CN116554300B - Polypeptide capable of interacting with clostridium difficile toxin TcdB and application thereof - Google Patents
Polypeptide capable of interacting with clostridium difficile toxin TcdB and application thereof Download PDFInfo
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- CN116554300B CN116554300B CN202310466044.2A CN202310466044A CN116554300B CN 116554300 B CN116554300 B CN 116554300B CN 202310466044 A CN202310466044 A CN 202310466044A CN 116554300 B CN116554300 B CN 116554300B
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Abstract
The invention provides a polypeptide which interacts with clostridium difficile toxin TcdB, and consists of 200 amino acids at the N-terminal (or called extracellular 21-220 amino acids, CD44Extracellular domain and CD 44-ECD) of human CD44 protein. The invention discovers that CD44 has the function of the TcdB cell receptor of clostridium difficile cytotoxin for the first time, uses CD44-ECD to block the combination of TcdB and macrophages, can obviously inhibit the macrophages from secreting IL-1 beta and IL-6, and relieves the damage of TcdB-mediated excessive inflammatory reaction to intestinal tissues, thereby effectively treating clostridium difficile infection and providing a new thought for treating clostridium difficile infection.
Description
Technical Field
The invention relates to the technical fields of protein engineering, molecular microbiology and infection immunology, in particular to a polypeptide capable of interacting with clostridium difficile toxin TcdB and application thereof, which can be used for treating clostridium difficile infection.
Background
Clostridium difficile (Clostridioides diffcile, CD) is the most predominant pathogen causing hospitalization for infectious diarrhea, and can cause varying degrees of diarrhea, fever, hematochezia, and even intestinal perforation and abdominal infections, and even death in patients. The CD toxicity is high, the antibiotics have high resistance, the number of multiple drug-resistant strains is increased, the recurrence rate after the infection is cured is high, the infection is a very difficult to treat, the continuous rise of the morbidity and the mortality of the infection brings serious threat to the health of the public, and a new treatment method needs to be explored.
The main effector of CD-mediated pathogenesis is its secretion of various toxins, of which toxin B (TcdB) is the most critical in the pathogenesis, and TcdB alone can induce death in animal models. TcdB enters the inside of host cells through receptor mediation, and causes apoptosis, denaturation, shrinkage, and cellulose and mucin exudation to form pseudomembranous inflammation; on the other hand, tcdB can also stimulate immune cells to release excessive inflammatory factors, trigger inflammatory cascade reactions to exacerbate damage to normal cells, and can cause enhanced intestinal mucosa and vascular permeability to lose body protection barrier, promote TcdB to enter the intestinal tissue lamina propria, and further exacerbate intestinal tissue damage.
TcdB proteins are large proteins containing 2366 amino acid sequences, largely divided into four domains, of which the combined repeat oligopeptide (CROPs, amino acids 1834-2366) domain and the recently discovered frizzled receptor binding domain (FBD, amino acids 1285-1804) are capable of binding to cell surface receptors. Among them, the receptor bound by CROPs domain is chondroitin sulfate proteoglycan CSPG4, and the receptor bound by FBD is frizzled FZDs, tissue factor pathway inhibitor TFPI, poliovirus receptor-like protein 3, PVRL3, etc., mediating TcdB to enter cell and exert its toxic action. The major receptors for TcdB are currently found mainly in intestinal epithelial cells or other tool cells (e.g. Hela cells) which do not mediate inflammatory responses in immune cells, and there are no relevant reports on immune cell receptor proteins used for TcdB. After TcdB removes the CROPs, which may not be the most dominant binding site, the affinity to host cells is still strong, and thus the present invention uses the FBD region for immune cell receptor protein screening.
Macrophages are common immune cells in intestinal tissues, and TcdB can stimulate the macrophages to secrete a large amount of pro-inflammatory cytokines (such as IL-1 beta, IL-6 and the like) while destroying intestinal epithelial cells, and the cytokines can kill normal intestinal epithelial cells, so that the damage to the intestinal tissues is further aggravated.
