Polyclonal antibody of enterohemorrhagic escherichia coli Intimin protein as well as preparation method and application of polyclonal antibody

Abstract

The invention relates to a polyclonal antibody of enterohemorrhagic Escherichia coli Intimin protein as well as a preparation method and application of the polyclonal antibody, and the polyclonal antibody of the enterohemorrhagic Escherichia coli Intimin protein can specifically recognize the enterohemorrhagic Escherichia coli Intimin protein and has relatively high titer; according to the present invention, with the enterohemorrhagic Escherichia coli Intimin protein polyclonal antibody, the infection process of enterohemorrhagic Escherichia coli can be deeply understood through the enterohemorrhagic Escherichia coli Intimin protein polyclonal antibody, such that the hereditary basis and the molecular mechanism of the enterohemorrhagic Escherichia coli infection host cell can be further researched; the method has double values for understanding pathogenesis and developing prevention and control means.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a polyclonal antibody against the intimin protein of enterohemorrhagic Escherichia coli, its preparation method, and its application. Background Technology

[0002] Escherichia coli (E. coli) is classified as either pathogenic or non-pathogenic. E. coli associated with human diseases are collectively known as diarrheagenic Escherichia coli (DEC), an important foodborne pathogen that easily infects humans. Infection can cause abdominal pain and diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, and other symptoms, threatening human health and safety. Enterohemorrhagic E. coli (EHEC), with its main serotype O157:H7, is a major foodborne pathogen that specifically colonizes the large intestine, leading to various diseases. The elderly and children are particularly susceptible; reports indicate that as few as 10 to 100 bacteria can cause human disease.

[0003] The EHEC infection process can be preliminarily divided into five stages: (1) reaching the infection site by crossing the gastrointestinal system; (2) initial adhesion and colonization of intestinal epithelial cells; (3) injection of effector proteins into colonic epithelial cells through the Type III secretion system (T3SS), causing cytoskeleton rearrangement; (4) tight adhesion between bacteria and colonic epithelial cells mediated by adhesinin; and (5) production of Shiga toxin, causing disease in the host. The EHEC infection process is strictly regulated by genes. EHEC colonizes the intestinal mucosa, inducing characteristic histopathological adhesion and effacing (A / E) damage. The characteristics of A / E damage are the local loss of microvilli at the edge of the intestinal brush hairs and the close adhesion between bacteria and host cells. The attached bacteria stimulate the cytoskeleton rearrangement of the intestinal epithelial cells, causing actin to aggregate around the bacteria and form a protruding cup-shaped base structure. The genes required for the formation of A / E lesions are located at the pathogenic island of the enterocyte disappearance (LEE). The LEE encodes a type III secretory system that can transmit various effector proteins (including Tir (translocated intimin receptor), EspF, EspG, EspH, and Map) and the intimin gene-encoded intimin protein. Intimin plays a crucial role in EHEC adhesion to intestinal epithelial cells, acting as a molecular bridge between bacteria and host cells. Tir, as an intimin receptor, interacts with intimin and recruits host proteins and induces actin rearrangement at the bacterial adhesion site, initiating the initial adhesion of EHEC to the intestine. The intimin gene-encoded protein, intimin, is essential for the formation of tight junctions between epithelial cells and is responsible for the tight junctions between bacteria and intestinal epithelial cells, thereby adhering the bacteria to the intestinal epithelial cells and initiating the infection process.

[0004] The symptoms of enterohemorrhagic Escherichia coli (EHEC) vary in severity, ranging from ordinary diarrhea to hemorrhagic colitis (HC) and even fatal hemolytic uremic syndrome (HUS). Therefore, EHEC detection is particularly important in order to better address food safety and healthcare infection issues caused by EHEC. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein, its preparation method, and its application.

[0006] The technical solution adopted in this invention is: a method for preparing polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein, using a recombinant protein containing a partial amino acid sequence of Intimin protein as shown in SEQ ID No. 1 as an antigen to prepare polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein.

[0007] Preferably, mice are immunized four times with a recombinant protein containing a portion of the amino acid sequence of the Intimin protein as an antigen, and the supernatant of mouse plasma is collected, which contains a polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein; the supernatant of mouse plasma samples can be used directly as a polyclonal antibody reagent against enterohemorrhagic Escherichia coli Intimin protein.

