CN116554300B - A polypeptide capable of interacting with Clostridium difficile toxin TcdB and its application - Google Patents

Abstract

The invention provides a polypeptide that interacts with Clostridium difficile toxin TcdB, which is composed of 200 amino acids from the N-terminus of human CD44 protein (or amino acids at positions 21-220 of the extracellular segment, CD44 Extracellular domain, CD44-ECD). This invention first discovered that CD44 has the function of a cell receptor for Clostridium difficile cytotoxin TcdB. Using CD44-ECD to block the combination of TcdB and macrophages can significantly inhibit the secretion of IL-1β and IL-6 by macrophages and relieve TcdB The mediated excessive inflammatory response damages intestinal tissue, thereby effectively treating Clostridium difficile infection and providing a new idea for the treatment of Clostridium difficile infection.

Description

A polypeptide capable of interacting with Clostridium difficile toxin TcdB and its application

Technical field

The present invention relates to the technical fields of protein engineering, molecular microbiology and infection immunology, and specifically relates to a polypeptide capable of interacting with Clostridium difficile toxin TcdB and its application, which can be used to treat Clostridium difficile infection.

Background technique

Clostridioides diffcile (CD) is the main pathogen causing hospitalization for infectious diarrhea. It can cause varying degrees of diarrhea, fever, blood in the stool, even intestinal perforation and abdominal infection, and even lead to patient death. CD is highly toxic, highly resistant to most antibiotics, has an increase in multi-drug-resistant strains, and has a high recurrence rate after infection has recovered. It is a very difficult to treat infectious disease, and its morbidity and mortality continue to rise, which has brought serious consequences to public health. A serious threat, new treatments are urgently needed.

The main effectors of the pathogenesis of CD are the various toxins secreted by it, among which toxin B (TcdB) is the most critical in the pathogenesis process. TcdB alone can induce death in animal models. TcdB enters the interior of host cells through receptor mediation, triggering cell apoptosis, degeneration, pyknosis, and exudation of fibrin and mucin to form pseudomembranous inflammation; on the other hand, TcdB can also stimulate immune cells to release excessive inflammatory factors, It triggers an inflammatory cascade, thereby aggravating the damage to normal cells, and can lead to increased permeability of the intestinal mucosa and blood vessels, resulting in the loss of the body's protective barrier, promoting TcdB to enter the lamina propria of intestinal tissue, further aggravating intestinal tissue damage.

TcdB protein is a huge protein containing 2366 amino acid sequences. It is mainly divided into four structural and functional domains, including the combined repeat oligopeptides (CROPs, amino acids 1834-2366) domain and the newly discovered Frizzled receptor binding domain (FBD) in recent years. , 1285-1804 amino acids) can bind to cell surface receptors. Among them, the CROPs domain-binding receptor is chondroitin sulfate proteoglycan CSPG4, and the FBD-binding receptor is Frizzled protein FZDs, tissue factor pathway inhibitor TFPI, poliovirus receptor-like protein 3 (PVRL3), etc., which mediate TcdB Enter cells and exert their toxic effects. At present, the main receptors of TcdB are mainly found in intestinal epithelial cells or other tool cells (such as HeLa cells). These receptors do not mediate inflammatory reactions in immune cells, and there is no relevant immune cell receptor protein used by TcdB. Report. After TcdB removes CROPs, the affinity with host cells is still strong, and CROPs may not be the most important binding site. Therefore, the present invention uses the FBD region to screen immune cell receptor proteins.

Macrophages are common immune cells in intestinal tissue. While TcdB destroys intestinal epithelial cells, it can also stimulate macrophages to secrete a large number of pro-inflammatory cytokines (such as IL-1β, IL-6, etc.), and these cytokines can Kill normal intestinal epithelial cells, thereby further aggravating intestinal tissue damage.

Contents of the invention

The present invention aims to explore receptor proteins that can interact with Clostridium difficile toxin TcdB (receptor proteins are generally cell transmembrane proteins, divided into extracellular segments, transmembrane regions and intracellular segments, in which the extracellular segment is followed by External antigen-binding part), and the extracellular segment of this protein blocks the binding of TcdB to macrophages, reduces the excessive inflammatory response mediated by macrophages, and thereby alleviates the impact of inflammatory factors secreted by macrophages on normal intestinal epithelial cells. damage as a potential drug for the treatment of Clostridium difficile infection.