Disclosure of Invention
The invention aims to explore a receptor protein capable of interacting with clostridium difficile toxin TcdB (the receptor protein is generally cell transmembrane protein and is divided into an extracellular section, a transmembrane region and an intracellular section, wherein the extracellular section is a part combined with external antigens), and the extracellular section of the protein is used for blocking the combination of the TcdB and macrophages, so that excessive inflammatory reaction mediated by the macrophages is lightened, and the injury of inflammatory factors secreted by the macrophages to normal intestinal epithelial cells is relieved, and the receptor protein is used as a potential medicament for treating clostridium difficile infection.
It is an object of the present invention to provide a polypeptide which interacts with clostridium difficile toxin TcdB.
To achieve the object of the present invention, a polypeptide of the present invention which interacts with clostridium difficile toxin TcdB is a polypeptide which is the N-terminal (or referred to as extracellular portion 21-220 amino acids, CD44Extracellular domain, CD44-ECD, or referred to as CD 44-N) of human CD44 protein 21-220 ) The amino acid sequence is shown as SEQ ID No.1, consists of 200 total amino acids at 21 st to 220 nd of the N end of CD44 protein, or is an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids.
The invention also provides a medicine for resisting clostridium difficile infection, the active ingredient of which is CD44-N 21-220 。
The invention also provides a polypeptide CD44-N 21-220 The application in preparing medicines for resisting clostridium difficile infection.
The present invention for the first time found that CD44 is the cellular receptor for clostridium difficile toxin TcdB. Because the CD44 full-length protein is larger (composed of 742 amino acids, the molecular weight is more than 80KD, the amino acid sequence is shown as SEQ ID No. 1), the application of the CD44 full-length protein is limited, and the interaction between TcdB and macrophages can be blocked by selecting the extracellular section of the CD44 full-length protein, so that the TcdB-mediated macrophages are inhibited from secreting IL-1 beta and IL-6, the damage of inflammatory reaction to intestinal tissues is relieved, and the infection of clostridium difficile is effectively treated. Given the indispensable role of TcdB in clostridium difficile infection and its effect of being able to significantly activate macrophage inflammation, CD44 would be an important target for the treatment of clostridium difficile infection.
CD44 is a type I transmembrane protein (GenBank: NP-000601.3) involved in cell-cell and cell-matrix interactions and signal transduction, which is expressed on most immune cells, inducing inflammatory responses upon antigen activation. The N-terminus of human CD44 has a 20 amino acid (aa) signal sequence with an N-terminal invariant portion ECD (aa 21-220), where ECD is the major portion that binds antigen. The ECD of human CD44 shares 76%, 86%, 83% and 79% identity with the corresponding mouse, rat, equine, canine and bovine CD44, respectively (Pure, e.and r.k.assian (2009) cell.signal.21:651;Ponta,H.et al. (2003) nat.rev.mol.cell biol.4:33.), and thus the ECD of human CD44 has wide application.
The technical scheme adopted for achieving the purposes of the invention is as follows: screening immune cell receptor protein by using an FBD region of clostridium difficile toxin TcdB, firstly prokaryotic expressing and purifying the FBD protein, then extracting cell membrane protein of human macrophage line THP-1 by using a kit, incubating and combining the two cell membrane proteins, sending a mass spectrum, and predicting and verifying that CD44 is a receptor of TcdB by adopting technologies such as bioinformatics, co-IP and the like. Further, the extracellular portion of CD44 was selected for relevant functional assays.
Compared with the prior art, the invention has the following beneficial effects and advantages:
1. the invention discovers that the CD44-ECD can block the combination of TcdB and CD44, thereby obviously inhibiting the macrophage mediated by TcdB from secreting inflammatory factors IL-1 beta and IL-6 and treating the injury of the inflammatory reaction mediated by the toxin TcdB to colon epithelial cells.
2. The invention discovers that CD44-ECD is only combined with a receptor through interfering toxin TcdB, does not have pressure for selecting antibiotics, can avoid the drug resistance problem brought by antibiotic therapy, and provides a new solution for overcoming the increasingly serious antibiotic drug resistance problem, large side effect and other problems of clinical clostridium difficile infection.