[0008] Preferably, polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein are obtained by enriching IgG subtype antibodies using protein A purification beads.

[0009] Preferably, a recombinant engineered bacterium capable of expressing a partial amino acid sequence of the Intimin protein as shown in SEQ ID No. 1 is constructed, the recombinant engineered bacterium is cultured and collected, and after sonication, a denaturing agent is added to transfer the recombinant protein including the partial amino acid sequence of the Intimin protein into the supernatant. The recombinant protein including the partial amino acid sequence of the Intimin protein is collected by purifying and separating the components of the supernatant; and the protein activity is then restored by dialysis.

[0010] The denaturing agent includes urea.

[0011] A method for preparing polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein.

[0012] Preferably, it is capable of specifically recognizing the Intimin protein of enterohemorrhagic Escherichia coli.

[0013] Preferably, the potency is 1:102400.

[0014] A diagnostic reagent comprising a polyclonal antibody against the enterohemorrhagic Escherichia coli Intimin protein.

[0015] Application of polyclonal antibodies or detection reagents for enterohemorrhagic Escherichia coli Intimin protein in the detection of enterohemorrhagic Escherichia coli

[0016] The advantages and positive effects of this invention are: the polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein can specifically recognize enterohemorrhagic Escherichia coli Intimin protein, and has the characteristics of high titer and good specificity. This polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein can be used for the detection of enterohemorrhagic Escherichia coli Intimin protein and subsequent research on the mechanism of enterohemorrhagic Escherichia coli Intimin protein infection of host cells as a clue. Attached Figure Description

[0017] Figure 1 Intimin target gene restriction fragment identification by gel electrophoresis;

[0018] Figure 2 Schematic diagram of plasmid structure;

[0019] Figure 3 Expression and affinity purification of recombinant PET-32a(+)-Intimin fusion protein;

[0020] Figure 4 ELISA method for determining the titer of polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein;

[0021] Figure 5 Western blot analysis of mouse serum before and after immunization with PET-32a(+)-Intimin fusion protein;

[0022] Figure 6 Western blot analysis of mouse serum before and after immunization with wild-type Intimin protein;

[0023] Figure 7 Adsorption experiment of EHEC O157:H7 wild-type strain on HeLa cells;

[0024] Figure 8 Fluorescent actin staining of HeLa cells by EHEC O157:H7 wild-type strain; a: Staining of HeLa cells in the control group; b: Staining of HeLa cells in the experimental group; c: Number of infected HeLa cells; d: Number of basal cells in HeLa cells. Detailed Implementation

[0025] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0026] This invention relates to a polyclonal antibody against enterohemorrhagic Escherichia coli (EHEC) intimin protein, its preparation method, and its application. The polyclonal antibody against EHEC intimin protein can specifically recognize the EHEC intimin protein and has a high titer. It can be prepared into a reagent for labeling intimin protein. Through the polyclonal antibody against EHEC intimin protein, we can gain a deeper understanding of the infection process of EHEC and further study the genetic basis and molecular mechanism of EHEC infection of host cells. This has dual value for understanding the pathogenesis and developing prevention and control measures.

[0027] A specific sequence was selected from the Intimin protein and used as an antigen to immunize mice. First, the genome was extracted from *Escherichia coli* enterohemorrhagic bacteria. Then, a portion of the Intimin gene from the target *Escherichia coli* was amplified using specific primers Intimin-F-XHOI and Intimin-R-BglII, as shown in SEQ ID No. 3 and SEQ ID No. 4. This amplified the partial amino acid sequence of the Intimin protein, as shown in SEQ ID No. 1 and the nucleotide sequence in SEQ ID No. 2.