The object of the present invention is to provide a polypeptide that interacts with Clostridium difficile toxin TcdB.

In order to achieve the purpose of the present invention, a polypeptide of the present invention that interacts with Clostridium difficile toxin TcdB is the N-terminus of human CD44 protein (or amino acids 21-220 of the extracellular segment, CD44 Extracellular domain, CD44-ECD, or Called CD44-N ^21-220 ), its amino acid sequence is shown in SEQ ID No. 1, consisting of a total of 200 amino acids from the 21st to 220th position of the N-terminus of the CD44 protein, or the sequence has been replaced, deleted or added with one or more Amino acids are functionally equivalent amino acid sequences.

The invention also provides a drug against Clostridium difficile infection, the active ingredient of which is CD44-N ^21-220 .

The present invention also provides the use of the polypeptide CD44-N ^21-220 in the preparation of drugs against Clostridium difficile infection.

The present invention discovered for the first time that CD44 is the cell receptor of Clostridium difficile toxin TcdB. Since the CD44 full-length protein is relatively large (composed of 742 amino acids, with a molecular weight of more than 80KD, and its amino acid sequence is shown in SEQ ID No. 1), its application is limited. Selecting its extracellular segment can block TcdB and macrophages. interaction, thereby inhibiting the TcdB-mediated secretion of IL-1β and IL-6 by macrophages, alleviating the damage to intestinal tissue caused by the inflammatory response, thereby effectively treating Clostridium difficile infection. Considering the indispensable role of TcdB in C. difficile infection and its ability to significantly activate macrophage inflammation, CD44 will be an important target for the treatment of C. difficile infection.

CD44 is a type I transmembrane protein (Gen Bank: NP_000601.3) that participates in cell-cell and cell-matrix interactions and signal transduction. It is expressed on most immune cells and induces inflammatory responses after activation by antigens. The N-terminus of human CD44 has a 20-amino-acid (aa) signal sequence and an N-terminal constant part ECD (aa 21-220), of which ECD is the main part that binds to antigen. The ECD of human CD44 shares 76%, 76%, 86%, 83% and 79% identity with the corresponding mouse, rat, equine, canine and bovine CD44 respectively (Pure, E. and R.K. Assoian (2009) Cell. Signal. 21:651; Ponta, H. et al. (2003) Nat. Rev. Mol. Cell Biol. 4: 33.), therefore the ECD of human CD44 has a wide range of uses.

The technical solution adopted to achieve the above object of the present invention is: use the FBD region of Clostridium difficile toxin TcdB to screen immune cell receptor proteins, first prokaryotically express and purify the FBD protein, and then use a kit to extract human macrophage cell line THP -1 cell membrane protein, after the two are incubated and combined, they are sent for mass spectrometry, and bioinformatics, co-IP and other technologies are used to predict and verify that CD44 is the receptor of TcdB. Furthermore, the extracellular segment of CD44 was selected for relevant functional testing.

Compared with the prior art, the beneficial effects and advantages of the present invention are:

1. The present invention found that CD44-ECD can block the combination of TcdB and CD44, thereby significantly inhibiting the secretion of inflammatory factors IL-1β and IL-6 by macrophages mediated by TcdB, and treating the inflammatory reaction mediated by toxin TcdB on colon epithelial cells. of damage.

2. The present invention found that CD44-ECD only interferes with the binding of toxin TcdB to the receptor, without the pressure of antibiotic selection, and can avoid the drug resistance problem caused by antibiotic therapy. It can be seen that the present invention can overcome the clinical emergence of Clostridium difficile infection. It provides new solutions to the increasingly serious problems of antibiotic resistance and side effects.

3. Animal experiment results show that after mice are infected with TcdB and treated with the CD44-ECD of the present invention, the inflammatory response is significantly reduced and the pathological changes of the colon are significantly alleviated. Overall, it has a good treatment for TcdB-infected mice. effect.