3. Animal experiment results show that after mice are infected by TcdB, the CD44-ECD provided by the invention has the advantages that inflammatory response is obviously reduced, pathological changes of colon are obviously lightened, and the mice infected by TcdB have good therapeutic effect on the whole.
Drawings
FIG. 1 is a gel electrophoresis diagram of successful extraction and purification of TcdB receptor binding domain FBD recombinant protein in example 1.
FIG. 2 shows the differential proteins related to inflammatory response screened by mass spectrometry in example 1.
FIG. 3 is a graph showing the validation of the binding of the receptor protein to FBD using co-immunoprecipitation in example 1.
FIG. 4 is a graph showing the affinity results of different doses of CD44-ECD for the FBD protein and the TcdB protein of example 1.
FIG. 5 is a graph of the percent binding of macrophages to toxin TcdB in the presence of CD 44-ECD.
FIG. 6 is a graph comparing the binding performance of CD44 to toxin TcdB in the presence of CD 44-ECD.
FIG. 7 is a graph comparing inhibition of secretion of inflammatory factors by macrophages stimulated by toxin TcdB in the presence of CD 44-ECD.
FIG. 8 is a graph comparing the inhibition of proliferation of colonic epithelial cells by toxin TcdB in the presence of CD 44-ECD. Wherein FIG. 8 (A) shows the proliferation of colon epithelial cells taken by the confocal high content imaging system, and FIG. 8 (B) shows the statistics of the proliferation of colon epithelial cells with respect to the cell area.
FIG. 9 is an immunofluorescent staining image of a TcdB intrarectal perfused colon tissue section of a mouse in the presence of CD 44-ECD: wherein FIG. 9 (A) is a diagram showing the expression of IL-1. Beta. Inflammatory factor, and FIG. 9 (B) is a diagram showing the expression of IL-6 inflammatory factor.
FIG. 10 is a HE staining plot of TcdB intrarectal perfused colon tissue of mice in the presence of CD 44-ECD.
FIG. 11 is a graph of histochemical scores of TcdB intrarectal perfused colon tissue of mice in the presence of CD 44-ECD: wherein FIG. 11 (A) is an overall view; FIG. 11 (B) shows the degree of epithelial destruction; FIG. 11 (C) is an inflammatory cell infiltration aspect; fig. 11 (D) is an edema aspect.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1 Mass Spectrometry techniques screening and verifying that CD44 is a receptor capable of binding to the FBD region of TcdB
1. FBD region protein expressing purified TcdB (abbreviated FBD protein)
According to the reference "Peng Chen et al, science.2018;360 The expression and purification of 664-669' method comprises the following steps:
1) The TcdB-FBD gene (residues 1285-1804) was cloned into a modified pET28a vector, the N-terminus of which was inserted with a 6 XHis-SUMO tag.
2) TcdB-FBD was expressed in E.coli BL21-Star (DE 3) (Invitrogen). Bacteria were cultured in LB medium containing kanamycin at 37 ℃. When OD is 600 When 0.8 is reached, the temperature is reduced to 16 ℃. Expression was induced with 1mM IPTG (isopropyl-b-D-thiogalactopyranoside) and continued overnight at 16 ℃. Cells were collected by centrifugation and stored at-80 ℃ until use.
3) Using Ni 2+ NTA (triacetate, qiagen) affinity resin TcdB-FBD protein was purified in 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 against 20mM HEPES,pH 7.5,150mM NaCl containing buffer at 4 ℃.
The results of electrophoresis of the TcdB-FBD protein carrying the 6 XHis-SUMO tag for pulldown and cell surface binding assays are shown in FIG. 1.
2. Extraction of macrophage membrane protein
1) The human macrophage cell line THP-1 cultured in large quantity was 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, washed once with Phosphate Buffer (PBS), scraped off with a cell scraper, collected by centrifugation, and the supernatant was aspirated off leaving a cell pellet for use.