[0028] SEQ ID No. 1:

[0029] MITHGCYTRTRHKHKLKKTLIMLSAGGLFFYVNQNSFANGENYFKLGSDSK

[0030] LLTHDSYQNRLFYTLKTGETVADLSKSQDINLSTIWSLNKHLYSSESEMMKA

[0031] APGQQIILPLKKLPFEYSALPLLGSAPLVAAGGVAGHTNKLTKMSPDVTKSN

[0032] MTDDKALNYAAQQAASLGSQLQSRSLNGDYAKDTALGIAGNQASSQLQAW

[0033] LQHYGTAEVNLQSGNNFDGSSLDFLLPFYDSEKMLAFGQVGARYIDSRFTAN

[0034] LGAGQRFFLPANMLGYNVFIDQDFSGDNTRLGIGGEYWRDYFKSSVNGYFR

[0035] MSGWHESYNKKDYDERPANGFDIRFNGYLPSYPALGAKLIYEQYYGDNVAL

[0036] FNSDKLQSNPGAATVGVNYTPIPLVTMGIDYRHGTGNENDLLYSMQFRYQFD

[0037] KSWSQQIEPQYVNELRTLSGSRYDLVQRNNNIILEYKKQDILSLNIPHDINGTE

[0038] HSTQKIQLIVKSKYGLDRIVWDDSALRSQGGQIQHSGSQSAQDYQAILPAYVQ

[0039] GGSNI

[0040] SEQ ID No.2:

[0041] ATGATTACTCATGGTTGTTATACCCGGACCCGGCACAAGCATAAGCTAAA

[0042] AAAAACATTGATTATGCTTAGTGCTGGTTTAGGATTGTTTTTTTATGTTAAT

[0043] CAGAATTCATTTGCAAATGGTGAAAATTATTTTAAATTGGGTTCGGATTCA

[0044] AAACTGTTAACTCATGATAGCTATCAGAATCGCCTTTTTTATACGTTGAAA

[0045] ACTGGTGAAACTGTTGCCGATCTTTCTAAATCGCAAGATATTAATTTATCG

[0046] ACGATTTGGTCGTTGAATAAGCATTTATACAGTTCTGAAAGCGAAATGAT

[0047] GAAGGCCGCGCCTGGTCAGCAGATCATTTTGCCACTCAAAAAACTTCCCT

[0048] TTGAATACAGTGCACTACCACTTTTAGGTTCGGCACCTCTTGTTGCTGCAG

[0049] GTGGTGTTGCTGGTCACACGAATAAACTGACTAAAATGTCCCCGGACGTG

[0050] ACCAAAAGCAACATGACCGATGACAAGGCATTAAATTATGCGGCACAAC

[0051] AGGCGGCGAGTCTCGGTAGCCAGCTTCAGTCGCGATCTCTGAACGGCGAT

[0052] TACGCGAAAGATACCGCTCTTGGTATCGCTGGTAACCAGGCTTCGTCACA

[0053] GTTGCAGGCCTGGTTACAACATTATGGAACGGCAGAGGTTAATCTGCAGA

[0054] GTGGTAATAACTTTGACGGTAGTTCACTGGACTTCTTATTACCGTTCTATG

[0055] ATTCCGAAAAAATGCTGGCATTTGGTCAGGTCGGAGCGCGTTACATTGAC

[0056] TCCCGCTTTACGGCAAATTTAGGTGCGGGTCAGCGTTTTTTCCTTCCTGCA

[0057] AACATGTTGGGCTATAACGTCTTCATTGATCAGGATTTTTCTGGTGATAAT

[0058] ACCCGTTTAGGTATTGGTGGCGAATACTGGCGAGACTATTTCAAAAGTAG

[0059] CGTTAACGGCTATTTCCGCATGAGCGGCTGGCATGAGTCATACAATAAGA

[0060] AAGACTATGATGAGCGCCCAGCAAATGGCTTCGATATCCGTTTTAATGGC

[0061] TATCTACCGTCATATCCGGCATTAGGCGCCAAGCTGATATATGAGCAGTA

[0062] TTATGGTGATAATGTTGCTTTGTTTAATTCTGATAAGCTGCAGTCGAATCC

[0063] TGGTGCGGCGACCGTTGGTGTAAACTATACTCCGATTCCTCTGGTGACGAT

[0064] GGGGATCGATTACCGTCATGGTACGGGTAATGAAAATGATCTCCTTTACT

[0065] CAATGCAGTTCCGTTATCAGTTTGATAAATCGTGGTCTCAGCAAATTGAAC

[0066] CACAGTATGTTAACGAGTTAAGAACATTATCAGGCAGCCGTTACGATCTG

[0067] GTTCAGCGTAATAACAATATTTCTGGAGTACAAGAAGCAGGATATTCT

[0068] TTCTCTGAATATTCCGCATGATATTAATGGTACTGAACACAGTACGCAGA

[0069] AGATTCAGTTGATCGTTAAGAGCAAATACGGTCTGGATCGTATCGTCTGG

[0070] GATGATAGTGCATTACGCAGTCAGGGCGGTCAGATTCAGCATAGCGGAAG

[0071] CCAAAGCGCACAAGACTACCAGGCTATTTTGCCTGCTTATGTGCAAGGTG

[0072] GCAGCAATATT

[0073] SEQ ID No.3Intimin-F-XHOI:

