SpaF polyclonal antibody and application thereof

Abstract

The invention discloses a SpaF polyclonal antibody and application thereof, and belongs to the technical field of biology. The SpaF polyclonal antibody is obtained by taking SpaF protein as an antigen, mixing the antigen with an adjuvant to prepare an immunogen, immunizing an animal, collecting positive serum, and performing separation and purification. The amino acid sequence of the SpaF protein is as shown in SEQ ID NO. 2. The polyclonal antibody can significantly inhibit the formation of a corynebacterium striatum biological membrane and the adhesion of A549 cells, can be used as a potential therapeutic target for corynebacterium striatum membrane-producing strain infection, especially a strong membrane-producing strain, and provides a new research direction for developing a novel therapeutic strategy for corynebacterium striatum infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a SpaF polyclonal antibody and application thereof. BACKGROUND

[0002] Corynebacterium striatum is an important gram-positive bacillus, and its pathogenicity is related to various virulence factors expressed or secreted by it, especially adhesion-related virulence factors. In the current study of the pathogenicity of Corynebacterium, adhesion to specific types of tissues and cells is a key step for many bacterial pathogens to successfully colonize the human body and cause infection. In recent years, there has been a significant increase in the number of reports of multiple drug-resistant (MDR) C. striatum infections. However, there are limited effective antimicrobial drugs available for the treatment of infections caused by this bacterium, and MDR strains often exhibit stronger biofilm formation ability, which can form mature biofilms on various surfaces and cause chronic infections, posing a great challenge to the clinical treatment of infectious diseases.

[0003] Among Corynebacterium, the spaDEF pilus gene cluster encoding product is spa-type pilus, which mediates the colonization and invasion of bacteria to host cells through specific interaction. Among them, spaF gene is significantly highly expressed in strong biofilm-producing C. striatum, and high levels of pilin protein encoded by spaF gene are detected in strong biofilm-producing strain EVs. SpaF gene can exist in free form and be secreted in vesicle form. Strong biofilm-producing strains all carry spaF gene, suggesting that this gene plays an important role in bacterial colonization and adhesion. The survival rate of SpaF-deficient mutant strains in macrophages is significantly reduced, indicating that the intracellular survival mechanism mediated by SpaF protein enables bacteria to escape recognition and elimination by the immune system of the body. Further studies have confirmed the importance of SpaF protein in systemic infection. In summary, SpaF protein is involved in multiple processes of biofilm formation, immune evasion and systemic infection of pathogenic bacteria. Pilus is an important virulence factor of Corynebacterium bacteria, which can mediate the adhesion of bacteria to host tissue cells, and is a key step in the infection of many bacterial pathogens to the host. However, there are few reports on the biological function of specific virulence factors of C. striatum, including spaDEF gene cluster.

[0004] However, there are few reports on SpaF in C. striatum, and its specific function is not clear. In several common gram-negative bacterial infection models, host-bacteria interaction is mediated by pilus on the surface of bacterial cells; there are relatively few reports on gram-positive bacteria, and a few research teams have reported the role of pilus in mediating the pathogenicity of C. diphtheriae, but there are few reports on C. striatum. SUMMARY

[0005] The SpaF polyclonal antibody and application thereof are provided to solve the problems of the prior art, and the SpaF polyclonal antibody is prepared by immunizing animals with SpaF protein as an immunogen, can significantly inhibit Corynebacterium striatum biofilm formation and A549 cell adhesion, can be used as a potential target for preventing and treating Corynebacterium striatum biofilm-related infections, especially strong membrane-producing strains, and provides a new research direction for developing a new treatment strategy for Corynebacterium striatum infection.

[0006] To achieve the above object, the application provides the following scheme.

[0007] The application provides a SpaF polyclonal antibody, which is prepared by taking SpaF protein as an antigen, mixing the antigen with an adjuvant to obtain an immunogen, then immunizing animals, collecting positive serum, and separating and purifying the positive serum.

[0008] Preferably, the amino acid sequence of the SpaF protein is shown as SEQ ID NO. 2.