2) Washing the cells: the cell pellet was lightly resuspended in a suitable ice-bath pre-chilled PBS, a small amount of cells were taken for counting, and the remaining cells were centrifuged at 600g for 5min at 4℃to pellet the cells. The supernatant was discarded, followed by centrifugation at 600g for 1 min at 4℃to pellet the residual liquid on the walls of the centrifuge tube and further pellet the cells, and the residual liquid was drained with maximum effort.
3) Cell pretreatment: 1ml of membrane protein extraction reagent A to which PMSF (phenylmethylsulfonyl fluoride is a serine protease inhibitor) was added just before use was added to 2000-5000 ten thousand cells, the cells were gently and sufficiently suspended, and the cells were left in an ice bath for 10-15min.
4) Identification of disruption of cells and disruption Effect: and (3) sequentially and repeatedly freezing and thawing the sample obtained in the previous step twice in liquid nitrogen and at room temperature, and then taking a small amount of sample to detect the cell disruption degree under a microscope.
5) Removal of nuclei and unbroken cells: centrifuge at 4 ℃,700g for 10 minutes, carefully collect the supernatant into a new centrifuge tube.
The supernatant was aspirated without touching the pellet-!
6) Precipitation of cell membrane fragments: centrifuge at 14000g for 30min at 4℃to pellet cell membrane debris.
7) Extracting membrane protein: centrifuge at 14000g for 10s at 4℃and drain the supernatant as much as possible. Adding membrane protein extraction reagent B, high-speed swirling for 5 seconds to re-suspend the sediment, and ice-bathing for 5-10min. The vortexing and ice bath were repeated 1-2 times to fully extract membrane proteins. Finally, the mixture is centrifuged for 5min at the temperature of 4 ℃ and 14000g, and the supernatant is collected to be the cell membrane protein solution for later use.
3. Preparation before mass spectrometry:
1) Mu.g of the purified FBD protein from step 1, the purchased commercial His tag protein and 20. Mu.l of Ni-NTA (Nickel NTA affinity chromatography medium) agarose beads were added to PBS and incubated for 4h at 4 ℃. Obtaining the FBD-Ni-NTA.
2) Adding the FBD-Ni-NTA and Ni-NTA into the cell membrane proteins extracted in the step 2 respectively, and incubating overnight at 4 ℃;
3) The Ni-NTA column was collected by centrifugation, washed twice with PBS, and bound protein was eluted with 200mM imidazole;
4) Eluted proteins were sent directly to the company for mass spectrometry.
4. Protein related to inflammation caused by FBD protein screened by mass spectrometry technology
Affinity Capture-MS is the most widely used method of protein interaction research at present. This method is also known as AP-MS (Affinity Purification coupled MS), affinity purification tandem mass spectrometry. The main principle of AP-MS is to purify the interaction protein of target protein and target protein from complex biological system based on the affinity of the affinity reagent and target protein, and then to perform mass spectrum detection to identify the interaction protein of target protein. Classical AP-MS experiments typically involve the following 4 key steps: 1) Lysis of cells or organelles; 2) Incubating the protein solution with the affinity microbeads; 3) Washing of non-specific binding and background proteins, elution of specific interaction proteins; 4) Mass spectrometry detection and analysis of interacting proteins.
According to the method, macrophage membrane protein is firstly extracted, then the micro beads of the FBD protein coupled with Ni-NTA and the control protein His are respectively incubated with the macrophage membrane protein, and after washing, the target protein in the macrophage membrane protein combined with the FBD protein and the His protein is different, so that the macrophage membrane protein can be detected by an AP-MS technology, and the credible value can be calculated according to the abundance of the detected target protein, and the sorting can be performed, so that the screened differential protein is obtained. The number of different proteins was large, but according to the results of the present test and the reports of the related literature, the total number of proteins related to inflammation was five, and the results are shown in fig. 2. Since the mass-screened differential protein is the only reference, further validation and confirmation of the target protein is required, and the three proteins with higher scores are to be selected for further co-immunoprecipitation validation.
5. Co-immunoprecipitation to verify receptor proteins binding to FBD
Co-immunoprecipitation is a classical method for studying protein interactions based on the specific interaction between antibodies and antigens, an effective method for determining the physiological interactions of two proteins in intact cells. The target protein obtained by the method is naturally combined with the protein of interest in cells, accords with the actual conditions in vivo, and has high reliability of the obtained results. This method is often used to determine whether two proteins of interest bind in vivo.