[0074] CTGCTGCTAAATTCGAACGCCAGCACATGGACAGCATGATTACTCATGGT

[0075] TGTTATA

[0076] SEQ ID No.4Intimin-R-BglII:

[0077] TTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGAATATTGCTGCCACC

[0078] TTGCACA

[0079] An expression vector containing the Intimin gene from *Escherichia coli* was constructed, and a recombinant engineered bacterium capable of expressing a partial amino acid sequence of the Intimin protein was prepared. After induction culture, a recombinant protein containing a partial amino acid sequence of the Intimin protein (PET-32a(+)-Intimin fusion protein) was prepared and purified. After ultrasonic disruption of the recombinant bacteria, the target protein PET-32a(+)-Intimin fusion protein was present in the protein precipitate. The precipitate was transferred to the supernatant by adding the denaturing agent urea. The target protein was collected by purifying and separating the components of the supernatant. Finally, the protein activity was restored by dialysis.

[0080] Mice were immunized with the PET-32a(+)-Intimin fusion protein as an antigen. After four immunizations, blood samples were collected from the mice, and serum containing polyclonal antibodies against enterohemorrhagic Escherichia coli (EHEC) Intimin protein was collected. Further, IgG subtype antibodies were enriched using protein A purification beads to obtain polyclonal antibodies against EHEC Intimin protein with higher purity. The titer of the polyclonal antibody against EHEC Intimin protein was as high as 1:102400. The preparation process of polyclonal antibodies against EHEC Intimin protein is simple, inexpensive, and fast, greatly saving time and material costs.

[0081] Polyclonal antibodies constructed using recombinant proteins containing a portion of the amino acid sequence of the Intimin protein (as shown in SEQ ID No. 3) as antigens can specifically recognize the Intimin protein, avoiding cross-reactions with other members of the Intimin protein family with similar sequences, thus preventing false positives. This significantly improves the accuracy of polyclonal antibodies as probes for detecting Intimin protein in enterohemorrhagic Escherichia coli.

[0082] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0083] Example 1: Preparation of a recombinant protein containing a portion of the amino acid sequence of the Intimin protein

[0084] 1.1 Preparation of the genome template of enterohemorrhagic Escherichia coli

[0085] Enterohemorrhagic Escherichia coli strains were inoculated into LB liquid medium and cultured overnight at 37°C to obtain bacterial culture. The genome of enterohemorrhagic Escherichia coli was extracted using a commercially available genomic kit.

[0086] 1.2 Amplification of a portion of the Intimin gene from enterohemorrhagic Escherichia coli

[0087] Prepare specific primers Intimin-F-XHOI and Intimin-R-BglII with sequences as shown in SEQ ID No.3 and SEQ ID No.4. Store the diluted Intimin-F-XHOI and Intimin-R-BglII primers at -20°C for later use, avoiding repeated freeze-thaw cycles.

[0088] SEQ ID No.3 Intimin-F-XHOI:

[0089] CTGCTGCTAAATTCGAACGCCAGCACATGGACAGCATGATTACTCATGGT

[0090] TGTTATA

[0091] SEQ ID No.4 Intimin-R-BglII:

[0092] TTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGAATATTGCTGCCACC

[0093] TTGCACA

[0094] Using the genome of enterohemorrhagic Escherichia coli as a template, PCR amplification was performed using specific primers Intimin-F-XHOI and Intimin-R-BglII.

[0095] PCR reaction system: Take a PCR tube, add 10.0 μL of 2×PCR ES Taq MasterMix, 1 μL of each primer (10 μM), 1.0 μL of template, and 7 μL of ultrapure water, and mix well;

[0096] The amplification program was as follows: pre-denaturation at 95℃ for 5 min; (denaturation at 95℃ for 30 s: annealing at 55℃ for 30 s; extension at 72℃ for 1 min) × 35 cycles; extension at 72℃ for 5 min. A partial fragment of the Intimin gene from enterohemorrhagic Escherichia coli was obtained by amplification.