Description of the drawings

Figure 1 is a gel electrophoresis picture of the TcdB receptor binding region FBD recombinant protein successfully extracted and purified in Example 1.

Figure 2 shows the differential proteins related to inflammatory response screened by mass spectrometry in Example 1.

Figure 3 shows the use of co-immunoprecipitation in Example 1 to verify the receptor protein binding to FBD.

Figure 4 is a graph showing the affinity results between CD44-ECD at different doses and FBD protein and TcdB protein in Example 1.

Figure 5 is a graph showing the binding percentage of macrophages to toxin TcdB in the presence of CD44-ECD.

Figure 6 is a graph comparing the binding properties of CD44 and toxin TcdB in the presence of CD44-ECD.

Figure 7 is a comparison chart of the inhibitory effect of toxin TcdB on stimulating macrophages to secrete inflammatory factors in the presence of CD44-ECD.

Figure 8 is a graph comparing the inhibitory effects of toxin TcdB on colon epithelial cell proliferation in the presence of CD44-ECD. Figure 8(A) shows the proliferation of colon epithelial cells captured by the confocal high-content imaging system, and Figure 8(B) is a statistical chart of colon epithelial cell proliferation relative to cell area.

Figure 9 is an immunofluorescence staining image of mouse colon tissue sections infused with TcdB intrarectally in the presence of CD44-ECD: Figure 9(A) is a picture of the expression of IL-1β inflammatory factors, and Figure 9(B) is IL -6 Expression diagram of inflammatory factors.

Figure 10 is a HE staining image of mouse colon tissue infused with TcdB intrarectally in the presence of CD44-ECD.

Figure 11 is a histochemical score chart of mouse colon tissue infused with TcdB intrarectally in the presence of CD44-ECD: Figure 11(A) shows the overall view; Figure 11(B) shows the degree of epithelial destruction; Figure 11 (C) shows the infiltration of inflammatory cells; Figure 11(D) shows the aspect of edema.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments.

Example 1 Mass spectrometry technology screens and verifies that CD44 is a receptor capable of binding to the FBD region of TcdB

1. Express the purified FBD region protein of TcdB (referred to as FBD protein)

Express and purify according to the method of reference "Peng Chen et al, Science.2018;360(6389):664-669". The specific process is as follows:

1) Clone the TcdB-FBD gene (residues 1285-1804) into the modified pET28a vector, and insert a 6×His-SUMO tag at its N-terminus.

2) TcdB-FBD is expressed in E. coli BL21-Star (DE3) (Invitrogen). Bacteria were cultured in LB medium containing kanamycin at 37°C. When OD ₆₀₀ reaches 0.8, the temperature drops to 16°C. Expression was induced with 1 mM IPTG (isopropyl-bD-thiogalactopyranoside) and maintained overnight at 16°C. Cells were collected by centrifugation and stored at -80°C until use.

3) Purify TcdB-FBD protein using Ni ²⁺ -NTA (nitrate triacetic acid, Qiagen) affinity resin in a buffer containing 50mM Tris, pH 8.0, 400mM NaCl and 40mM imidazole. Proteins were eluted with high imidazole buffer (50mM Tris, pH 8.0, 400mM NaCl, 300mM imidazole) and then dialyzed at 4°C against a buffer containing 20mM HEPES, pH 7.5, 150mM NaCl.

The electrophoresis results of TcdB-FBD protein are shown in Figure 1. The protein carries 6×His-SUMO tag for pull-down and cell surface binding analysis.

2. Extraction of macrophage membrane proteins

1) Mass-cultured human macrophage cell line THP-1 cells were induced with PMA (phorbol 12-myristate 13-acetic acid) at a concentration of 100ng/mL for 48 hours to differentiate THP-1 cells into macrophages. Wash once with phosphate buffer saline (PBS), scrape the cells with a cell scraper, collect the cells by centrifugation, aspirate the supernatant, and leave the cell pellet for later use.

