Antibodies against Escherichia coli O157:H7 specific proteins and their application in the enrichment or isolation of this bacterium

Abstract

The present invention discloses antibodies against Escherichia coli O157:H7 specific proteins and their application in the enrichment or isolation of this bacterium, belonging to the field of biotechnology. The present invention screened specific antibodies that respectively recognize the LPS and fliC of Escherichia coli O157:H7. The two antibodies were respectively conjugated to magnetic beads, and the two magnetic beads were used for the enrichment or isolation of Escherichia coli O157:H7. They can bind to different parts of Escherichia coli, form a synergistic effect, and can improve the coverage rate and reduce off-target effects. The present invention can directly enrich or isolate Escherichia coli O157:H7 from feces for the sequencing or culturing of this bacterium.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an antibody against a specific protein of Escherichia coli O157:H7 and an application thereof in enriching or separating the bacterium. Background Art

[0002] Numerous studies have shown that the intestinal microbiome is widely involved in the host's neurological, physiological, metabolic, and immune processes and physiological activities, and is closely related to the host's health. The extraction and isolation of a specific natural microorganism or class of natural microorganisms from the feces of healthy individuals or patients plays a significant role in elucidating the relationship between microorganisms and disease. Furthermore, the isolation and removal of pathogenic bacteria in fecal transplantation also provides a new direction for fecal transplantation. By coupling antibodies that recognize microbial surface proteins to magnetic beads, then sorting and collecting them using a magnetic field, and then undergoing antigen-antibody dissociation, the target microorganism can be obtained from the feces or harmful microorganisms can be removed.

[0003] Escherichia coli, also known as Escherichia coli, is 0.5 x 1-3 microns in size. Discovered by Escherichia coli in 1885, it possesses peritrichous flagella and is a conditionally pathogenic bacterium. Under certain conditions, it can cause gastrointestinal infections and localized infections of various tissues and organs, including the urinary tract, in humans and a variety of other animals. E. coli is a normal inhabitant of the animal intestines, entering the intestines of infants after birth with breastfeeding and remaining with humans throughout their lives. A small percentage of these bacteria can cause disease under certain conditions. The infection-causing properties of E. coli serotypes are primarily due to the presence of fimbrial antigens and pathogenic toxins. E. coli O157:H7 is a species of E. coli commonly found in the intestines of warm-blooded animals such as cattle. This species releases a potent toxin that can cause severe intestinal symptoms, such as bloody diarrhea.

[0004] The O-antigen is a polysaccharide composed of repeating polysaccharide units, expressed on the bacterial outer membrane. It is one of the most common antigens of Escherichia coli and a primary means of identifying E. coli. The major surface antigens of E. coli O157:H7 are the cell wall lipopolysaccharide (LPS; O-antigen) and the capsular polysaccharide (PS; K-antigen). These polysaccharides are synthesized on the bacterial cytoplasmic membrane. LPS is transported to the outer membrane where it resides, while PS is located on the outer layer of the O-antigen. LPS consists of lipid A covalently linked to a core oligosaccharide, which is covalently linked to an O-specific polysaccharide. The O-specificity of the LPS determines the bacterial O-specificity and is specifically bound to E. coli O157:H7 by anti-O157 LPS antibodies. The flagellin H7 antigen is a key virulence factor of enterohemorrhagic E. coli O157:H7. Its encoding gene, fliC, consists of three main regions: the highly conserved C1 and C2 regions at either end, and the central, hypervariable C3 region. The H7 flagellum plays an important role in initiating the binding of E. coli O157:H7 to the intestinal mucosa, mucus, or both.

[0005] The main principle of the immunomagnetic bead method for separating E. coli O157:H7 is to selectively capture E. coli O157:H7 using magnetic beads coated with O157-specific antibodies, and then complete the mechanical movement through magnetic technology to separate E. coli O157:H7. The existing immunomagnetic bead method for separating E. coli O157:H7 uses polyclonal antibodies or a certain monoclonal antibody, which has low separation efficiency and cannot directly separate E. coli O157:H7 from fecal samples. The fecal sample must first be enriched and cultured. The present invention uses two antibodies for composite screening to produce a synergistic effect, which can improve the separation efficiency and make it possible to directly separate O157:H7 from fecal samples. Summary of the Invention

[0006] The purpose of the present invention is to solve the shortcomings and deficiencies of the prior art and provide antibodies to Escherichia coli O157:H7-specific proteins and their use in enriching or separating the bacteria.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The antibodies to the Escherichia coli O157:H7 specific protein provided by the present invention are LPS antibodies and fliC antibodies.