[0097] 1.3 Construction of recombinant expression vectors

[0098] The obtained partial fragment of the enterohemorrhagic Escherichia coli Intimin gene was ligated into the prokaryotic expression vector pET-32a(+).

[0099] The Intimin target gene obtained by PCR was subjected to double enzyme digestion. The digestion system was as follows: 1000 ng of the Intimin target gene fragment, 1 μL of ndeI enzyme, 1 μL of BamHI enzyme, 5 μL of 10× buffer, and the remainder was made up to 50 μL with water. The reaction parameters were: 37℃, 40 min; storage at 4℃. The Intimin target gene digested fragments were identified by nucleic acid gel electrophoresis as follows. Figure 1 As shown, M represents a marker of size 2000; lane 1 is an Intimin DNA digestion fragment; lane 2 is a Pet-32a(+) digestion fragment.

[0100] The prokaryotic expression vector pET-32a(+) was double-digested with enzymes. The enzyme digestion system for the Intimin expression vector was as follows: 1000 ng of prokaryotic expression vector pET-32a(+), 1 μL of XHOI enzyme, 1 μL of BglII enzyme, 5 μL of 10x buffer, and the remainder was made up to 50 μL with water. The reaction parameters were: 37℃ for 30 min and stored at 4℃.

[0101] The digested Intimin target gene fragment was ligated into the prokaryotic expression vector pET-32a(+). The ligation system consisted of 30 ng of the Intimin target gene fragment, 100 ng of the prokaryotic expression vector pET-32a(+), 2 μL of T4 ligase, and water to a final volume of 10 μL. The reaction parameters were 22℃ for 16 h. The schematic diagram of the constructed plasmid structure is shown below. Figure 2 As shown.

[0102] 1.4 Construction of recombinant expression strains

[0103] The obtained recombinant expression vector was transferred into DH5α competent cells; 10 μL of the recombinant expression vector was added to the DH5α competent cells, and the cells were incubated on ice for 30 min, followed by heat shock at 42℃ for 45 s, and then incubated on ice for 5 min. 1 mL of LB liquid medium was added, and the cells were incubated on a shaker at 37℃ and 180 rpm for 1 h.

[0104] The fully revived DH5α cells were spread on a solid plate containing ampicillin (Amp, 50 ng / mL) and incubated at 37°C. Single colonies were picked for PCR identification. The successfully identified colonies were then placed in LB liquid medium containing Amp antibiotic and incubated overnight at 37°C. The plasmids were extracted the next day according to the plasmid extraction kit.

[0105] The protein expression vector carrying the Intimin target gene was electroporated into strain BL21, and 900 μL of BL liquid medium was added. The culture was incubated at 37°C for 1 h, and then plated onto solid plates containing Amp antibiotic and incubated overnight at 37°C. Single colonies were picked, inoculated into LB medium, and stored at -80°C. The recombinant strain obtained was named BL21-pET32a-Intimin. Sequence identification of strain BL21-pET32a-Intimin was performed. After extracting the genomic template, amplification was performed using identification primers. The primer sequences are as follows:

[0106] SEQ ID No.5pET-32a-F:CTTGTCGACGGAGCTCGAAT

[0107] SEQ ID No.6pET-32a-R:CTTCCCCATCGGTGATGTCG

[0108] Sequencing of the amplification products confirmed that the strain contained the gene sequence of the target protein.

[0109] 1.5 Preparation of recombinant proteins containing partial amino acid sequences of Intimin protein

[0110] Single clones of the recombinant expression strain BL21-pET32a-Intimin were inoculated into 20 mL of LB liquid medium containing Amp antibiotic and cultured overnight at 37°C. The culture was then expanded by inoculating 200 mL of LB liquid medium containing Amp antibiotic at a 1:100 ratio. When the absorbance (A) at 600 nm reached 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 M, and the culture was incubated at 16°C for 16 h. The bacterial cells were collected, washed twice with PBS, and resuspended in 20 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, H2O). Lysozyme and 1 mM PMSF were added to the sample, and the mixture was placed on ice and sonicated to disrupt the bacterial cells.