1) A large amount of cultured human macrophage cell line THP-1 was induced with PMA at a concentration of 100ng/mL for 48 hours to differentiate THP-1 cells into macrophages, and the FBD-his protein obtained in step 1 was added to stimulate for 2 hours to make no stimulation as a control group.
2) Washed once with Phosphate Buffered Saline (PBS), added to RIPA lysate and incubated overnight at 4 ℃.
3) After the incubation time, the FBD-his Protein and other Protein complexes are pulled by adding Protein-Ni magnetic beads, and incubated for 2 hours at 4 ℃.
4) The magnetic bead complex is pulled out by a magnetic frame, PBST (phosphate Tween buffer) is washed twice, eluent is added, the pulled-out complex is eluted, and if the Protein complex contains target Protein which can interact with FBD Protein, conjugates of the target Protein, FBD-his and Protein-Ni magnetic beads can be formed. The complex was electrophoresed by adding SDS-Loading Buffer, and western blot was performed using different CD44, HSP90AB1 and IGHA1 antibodies (Thermofish company, accession numbers 14-0441-82, MA5-33168 and MA5-31774, respectively), and the results were shown in FIG. 3, in which the Protein-Ni magnetic beads coupled to the FBD-His proteins were not pulled to the HSP90AB1 and IGHA1 proteins, but were pulled to the CD44 proteins, the Protein-Ni magnetic beads coupled to the His proteins of the control group were not pulled to any proteins, and the Input group was a normal cell group to which no proteins were added, indicating that the cells of each group contained the proteins to be pulled, i.e., the positive control group. Na/K ATPase is an internal reference control of cell membrane proteins, and shows the reliability of the result, which is the conventional requirement of a western blot experiment.
The above results indicate that FBD can interact with CD44, i.e. both bind at the cellular level.
6. The affinity results for different doses of CD44-ECD with the FBD protein and the TcdB protein are shown.
1) The FBD protein or TcdB protein, which prokaryotic expressed purified TcdB, was dissolved in a coating buffer (ph 9.6 carbonate buffer: 40mM Na 2 CO 3 ，60mMNaHCO 3 ) And coated on 96-well ELISA plates, and PBS was used for washing the plates after overnight at 4 ℃.
2) Fc-tagged CD44-ECD protein polypeptide (Sinobiologic Co., cat. No. 12211-H02H, amino acids 21-220 of the extracellular portion of human CD44 protein, CD44Extracellular domain, CD44-ECD, or CD 44-N) 21 -220 ) The amino acid sequence of the CD44-ECD is shown as SEQ ID No.2, 12 concentration gradients (0-1024 nM) are set for the Fc labeled CD44-ECD, and the affinity of the Fc labeled CD44-ECD with the target protein of the FBD or TcdB protein (500 ng/mL) is detected at different concentrations;
3) As shown in FIG. 4, it is clear from FIG. 4 that the OD value increases with the increase in the concentration of CD44-ECD at a fixed concentration of the target protein, but with the concentration of CD44-ECDIncreasing its OD also gradually goes to plateau. Affinity value K D And (3) performing calculation, and displaying the result: k of FBD D K of TcdB protein with a value of 82.08 +/-1.372 nM D The value was 77.98.+ -. 1.421nM. K (K) D Values were on the order of nM, showing that CD44-ECD has a higher affinity for either FBD or TcdB proteins.
The above results demonstrate that CD44 is a receptor capable of binding to the FBD region of TcdB.