[0009] Preferably, the antigen and physiological saline are diluted at a mass-volume ratio of 4 μg:(2-5) μl, and then mixed with the adjuvant in an equal volume to obtain the immunogen.

[0010] Preferably, the separation and purification is performed by using a HiTrap rProtein AFF affinity chromatography column.

[0011] The application also provides application of the SpaF protein in any of the following aspects:

[0012] (1) application in preparation of a medicine for inhibiting Corynebacterium striatum biofilm formation;

[0013] (2) application in preparation of a medicine for treating Corynebacterium striatum infection.

[0014] The application also provides application of the SpaF protein in preparation of a medicine for inhibiting cell adhesion.

[0015] Preferably, the amino acid sequence of the SpaF protein is shown as SEQ ID NO. 2.

[0016] The application also provides application of the SpaF polyclonal antibody in any of the following aspects:

[0017] (1) application in preparation of a medicine for inhibiting Corynebacterium striatum biofilm formation;

[0018] (2) application in preparation of a medicine for treating Corynebacterium striatum infection.

[0019] The application further provides application of the polyclonal antibody of SpaF in preparation of a medicine for inhibiting cell adhesion.

[0020] The application further provides a medicine taking the polyclonal antibody as a main effective component.

[0021] The application discloses the following technical effects:

[0022] The application provides a polyclonal antibody for a SpaF pilus gene, the polyclonal antibody can significantly inhibit the formation of a Corynebacterium striatum biofilm and the adhesion of A549 cells, can be used as a potential treatment target for infection of a Corynebacterium striatum biofilm-producing strain, especially a strong biofilm-producing strain, and provides a new research direction for developing a new treatment strategy for Corynebacterium striatum infection. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0024] Figure 1 Preparation flow chart of rabbit anti-SpaF polyclonal antibody;

[0025] Figure 2 SDS-PAGE detection results of a small amount of protein expression sample, wherein M is Marker, 1 is a non-induced control (BL21), and 2-3 are IPTG-induced (BL21);

[0026] Figure 3 SDS-PAGE detection results of a large amount of protein expression and purified sample, wherein M is Marker, 1 is whole bacteria after ultrasonic treatment, 2 is supernatant after ultrasonic treatment, and 3 is precipitate after ultrasonic treatment;

[0027] Figure 4 Recombinant protein purity analysis result chart;

[0028] Figure 5 Rabbit anti-SpaF polyclonal antibody titer detection results;

[0029] Figure 6 SDS-PAGE detection SpaF antibody purity results, wherein 1 is SpaF antibody, and 2 is Marker;

[0030] Figure 7 Antagonistic effect of SpaF recombinant protein on biofilm formation of six strong biofilm-producing Corynebacterium striatum strains;

[0031] Figure 8Inhibition effect of rabbit anti-SpaF polyclonal antibody on biofilm of 6 strains of strong membrane band bacillus;

[0032] Figure 9 Inhibition effect of rabbit anti-SpaF polyclonal antibody on biofilm of 6 strains of common clinical pathogenic bacteria (Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis);

[0033] Figure 10 Inhibition activity of SpaF recombinant protein on bacterial adhesion to cells; A: adhesion number, B: cell adhesion rate; wherein Control, positive control group; Adherent bacteria (CFU / ml), adhesion number; Cell adhesive rate (%), cell adhesion rate; 20 μg / ml, 100 μg / ml, indicating the concentration of SpaF recombinant protein;

[0034] Figure 11 Inhibition activity of SpaF polyclonal antibody on bacterial adhesion to cells; A: adhesion number, B: cell adhesion rate; wherein Control, positive control group; Adherent bacteria (CFU / ml), adhesion number; Cell adhesive rate (%), cell adhesion rate; 1:100; 1:50; 1:20, indicating the ratio of SpaF antibody to bacteria. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not restrictive of the application. It should be understood that the detailed description and specific examples, while indicating certain aspects of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.