[0111] The ultrasonically disrupted bacterial cells were ultracentrifuged at 12,000 rpm, 4°C, for 30 min. The supernatant was removed, and protein denaturation solution (50 mM NaH2PO4, 300 mM NaCl, 8 M CO(NH2)2, H2O) was added to dissolve the centrifuged precipitate. The disrupted bacterial cell solution was then placed in a chromatography cabinet at 4°C and rotated overnight for denaturation. The overnight denatured sample was centrifuged (parameters: 12,000 rpm, 30 min, 4°C), and the collected supernatant was collected.

[0112] Recombinant proteins containing a portion of the amino acid sequence of Intimin protein were affinity purified. ddH2O and 5 mL of denaturing His resin were added to an empty purification column, and the column was incubated overnight at 4°C in a chromatography cabinet. The column was equilibrated with protein denaturing buffer, and the peristaltic pump was set to a speed of 7.5 mL / min for 30 min. The prepared sample was added to the purification column, and the sample and purification resin were incubated using a peristaltic pump at a speed of 6 mL / min for 5 h. After sufficient incubation, denaturing elution buffer I (50 mM NaH2PO4, 300 mM NaCl, 2 mM imidazole, 8 M urea) was added to wash away contaminating proteins. The elution buffer was the first elution buffer. Coomassie Brilliant Blue G-250 was used to identify whether there were still contaminating proteins in the first elution buffer. Washing continued until the effluent could no longer turn G250 blue. Denaturing elution buffer II (50 mM NaH2PO4, 300 mM NaCl, 15 mM imidazole, 8 M urea) was added to the purification column to competitively wash away contaminating proteins. This was the second elution buffer. Elution continued until the second elution buffer no longer turned the G250 blue. Then, denaturing elution buffer (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, 8 M urea) was added to the purification column to elute the target protein. This was the third elution buffer. Elution continued until the effluent from the third elution buffer no longer turned the G250 blue. All of the third elution buffer was collected and dialyzed to remove small molecule impurities such as imidazole, yielding the target protein PET-32a(+)-Intimin fusion protein. The protein concentration was determined using the BCA method, and the protein was aliquoted (50 μL / tube) and stored at -80°C. Repeated freeze-thaw cycles should be avoided before use.

[0113] The results of the detection of expression and affinity purification of recombinant PET-32a(+)-Intimin fusion protein are as follows: Figure 3 As shown, 1 is the protein precipitated after induction by isopropyl-β-D-thiogalactosidase (IPTG); 2 is the first elution buffer; 3 is the second elution buffer; and 4-7 are the third elution buffers.

[0114] Example 2: Preparation of polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein

[0115] Polyclonal antibodies were prepared by immunizing female mice around six weeks old with the refolded PET-32a(+)-Intimin fusion protein four times. The specific steps are as follows:

[0116] Initial immunization: 100 μL of complete Freund's adjuvant and 100 μL of PET-32a(+)-Intimin fusion protein (concentration approximately 1 mg / mL) -1The mixture was thoroughly mixed, ultrasonically broken down, and emulsified. Multiple subcutaneous injections were then administered into the abdomen of mice. Secondary immunization: Two weeks after the initial immunization, 100 μL of incomplete Freund's adjuvant was mixed with 100 μL of PET-32a(+)-Intimin fusion protein (approximately 1 mg / mL). -1 The mixture was thoroughly mixed, ultrasonically broken up and emulsified, and then injected subcutaneously at multiple points in the abdomen of mice. Three immunizations: One week after the second immunization, the same method as the second immunization was administered. Four immunizations: One week after the third immunization, the same method as the second immunization was administered.

[0117] One week after four immunization treatments, polyclonal antibodies were collected. Blood samples from mice were collected into 1.5 mL centrifuge tubes using the enucleation method and incubated overnight at 4°C with the tube tilted. The supernatant serum containing polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein was collected at 4000 g, 4°C, and 15 min, and aliquoted in small quantities and stored at -80°C for later use.