2) Wash cells: Gently resuspend the cell pellet in an appropriate amount of ice-cooled PBS, take a small amount of cells for counting, and centrifuge the remaining cells at 4°C and 600g for 5 minutes to pellet the cells. Discard the supernatant, and then centrifuge at 4°C and 600g for 1 minute to precipitate the remaining liquid on the wall of the centrifuge tube and further precipitate the cells, and try your best to absorb the remaining liquid.

3) Cell pretreatment: Add 1 ml of membrane protein extraction reagent A with PMSF (phenylmethylsulfonyl fluoride, a serine protease inhibitor) added before use to 20-50 million cells, and gently and fully suspend the cells. Leave in ice bath for 10-15min.

4) Identification of cell disruption and disruption effect: freeze and thaw the sample in the previous step twice in liquid nitrogen and room temperature, and then take a small amount of sample to detect the degree of cell disruption under a microscope.

5) Remove cell nuclei and unbroken cells: centrifuge at 700g for 10 minutes at 4°C and carefully collect the supernatant into a new centrifuge tube.

Do not touch the sediment when aspirating the supernatant!

6) Precipitate cell membrane fragments: Centrifuge at 14000g for 30 minutes at 4°C to precipitate cell membrane fragments.

7) Extract membrane proteins: Centrifuge at 14000g for 10 seconds at 4°C, and try to absorb as much supernatant as possible. Add membrane protein extraction reagent B, vortex at high speed for 5 seconds to resuspend the pellet, and keep in ice bath for 5-10 minutes. Repeat vortexing and ice bathing 1-2 times to fully extract membrane proteins. Finally, centrifuge at 14,000g for 5 minutes at 4°C, and collect the supernatant as the cell membrane protein solution, which is set aside for use.

3. Preparation before measuring mass spectrometry:

1) Add 10 μg of the FBD protein purified in step 1, the purchased commercial His-tagged protein and 20 μl of Ni-NTA (nickel NTA affinity chromatography medium) agarose beads into PBS, and incubate at 4°C for 4 hours. Get FBD-Ni-NTA.

2) Add the above FBD-Ni-NTA and Ni-NTA to the cell membrane proteins extracted in step 2, and incubate at 4°C overnight;

3) Collect the Ni-NTA column by centrifugation, wash it twice with PBS, and use 200mM imidazole to elute the bound protein;

4) The eluted protein is sent directly to the company for mass spectrometry.

4. Proteins related to inflammation caused by FBD protein screened by mass spectrometry technology

Affinity Capture-MS is currently the most widely used protein interaction research method. This method is also called AP-MS (Affinity Purification coupled MS), that is, affinity purification tandem mass spectrometry analysis. The main principle of AP-MS is to purify the target protein and its interacting proteins from complex biological systems based on the affinity between the affinity reagent and the target protein, and then perform mass spectrometry detection to identify the interacting proteins of the target protein. . Classic AP-MS experiments usually include the following four key steps: 1) lysis of cells or organelles; 2) incubation of protein solution and affinity beads; 3) cleaning of non-specific binding and background proteins as well as specific interacting proteins. Elution; 4) Mass spectrometry detection and analysis of interacting proteins.

The present invention is carried out according to the above steps. First, macrophage membrane proteins are extracted, and then the microbeads of FBD protein coupled to Ni-NTA and the control protein His are incubated with them respectively. After cleaning, the macrophage membrane proteins are combined with FBD protein and His protein. Different target proteins in the protein can be detected by AP-MS technology, and the confidence value can be calculated and sorted based on the abundance of the detected target proteins, which is the screened differential protein. There are many differential proteins, but according to the test results and relevant literature reports, there are five proteins related to inflammation. The results are shown in Figure 2. Since the differential proteins screened by mass spectrometry are only for reference, the target proteins need to be further verified and confirmed. It is planned to select the three proteins with higher scores for further co-immunoprecipitation verification.

5. Co-immunoprecipitation to verify the receptor protein binding to FBD

Co-immunoprecipitation is a classic method for studying protein interactions based on the specific interaction between antibodies and antigens. It is an effective method to determine the physiological interaction of two proteins in intact cells. The target protein obtained by this method is naturally combined with the protein of interest in cells, which is consistent with the actual situation in the body, and the results obtained are highly credible. This method is commonly used to determine whether two target proteins bind in vivo.