[0009] The amino acid sequences of the CDR and FR regions of the LPS antibody light chain are shown below:

[0010] FR-L1:EVTITC,

[0011] CDR-L1:SASSSVTSMH,

[0012] FR-L2: WYLLKPGQSPKLLIY,

[0013] CDR-L2:STSNLAS,

[0014] FR-L3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC,

[0015] CDR-L3:QQRSSYPFT,

[0016] FR-L4: FGGGTKLEIK.

[0017] The amino acid sequences of the LPS antibody heavy chain CDR and FR regions are shown below:

[0018] FR-H1: EVKLVESGAELVKPGASVKLSCKASGYTFT,

[0019] CDR-H1:NYWMH,

[0020] FR-H2: WVKQRPGQGLEWIG,

[0021] CDR-H2: EIKPGNGRTNYNEKFST,

[0022] FR-H3:KATLTVDKSSNTAYIQLSSLTSEDSAVYYCSR,

[0023] CDR-H3:EGDYDNPAYFPY,

[0024] FR-H4:WGQGTLVTVSA.

[0025] The amino acid sequences of the fliC antibody light chain CDR and FR regions are shown below:

[0026] FR-L1: DILITQTPLSLPVSLGDQASISC,

[0027] CDR-L1:RSSQRIVHSNGNTYLE,

[0028] FR-L2: WYLLKPGQSPKLLIY,

[0029] CDR-L2:KVFNRFS,

[0030] FR-L3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC,

[0031] CDR-L3: FQGSHVPPT,

[0032] FR-L4: FGGGTQ.

[0033] The amino acid sequences of the fliC antibody heavy chain CDR and FR regions are shown below:

[0034] FR-H1: QVQLKESGPGILQPSQTLTLTCSFSGFSLS,

[0035] CDR-H1:TSGMGVS,

[0036] FR-H2: WIRQPSGKGLEWLA,

[0037] CDR-H2: HIYWDDDKRYIPSLMS,

[0038] FR-H3:RLTISKDASGNQVFLKITSVDTADSATYYCAR,

[0039] CDR-H3: TDTSALHWYFDV,

[0040] FR-H4: WGAGTTVTVSS.

[0041] The present invention also provides the use of the LPS antibody and fliC antibody in the enrichment or separation of Escherichia coli O157:H7.

[0042] The present invention also provides the use of magnetic beads coated with the aforementioned LPS antibody and magnetic beads coated with the aforementioned fliC antibody for the enrichment or isolation of Escherichia coli O157:H7. The antibody-coated magnetic beads are preferably obtained by coupling the antibody to carboxyl magnetic beads using EDC and NHS; the magnetic beads preferably have a particle size of 200 nm.

[0043] The present invention also provides a method for enriching or isolating E. coli O157:H7, which utilizes magnetic beads coated with the aforementioned LPS antibody and magnetic beads coated with the aforementioned fliC antibody to enrich or isolate E. coli O157:H7 in a sample to be tested. Furthermore, the method comprises the following steps: adding the magnetic beads coated with the aforementioned LPS antibody and the magnetic beads coated with the aforementioned fliC antibody to a sample to be tested, incubating the beads to allow E. coli O157:H7 to bind to the magnetic beads, and separating the beads using magnetic force; resuspending the separated magnetic beads, adding papain, and incubating the beads to dissociate the antibodies from the magnetic beads; then magnetically adsorbing the beads, resulting in a supernatant that is a suspension of enriched or isolated E. coli O157:H7. The sample to be tested may include a fecal sample; and the solution used to resuspend and isolate the magnetic beads may include physiological saline, 0.1 mol / L PBS, 0.2 mol / L EDTA, 0.01 mol / L cysteine, and the like.

[0044] The present invention has the following advantages and beneficial effects compared to the prior art:

[0045] The present invention screened and obtained specific antibodies that recognize LPS and fliC of E. coli O157:H7, respectively. The two antibodies were coupled to magnetic beads, which were then used to enrich or isolate E. coli O157:H7. The beads bind to different sites of E. coli, creating a synergistic effect that increases coverage and reduces off-target effects.

[0046] The Enterobacter O157:H7 enriched or separated from feces in the present invention can be used for bacterial species identification and sequencing, and can also be used for the cultivation of the bacteria.