[0118] The polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein was enriched with IgG subtype antibodies using protein A purification beads to obtain antibodies with higher specificity. The polyclonal antibody against Intimin protein was mixed with binding buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0-7.2) at a volume ratio of 1:10, and added to a binding column pre-equilibrated with protein A packing material. After binding at 4°C for 4 hours, the column was washed with binding buffer until the G-250 color remained unchanged. Finally, elution was performed with elution buffer (100 Mm sodium citrate, pH 2.5). Neutralization solution was added to the collection tube beforehand to obtain polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein with higher purity.

[0119] The titer of the polyclonal antibody against the prepared enterohemorrhagic Escherichia coli Intimin protein was determined by ELISA, and the titer of the polyclonal antibody was 1:102400.

[0120] Example 3: Validation of the immunogenicity of polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein

[0121] Mice were used as immunization subjects. First, mouse serum was collected. The partial amino acid sequence of Intimin protein was extracted from the recombinant expression strain BL21-pET32a-Intimin constructed in Example 1 (according to the extraction method in Example 1). This protein was used as an antigen and injected into mice. After immunization, mouse serum was collected.

[0122] Using the polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein prepared in Example 2 as a probe, Western blot was performed on mouse serum samples before and after immunization (antibody dilution factor of 2000). The results are as follows: Figure 5 As shown in the figure, the left vertical axis represents the standard molecular weight of the protein. "Pre-immuune" refers to the serum before immunization for detecting enterohemorrhagic Escherichia coli Intimin protein, and "Affinity purified" refers to the serum after antigen-antibody purification for detecting enterohemorrhagic Escherichia coli Intimin protein. It can be seen that the polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein can bind to a portion of the Intimin protein's amino acid sequence.

[0123] The binding affinity of a polyclonal antibody to enterohemorrhagic Escherichia coli (EHEC) intimin protein was determined. Wild-type EHEC O157:H7 cells were collected, sonicated, and the supernatant was purified to obtain intact intimin protein. The obtained intact intimin protein was injected into mice using the method described above. Serum samples were collected before and after injection. Western blot analysis was performed using the polyclonal antibody against EHEC intimin protein as a probe (intimin-specific detection results, antibody dilution factor 2000). The results are shown below. Figure 6 As shown in the figure, polyclonal antibodies against the enterohemorrhagic Escherichia coli Intimin protein can bind to the wild-type Intimin protein.

[0124] Example 4: Functional verification of polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein

[0125] 4.1 Cell adhesion experiment

[0126] Take cultured HeLa cells, remove the culture medium, wash the cells three times with preheated PBS, add 2 mL of trypsin to digest them thoroughly, remove the trypsin, add fresh DMEM culture medium and mix well by pipetting, dispense 2 mL into 6-well cell culture plates, and culture until the cell coverage at the bottom of the well is about 90%.

[0127] Wild-type enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) strain was inoculated into 20 mL of LB medium and cultured overnight at 37°C. The overnight culture was then transferred to DMEM medium at a ratio of 1:100 and cultured in a shaker at 37°C and 180 rpm until the OD600 reached 0.6.

[0128] Remove the prepared HeLa cells, discard the culture medium from the 6-well plate, and wash three times with PBS. Add serum-free and antibiotic-free DMEM medium to three wells, along with EHEC O157:H7 at an MOI of 1:100 as a control group; add DMEM medium containing 5% serum (a polyclonal antibody containing enterohemorrhagic Escherichia coli Intimin protein prepared in Example 2), along with EHEC O157:H7 at an MOI of 1:100 as a treatment group, and incubate at 37°C in a 5% CO2 incubator for 3 hours.

[0129] Three hours after cell infection, the cells were washed three times with PBS, and then 1 mL of 0.1% SDS was added to each well, followed by uniform pipetting to fully lyse the cells. Serial dilutions were then performed, and the cells were plated and incubated at 37°C for 16 hours. Single colonies were counted, and the results are shown below. Figure 7 As shown in the figure. Adsorption experiments of wild-type EHEC O157:H7 on HeLa cells revealed that the addition of a polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein significantly inhibited the adhesion of EHEC O157:H7 to HeLa cells, thus achieving a protective effect.

[0130] 4.2 Fluorescent actin staining

[0131] After fully digesting HeLa cells with trypsin, discard the trypsin, add 5 mL of DMEM medium to resuspend the cells, mix well, and add 0.5-1 mL of the mixture to 12 mL of DMEM medium. Mix well and then add 2 mL of the mixture to each well of a six-well plate (with a cell spreader placed at the bottom of each well). Incubate at 37°C for 24 h. The cell coverage on the spreader should be approximately 50-60%.