1) A large number of cultured human macrophage cell line THP-1 cells were induced with PMA at a concentration of 100ng/mL for 48 hours to differentiate the THP-1 cells into macrophages. The FBD-his protein obtained in step 1 was added to stimulate for 2 hours. No stimulation is the control group.

2) Wash once with phosphate buffer saline (PBS), add RIPA lysis buffer and incubate overnight at 4°C.

3) After the incubation time is up, add Protein-Ni magnetic beads to pull out the FBD-his protein and other protein complexes, and incubate at 4°C for 2 hours.

4) Use a magnetic stand to pull out the magnetic bead complex, wash it twice with PBST (phosphate Tween buffer), add eluent, and elute the pulled complex. If the protein complex contains proteins that can interact with FBD If the target protein acts on it, a conjugate of the target protein, FBD-his and Protein-Ni magnetic beads can be formed. Add SDS-Loading Buffer for electrophoresis of the above complex, and use different CD44, HSP90AB1, and IGHA1 antibodies (Thermofisher Company, product numbers: 14-0441-82, MA5-33168, MA5-31774 respectively) to perform western blot to verify the properties of the complex. The formation situation, the results are shown in Figure 3. The Protein-Ni magnetic beads coupled to FBD-his protein did not pull out HSP90AB1 and IGHA1 proteins, but CD44 protein was pulled out. The Protein-Ni magnetic beads coupled to the His protein in the control group No protein was extracted, and the Input group was a normal cell group without any protein added, indicating that the cells in each group this time contained the protein to be extracted, which is the positive control group. Na/K ATPase is an internal reference control for cell membrane proteins, indicating the reliability of this result. This is a routine requirement for western blot experiments.

The above results show that FBD can interact with CD44, that is, the two can combine at the cellular level.

6. The affinity results of different doses of CD44-ECD with FBD protein and TcdB protein.

1) Dissolve the FBD protein or TcdB protein of prokaryotic expression and purification of TcdB in the coating buffer (pH9.6 carbonate buffer: 40mM Na ₂ CO ₃ , 60mM NaHCO ₃ ) and coat it on a 96-well ELISA plate, 4 ℃ overnight and then wash the plate with PBS.

2) Purchase Fc-labeled CD44-ECD protein peptide (Sinobiological Company, Cat. No. 12211-H02H, namely amino acids 21-220 of the extracellular domain of human CD44 protein ^{, CD44 Extracellular domain, CD44-ECD, or CD44-N 21-220 )} , the amino acid sequence of CD44-ECD is shown in SEQ ID No. 2, Fc-labeled CD44-ECD was set up in 12 concentration gradients (0-1024nM), and different concentrations of Fc-labeled CD44-ECD and FBD or TcdB protein ( 500ng/mL) affinity of target protein;

3) The results are shown in Figure 4. It can be seen from Figure 4 that at a fixed concentration of target protein, as the concentration of CD44-ECD increases, its OD value also increases, but as the concentration of CD44-ECD increases, the Its OD value is also gradually approaching the platform. The affinity value K _D was calculated and the results showed that the K _D value of FBD was 82.08±1.372nM and the K _D value of TcdB protein was 77.98±1.421nM. K _D values are all in the order of nM, showing that CD44-ECD has a higher affinity for FBD or TcdB protein.

The above results prove that CD44 is a receptor that can bind to the FBD region of TcdB.

Example 2 Experiment on the mechanism of CD44-ECD blocking the binding of TcdB toxin to macrophages

1. The binding percentage of macrophages to toxin TcdB in the presence of CD44-ECD.

1) Cultured human macrophage cell line THP-1 cells (1×10 ⁶ cells/well) were subcultured and seeded in six-well plates, and induced with PMA at a concentration of 100ng/mL for 48 hours to differentiate THP-1 cells into macrophages. , washed once with phosphate buffer saline (PBS) to obtain three groups of macrophages;

2) Change to fresh culture medium, add TcdB protein to two groups of culture media respectively, and add CD44-ECD (concentration of 10 μg/mL) to one group of culture media containing TcdB protein (concentration: 10 μg/mL) for blocking. Incubate in cell culture incubator for 3 hours;