[0036] It should be understood that the terms used in the present application are merely for the purpose of describing particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value within any stated value or stated range, and any other stated value or intermediate value within the stated range, is also included within the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not an admission that it is prior art to the present application.

[0038] Many modifications and variations of the present application described herein will be apparent to those of ordinary skill in the art from the foregoing description. Other embodiments of the application will be apparent to those of ordinary skill in the art from the foregoing description. The description and embodiments of the present application are exemplary only, and are not intended to be limiting.

[0039] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0040] Example 1: Preparation of SpaF polyclonal antibody

[0041] The flow chart of preparation of rabbit anti-SpaF polyclonal antibody is shown in Figure 1 The specific operation is as follows:

[0042] 1. Experimental animals

[0043] In the present application, 2 New Zealand white rabbits for immunization were purchased from Beijing Changyang Xishan Breeding Farm (Experimental Animal Production License: SCXK [Jing] 2021-0008).

[0044] 2. Cell source

[0045] The human alveolar epithelial A549 cell line used in the present application was purchased from BeNa Culture Collection (BNCC) North-Na Link Biotechnology Co., Ltd.

[0046] 3. Cloning, expression, purification and identification of recombinant differential proteins

[0047] 3.1 Differential protein expression identification

[0048] The SpaF protein coding gene fragment sequence was searched with the Corynebacterium striatum clinical strain (CS-1, strong membrane-producing strain) gene sequence (GenBank number: SAMN35779365 CS-1) as the reference sequence. The synthesized gene fragment contains an upstream EcoRI restriction endonuclease site and a downstream XhoI restriction endonuclease site.

[0049] The SpaF gene sequence (SEQ ID NO. 1) is as follows:

[0050]

[0051] The amino acid sequence of SpaF (SEQ ID NO. 2) is as follows:

[0052] SCVEKGFINIPDTKGVDYFIGGERKSAGQHFYEGQKTTVKVTAKPQNGYQIAAGARTEWSFDFSGQVKNCSDSSEDPKPPAVEGRHFDAIGHDFAVESDPVDPKNPNAFTAKVTEESRMKYATVRIETDATPLEAQQYNLELDKIESGVTLQKRNVNIGNGFITMEVVPVKDGKPVDSAVVPKDAVFTFTNNFSQNKNLKVTLDVYGEKKGEPKPPIISPPDGADWVHGRVPNPPMPQRCGLRIAVVADLSTSLNYADSNGFTESKKAANALIDSLAGTPAELGIYNFAGSAPRNPRGSTHNENPPYISLQSDDGVSRAKGFVSNWDGNGSTNWEAGLKQVAAGNYD.

[0053] The enzyme-cut spaF gene fragment was ligated with the pET30a vector by T4 DNA ligase, and then transformed into BL21-Codon Plus (DE3) competent cells, with the specific process as follows:

[0054] (1) The BL21 competent cells stored at -80°C were thawed on ice.

[0055] (2) After gently mixing the recombinant plasmid with the competent cells, the mixture was incubated in ice bath for 30 min.

[0056] (3) 42°C heat shock for 90 s.

[0057] (4) After ice bath incubation for 2 min, 800 μl of LB liquid medium without antibiotics was added to the system.

[0058] (5) 37°C culture for 45 min.

[0059] (6) After centrifugation at 5000 rpm for 3 min, most of the supernatant was discarded, and about 100-150 μl of culture medium was used to resuspend the bacterial cells, which were evenly spread on the LB agar plate containing antibiotics.

[0060] (7) The plated plate was placed at room temperature until the surface was dry, and then placed in a 37°C constant temperature incubator for inverted culture for 12-16 hours.

[0061] (8) Single colonies were picked from the transformation plate and inoculated into 1.5 ml LB liquid medium containing 50 μg / ml kanamycin and incubated at 37 °C, 200 rpm.

[0062] (9) The culture was induced with IPTG (0.5 mM) when the OD reached 0.6-0.8 and incubated at 37 °C, 200 rpm for 2 hours.

