Study On The Pathogenic Mechanism Of Xinjiang Cattle Origin Non-O157 STEC H34 Strains Stx1A And Stx1B Effect On HIEC Cells

Shiga toxin-producing Escherichia coli(STEC)is a class of highly pathogenic food-borne pathogens that carry one or two Shiga toxin genes encoded by the prophage,which seriously threatens human health.Xinjiang is one of the five major pastoral areas in China.Cattle and sheep breeding is the leading industry in the development of modern animal husbandry in Xinjiang.Cattle and sheep are the natural hosts of STEC strains,and their carrying rate can be as high as 71%.For the study of the main Shiga toxin protein Stx1 and its subunit protein of non-O157 STEC strain from Xinjiang cattle,to provide scientific basis for the pathogenesis of human intestinal epithelial cells(HIEC).The content have(1)we have analyzed the Stx genotypes of non-O157 STEC from Xinjiang cattle and sheep.(2)we have used non-O157 STEC H34 strains and expressed Stx1(Stx1A+Stx1B),Stx1 A,and Stx1 B proteins to effect on HIEC cells,Observe cell apoptosis,detect the gene expression and protein expression of receptor proteins and actin(Gb3,Gb4,NMII,Cdc42),at the same time,analyze the expression of immune-related genes.(3)we construct Stx1,Stx1 A and the fluorescent recombinant plasmid of Stx1 B and transfect into HIEC cells,and observe of the expression and localization of Stx1,Stx1 A and Stx1 B proteins in HIEC cells use a confocal microscope.The main research contents and results are as follows:1.Analysis of Stx subtypes of non-O157 STEC strains from Xinjiang: 56 strains of non-O157 STEC strains from cattle and sheep isolated and identified in this laboratory,Extraction of DNA template,amplify the full-length genes of Stx1 and Stx2,after sending it to the company for sequencing,analyze of the Stx1 and Stx2 gene subtypes.Of the 56 non-O157 STEC strains,31(55.4%)strains coded for Stx1,only 3(5.4%)strains coded for Stx2,and 22(39.3%)strains coded for Stx1+Stx2 at the same time.Analysis of sequencing results that there are only 2 subtypes of 53 Stx1,namely Stx1a(66%)and Stx1c(34%),and there were 3subtypes of 25 Stx2,namely Stx2a(56.0%),Stx2b(20.0%)and Stx2c(24.0%).There are a total of 8 Stx gene subtype combinations in 56 non-O157 STEC strains,namely Stx1a(30.4%),Stx1c(25.0%),Stx2a(1.8%),Stx2b(1.8%),Stx2c(1.8%),Stx1a+Stx2a(23.2%),Stx1a+Stx2c(8.9%)and Stx1c+Stx2b(8.9%).Among Stx1 a is the most common in the combination.This gene is selected as the research object,we detected the expression of related genes and localization in HIEC cells of HIEC cells infected with Stx1 a subtype of non-O157 STEC H34 strain from Xinjiang cattle.2.Gene expression of non-O157 STEC H34 strain,Stx1 A and Stx1 B proteins effect on HIEC cells:we construct prokaryotic expression vectors of PEGX-6P-1-Stx1 A and PEGX-6P-1-Stx1 B,and purified Stx1 A and Stx1 B proteins.The optimal concentrations of Stx1 A,Stx1B and Stx1(Stx1A+Stx1B)effect on HIEC cells were 10.25 ?g,10.8 ?g and 13.8 ?g,respectively,by CCK-8 method.Then by q PCR and Western Blot detect of non-O157 STEC H34 strains,Stx1,Stx1 A and Stx1 B proteins effect on HIEC cells.The receptor proteins Gb3,Gb4 and actin Cdc42 were up-regulated in effect of H34 strain,Stx1 and Stx1 A protein.My Hc-IIA and My Hc-IIB are up-regulated by H34 strain.The immune-related genes IL-1?,TNF-a,TNF-R1,TLR4 and TLR5 are up-regulated in H34 strain,Stx1,Stx1 A and Stx1 B protein,and the IL1R1 was down-regulated under the action of Stx1 protein.3.Localization of Shiga toxin Stx1,Stx1 A and Stx1 B proteins in HIEC cells: The fluorescent recombinant eukaryotic vectors p LVML-GFP-Stx1,p LVML-GFP-Stx1 A,p LVML-GFP-Stx1 B were successfully constructed.It was transfected into HIEC cells,and the fluorescent plasmid p LVML-GFP was used as the transfection control.By confocal fluorescence microscope,we observe the fluorescent recombinant plasmid p LVML-GFP-Stx1 A is distributed on the organelles of the cytoplasm and wrapped around the nucleus,the fluorescent recombinant plasmids p LVML-GFP-Stx1 and p LVML-GFP-Stx1 B are distributed throughout the cell(distributed in both the cytoplasm and the nucleus).In summary,this experiment successfully determined the way Stx1 and its subunits enter HIEC cells,and at the same time determined the localization of Stx1,Stx1 A,and Stx1 B proteins in HIEC cells,which is the effect of Stx1 a subunits of non-O157 STEC strains on HIEC cells.The disease mechanism provides data support and provides a scientific basis for the prevention and treatment of the disease.