3) After washing three times with PBS, remove non-specifically bound proteins, scrape the cells with a cell scraper, collect the cells by centrifugation, and aspirate the supernatant;

4) Add primary antibody (rabbit-derived anti-TcdB antibody, Abcam Company, product number ab270452), and incubate on ice for 1 hour;

5) After washing with PBS, add the fluorescently labeled anti-rabbit secondary antibody to the corresponding groups of cells above at the dilution concentration according to the antibody instructions, and incubate on ice for 1 hour;

6) Wash once with PBS, resuspend and test on the machine, and make statistics on the binding percentage (Bindingpercentage) results detected by flow cytometry.

Test results: The results are shown in Figure 5. CD44-ECD can block the binding of toxin TcdB to macrophages.

2. Comparison of the binding properties of CD44 and toxin TcdB in the presence of CD44-ECD.

1) Coat TcdB toxin (500ng/mL) on the ELISA plate, place it in a humid box at 37°C and incubate it for 1 hour, then wash it 5 times with PBST;

2) Add 200 μL salmon sperm DNA blocking solution (100 μg/mL) and incubate at 37°C for 1 hour;

3) After washing three times with PBST, add CD44-ECD at a concentration of 300nM, and incubate in a humid box at 37°C for 1 hour;

4) Add His-tagged CD44 (500ng/mL) respectively and incubate at 37°C for 1 hour;

6) Wash 3 times with PBST, add 100 μL of 1:3000 diluted horseradish peroxidase (HRP)-labeled anti-his antibody to each well, and incubate in a 37°C humid box for 30 minutes;

7) After washing three times with PBST, add TMB chromogenic solution for color development, and incubate at 37°C for 8 minutes;

8) After adding the ELISA stop solution, detect OD _{450 nm} with a microplate reader.

Test results: The results are shown in Figure 6. It can be seen from Figure 6 that the binding between CD44 and toxin TcdB is significantly weakened after adding CD44-ECD. CD44-ECD can block the binding between CD44 and toxin TcdB, that is, CD44-ECD blocks the passage of Block the binding of toxin TcdB to the CD44 receptor, thereby inhibiting the binding of TcdB to macrophages.

Example 3 CD44-ECD significantly inhibits TcdB stimulation of THP-1-derived macrophages to secrete inflammatory factors, thereby reducing the damage of inflammatory response to intestinal epithelial cells Caco-2

1. CD44-ECD significantly inhibits TcdB from stimulating THP-1-derived macrophages to secrete inflammatory factors.

1) Use PMA at a concentration of 100ng/mL to induce THP-1 cells for 48 hours to differentiate into macrophages. Use TcdB and TcdB+CD44-ECD to stimulate macrophages respectively. After 48 hours of stimulation, collect the cell culture supernatant. Add the clear and diluted gradient concentration standards to the wells (100 μL/well) respectively, and incubate in a 37°C incubator for 90 minutes;

2) Add 350 μL of washing solution to each well, shake for 2 minutes and shake off the liquid, pat dry on thick absorbent paper, repeat washing the plate 4 times and shake off the liquid in the well;

3) Dilute the biotin-labeled IL-1β or IL-6 antibody with the antibody diluent at 1:100, add it to the well after dilution (100 μL/well), incubate in a 37°C incubator for 60 minutes, and then wash the plate 4 times. The method of washing the plate is the same as step 2);

4) Dilute the enzyme conjugate (horseradish peroxide-labeled streptavidin) and the enzyme conjugate diluent at 1:100, add it to the well after dilution (100 μL/well), and incubate in a 37°C incubator. After 30 minutes, wash the plate 5 times. The method of washing the plate is the same as step 2);

5) Add 100 μL/well of chromogenic reagent, protect from light, and incubate in a 37°C incubator for 10 minutes;

6) Add 100 μL/well of stop solution, mix and measure the OD450 value immediately;

7) Use Excel software to draw a standard curve from the obtained results, convert this to obtain the sample value, and perform statistical analysis;

Test results: The results are shown in Figure 7. CD44-ECD significantly inhibited TcdB-stimulated macrophages to secrete IL-1β and IL-6, and the results were statistically significant.