[0063] (10) 1 ml of the induced culture was collected by centrifugation at 12,000 rpm for 1 min. The supernatant was discarded and the bacterial pellet was resuspended in 50-100 μl of 10 mM Tris-HCl (pH 8.0) and an equal volume of 2x loading buffer was added. The sample was boiled in a water bath for 5 min and then analyzed by SDS-PAGE.

[0064] (11) 1-2 μl of the activated bacterial solution was inoculated into 5 ml of LB medium containing 50 μg / ml kanamycin and incubated at 37 °C, 200 rpm.

[0065] (12) The activated bacterial solution was inoculated into 250 ml of LB medium containing 50 μg / ml kanamycin and incubated at 37 °C, 200 rpm until the logarithmic growth phase. Then, 0.5 mM IPTG was added and the expression was induced at 16 °C for 12-16 hours.

[0066] (13) The bacteria were collected by centrifugation at 8000 rpm for 6 min and the supernatant was discarded.

[0067] (14) The bacterial pellet was resuspended in 20-30 ml of 10 mM Tris-HCl buffer (pH 8.0) and treated with an ultrasonic disrupter (power 500 W) with the following parameters: 180 cycles, 5 s of sonication and 5 s of interval.

[0068] (15) To identify the protein expression form, 100 μl of the ultrasonic disrupted bacterial solution was centrifuged at 12,000 rpm for 10 min to separate the supernatant and the pellet. 50 μl of the supernatant was transferred to a new EP tube and the pellet was resuspended in 50 μl of 10 mM Tris-HCl (pH 8.0) buffer. The samples were analyzed by SDS-PAGE.

[0069] 3.2 Purification of differential protein expression

[0070] (1) The nickel affinity chromatography column (Ni Sepharose 6 Fast Flow, GE Healthcare) was washed with deionized water until the pH of the effluent reached 7.0.

[0071] (2) Nickel was attached to the column at pH 2-3.

[0072] (3) The column was washed with deionized water to pH 7.0.

[0073] (4) Equilibrate the nickel affinity column with 10 mM Tris-HCl buffer (pH 8.0), and the equilibration volume is about 100 ml.

[0074] (5) Equilibrate the nickel column with 10 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl, and the equilibration volume is about 50 ml.

[0075] (6) After dilution, load the sample, and ensure that the final concentration of NaCl in the sample is 0.5 M.

[0076] (7) After loading is completed, rinse the column with 10 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl.

[0077] (8) Perform stage elution with 10 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl and gradient imidazole concentration (15 mM, 60 mM, 300 mM), and collect the protein elution peaks at each concentration to obtain the purified SpaF protein.

[0078] (9) Detect the protein purification effect by SDS-PAGE method: cut the suspected protein band from the gel block, and after trypsin digestion, use LC-MS / MS technology to identify the potential SpaF protein by mass spectrometry.

[0079] (10) Search and analyze the obtained sequence by Mascot software, and analyze the purity of the recombinant protein by Image Lab / Bio-Rad.

[0080] 3.3 Experimental results

[0081] As shown in Figure 2 and Figure 3 , the recombinant expression vector pET-30a-SpaF can be efficiently expressed after transformation of E. coli competent cells. The purified recombinant protein was identified by LC-MS / MS method, and it was confirmed to be SpaF protein. As shown in Figure 4 , the protein purity is 85.3%. According to the sequence of the target protein coding gene, the expected relative molecular mass is 45 000. The molecular weight of the purified protein is slightly different from the expected molecular weight, which may be related to the protein modification during protein expression.

[0082] 4, Preparation of differential protein polyclonal antibody

[0083] 4.1 Experimental method

[0084] (1) New Zealand white rabbits weighing about 2.0 kg were selected for immunization experiments. 1-2 ml of negative control serum was collected from the marginal vein before immunization.

[0085] (2) 400 μg of the purified SpaF recombinant protein was taken as an antigen, diluted with normal saline to 200-500 μl, mixed with an equal volume of Freund's adjuvant (complete adjuvant for the first immunization and incomplete adjuvant for the booster immunization) to form a stable emulsion by homogenization.

