| 1. Background and ObjectiveEnterohemorrhagic Escherichict coli O157:H7(EHEC O157:H7) are a very diverse species of bacteria found naturally in the intestinal tract of many animal species. A subset of E. coli is capable of causing enteric/diarrhoeal disease. A different subset cause extra-intestinal disease, including urinary tract infection (UTI), haemorrhagic colitis (HC), sepsis and neonatal meningitis, which are classified as extra intestinal pathogenic E. coli. Riley noticed a serious food-borne hemorrhagic colitis caused by EHEC O157:H7in Michigan and Oregan for the first time in1983. After this, there always are cases dispersive or roost of EHEC O157:H7. According to the world health organization (WHO)’s reports, there was a outbreak involving5000peoples of EHEC O157:H7in Walkerton of Canada, in this outbreak,27peoples went to hospital, and5peoples died. By report, the cause of this outbreak is the water supply system in Walkerton was polluted. Nowadays the prevalance of EHEC O157:H7frequently breaks out all over the world and threatens human’s public health.The mechanism of EHEC O157:H7is still unclear. Toxin, Plasmid pO157and the locus of enterocyte effacement (LEE) are the maining genes causing disease. The classic feature of pathogenisis in EHEC is attaching to host cell membrane firmly and forming an attaching-and-effacing (A/E) lesion. The virulence genes of EHEC are located on the pathogenic iland LEE. There are as many as20different effect proteins, in witch, the role of EspF is most important of all, and is Is multifunction. EspF is considered to destroy the barrier of intestinal epithelial cells. It can target to motochondria through its mitochondrial targeting signal (MTS) area in N terminal, initiating the program of the early aptosis in host cells. Substitution of the16th leucine with glutamic acid EspF (L16E) completely abolished EspF activity, suggesting that the16th leucine in the MTS is a critical amino acid for EspF function. Nucleolar accumulation happened in later period of infection, and this process is dominated by mitochondria. According to the recent study, EspF cam bind to some protein of host cells by SH3and EVH1domians that interfered the signal transmission, leading to the closely connection of epithelial cells destroyed, microvilli disappeared, intestinal epithelium cells skeleton rearranged, actin gathered and typical pedestal formed. In academic, EspF is deemed to the Swiss army knife of bacterial pathogenesis, it is very crucial for clearous of pathogenesis by EHECRecent years, there is further research about EspF, research shows that it can destroy the closely connection of epithelial cells, microvilli disappeared, reduce TEER of host cells. But the mechanism of EHEC O157:H7is still unclear. In previous study of this laboratory, we had constructed espF gene defect mutant in EHEC successfully by OL-PCR and homologous recombination. In this study, we preparation and primary purification of polyclonal antibodies against EspF of Enterohaemorrhagic Escherichia coli O157:H7. In order to reveal the fuction of EspF in EHEC.2. Methods(1)Preparation and primary purification of polyclonal antibodies against EspF of Enterohaemorrhagic Escherichia coli O157:H7①Antigens design and synthesis Forecast hydrophilic amino acids by Hopp-Woods and Kyte-Doolittle, forecast Flexible and predict the surface possibility by Karplus-Schultz and Emini, forecast Potential B cells epitope by Jameson-Wolf and Wolf’s antigen index method, using Protean of DNAStar. Select advantage section of B cells epitope, then couple KLH to its C-terminal. Designed antigen is synthesized by Shanghai Jill biochemical Co. LTD.(2) Preparation and preliminary purification of the polyclonal antibody There are two New Zea land white rabbits(Weight1.5～2.5kg, and named1,2). Took blood from ear margin veins before immunization, separation serum, save at-20℃as contrast. Immunize rabbits at1,28,42,66day, and the dosage is lmg per rabbit. Antigenic Protein is dissolved in normal saline, it’s emulsified uniformly with Freund’s complete adjuvant in primary immune, inject2ml per rabbit at back and shoulder in skin.In promote immunization, Antigenic Protein is emulsified uniformly with Freund’s incomplete adjuvant, inject2ml per rabbit at shoulder and leg in muscles. Took blood from ear margin veins after second promote immunization, detect valence of antibody by ELISA to checkout there is antibody production. Detect valence of antibody by ELISA10days after last immunization. Take whole blood from heart, separation serum, save at-20℃in subpackage. Valence of antibody was detected by ELISA, specificity was tested by western blot. Polyclonal antiserum was purified by caprylic acid-ammonium sulfate precipitation, then, the purity of antibodies was detected by SDS-PAGE.