2. In the presence of CD44-ECD, the toxin TcdB activates and stimulates macrophages, which can reduce the inhibitory effect of inflammatory response on colon epithelial cell proliferation.

1) The colon epithelial Caco-2 cells growing in the logarithmic phase were subcultured into a 24-well plate at 2 × 10 ⁵ cells/well (500 μL, 4 × 10 ⁵ cells/mL), and cultured in an incubator for 48 hours; Add TcdB and TcdB+CD44-ECD respectively, and continue culturing for 48 hours;

2) Use PMA at a concentration of 100ng/mL to induce THP-1 cells for 48 hours to differentiate into macrophages. Stimulate macrophages with TcdB and TcdB+CD44-ECD respectively. After 6 hours of stimulation, wash three times with PBS and scrape with a cell scraper. Remove the cells, collect them by centrifugation, aspirate the supernatant, transfer the collected cells into Transwell dishes at a ratio of 1:1, and place them into the above-mentioned 24-well plates to co-culture with Caco-2 cells. Since macrophages have been stimulated by TcdB and TcdB+CD44-ECD respectively, they will produce different concentrations of inflammatory factors IL-1β and IL-6, resulting in different degrees of inhibition of the proliferation of colon epithelial Caco-2 cells;

3) Use the confocal high-content imaging analysis system to observe the cell growth status, and use the system's built-in function to detect the cell area.

Test results: The cell growth status diagram is shown in Figure 8. Figure 8(A) shows the proliferation of colon epithelial cells captured by the confocal high-content imaging system. Figure 8(B) shows the statistics of colon epithelial cell proliferation relative to cell area. picture. As can be seen from Figure 8, IL-1β and IL-6 cytokines, as pro-inflammatory factors, will aggravate the damage of normal tissues, while CD44-ECD can significantly inhibit the TcdB-mediated inflammatory response, thus relieving the inhibitory effect on cell proliferation.

Example 4 In vivo mouse experiment of CD44-ECD

1. Construction and grouping of mouse models

1) 18 female BALB/c mice (age: 6-8 weeks, purchased from the Experimental Animal Center of Hubei Medical University) were randomly divided into 3 groups, 6 mice in each group;

2) Combine 500 pM CD44-ECD with 100 pM TcdB at 37°C for 2 hours, wash twice with PBS, and resuspend in 100 μL PBS to prepare an infection reagent containing CD44-ECD;

3) Treat each group of mice for intrarectal infusion infection according to the following table:

Table 2. Grouping and infection treatment of BALB/c mice

During infection treatment, mice were anesthetized with 1% sodium pentobarbital (use concentration: 50 mg/kg), a 1 mm diameter tube was inserted into the rectum (~4 cm) from the anus and the infection reagent was injected, followed by medical anastomotic glue. The anus was sealed to prevent the leakage of toxins. During the experiment, the mice in each group had free access to drinking water and food (regular diet);

2. Immunofluorescence detection of mouse colon tissue

1) Dewax the paraffin sections to water: Place the sections in sequence in xylene I 15min - xylene II 15min - absolute ethanol I 5min - absolute ethanol II 5min - 85% alcohol 5min - 75% alcohol 5min - wash with distilled water;

2) Antigen retrieval: Place tissue slices in a repair box filled with EDTA antigen retrieval buffer (pH9.0) and perform antigen retrieval in a microwave oven. After medium to boiling, turn off the power and wait for 10 minutes. During this process, Excessive evaporation of the buffer should be prevented and the slides must not be dried. After natural cooling, place the slides in PBS (pH 7.4) and shake them on a destaining shaker for 3 times, 5 minutes each time;

3) BSA sealing: After shaking the section slightly, use a histochemistry pen to draw a circle around the tissue (to prevent the antibody from flowing away), drop 3% BSA in the circle to evenly cover the tissue, and seal at room temperature for 30 minutes;

4) Add primary antibody: Gently shake off the blocking solution, and drop the primary antibody prepared in PBS in a certain proportion on the section (primary antibody for macrophage surface marker CD68, cytokines IL-1β and IL-6 in the tissue) ), place the slices flat in a humidified box and incubate at 4°C overnight (add a small amount of water to the humidified box to prevent antibody evaporation);