[0132] EHEC O157:H7 1:1000 from glycerol tubes was inoculated into LB medium and cultured overnight for 12 h. The overnight cultured EHEC O157:H7 1:100 was then transferred to serum-free DMEM medium and cultured until the OD600 reached 0.6-0.8. The bacterial cells were collected, washed three times with DMEM medium, and the bacterial concentration was adjusted to OD600 of 0.4.

[0133] Remove the DMEM medium from the 6-well plate, add 100 μL of EHEC O157:H7 bacterial culture to each well, and add fresh DMEM medium to bring the volume to 2 mL. Wells containing serum-free and antibiotic-free DMEM medium serve as the control group, while wells containing DMEM medium containing 5% serum (containing a polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein) serve as the experimental group. Incubate the 6-well plate at 37°C with 5% CO2 for 3 h. Remove the medium and wash twice with preheated PBS. Fix with 3.7-4% paraformaldehyde at room temperature for 10 min, then wash twice with PBS. Add 2 mL of 0.1% Triton X-100 solution, permeate at room temperature for 20 min, and wash twice with PBS. Aspirate any remaining PBS from the 6-well plate, add 200 μL of FITC-Phalloidin to the slide, and incubate in the dark for 30-60 min. Wash 2-3 times with PBS, treat with RNase, and incubate at 37°C for 1.5 h.

[0134] Remove the smear using a hook and tweezers, quickly dry the bottom with lens paper, transfer it to a new six-well plate, add 200 μL of Propidium Iodide to the smear, and incubate in the dark for 15 min. Wash 2-3 times with PBS, remove the smear using a hook and tweezers, quickly dry the bottom with lens paper, place the cell-containing side onto a slide containing Fluoromount-G anti-quenching fluorescent mounting medium, gently wipe dry, and store in the dark at 4°C. Observe using a laser confocal microscope under 40x magnification. The results are as follows. Figure 8 As shown in the figure. Comparison of data from the control group and the experimental group shows that the number of HeLa cells infected decreased after the addition of polyclonal antibodies, and EHEC O157:H7 formed fewer substrates on HeLa cells.

[0135] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for preparing a polyclonal antibody against Intimin protein of enterohemorrhagic Escherichia coli, characterized in that: A polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein was prepared using a recombinant protein containing a partial amino acid sequence of the Intimin protein as shown in SEQ ID No. 1 as an antigen.

2. The method for preparing polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein according to claim 1, characterized in that: Mice were immunized four times with a recombinant protein containing a portion of the amino acid sequence of the Intimin protein as an antigen. The supernatant of the mouse plasma was collected, which contained polyclonal antibodies against the Intimin protein of enterohemorrhagic Escherichia coli.

3. The method for preparing polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein according to claim 2, characterized in that: Polyclonal antibodies against enterohemorrhagic Escherichia coli Intimin protein were obtained by enriching IgG subtype antibodies using protein A purification beads.

4. The method for preparing a polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein according to claim 1, characterized in that: A recombinant engineered bacterium capable of expressing a partial amino acid sequence of the Intimin protein as shown in SEQ ID No. 1 was constructed. The recombinant engineered bacterium was cultured and collected. After sonication, a denaturing agent was added to transfer the recombinant protein containing the partial amino acid sequence of the Intimin protein into the supernatant. The recombinant protein containing the partial amino acid sequence of the Intimin protein was collected by purifying and separating the components of the supernatant. Protein activity is then restored through dialysis.

5. The polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein prepared by the method described in any one of claims 1-4.

6. The polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein according to claim 5, characterized in that: It can specifically recognize the Intimin protein of enterohemorrhagic Escherichia coli.

7. The polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein according to claim 5, characterized in that: The potency is 1:102400.

8. A detection reagent, characterized in that: A polyclonal antibody including the enterohemorrhagic Escherichia coli Intimin protein as described in any of claims 5-7.

9. The use of the polyclonal antibody against enterohemorrhagic Escherichia coli Intimin protein as described in any one of claims 5-7 or the detection reagent as described in claim 8 in the detection of enterohemorrhagic Escherichia coli.

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