(2)Location of EspF of EHEC during its clinging to lovo Vaccination lovo cells to confocal petri dishes, incubation cells to80%of the dish. Incubation EHEC O157:H7(wild plants and espF mutations (△espF)) at37℃, shaking12h. Bacterial affect cells for2h (cells:bacteria=1:100). Wash three times with PBS, and10min/time. Fixed with4%of Polyphosphate formaldehyde for30min. Permeate for20min with0.05%triton. Wash two times with PBS,5min/time. Closed1h with PBS with1%BSA. Incubation18h with dilute antibody at4℃. Wash three times with PBS, and10min/time. Avoid light incubation45min at room temperature with fluorescent antibody. Wash three times with PBS, and10min/time. Incubation3-5min at room temperature with DAPI. Wash two times with PBS,5min/time. Taking pictures and analyze using laser confocal microscope. (3)Bacterial adhesion experiment Preparation cell slides of climb, bacterial affect cells for2h, wash two times with PBS, fixed2h with2.5%glutaraldehyde at4℃. Microscopic at electron microscopy room.(4) Detection cell apoptosis by testing the mitochondrial membrane potential Vaccination lovo cells to24hole cell culture plate, incubation cells to80%of the hole. Incubation EHEC O157:H7(wild plants and espF mutations (△espF)) at37℃, shaking12h.Bacterial affect cells for0.5,1.5,2.5h. Wash three times with PBS, and10min/time. Then operate according to the instruction and operation manual of JC-1. Take pictures with a fluorescent microscope.3. Results(1)Preparation and purification polyclonal antibody against EspF protein of EHEC O157:H7Valence of antibody was detected by ELISA, The titer of the antiserum obtained in this experiment was up to1:2048000, specificity was confirmed by western blot. Polyclonal antiserum was purified by caprylic acid-ammonium sulfate precipitation, then, the purity of antibodies was detected by SDS-PAGE. So we obtained antibodies with specificity against the protein EspF of EHECO157:H7.(2) EspF protein of EHEC targets to motochondria of host cell Green fluorescent signal of blank control was not detected. Common orientation phenomenon between green fluorescent signal and mtHsp70was not observed in espF mutation, while wild type was. It can be further proved in fluorescent signal strength in straight lines:Curve of green and red fluorescence intensity in straight lines did not show correlation in espF mutation, while they did show correlation in wild type. We analyse the ubiety between nucleus and EspF protein by the same method, there are always negative correlation either the espF mutation or wild type. So EspF protein did not combine with nuclear, neither the espF mutation nor wild type. (3)Bacterial adhesion experiment Bacterial affect lovo cells for3h, then observe through Electronic Microscopy, in wild type, Cell membrane is fuzzy, there are a large number of bacterial on cell membrane surface,cells are integrated; while in espF mutation, cell membrane is clear, there is little bacterial on cell membrane surface, and there are not structural damage in cell membrane.(4)Detection cell apoptosis by testing the mitochondrial membrane potential Bacterial affect cells for0.5h, there are obvious mitochondrial membrane potential change either the espF mutation or wild type. Bacterial affect cells for1.5,2.5h, there is more obvious change in wild type then espF mutation.4. Conclusions(1)Obtained antibodies with high-titer and specificity against the protein EspF of EHECO157:H7.(2)EspF protein is target to motochondria during its infection, and N terminal is its mitochondrial targeting signal (MTS) area. EspF protein did not combine with nuclear, neither the espF mutation nor wild type.(3)The deletion mutantion of espF gene reduce the diffusedly adherent of EHEC O157:H7to host cell.(4)The toxicity to mitochondria of wild type is much greater than espF mutation. |
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