The specific mechanisms of interferon regulatory factor 8(IRF8)in porcine intestinal in-nate immunity and resistance to enteric virus infection remain to be elucidated.To investigate the immunoregulatory role of IRF8,establishing an IRF8 gene knockout porcine intestinal epithelial cell(IPEC-J2)monoclonal cell line is of significant importance.This study initially aimed to obtain recombinant adeno-associated virus rAAV-sgIRF8-eGFP capable of knocking out the IRF8 gene through co-transfection of HEK-293T cells with three plasmids.Subsequently,IPEC-J2 cells were infected with the virus,and those expressing eGFP were selected by flow cytometry and cultured to form monoclonal cell lines.These cell lines were then identified by Sanger sequencing and West-ern blot techniques.Lastly,qPCR analysis was used to measure the expression levels of interferon factors IFN-α,IFN-β,IFN-γ and IFN-λ,providing preliminary insights into the impact of IRF8 gene knockout on IPEC-J2 cell immunity.The results demonstrated successful generation of rAAV-sgIRF8-eGFP,which successfully infected IPEC-J2 cells leading to eGFP fluorescence.Flow cytometry followed by cell culture led to the establishment of two monoclonal cell lines,IRF8-KO1 and IRF8-KO3.Sanger sequencing revealed a five-base deletion in IRF8-KO1 and a seven-base dele-tion in IRF8-KO3.Western blot confirmed the absence of IRF8 protein expression in IRF8-KO1,making it an ideal candidate monoclonal cell line.qPCR analysis of interferon factors indicated sig-nificant decrease in IFN-γ(P＜0.05)and IFN-λ(P＜0.01)transcription level in IRF8-knockout cells,while the transcription levels of IFN-α and IFN-β remained relatively unchanged.This study successfully established an IRF8 gene knockout IPEC-J2 monoclonal cell line,providing a founda-tion for further research on IRF8-related porcine intestinal immune regulation and mechanisms of intestinal virus infection.

The specific mechanisms of interferon regulatory factor 8(IRF8)in porcine intestinal in-nate immunity and resistance to enteric virus infection remain to be elucidated.To investigate the immunoregulatory role of IRF8,establishing an IRF8 gene knockout porcine intestinal epithelial cell(IPEC-J2)monoclonal cell line is of significant importance.This study initially aimed to obtain recombinant adeno-associated virus rAAV-sgIRF8-eGFP capable of knocking out the IRF8 gene through co-transfection of HEK-293T cells with three plasmids.Subsequently,IPEC-J2 cells were infected with the virus,and those expressing eGFP were selected by flow cytometry and cultured to form monoclonal cell lines.These cell lines were then identified by Sanger sequencing and West-ern blot techniques.Lastly,qPCR analysis was used to measure the expression levels of interferon factors IFN-α,IFN-β,IFN-γ and IFN-λ,providing preliminary insights into the impact of IRF8 gene knockout on IPEC-J2 cell immunity.The results demonstrated successful generation of rAAV-sgIRF8-eGFP,which successfully infected IPEC-J2 cells leading to eGFP fluorescence.Flow cytometry followed by cell culture led to the establishment of two monoclonal cell lines,IRF8-KO1 and IRF8-KO3.Sanger sequencing revealed a five-base deletion in IRF8-KO1 and a seven-base dele-tion in IRF8-KO3.Western blot confirmed the absence of IRF8 protein expression in IRF8-KO1,making it an ideal candidate monoclonal cell line.qPCR analysis of interferon factors indicated sig-nificant decrease in IFN-γ(P＜0.05)and IFN-λ(P＜0.01)transcription level in IRF8-knockout cells,while the transcription levels of IFN-α and IFN-β remained relatively unchanged.This study successfully established an IRF8 gene knockout IPEC-J2 monoclonal cell line,providing a founda-tion for further research on IRF8-related porcine intestinal immune regulation and mechanisms of intestinal virus infection.

Objective To investigate the effect of dexmedetomidine pretreatment on Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway during myocardial injury induced by liver ischemia-reperfusion (I/R) in rats.Methods Twenty-four healthy adult male SpragueDawley rats,aged 8-10 weeks,weighing 220-250 g,were divided into 3 groups (n=8 each) using a random number table method:sham operation group (group S),liver I/R group (group I/R),and dexmedetomidine pretreatment group (group D).The portal vein,superior and inferior vena cava,subhepatic inferior vena cava and hepatic artery were clamped,and the liver was perfused with 4 ℃ lactated Ringer's solution for 60 min through the portal vein to establish the model of liver cold I/R in anesthetized rats.Dexmedetomidine 100 μg/kg was intraperitoneally injected at 30 min before ischemia in group D.Blood samples were collected at 8 h of reperfusion from the inferior vena cava for determination of serum cardiac troponin Ⅰ (cTnⅠ) and heart-type fatty acid binding protein (H-FABP) concentrations (using the automatic biochemistry analyzer),tumor necrosis factor-alpha (TNF-α) and high-mobility group box 1 protein (HMGB1) concentrations (by enzyme-linked immunosorbent assay).The rats were then sacrificed,andhearts were harvested for examination of histopathological changes (with a light microscope) and for determination of the malondialdehyde (MDA) content (using thiobarbituric acid method) and superoxide dismutase (SOD) activity (by xanthine oxidase method),and expression of phosphorylated STAT1 and STAT3 (p-STAT1,p-STAT3) and phosphorylated JAK2 (p-JAK2) in myocardial tissues (by Western blot).Results Compared with group S,the serum concentrations of cTnI,H-FABP,TNF-α and HMGB1 were significantly increased,the MDA content was increased,the SOD activity was decreased,and the expression of p-JAK2,p-STAT1 and p-STAT3 was up-regulated in I/R and D groups (P＜0.05).Compared with group I/R,the serum concentrations of cTnI,H-FABP,TNF-α and HMGB1 were significantly decreased,the MDA content was decreased,the SOD activity was increased,the expression of pJAK2,p-STAT1 and p-STAT3 was down-regulated (P＜0.05),and pathological changes of myocardium were significantly attenuated in group D.Conclusion The mechanism by which dexmedetomidine pretreatment mitigates myocardial injury induced by liver cold I/R may be related to inhibiting activation of JAK/STAT signaling pathway in rats.

Objective To construct a prokaryotic expression system containing the dense granule protein 4(GRA4) of Toxoplasma gondii,purify the expressed protein and detect its immunogenicity.Methods The specific fragment of GRA4 gene was amplified by PCR.After subcloning the prokaryotic expression recombinant pET,GRA4,the expressed product was purified with His?BindTM affinity chromatography and analyzed by Western blot.BALB/c mice were immunized with the GRA4 recombinant protein,and the antibody IgG titer was detected by ELISA.Results The pET,GRA4 prokaryotic expression system was obtained.The MW of the expressed protein was Mr 40 000 and formed in inclusion body.After purification,the recombinant protein could be specifically recognized by the T.gondii infected rabbit serum.Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.Conclusion The pET,GRA4 recombinant protein was successfully expressed and purified,which shows the immunogenicity.