5) Add secondary antibodies: Place the slides in PBS (pH 7.4) and wash 3 times on a destaining shaker, 5 minutes each time. After the slices are slightly dried, a secondary antibody of the same species as the primary antibody is dropped into the circle to cover the tissue, and incubated in the dark for 50 minutes;

6) DAPI counterstained cell nuclei: Place the slides in PBS (pH 7.4) and shake and wash on a destaining shaker 3 times, 5 minutes each time. After the slices are slightly dried, DAPI dye solution is added dropwise into the circle, and incubated at room temperature for 10 minutes in the dark;

7) Sealing: Place the slides in PBS (pH 7.4) and shake on a destaining shaker for 3 times for 5 minutes each time. Dry the sections briefly and then mount them with anti-fluorescence quenching mounting medium;

8) Microscopic examination and photography: observe the sections under a confocal microscope and collect images;

Test results: The results are shown in Figure 9. Compared with the TcdB group, the TcdB+CD44-ECD group has a lower proportion of macrophage surface marker CD68-positive cells in the tissue, which can reduce macrophage infiltration and inhibit IL- 1β (Figure 9A) and IL-6 (Figure 9B) secretion. This trend is consistent with the results shown in Figure 7, further proving that CD44-ECD can significantly reduce TcdB-mediated macrophage secretion of IL-1β and IL-6 and other inflammatory conditions. factor level.

3. HE staining of mouse colon tissue

1) Preparing slices: Wash the dissected colons of the mice with intrarectal instillation in step 1 of this example with PBS, and embed the colon tissue fixed with 4% paraformaldehyde in conventional paraffin, and then make 4 μm slices;

2) Drying the slices: Place the slices in hot water and iron them flat, then attach them to the glass slide and place them in a 45°C constant temperature oven to dry;

3) Dewax the paraffin sections to water: Place the sections into xylene I 10min - xylene II 10min - absolute ethanol I 5min - absolute ethanol II 5min - 95% alcohol 5min - 90% alcohol 5min - 80% alcohol 5min - 70% alcohol 5min-wash with distilled water;

4) Hematoxylin staining of cell nuclei: Stain the sections with hematoxylin for 6 minutes, wash with tap water, then differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, return to blue with 0.6% ammonia, and rinse with running water;

5) Eosin staining of cytoplasm: Stain the sections in eosin staining solution for 2 minutes;

6) Dehydration and sealing: Place the slices in sequence in 95% alcohol I 5min-95% alcohol II 5min-absolute ethanol I 5min-absolute ethanol II 5min-xylene I 5min-xylene II 5min to dehydrate and make them transparent, and remove the slices from xylene Let it dry for a while, then seal it with neutral gum;

7) Upright microscope examination: The stained sections were scored by two pathologists based on the overall appearance, epithelial destruction, inflammatory cell infiltration, and edema degree on a scale of 0 to 3 (mild to severe);

Test results:

The HE staining picture of the mouse colon tissue is shown in Figure 10. The overall view of the mouse colon tissue, the degree of epithelial destruction, inflammatory cell infiltration, edema and other aspects of the score are shown in Figure 11. Figure 10 shows the colon epithelium of the TcdB stimulation group. Destruction, obvious submucosal edema, the lamina propria secreted a large number of inflammatory cells, and the overall colon tissue showed a state of hemorrhagic edema. However, after CD44-ECD treatment, the overall view of the mouse colon tissue (Figure 11A) and the degree of epithelial destruction (Figure 11B) , inflammatory cell infiltration (Figure 11C), edema (Figure 11D) and other aspects were significantly improved.

Claims (2)

1. The application of the polypeptide capable of interacting with clostridium difficile toxin TcdB in preparing a medicine for resisting clostridium difficile infection is characterized in that the polypeptide capable of interacting with clostridium difficile toxin TcdB is the 21 st-220 th amino acid sequence shown as SEQ ID No. 1.

2. A medicament against clostridium difficile infection, wherein the active ingredient is a polypeptide according to claim 1.