Example 2 mechanism assay for CD44-ECD blocking binding of TcdB toxin to macrophages
1. Percentage of binding of macrophages to toxin TcdB in the presence of CD 44-ECD.
1) Cultured human macrophage cell line THP-1 (1X 10) 6 Individual/well) were subcultured in six well plates, and induced with PMA at a concentration of 100ng/mL for 48h to differentiate THP-1 cells into macrophages, which were washed once with Phosphate Buffered Saline (PBS) to obtain three groups of macrophages;
2) Fresh culture medium is replaced, tcdB protein is added into two groups of culture medium respectively, one group of culture medium containing TcdB protein (with the concentration of 10 mug/mL) is added into CD44-ECD (with the concentration of 10 mug/mL) for blocking, and the culture medium is incubated for 3 hours in a cell incubator;
3) After three times of PBS washing, nonspecific binding proteins are removed, cells are scraped by a cell scraper, cells are collected by centrifugation, and the supernatant is sucked;
4) Adding primary antibody (anti-TcdB antibody of rabbit origin, abcam company, cat# ab 270452), and incubating on ice for 1h;
5) After PBS washes, adding the fluorescent labeled anti-rabbit secondary antibody into the corresponding groups of cells according to the dilution concentration of the antibody specification, and incubating on ice for 1h;
6) The PBS was washed once, resuspended and then run on a machine to count the percentage of binding (Binding percentage) detected by flow cytometry.
Test results: as shown in FIG. 5, CD44-ECD blocks the binding of toxin TcdB to macrophages.
2. Comparison of binding properties of CD44 to toxin TcdB in the presence of CD 44-ECD.
1) Coating TcdB toxin (500 ng/mL) on an ELISA plate, placing the ELISA plate in a 37 ℃ wet box for incubation for 1h, and washing PBST for 5 times;
2) 200. Mu.L salmon sperm DNA blocking solution (100. Mu.g/mL) was added and incubated at 37℃for 1h;
3) After PBST is washed 3 times, CD44-ECD with the concentration of 300nM is added respectively, and the mixture is placed in a wet box at 37 ℃ for incubation for 1h;
4) His-tagged CD44 (500 ng/mL) was added separately and incubated for 1h at 37 ℃;
6) PBST was washed 3 times, 100. Mu.L of a 1:3000 dilution of horseradish peroxidase (HRP) -labeled anti-his antibody was added to each well, and incubated in a 37℃wet box for 30min;
7) PBST is washed for 3 times, TMB chromogenic solution is added for chromogenic reaction, and incubation is carried out for 8min at 37 ℃;
8) Detection of OD by ELISA after addition of ELISA stop solution 450 nm 。
Test results: as a result, as shown in FIG. 6, it is evident from FIG. 6 that the binding of CD44 to the toxin TcdB was significantly weakened after the addition of CD44-ECD, and that CD44-ECD was able to block the binding of CD44 to the toxin TcdB, i.e., CD44-ECD inhibited the binding of TcdB to macrophages by blocking the binding of the toxin TcdB to the CD44 receptor.
Example 3CD44-ECD significantly inhibits TcdB stimulation of THP-1-derived macrophages to secrete inflammatory factors to reduce injury of the intestinal epithelial cells Caco-2 by the inflammatory response
1. CD44-ECD significantly inhibits TcdB from stimulating THP-1-derived macrophages to secrete inflammatory factors
1) Inducing with PMA with concentration of 100ng/mL for 48h to differentiate THP-1 cells into macrophages, respectively stimulating macrophages with TcdB, tcdB+CD44-ECD, collecting cell culture supernatant after 48h stimulation, respectively adding the collected supernatant and diluted gradient concentration standard into holes (100 mu L/hole), and incubating in a 37 ℃ incubator for 90min;
2) Adding 350 mu L of washing liquid into each hole, shaking the holes for 2min, then throwing out the liquid, drying the holes by beating on thick overlapped absorbent paper, repeatedly washing the plates for 4 times, and throwing out the liquid in the holes;
3) Biotin-labeled IL-1 beta or IL-6 antibodies were combined with antibody dilutions at 1: diluting 100, adding into a hole (100 mu L/hole) after diluting, incubating in a 37 ℃ incubator for 60min, and washing the plate for 4 times, wherein the plate washing method is the same as that in the step 2);
4) Diluting enzyme conjugate (horseradish peroxide labeled streptavidin) and enzyme conjugate diluent at a ratio of 1:100, adding the diluted mixture into a hole (100 mu L/hole), incubating the diluted mixture in a 37 ℃ incubator for 30min, and washing the plate for 5 times, wherein the plate washing method is the same as that in the step 2);
5) Adding 100 mu L/hole of a color reagent, keeping away from light, and incubating in a constant temperature oven at 37 ℃ for 10min;
6) Adding 100 mu L/hole of stop solution, and measuring OD450 value immediately after mixing uniformly;
7) Drawing a standard curve of the obtained result through Excel software, converting the standard curve to obtain a sample value, and carrying out statistical analysis;
test results: the results are shown in FIG. 7, where CD44-ECD significantly inhibited TcdB stimulated macrophages to secrete IL-1 beta and IL-6, and the results were statistically significant.