[0086] (3) The emulsified immunogen was injected subcutaneously through multiple points on the back, with 8-10 injection points.

[0087] (4) Negative serum was collected by the ear vein blood collection method. Before blood collection, the rabbit ear vein was wiped with a 75% alcohol cotton ball until the blood vessel was fully dilated.

[0088] (5) The ear vein was punctured with a sterile syringe to collect 1-2 ml of negative control serum.

[0089] (6) After the whole blood sample was left at room temperature for 30-120 min, the serum was separated by centrifugation at 5000 rpm for 10 min.

[0090] (7) After the first immunization, booster immunization was performed every 2 weeks, for a total of 3 times.

[0091] (8) 35 ml of polyclonal positive serum was collected from the carotid artery after 1 week: The rabbit was anesthetized with anesthetic (sodium pentobarbital, 30 mg / kg). After the experimental rabbit was anesthetized and fixed, the carotid artery beside the trachea was surgically exposed, clamped with a hemostat, and then cut to collect blood, which was centrifuged at 5000 rpm for 10 min (repeated twice) to separate the serum.

[0092] (9) The HiTrap rProtein A FF affinity chromatography column was used for antibody purification, with 0.1 M glycine-HCl, pH 3.0 as the eluent.

[0093] (10) Goat anti-rabbit IgG-HRP was selected as the enzyme-labeled secondary antibody, and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). The serum dilution corresponding to 50% of the maximum absorbance was used as the titer determination standard. Double-wavelength (450 and 630 nm) detection was used to record data and perform chart analysis.

[0094] (11) The antibody concentration was determined by ultraviolet spectrophotometry, and the antibody purity was analyzed by SDS-PAGE electrophoresis.

[0095] 4.2 Experimental results

[0096] SpaF recombinant protein immunized New Zealand white rabbits, successfully obtained antisera. After purification, ELISA titer detection (with 50% of the maximum absorbance corresponding to the dilution standard), UV spectrophotometer at double wavelength (450 nm and 630 nm) conditions to detect A value, SDS-PAGE analysis of antibody purity. As shown in Figure 5 , the antibody titer reached 1:12800. Figure 6 For SDS-PAGE detection of antibody purity results, the results showed that the purity of SpaF polyclonal antibody reached more than 90%.

[0097] Example 2: Biofilm inhibition activity detection results

[0098] 1. Experimental method

[0099] 1.1 SpaF recombinant protein biofilm antagonistic experiment

[0100] Crystal violet staining method was used to detect the biofilm inhibition effect of SpaF recombinant protein and polyclonal antibody on strong biofilm-producing C. striatum clinical strains. The following formula was used to calculate the biofilm inhibition percentage:

[0101] (1) SpaF recombinant protein biofilm antagonistic experiment:

[0102] Biofilm inhibition percentage (%) = (A 620nm [positive control] - A 620nm [SpaF recombinant protein]) / (A 620nm [positive control] - A 620nm [negative control]) x 100%;

[0103] (2) SpaF polyclonal antibody biofilm inhibition experiment:

[0104] Biofilm inhibition percentage (%) = (A 620nm [positive control] - A 620nm [SpaF polyclonal antibody]) / (A 620nm [positive control] - A 620nm [negative control]) x 100%.

[0105] The basic experimental steps are as follows:

[0106] (1) Sterile cotton swab was used to pick single colony and inoculate in 5-6 ml TSB culture solution, 37℃ shaking culture overnight to logarithmic phase.

[0107] (2) 5 μg / ml and 10 μg / ml SpaF recombinant protein were added to 96-well polystyrene plates, respectively, and incubated at 37℃ for 30 min. Then the SpaF protein solution was discarded, and the wells were washed with PBS buffer for 3 times to remove the unbound protein.

[0108] (3) Prepare a bacterial solution with 0.5 Mcf turbidity (10 8 CFU / ml), dilute 100 times to 10 6 CFU / ml, add 200 μl of the mixed and diluted bacterial suspension to each well, and the positive control group is a growth control well without SpaF protein, and the blank group is simply added with TSB medium.

