The Inhibition And Mechanism Of Prokaryotic Expression Of Porcine IFN-? Against Transmissible Gastroenteritis Virus

Transmissible gastroenteritis(TGE)is a highly contagious swine enteric disease,which caused by Transmissible gastroenteritis virus with high morbidity and mortality.TGEV can infect any pigs of any age,mainly infected with newborn piglets,causing acute diarrhea in piglets,which can cause huge economic losses.Although there are many vaccines for preventing TGEV on the market,but there is currently no effective preparation for the treatment of TGEV infection.This study was conducted by prokaryotic expression of porcine IFN-?(Interferon-?,IFN-?).IFN-? anti-TGEV activity and related mechanisms.IFN-? has the ability to modulate host immune responses,it was originally discovered to have antiviral activity,but currently IFN-? is poorly understood in inhibiting TGEV infection.In this study,the E.coli prokaryotic expression system was used to express and purify IFN-?.The system has the advantages of easy operation,large production volume,high speed and low cost,and is the most widely used expression system at present.In this research,the optimized Fc fragment was cloned into the prokaryotic expression vector pET-21 b to obtain the pET-21b-Fc recombinant vector,and then the IFN-? clone was ligated into the pET-21b-Fc vector,finally the recombinant plasmid pET-21b-was successfully obtained.pET-21b-Fc was transformed into E.coli Shuffle expressing strain,induced to express by conditional exploration,then the supernatant of the cell was purified by Protein A.This study confirmed that IFN-? can inhibit the replication of TGEV in ST cells and IPEC-J2 cells in a dose-dependent manner by qPCR,TCID50,Western-Blot,and indirect immunofluorescence manners.Further exploration of its mechanism revealed that IFN-? significantly up-regulated the expression of porcine interferon regulatory factor 1(poIRF1),and that poIRF1 can affect The IFN-?-mediated signaling pathway was found after knockdown of poIRF1.It can completely eliminate the inhibitory effect of IFN-? on TGEV,and it can be concluded that IFN-? inhibits TGEV replication mainly through the poIRF1 signaling pathway.In addition,we using type I IFN chromosome deletion strain,Vero E6 cells demonstrated that IFN-? direct anti-TGEV activity is IFN-?/?-independent,but IFN-? showed more potent inhibition of TGEV infection in ST cells and IPEC-J2 cells than in Vero E6 cells,whereas Vero E6 was a type I interferon-deficient strain,indicating that type I interferon is in type II interferon Inhibition of TGEV replication plays a role,and detection of IFN-? after treatment of ST cells shows that IFN-? can induce up-regulation of type I interferon,while co-treatment of IFN-? and IFN-? is compatible with TGEV.Inhibition has a very pronounced synergistic effect,suggesting that IFN-? can synergize with type I interferon to induce anti-TGEV replication by inducing type I interferon expression.In this study,we successfully expressed IFN-? by E.coli and found that IFN-? can inhibit the replication of TGEV,and its inhibition is mainly mediated by poIRF1,and IFN-? can induce the expression of type I interferon,with type I interferon.There is a synergistic effect of inhibiting TGEV replication,which lays a foundation for the prevention and treatment of TGEV.