[0047] The present invention can directly remove pathogenic Escherichia coli O157:H7 in feces, while selective culture medium screening cannot achieve this purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the SDS-PAGE identification diagram of the purified antibodies G2, F5, H6 and C8. DETAILED DESCRIPTION

[0049] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be considered equivalent replacement methods and shall be included in the scope of protection of the present invention.

[0050] Example 1 Expression and purification of fliC protein

[0051] Escherichia coli BL21 was used to express 337-489aa of the Escherichia coli O157:H7 cell wall H7 flagellin fliC (Uniprot accession number Q7DBI0). The codon-optimized encoding nucleotide sequence of the fliC protein 337-489aa is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2. The codon-optimized encoding nucleotide sequence of the fliC protein 337-489aa was ligated to pET28a to construct pET28a-fliC. The recombinant vector pET28a-fliC was transformed into the Escherichia coli BL21 host. The induced culture medium of the Escherichia coli BL21 transformed with pET28a-fliC was collected, the culture medium was ultrasonically disrupted, and the supernatant after centrifugation was purified by nickel column to obtain the protein. The protein A280 concentration was calculated using an ultramicrospectrophotometer, and the protein purity was analyzed by SDS-PAGE. The results are shown in Table 1 below.

[0052] The codon-optimized nucleotide sequence encoding the fliC protein 337-489aa (SEQ ID NO. 1): AAAGCTG CTAGCGAAGGTTCTGATGGTGCCTCTCTGACTTTCAACGGTACTGAATATACTATCGCTAAGGCCACTCCGGCTACCACTACTCCAGTTGCTCCTCTGATCCCGGGTGGTATTACGTATCAGGCGACCGTGAGCAAGGACGTTGTCCTGTCCGAAACTAAAGCTGCGGCTGCTACCTCTTCCATTACTTTTAACTCCGGCTACTGTCCAAAACCATCGGCTTCA CCGCAGGTGAATCCTCTGATGCTGCTAAGTCCTACGTAGACGACAAAGGCGGTATTACTAACGTAGCTGACTATACCGTAAGCTATTCCGTGAACAAAGACAACGGCTCTGTTACCGTTGCGGGTTACGCTAGCGCAACTGATACCAACAAAGACTACGCGCCGGCTATCGGTACCGCCGTGAATGTTAACTCTGCGGGTAAGATCACGACCGAAACCACCTCC.

[0053] Amino acid sequence of fliC protein 337-489aa (SEQ ID NO. 2): KAASEGSDGASLTFNGTEYTIAK ATPATTTPVAPLIPGGITYQATVSKDVVLSETKAAAATSSITFNSGVLSKTIGFTAGESSDAAK SYVDDKGGITNVADYTVSYSVNKDNGSVTVAGYASATDTNKDYAPAIGTAVNVNSAGKITT ETTS.

[0054] Table 1

[0055] Protein name Purification tags Protein concentration Protein purity fliC His 2.2mg / ml 92％

[0056] Example 2 Mouse immunization and antiserum titer detection and hybridoma cell screening

[0057] O157 LPS (purchased from Creative Diagnostics) and purified fliC protein were mixed with Freund's complete adjuvant and emulsified in a blender before immunization. The first immunization dose was 50 μg protein, and the second, second, third, and fourth immunization doses were also 50 μg protein. Immunizations were conducted every two weeks. After 3-4 immunizations, 2 mL of blood was collected from the ear vein of the mice for serum antibody titer. A spleen cell suspension was prepared and washed with PBS. It was then mixed with SP2 / 0 cells at a ratio of 10:1 spleen cells:SP2 / 0 cells. The mixture was centrifuged at 1000 rpm for 5 minutes, then dripped dry and gently tapped to loosen cell clumps. 1 mL of PEG-1450 was added in a 37°C water bath. After addition, the mixture was incubated at 37°C for 2 minutes. Then, 20 mL of RPMI-1640 stop solution was slowly added along the tube wall. After cell fusion was terminated, the cells were centrifuged at 800 rpm for 5 minutes, and the remaining liquid was aspirated. The screened cells were diluted to 1 cell / well by limiting dilution method using HT medium, and the cells were plated in 96-well cell culture plates. 4 The supernatant was added to an ELISA plate pre-coated with LPS and fliC protein, and the titer was measured by OD450. Positive cells were then sampled and subcloned again. Once all supernatants from all wells were positive, the same monoclonal screening process was repeated until all positive results were obtained after limiting dilution. This confirmed that a positive cell line had been screened and hybridoma cells were obtained. Hybridomas with the highest antibody titers were screened. Cells that recognized O157 LPS were numbered G2 and F5, and those that recognized fliC were numbered H6 and C8. The corresponding antibody titer test results are shown in Table 2.