[0109] (4) After incubation at 37°C for 24 h, the biofilm formation ability is detected according to the following steps: ① In a conical flask, add 400 ml of deionized water, accurately weigh 12 g of tryptone soy broth (TSB) powder, dissolve, seal, and sterilize at 121°C for 15 min. ② Take the TSB liquid medium from the high-pressure steam sterilization pot, and when it returns to room temperature, place it in a biological safety cabinet, 15 ml of centrifuge tube is divided into 5-6 ml of TSB per tube. Using a disposable sterile cotton swab, pick a single colony of Ochrobactrum anthropi from a freshly prepared Columbia blood plate, transfer it to a 1.5 ml sterile centrifuge tube, and incubate at 37°C, 220 rpm in a shaking incubator overnight to the logarithmic growth phase. ③ Adjust the bacterial suspension to 0.5 Mcf (equivalent to 1×10 8 CFU / ml) with sterile saline, dilute 100 times with TSB medium, and prepare a working concentration of 1×10 6 CFU / ml. Take 200 μl of the bacterial solution and inoculate each well of the 96-well plate, and place it in a 37°C, 5% CO2 incubator for 24 h. Each test strain has four replicates. ④ After the incubation is terminated, each well is washed with 200 μl of PBS buffer (0.01M, pH 7.2), gently shaken for 3 times, and left at room temperature to remove the residual liquid. ⑤ Add 200 μl of 99% methanol for 15 min, discard the methanol and dry. ⑥ Add 200 μl of 2% (w / v) crystal violet solution to each well, stain at room temperature for 5 min, remove the staining solution, wash each well 3 times with 0.01M PBS (pH 7.4) 200 μl, remove the unbound dye, remove the residual water in the well plate, and leave it at room temperature until it is completely dry. ⑦ Add 160 μl of 33% (v / v) glacial acetic acid solution to each well, dissolve the biofilm for 10-30 min, and use an enzyme-labeled instrument to measure the absorbance value at 620 nm wavelength, calculate the average value, and the negative control group is TSB culture medium without bacterial solution.

[0110] The evaluation criteria for biofilm formation ability refer to previous studies: when OD value ≤ critical value ODc, it is determined that no biofilm is formed; if ODc < OD ≤ 2ODc, it is determined that weak biofilm formation occurs; if 2ODc < OD ≤ 4ODc, it is determined that moderate biofilm formation occurs; if OD > 4ODc, it is determined that strong biofilm formation occurs. The critical OD value (ODc) is the absorbance value of the blank control group at 620 nm.

[0111] 1.2 SpaF polyclonal antibody biomembrane inhibition assay

[0112] Prepare the bacterial suspension according to the method described in "1.1". Add 200 μl of the diluted bacterial suspension to each well of a 96-well polystyrene plate. Add SpaF polyclonal antibody at dilutions of 1:100, 1:200, and 1:400, respectively. The positive control group is a bacterial suspension without SpaF polyclonal antibody. Each experiment is performed in triplicate. The A value per well is calculated based on the results before and after SpaF polyclonal antibody treatment. 620nm The degree of change in the value is used to determine its biofilm inhibitory activity.

[0113] To further evaluate the specificity of SpaF polyclonal antibody in inhibiting biofilm formation, this invention simultaneously tested its effect on biofilm formation by various common clinical pathogens, such as Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. Bacterial suspensions were prepared according to the method described in "1.1," and the diluted bacterial suspension was added to 96-well polystyrene plates, 200 μl per well, with SpaF polyclonal antibody added at a 1:100 dilution. The control group consisted of bacterial suspensions without SpaF antibody. Each experiment was conducted in triplicate. The results were calculated based on the A value per well before and after SpaF polyclonal antibody treatment. 620nm The degree of change in the value is used to determine its biofilm inhibitory activity.

