Avian- and mammalian-derived antibodies against adherence-associated proteins inhibit host cell colonization by Escherichia coli O157:H7

AIM: To evaluate the potential for polyclonal antibodies targeting enterohaemorrhagic Escherichia coli (EHEC) virulence determinants to prevent colonization of host cells by E. coli O157:H7. METHODS AND RESULTS: Rats and laying hens were immunized with recombinant proteins from E. coli O157:H7, EspA, C-terminal intimin or EscF. Rat antisera (IgG) or chicken egg powders (IgY) were assessed for their ability to inhibit growth and colonization-associated processes of E. coli O157:H7. Mammalian antisera with antibodies to intimin, EspA or EscF effectively reduced adherence of the pathogen to HeLa cells (P<0.05) and prevented type III secretion of Tir. Similarly, HeLa cells treated with chicken egg powder containing antibodies against intimin or EspA were protected from EHEC adherence (P<0.05). Neither egg nor rat antibody preparations had any antibacterial effect on the growth of EHEC (P>0.05). CONCLUSIONS: Antibody preparations targeting EHEC adherence-associated factors were effective at preventing adhesion and intimate colonization-associated events. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates that immunotherapy with anti-adherence antibodies can reduce E. coli O157:H7 colonization of host cells. Passive immunization with specific antibodies may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle or humans.     

Use of virulence factor-specific egg yolk-derived immunoglobulins as a promising alternative to antibiotics for prevention of attaching and effacing Escherichia coli infections

Using a porcine ileal in vitro organ culture model, we have demonstrated that egg yolk-derived antibodies specific for the attaching and effacing Escherichia coli (AEEC) virulence factors intimin and translocated intimin receptor (Tir), but not those specific for the AEEC-secreted proteins EspA, EspB and EspD, significantly reduced the bacterial adherence of the porcine enteropathogenic E. coli strain ECL1001, formerly 86-1390. Moreover, antibodies specific for intimin and Tir also significantly reduced bacterial adherence of heterologous AEEC strains, including human, bovine and canine enteropathogenic E. coli strains, as well as of O157:H7 Shiga toxin-producing E. coli strains in this model. In addition, we demonstrated that the oral administration of these anti-intimin antibodies significantly reduced the extent of attaching and effacing lesions found in the small intestine of weaned pigs challenged with the porcine enteropathogenic E. coli strain ECL1001. Overall, our results underline the potential use of specific egg yolk-derived antibodies as a novel approach for the prevention of AEEC infections.     

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.     

Use of Antibody Responses against Locus of Enterocyte Effacement (LEE)-Encoded Antigens To Monitor Enterohemorrhagic Escherichia coli Infections on Cattle Farms

Enterohemorrhagic Escherichia coli (EHEC) is a significant zoonotic pathogen causing severe disease associated with watery and bloody diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome (HUS) in humans. Infections are frequently associated with contact with EHEC-contaminated ruminant feces. Both natural and experimental infection of cattle induces serum antibodies against the LEE-encoded proteins intimin, EspA, EspB, and Tir and the Shiga toxins Stx1 and Stx2, although the latter are poorly immunogenic in cattle. We determined whether antibodies and/or the kinetics of antibody responses against intimin, Tir, EspA, and/or EspB can be used for monitoring EHEC infections in beef cattle herds in order to reduce carcass contamination at slaughter. We examined the presence of serum antibodies against recombinant O157:H7 E. coli intimin EspA, EspB, and Tir during a cross-sectional study on 12 cattle farms and during a longitudinal time course study on two EHEC-positive cattle farms. We searched for a possible correlation between intimin, Tir, EspA, and/or EspB antibodies and fecal excretion of EHEC O157, O145, O111, O103, or O26 seropathotypes. The results indicated that serum antibody responses to EspB and EspA might be useful for first-line screening at the herd level for EHEC O157, O26, and most likely also for EHEC O103 infections. However, antibody responses against EspB are of less use for monitoring individual animals, since some EHEC-shedding animals did not show antibody responses and since serum antibody responses against EspB could persist for several months even when shedding had ceased.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.     

Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157:H7 strains in environmental samples

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield "fingerprint" patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples.     

Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

Background

Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry.

Methods/Principal Findings

We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments.

Conclusions

Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis.     

EspE, a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine-phosphorylated 90 kDa protein

Shiga toxin-producing Escherichia coli (STEC), enteropathogenic E . coli (EPEC) and some strains of Hafnia alvei are capable of inducing attaching and effacing (A/E) lesions, characterized by tight apposition of the bacteria to the eukaryotic membrane and formation of actin-based pedestals. In this study, we report on the identification of EspE, a novel secreted 80 kDa protein of A/E bacteria. During infection, EspE is delivered into the cytoplasm of the infected host cell, where it is detected as a higher-molecular-weight form of 90 kDa. We present evidence that translocated EspE becomes tyrosine phosphorylated and that this modified form of EspE may be identical to Hp90, the putative receptor of EPEC intimin. Bacteria of the classic enterohaemorrhagic E . coli (EHEC) serotype O157:H7 fail to induce a tyrosine phosphorylation of EspE and differ in this respect from other A/E bacteria. Translocated EspE, whether tyrosine phosphorylated or not, becomes incorporated into the bacteria-induced cytoskeletal structures, where it normally colocalizes with filamentous actin. EPEC are also able to induce 'pseudopods', elongated pedestals that have recently been implicated in a novel kind of actin-based motility. EspE is enriched at the tip of these structures, suggesting its involvement in the process of actin dynamics, which is triggered during the attaching and effacing process.     

Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.     

A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7

The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Polarity of enteropathogenic Escherichia coli EspA filament assembly and protein secretion

Type III secretion systems (TTSS) are sophisticated macromolecular structures that play an imperative role in bacterial infections and human disease. The TTSS needle complex is conserved among bacterial pathogens and shows broad similarity to the flagellar basal body. However, the TTSS of enteropathogenic and enterohemorrhagic Escherichia coli, two important human enteric pathogens, is unique in that it has an approximately 12-nm-diameter filamentous extension to the needle that is composed of the secreted translocator protein EspA. EspA filaments and flagellar structures have very similar helical symmetry parameters. In this study we investigated EspA filament assembly and the delivery of effector proteins across the bacterial cell wall. We show that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament. Moreover, EspA filament length is modulated by the availability of intracellular EspA subunits. Finally, we provide direct evidence that EspA filaments are hollow conduits through which effector proteins are delivered to the extremity of the bacterial cell (and subsequently into the host cell).     

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were 相似文献   

Rearrangements of actin cytoskeleton during infection with Escherichia coli O157 in macrophages

The lamina propria of the large intestine is rich in macrophages, and they might be one of the first lines of the host defense in enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection. Although macrophages were infected with them, they can survive the EHEC O157 infection. We examined the structural rearrangements of the actin cytoskeleton during the microbial infection process. Macrophage actin filaments were rearranged in the following sequence; 1) disappearance of the actin filament bundles in the cytoplasm, 2) accumulation of actin filaments under the cell surface, and 3) construction of actin networks underlying the endosome membrane. Before infection, actin filaments were distributed under the cell surface and in bundles located in the macrophage cytoplasm. Within 2 min, infection caused a rapid and marked loss of the actin filament bundles that had run parallel to the long axis of the cell. Concomitant with the loss, actin filaments became more markedly distributed under the cell surface. In the formation of the endosome, new networks of actin filaments were constructed below the phagosome membrane. The networks contained a large amount of actin as well as a fodrin-like immunoreactivity. The thickness of the networks reached about 400 nm under the phagosome membrane. The actin networks disappeared again after the bacterial digestion. The results of this study showed that actin filaments undergo three major rearrangements of the actin filaments during the infection in macrophages, and suggested that the third rearrangement is mediated by actin-binding proteins, such as a fodrin-like molecules. These morphological changes in macrophages were not clear after infection with other strains of Escherichia coli.     

SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD

Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.     

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.     

QCM immunosensor detection of Escherichia coli O157:H7 based on beacon immunomagnetic nanoparticles and catalytic growth of colloidal gold

In this paper, we describe a novel method for detecting Escherichia coli (E. coli) O157:H7 by using a quartz crystal microbalance (QCM) immunosensor based on beacon immunomagnetic nanoparticles (BIMPs), streptavidin-gold, and growth solution. E. coli O157-BIMPs were magnetic nanoparticles loaded with polyclonal anti-E. coli O157:H7 antibody (target antibody, T-Ab) and biotin-IgG (beacon antibody, B-Ab) at an optimized ratio of 1:60 (T-Ab:B-Ab). E. coli O157:H7 was captured and separated by E. coli O157-BIMPs in a sample, and the streptavidin-gold was subsequently conjugated to E. coli O157-BIMPs by using a biotin-avidin system. Finally, the gold particles on E. coli O157-BIMPs were enlarged in growth solution, and the compounds containing E. coli O157:H7, E. coli O157-BIMPs, and enlarged gold particles were collected using a magnetic plate. The QCM immunosensor was fabricated with protein A from Staphylococcus aureus and monoclonal anti-E. coli O157:H7 antibody. The compounds decreased the immunosensor's resonant frequency. E. coli O157-BIMPs and enlarged gold particles were used as "mass enhancers" to amplify the frequency change. The frequency shift was correlated to the bacterial concentration. The detection limit was 23 CFU/ml in phosphate-buffered saline and 53 CFU/ml in milk. This method could successfully detect E. coli O157:H7 with high specificity and stability. The entire procedure for the detection of E. coli O157:H7 took only 4 h.     

肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果

目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）0157：H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法：采用PCR技术从EHEC0157：H7基因组中扩增espA和espB基因,连接至pET-22b（4-）载体上,转化至宿主细胞大肠杆菌BL21（DE3）,经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100％;得到了纯度为95％以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为10^6。结论：重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。