Preliminary Study On The Effect Of TGEV Infection On The Activity Of NHE3

Transmissible gastroenteritis(TGE)is a highly contagious enteric infectious disease caused by transmissible gastroenteritis virus(TGEV),which mainly infects piglets within 2 weeks,Severe diarrhea,vomiting and dehydration are common symptoms in sick piglets,and the death rate can reach 100%.TGEV is an important cause of diarrhea death of newborn piglets in large-scale pig farms,which has brought serious losses to the healthy development of the world pig industry.Because the pathogenesis of TGEV has not been fully explored,the prevention and treatment of TGEV is still difficult.Diarrhea is closely related to the function of Na~+/H~+exchange protein NHE3 in the brush border membrane of intestinal epithelial cells to transport Na~+,In addition,the transmissible gastroenteritis virus mainly infects the small intestine through the digestive tract,and TGEV replicates in a large number of small intestine epithelial cells.At this time,it will destroy the sodium transport function of epithelial cells,resulting in the increase of intestinal osmolality and diarrhea.Our previous study showed that the expression of NHE3 and the activity of NHE3 on the surface of intestinal epithelial cells could be reduced by TGEV infection(IPEC-J2),the absorption of extracellular Na~+by intestinal epithelial cells could be reduced.The results showed that NHE3 may play an important role in the mechanism of diarrhea induced by TGEV.However,whether the results are the same in the real environment of piglets remains to be determined.Under normal physiological conditions,NHE3 circulates between the plasma membrane and the inner body of the cell core,and finally transports to the NHE3 at the top of the plasma membrane to play its biological activity.However,how TGEV regulates the circulation of NHE3 after infecting IPEC-J2 cells has not been explored.Therefore,we first used IPEC-J2 cells as the infection model,and analyzed the differential expression of proteins by analyzing the histone data of TGEV infected group and uninfected group,screening signal molecules regulating the activity of NHE3.The effect of NHE3 activity after TGEV infection was confirmed on Precision-cut intestinal slices and IPEC-J2 cells.Then,the expression of Clathrin was detected to verify whether TGEV infection regulated NHE3 activity through clathrin.The research contents are as follows:1.Proteomic study of IPEC-J2 cells infected by TGEVIn order to understand the effect of TGEV infection on the protein expression of IPEC-J2 cells,the quantitative proteomics of infected and uninfected IPEC-J2 cells were studied by non-standard quantitative technology and quantitative proteomics technology based on mass spectrometry.A total of 5143.0 proteins were identified,of which 3936.0 contained quantitative information.Among the quantitative proteins,559proteins were up-regulated and 473 proteins were down regulated in TGEV vs NC group.Based on the above data,a systematic bioinformatics analysis(protein function annotation)was carried out for all identified proteins,and functional classification,functional enrichment and clustering analysis based on functional enrichment were carried out for all differentially expressed proteins.The results show that TGEV infection can down-regulate the expression of NHE3,and the protein pathways related to membrane transport and endocytosis are differentially expressed.Therefore,based on the above information,we speculate that TGEV infection may be involved in regulating NHE3 activity by inducing host cell signaling molecules.2.Detection of NHE3 expression after TGEV infectionThe small intestine of healthy 1-week-old piglets was washed with cold KHB buffer and then filled with 3%low melting point agarose,which was put into cold KHB buffer and then covered with low melting point agarose again after coagulation.The tissue block was cut and transferred to sterile tissue slicer to prepare precise-cut intestine slice.Slices were selected and cultured in WME medium.Atp test kit and living dead cell staining kit were used to detect the activity of slices,and HE staining was used to detect the tissue integrity of slices.RT-PCR was used to detect the virus infection and immunofluorescence was used to detect the expression of NHE3 in small intestine.The expression of NHE3 protein on the surface of IPEC-J2 cells was detected by immunofluorescence,and the transformation rate of NHE3 mRNA was detected by qRT-PCR.The results showed that TGEV could infect PCIs successfully,and the activity of small intestine decreased to half at 24 hours.With the increase of infection time,the integrity of small intestine decreased gradually,the villi of small intestine almost fell off after 24 hours,and the expression of NHE3 in small intestine decreased after TGEV infection.In IPEC-J2 cells,TGEV infection can reduce the expression of NHE3 on the membrane surface and the conversion of NHE3.3.The effect of TGEV infection on clathrin and the endocytosis of NHE3 in IPEC-J2cellsFirst,the growth kinetics of TGEV on IPEC-J2 was detected by absolute Quantitative Real-time PCR,and the protein expression of NHE3 in endocytic vesicles was detected by biotinylation.Western blot was used to detect the expression of clathrin and phosphorylated Ezrin,and Quantitative Real-time PCR was used to detect the relative mRNA level of clathrin.The results showed that the best detection time of TGEV mRNA was 12h after infection.After TGEV infection,NHE3 protein expression in endocytic vesicles increased,also increased the expression of phosphorylated ezrin and Clathrin protein expression and mRNA transcription level decreased.