Study On The Mechanism Of Anti-TGEV Infection Of Lactobacillus Plantarum Supernatant Based On JAK-STAT1 Signaling Pathway

Transmissible gastroenteritis（TGE）is an acute,highly contagious infection caused by severe diarrhea,vomiting and dehydration caused by Transmissible gastroenteritis virus（TGEV）.Sexual disease.It has a very high mortality rate for piglets within two weeks of age,which brings great harm to the pig industry.The existing TGEV vaccine does not produce the desired control effect,so it is particularly important to control porcine transmissible gastroenteritis with new methods.As a ubiquitous strain in the intestinal tract of animals,probiotics have various functions such as promoting intestinal peristalsis and maintaining intestinal homeostasis.At present,studies have shown that a variety of probiotics can exert antiviral effects.In the previous stage,our group successfully isolated and identified a Lactobacillus plantarum,which was named LP-1.It was found that ST cells can significantly inhibit TGEV infection after treatment with the supernatant（LP-1S）.However,whether LP-1S can act on IPEC-J2 cells and inhibit the proliferation of TGEV in this cell has not been reported in the literature.This experiment explores whether LP-1S can inhibit the replication of TGEV in this cell by inhibiting the effect of IGEV on intracellular replication of TGEV,and further detecting the infection of TGEV cells at different time points after treatment of LP-1S-treated IPEC-J2 cells.Induction of IFN-βlevel,intracellular STAT1 phosphorylation,p-STAT1 nuclear translocation and the transcription and protein expression of some ISGs,preliminary clarification of LP-1S exerts anti-TGEV by affecting JAK-STAT1 signaling pathway in IPEC-J2 cells.The mechanism of action.The main research contents are as follows:1、Replication of TGEV in IPEC-J2 cells after treatment with Lactobacillus plantarum Supernatant（1）MTT assay showed that 1/4-fold dilution of LP-1S was the largest non-toxic dose of IPEC-J2 cells;using Wstern-blot method,it was found that LP-1S was concentration-dependent in anti-TGEV effect in IPEC-J2 cells.Sex,the optimal concentration dose is 1/4 fold dilution.（2）1/4 fold of LP-1S was injected into IPEC-J2 cells and infected with TGEV GFP Miller（MOI=0.1）strain.The fluorescence intensity of TGEV GFP Miller strain was observed by inverted fluorescence microscopy at different time points,and LP-1S was found.The number of fluorescence in the pretreatment group was significantly lower than that in the untreated group.The results of TCID₅₀ showed that there was a significant difference between the 24h and 48h LP-1S treatment groups compared with the simultaneous TGEV challenge group（P<0.01）.Absolute fluorescence quantification and Western-blot were used to detect the viral copy number and N protein expression of TGEV Miller N gene in IPEC-J2 cells,respectively.It was found that the12h LP-1S pretreatment group had significant difference in N gene copy number compared with TGEV challenge group.（0.01<P<0.05）,there was no significant difference in the expression of N protein（0.05<P）.There were significant differences in N gene copy number and protein expression between the two groups at 24h and 48h（P<0.01）.2、The IFN induction,STAT1 phosphorylation level and nuclear translocation condition in IPEC-J2 cells after treatment with Lactobacillus plantarum Supernatant.The IPEC-J2 cells were divided into three groups:the Lactobacillus plantarum supernatant pretreatment,the challenge group and the blank control group were collected at different time points,and the cell supernatant was collected by IFN-βELISA reagent.The levels of IFN-βshowed that the production of IFN-βwater in IPEC-J2 cells induced by Lactobacillus plantarum pretreatment group was significantly different（0.01<P<0.05）.The phosphorylation level of STAT1 protein in three groups of different time points was detected by Western-blot technique.The STAT1phosphorylation level in the late TGEV（24h-48h）was significantly different from that in the TGEV-infected group（P<0.01）.Indirect IIFA showed that the nuclear translocation level of p-STAT1 was significantly different after TGEV infection in the12h LP-1S treatment group（0.01<P<0.05）,and the TGEV group was infected in the LP-1S treatment group at 24h and 48h.There was a significant difference in the level of p-STAT1 nuclear translocation between the TGEV direct infection group（P<0.01）.3、The Transcriptional level and protein expression of partial ISGs in IPEC-J2cells after treatment with Lactobacillus plantarum SupernatantIPEC-J2 was divided into three groups as described above,and relative transcription was used to quantitatively detect the transcription level of intracellular partial ISGs at different time points.The results showed that after TGEV infection（24h⁴8h）,the transcription level of LP-1S pretreatment group was significantly different from that of TGEV challenge group（0.01<P<0.05）.The transcription levels of Mx1 and Mx2 in the two groups were detected.The highest value was reached at 24h,and the transcription levels of PKR,ZAP,OASL and ISG15 were positively correlated with time and peaked at 48h.The results of Western-blot showed that the expression of PKR protein in the TGEV group was significantly different at 24h and 48h after treatment with 24h LP-1S（P<0.01）.The expression of ZAP protein was compared with TGEV infection group at 24h and 48h.There was a significant difference（0.01<P<0.05）.The protein expression of ZAP and PKR was consistent with the mRNA transcription trend.In addition,there was no significant difference in the p-PKR/PKR ratio between the two groups at different time points（24h,48h）.The ratio of p-PKR/β-Tubulin in the challenge group was significantly higher than that in the TGEV challenge group（0.01<P<0.05）.The ratio of p-PKR/β-Tubulin in the challenge group was significantly higher than that in the challenge group after 48h LP-1S treatment.TGEV challenge group（0.01<P<0.05）.