentially,the strain identified as positive was expanded and induced,the induction conditions were repeatedly optimized,the soluble protein HIS-IFIT5 was prepared,and as an antigen immune BALB/c mouse,polyclonal antibody were prepared,and indirect ELISA detected the potency of multiple antibody,and the results showed that the homemade multi-antibody titer was above 10~4.The specificity of the polyclonal antibody was detected by WB,results showed that the homemade mouse multi-antibody serum was well able to recognize exogenous and endogenous IFIT5 proteins.DF-1 cells were plated,seeded with ALV-J at a density of 80%,and detected with q PCR and WB,and the results showed that ALV-J infection with DF-1 cells could cause IFIT5 transcriptional levels were up regulated,but there is no noticeable change in WB results.Continue to perform infection experiments in vivo for verification,prepare protein samples with ALV-J-infected chicken spleen tissue,and find that ALV-J infection with chicken embryos by WB detection can cause upregulation of IFIT5 expression levels within spleen tissue cells.To study the relationship between chicken IFIT5 and ALV-J replication,we analyzed the dynamic expression of IFIT5 after ALV-J infection.The IFIT5 gene was connected to the pcDNA5-flag vector,and the homemade IFIT5 polyclonal antibody was used as the primary antibody,and the HPR-sheep anti-mouse antibody was the secondary antibody,and the WB was successfully constructed with expressed plasmid.After the DF-1 cells are plated,the density is higher than 85%,and the ALV-J is seeded24 h after the liquid change,these cells are collected at different time points,and the q PCR and WB are used for detection.The results showed that overexpression of IFIT5 can promote the replication of ALV-J in DF-1 cells,if the IFIT5 gene is knocked out and connected to the PX459vector,the DF-1 cells are plated with a density of more than 85%for transfection,the ALV-J is inoculated 24h after the liquid change,the cells are collected at different time points,and the WB detection is performed,and the results show that knockout IFIT5can inhibit the replication of ALV-J in DF-1 cells.When we examined whether the replication level of ALV-J would continue to rise with the increase of IFIT5 dose,we found that the DF-1 cell state was poor and the number of cells decreased after if IFIT5 transfection increased,which may be that IFIT5will affect DF-1 proliferation.After the DF-1 cells were expanded and cultured,three24-well plates were laid at the same time,and the cell proliferation of 6d was determined,and 6 replicates were made in each group,which were verified by MTT experiments,and the results showed that over expression of IFIT5 would indeed significantly inhibit the proliferation of DF-1 cells within 1-5 days.Taking chicken IFIT5 as the research object,this project analyzes its dynamic changes in the ALV-J infection process and its important role in ALV-J replication,and provides a new direction for the development of anti-ALV-J drugs.