[0058] Table 2

[0059]

[0060] Example 3 Serum-free culture of hybridoma cells and purification of antibodies

[0061] The hybridoma cells screened above are revived in the presence of serum. During several consecutive cell passages, the serum ratio is gradually reduced (e.g., from 20% serum to 15% serum to 10% serum and then to serum-free) to allow the hybridoma cells to adapt and continue to grow. When preparing monoclonal antibodies, the hybridoma cells are revived in serum-free medium. When the cells are in good condition, the amount of medium and the container used for culturing the cells are increased. When a large number of cells die, the liquid is collected and centrifuged to obtain the supernatant, which is collected by filtration. The sample to be purified is loaded onto a Protein A agarose affinity chromatography column at a flow rate of 0.5 mL / min to allow the antibody to bind to Protein A. Finally, the antibody is eluted with an eluent to obtain the antibody. Its purity is identified by SDS-PAGE. The results are shown in FIG. Figure 1 .

[0062] Example 4 Magnetic bead separation and E. coli O157:H7 enrichment

[0063] Replace the buffer of antibodies G2, F5, H6, and C8 with 15mM MES buffer (pH 6.0) and dilute the antibody concentration to 2mg / mL in 15mM MES buffer (pH 6.0). Wash 1mg of carboxyl magnetic beads of different particle sizes (50nm, 200nm, 1000nm) three times with MES buffer and resuspend in 0.1mL MES buffer to 10mg / mL. Weigh EDC and NHS and dissolve them in 15mM MES buffer (pH 6.0) to prepare 20mg / mL EDC and 24mg / mL NHS. Add 50μL EDC and 50μL NHS to the washed beads and activate them at 25°C for 30min. 100 μg of antibody was mixed with 1 mg of activated carboxyl magnetic beads (particle size 50 nm, 200 nm and 1000 nm), reacted at 25°C for 16 h, magnetically separated, the supernatant magnetic beads were aspirated and washed two to three times with saline, and resuspended with saline.

[0064] Mix G2, F5, H6, and C8 magnetic beads in a 1:1 mass ratio to create mixed magnetic beads: G2+H6, G2+C8, F5+H6, and F5+C8. Add 1 mg of G2, F5, H6, or C8 magnetic beads, or a mixed magnetic bead mixture, to 5 mL of a stool suspension containing E. coli O157:H7 (5 g of stool suspended in 5 mL of normal saline). Incubate at 37°C for 2 hours, then separate the beads using a magnetic stand. Remove any unbound microorganisms and the supernatant. The magnetic beads bound to E. coli O157:H7 were then resuspended in physiological saline. The separated magnetic beads were mixed with 0.05% papain (S10011, Yuanye Biotechnology), an antibody label removal reagent, and incubated at 37°C for 3 hours to cleave the Fc and Fab of the mouse monoclonal antibody, separating the magnetic beads from the E. coli O157:H7. The beads were then collected using a magnetic rack, and the supernatant was the E. coli O157:H7 suspension. The E. coli O157:H7 cells were diluted and added dropwise to a hemocytometer and counted under a microscope. The results are shown in Table 3. The best enrichment of E. coli O157:H7 was achieved when the antibody combination was G2 + C8 and the magnetic beads had a particle size of 200 nm.

[0065] Table 3

[0066]

[0067]

[0068] Example 5 Cultivation and Identification of Escherichia coli O157:H7

[0069] The isolated E. coli O157:H7 was diluted to 10 3 -10 4 The strain was then plated onto LB solid medium and incubated anaerobically at 37°C for 48 hours. Colony morphology was then observed. Twenty individual colonies were selected and used as templates for PCR amplification. Primers were designed using the E. coli O157:H7-specific gene rfbE, targeting a 277-bp target fragment. The upstream primer sequence was 5′-ACCAGTTTACCAACCGTCA-3′, and the downstream primer sequence was 5′-GGGTGGCTCCTGT GTATTT-3′.

[0070] The PCR reaction system consisted of 1 μL DNA template (10 ng / μL), 2 μL each of upstream and downstream primers (10 μmol / L), 5 μL of 10× PCR Buffer, 4 μL of dNTPs (2.5 mmol / L), 0.5 μL of Taq enzyme (5 U / μL), and 35.5 μL of ddH2O. PCR reaction conditions were: 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 56°C for 1 min, and 72°C for 25 s, and finally 72°C for 10 min. After completion of the reaction, the results were confirmed by agarose gel electrophoresis. The target bands were purified and sequenced. BLAST comparison of the sequencing results against the NCBI database revealed that the rfbE gene sequences of the 20 isolates shared 100% identity with E. coli O157:H7 (GenBank: AE005174.2), confirming that the isolates were E. coli O157:H7.