2. The inhibition of colonic epithelial proliferation by inflammatory response can be reduced by activation of the toxin TcdB in the presence of CD44-ECD, which stimulates macrophages.
1) Colon epithelial cells Caco-2 cells grown in log phase were grown at 2X 10 5 Each well (500. Mu.L, 4X 10) 5 The culture medium is planted in 24 pore plates in a passaging way, and the culture medium is cultivated in an incubator for 48 hours; then, tcdB and TcdB+CD44-ECD are added respectively, and the culture is continued for 48 hours;
2) Inducing with PMA at concentration of 100ng/mL for 48h to differentiate THP-1 cells into macrophages, stimulating macrophages with TcdB, tcdB+CD44-ECD respectively, washing with PBS three times after 6h stimulation, scraping off cells with a cell scraper, centrifuging to collect cells, sucking off supernatant, and collecting the cells according to the following formula 1:1 into a Transwell dish, and placing the Transwell dish into the 24-well plate to co-culture with Caco-2 cells. Since macrophages have been stimulated by TcdB, tcdb+cd44-ECD, respectively, different concentrations of inflammatory factors IL-1β and IL-6 are produced, resulting in different degrees of inhibition of proliferation of colonic epithelial Caco-2 cells;
3) The confocal high content imaging analysis system is used for observing the growth state of the cells, and the cell area is detected by the self-contained function of the system.
Test results: the cell growth state diagram is shown in fig. 8, wherein fig. 8 (a) is a graph showing the proliferation of colon epithelial cells taken by the confocal high content imaging system, and fig. 8 (B) is a statistical graph showing the proliferation of colon epithelial cells versus the cell area. As can be seen from FIG. 8, IL-1 beta and IL-6 cytokines act as pro-inflammatory factors, exacerbating normal tissue damage, while CD44-ECD significantly inhibits TcdB-mediated inflammatory responses, thus alleviating the inhibition of cell proliferation.
Example 4 mouse in vivo experiments with CD44-ECD
1. Construction and grouping of mouse models
1) 18 female BALB/c mice (age: 6-8 weeks, purchased from the laboratory animal center of the Hubei pharmaceutical laboratory) were randomly divided into 3 groups of 6 animals each;
2) Combining 500pM of CD44-ECD with 100pM of TcdB at 37 ℃ for 2 hours, washing twice by PBS, and re-suspending by 100 mu L of PBS to prepare an infectious agent containing the CD 44-ECD;
3) Each group of mice was treated for intrarectal perfusion infection according to the following table:
TABLE 2 grouping and infection treatment of BALB/c mice
At the time of infection treatment, mice were anesthetized with 1% sodium pentobarbital (using a concentration of 50 mg/kg), a tube of 1mm diameter was inserted from the anus into the rectum (-4 cm) and the infectious agent was injected, and then the anus was sealed with a medical anastomosis glue to prevent toxin from leaking, and drinking water and food (regular diet) were freely available to each group of mice during the experiment;
2. immunofluorescence detection of colon tissue of mice
1) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I15 min-xylene II 15 min-absolute ethanol I5 min-absolute ethanol II 5min-85% alcohol 5min-75% alcohol 5 min-distilled water for washing;
2) Antigen retrieval: the tissue slice is placed in a repair box filled with EDTA antigen repair buffer solution (pH9.0) for antigen repair in a microwave oven, and after the medium fire is switched off to boiling, the medium fire is switched off for 10min, and the buffer solution is prevented from being excessively evaporated in the process, and the slice is not dried. Naturally cooling, placing the slide in PBS (pH 7.4), and shaking and washing on a decolorizing shaking table for 3 times, each time for 5min;
3) BSA blocking: after the slices are slightly thrown, a histochemical pen is used for drawing circles around the tissues (preventing antibodies from flowing away), 3% BSA is added dropwise into the circles to uniformly cover the tissues, and the tissues are sealed for 30min at room temperature;