[0071] Example 6 Sequencing of Hybridoma Cells G2 and C8

[0072] G2 and C8 hybridoma cells were cultured and lysed, and total RNA and mRNA were extracted from the lysates. The mRNA was reverse transcribed into cDNA using random hexamer primers (5'-Pd(NNNNNN)-3'N=G, A, T or C), and then amplified by two rounds of nested PCR: the first-strand cDNA was used as a template, the forward primer was a sequence complementary to the corresponding heavy chain and light chain leader sequence, and the reverse primer was a sequence within the constant region of the heavy chain and light chain.

[0073] Heavy chain forward primer: CGGCCCAGCCGGCC, heavy chain reverse primer: TGAACCGCCTCCACC;

[0074] Light chain forward primer: GGTTCCACTGGT, light chain reverse primer: GTGCAGCATCAGC.

[0075] The PCR amplification program was as follows: denaturation at 94°C for 2 min; denaturation at 94°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 60 s, and 40 PCR cycles; and final extension at 72°C for 5 min.

[0076] In the second round, gene products with restriction enzyme sites (EcoRI and HindIII) were amplified and connected to the pMD19-T cloning vector. Then, through sequencing and analysis, the light chain and heavy chain variable region sequences of antibodies G2 and C8 were obtained.

[0077] Heavy chain forward primer: TGAATTCCGGCCCAGCCGGCC, heavy chain reverse primer: TAAGCTTTGAACCGCCTCCACC;

[0078] Light chain forward primer: TGAATTCGGTTCCACTGGT, light chain reverse primer: TAAGCTTGTGCAGCAT CAGC.

[0079] The nucleotide sequence encoding the light chain of monoclonal antibody G2 is as follows: GAAGTGACCATCACCTGTTCCGC CTCTTCCTCCGTTACCTCCATGCATTGGTATCTGCTGAAACCGGGCCAGTCCCCAAAACTGCTGATCTACAGCACCTCCAACCTGGCTTCTGGTGTTCCGGATCGTTTCTCTGGTTCCGGTTCTGGTACCGACTTCACCCTGAAGATCAGCCGCGTGGAAGCAGAAGACCTGGGCGTTTACTATTGCCAACAGCGCAGCTCCTACCCATTCACCTTTGGTGGCGGCACCAAACTGGAAATCAAA (SEQ ID NO. 3).

[0080] The amino acid sequences of the light chain CDR and FR regions of monoclonal antibody G2 are as follows:

[0081] FR-L1:EVTITC,

[0082] CDR-L1:SASSSVTSMH,

[0083] FR-L2: WYLLKPGQSPKLLIY,

[0084] CDR-L2:STSNLAS,

[0085] FR-L3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC,

[0086] CDR-L3:QQRSSYPFT,

[0087] FR-L4: FGGGTKLEIK.

[0088] The coding nucleotide sequence of the monoclonal antibody G2 heavy chain is as follows: GAGGTGAAGCTGGTGGAGTCTG GGGCTGAACTGGTGAAGCCTGGGGCCTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAAACCTGGCAACGGTCGTACTAACTACAATGAGAAGTTCTC GACCAAGGCCACTCTGACTGTAGACAAATCCTCCAACACAGCCTACATACAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCAAGGGAGGGGGATTATGATAACCCGGCCTACTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO.4).

[0089] The amino acid sequences of the heavy chain CDR and FR regions of monoclonal antibody G2 are as follows:

[0090] FR-H1: EVKLVESGAELVKPGASVKLSCKASGYTFT,

[0091] CDR-H1:NYWMH,

[0092] FR-H2: WVKQRPGQGLEWIG,

[0093] CDR-H2: EIKPGNGRTNYNEKFST,

[0094] FR-H3:KATLTVDKSSNTAYIQLSSLTSEDSAVYYCSR,

[0095] CDR-H3:EGDYDNPAYFPY,

[0096] FR-H4:WGQGTLVTVSA.