4) Adding an antibody: gently throwing away the sealing liquid, dripping the primary antibody (primary antibody of macrophage surface marker CD68, cytokine IL-1 beta and IL-6) prepared by PBS according to a certain proportion on the slice, horizontally placing the slice in a wet box, and incubating overnight at 4 ℃ (adding a small amount of water in the wet box to prevent the antibody from evaporating);
5) Adding a secondary antibody: the slides were washed 3 times in PBS (pH 7.4) for 5min each on a decolorizing shaker. Slightly spin-drying the slices, then dripping secondary antibody covering tissues corresponding to the primary antibody in the circles, and incubating for 50min in a dark place;
6) DAPI counterstaining nuclei: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. Dripping DAPI dye solution into the ring after the slices are slightly dried, and incubating for 10min at room temperature in a dark place;
7) Sealing piece: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. The slices are slightly dried and then sealed by anti-fluorescence quenching sealing tablets;
8) And (5) microscopic examination and photographing: the slice is observed and images are acquired under a confocal microscope;
detection result: as shown in FIG. 9, the ratio of CD68 positive cells of the macrophage surface marker in the tissue is lower than that of the TcdB group, so that macrophage infiltration can be reduced, and secretion of IL-1 beta (FIG. 9A) and IL-6 (FIG. 9B) can be inhibited, and the trend is consistent with the result shown in FIG. 7, so that the CD44-ECD can obviously reduce the level of inflammatory factors such as IL-1 beta, IL-6 and the like secreted by the TcdB-mediated macrophages.
3. HE staining of colon tissue in mice
1) Slicing: the colonoscopy PBS of the intrarectal instillation experimental mice in step 1 of this example was washed, 4% paraformaldehyde fixed colon tissue was paraffin-embedded conventionally, and then 4 μm sections were prepared;
2) Slicing and drying: putting the slices into hot water for ironing, then attaching the slices onto a glass slide, and putting the glass slide into a 45 ℃ incubator for drying;
3) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I10 min-xylene II 10 min-absolute ethanol I5 min-absolute ethanol II 5min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5 min-distilled water for washing;
4) Hematoxylin-stained nuclei: the slices are stained with hematoxylin for 6min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, washed with tap water, and rinsed with 0.6% ammonia water to turn blue;
5) Eosin-stained cytoplasm: the slice is dyed in eosin dye solution for 2min;
6) And (3) removing the water sealing piece: sequentially placing the slices into 95% alcohol I5 min-95% alcohol II 5 min-absolute alcohol I5 min-absolute alcohol II 5 min-xylene I5 min-xylene II 5min for dehydration and transparency, taking out the slices from the xylene, slightly airing, and sealing the slices with neutral resin;
7) And (5) performing microscopic examination by a positive microscope: stained sections were scored by two pathologists on a scale of 0 to 3 (mild to severe) for general appearance, epithelial destruction, inflammatory cell infiltration, degree of edema;
detection result:
the HE staining pattern of the colon tissue of the mice is shown in FIG. 10, the scores of the colon tissue of the mice in terms of overall appearance, degree of epithelial destruction, inflammatory cell infiltration, edema and the like are shown in FIG. 11, FIG. 10 shows that the TcdB stimulated group has the colon epithelial destruction, obvious submucosa edema, and the lamina propria secretes a large amount of inflammatory cells, and the overall colon tissue presents a state of bloody edema, but the scores of the colon tissue of the mice in terms of overall appearance (FIG. 11A), the degree of epithelial destruction (FIG. 11B), the inflammatory cell infiltration (FIG. 11C), the edema (FIG. 11D) and the like are all significantly improved after the CD44-ECD treatment is given.
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.
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