[0097] The nucleotide sequence encoding the light chain of monoclonal antibody C8 is as follows: GACATTCTGATTACTCAAACTCC GCTGTCTCTGCCGGTTTCTCTGGGTGATCAGGCATCTATCTCCTGCCGTTCTAGCCAGCGTATCGTTCACAGCAACGGCAACACCTATCTGGAGTGGTATCTGCTGAAACCAGGCCAGTCTCCAAAACTGCTGATCTACAAAGTGTTCAACCGCTTCTCCGGTGTTCCGGATCGTTTCTCTGGTTCTGGTTCCGGTACGGACTTCACTCTGAAAATCAGCCGCGTGGAAGCGGAAGATCTGGGCGTTTACTACTGTTTCCAAGGCTCTCATGTTCCGCCAACGTTCGGTGGTGGCACCCAG (SEQ ID NO. 5).

[0098] The amino acid sequences of the CDR and FR regions of the monoclonal antibody C8 light chain are as follows:

[0099] FR-L1: DILITQTPLSLPVSLGDQASISC,

[0100] CDR-L1:RSSQRIVHSNGNTYLE,

[0101] FR-L2: WYLLKPGQSPKLLIY,

[0102] CDR-L2:KVFNRFS,

[0103] FR-L3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC,

[0104] CDR-L3: FQGSHVPPT,

[0105] FR-L4: FGGGTQ.

[0106] The coding nucleotide sequence of the monoclonal antibody C8 heavy chain is as follows: CAGGTGCAGCTGAAGGAGTCTG GCCCTGGGATATTGCAGCCCTCCCAGACCCTCACTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATATCCCATCCCTGA TGAGCCGGCTCACAATCTCCAAGGATGCCTCCGGCAACCAGGTATTCCTCAAAATCACCAGTGTGGACACTGCAGATTCTGCCACATACTACTGTGCTCGAACTGATACTTCGGCTCTCCACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO.6). The amino acid sequences of the CDR and FR regions of the heavy chain of monoclonal antibody C8 are as follows: FR-H1: QVQLKESGPGILQPSQTLTLTCSFSGFSLS, CDR-H1: TSGMGVS,

[0107] FR-H2: WIRQPSGKGLEWLA,

[0108] CDR-H2: HIYWDDDKRYIPSLMS, FR-H3: RLTISKDASGNQVFLKITSVDTADSATYYCAR, CDR-H3: TDTSALHWYFDV, FR-H4: WGAGTTVTVSS.

Claims

1. An antibody against an Escherichia coli O157:H7 specific protein, characterized in that: The antibody described above is a monoclonal antibody against LPS, and its light chain CDR regions have the following amino acid sequences: CDR-L1: SASSSVTSMH, CDR-L2: STSNLAS, CDR-L3: QQRSSYPFT, and the heavy chain CDR regions have the following amino acid sequences: CDR-H1: NYWMH, CDR-H2: EIKPGNGRTNYNEKFST, CDR-H3: EGDYDNPAYFPY.

2. A nucleic acid encoding the antibody according to claim 1.

3. A magnetic bead coated with the antibody according to claim 1.

4. An antibody against an Escherichia coli O157:H7 specific protein, characterized in that: The antibody described above is a monoclonal antibody against fliC, and its light chain CDR regions have the following amino acid sequences: CDR-L1: RSSQRIVHSNGNTYLE, CDR-L2: KVFNRFS, CDR-L3: FQGSHVPPT, and the heavy chain CDR regions have the following amino acid sequences: CDR-H1: TSGMGVS, CDR-H2: HIYWDDDKRYIPSLMS, CDR-H3: TDTSALHWYFDV.

5. A nucleic acid encoding the antibody according to claim 4.

6. A magnetic bead coated with the antibody according to claim 4.

7. Use of the antibody according to claim 1 or 4 or the magnetic bead according to claim 3 or 6 in the enrichment or separation of Escherichia coli O157:H7.

8. A method for enriching or isolating Escherichia coli O157:H7, characterized in that: Enrich or separate Escherichia coli O157:H7 in a test sample using the magnetic beads according to claims 3 and 6.

9. The method according to claim 8, wherein: It includes the following steps: adding the magnetic beads according to claims 3 and 6 to the test sample for incubation to allow Escherichia coli O157:H7 to bind to the magnetic beads, and separating the magnetic beads using magnetic force; after resuspending the separated magnetic beads, adding papain for incubation to dissociate the antibody from the magnetic beads, and then adsorbing the magnetic beads using magnetic force, and the supernatant is the enriched or separated suspension of Escherichia coli O157:H7.

10. The method according to claim 8 or 9, characterized in that: The test sample described above includes fecal samples; the particle size of the magnetic beads is 200 nm.

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