[0114] 2. Experimental Results

[0115] Compared with the control group, 96-well cell culture plates pretreated with SpaF recombinant protein (5 μg / ml and 10 μg / ml) for 30 min showed increased A production of 6 (100%, 6 / 6) highly membranous Corynebacterium strains. 620nm The value was significantly reduced (P < 0.05); the biofilm inhibition rate of 5 μg / ml SpaF recombinant protein ranged from 61.38% to 71.35%, and that of 10 μg / ml SpaF recombinant protein ranged from 69.13% to 79.68%. Compared with the 5 μg / ml SpaF recombinant protein pretreatment group, the 10 μg / ml SpaF recombinant protein group showed a stronger inhibitory effect (P < 0.05). However, the inhibitory effect of SpaF recombinant protein on Corynebacterium striatum biofilm showed inter-strain differences; see [link to results]. Figure 7 .

[0116] Compared with the control group, the biofilm formation ability of all six Corynebacterium striatum strains was inhibited by rabbit anti-SpaF polyclonal antibody (1:100, 1:200, and 1:400 dilutions), but the degree of inhibition varied among strains, with biofilm inhibition rates ranging from 3.73% to 47.05%. The high-concentration polyclonal antibody group (1:100) showed a stronger overall biofilm inhibition effect than the low-concentration groups (1:200 and 1:400). At a 1:100 dilution, the biofilm formation ability of 100% (6 / 6) of the strains was significantly reduced (P < 0.05); at a 1:200 dilution, the biofilm formation ability of 66.67% (4 / 6) of the strains was significantly reduced (P < 0.05); and at a 1:400 dilution, the biofilm formation ability of 0% (0 / 6) of the strains was significantly reduced (P < 0.05). The biofilm inhibitory activity of SpaF polyclonal antibody against clinical Corynebacterium striatum strains showed an overall concentration-dependent characteristic. See Figure 8 .

[0117] Compared with the control group, rabbit anti-SpaF polyclonal antibody (1:100 dilution) did not significantly inhibit the biofilm formation ability of various common clinical pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis). This result confirms the specificity of the SpaF polyclonal antibody in inhibiting the biofilm formation of Corynebacterium stearothermum, suggesting that it may exert its effect by targeting the biofilm formation pathway specific to Corynebacterium stearothermum, rather than broadly interfering with the bacterial biofilm formation mechanism. These findings provide important evidence for the subsequent development of specific anti-biofilm strategies against Corynebacterium stearothermum. See Figure 9 .

[0118] Example 3: Results of Cell Adhesion Inhibition Activity Assay

[0119] 1. Experimental Method:

[0120] 1.1 Cell Culture

[0121] (1) Remove the frozen A549 cells from liquid nitrogen and immediately place them in a 37°C water bath for rapid thawing. Transfer the cell suspension to a 15ml centrifuge tube containing complete culture medium in a biosafety cabinet and collect the cells by centrifugation at 1000rpm for 5min.

[0122] (2) Discard the supernatant, resuspend the cell pellet in complete culture medium, transfer it to a cell culture flask, and incubate it in a 37°C, 5% CO2 incubator.

[0123] (3) Observe the cell morphology and growth status daily using an inverted microscope, and perform subculture when the cell fusion reaches 80-90%.

[0124] (4) Remove the culture supernatant in a clean bench, wash the cells once with sterile PBS (0.01M, pH 7.2), add 1-2 ml of trypsin digestion solution and shake gently to mix. Observe under an inverted microscope until the intercellular spaces increase and the cells begin to detach, then immediately add fresh culture medium to stop digestion.

[0125] (5) Use a sterile dropper to gently blow the cells to completely detach them, transfer the cell suspension to a 15ml centrifuge tube, and centrifuge at 1000rpm for 5min to collect the cells.

[0126] (6) After aspirating the supernatant, add fresh culture medium to resuspend the cells, seed them into a new culture flask at a ratio of 1:3, add an appropriate amount of culture medium, and place them in a 37℃, 5% CO2 incubator for continued culture.

[0127] 1.2 SpaF recombinant protein antagonistic experiment against Corynebacterium striatum cell adhesion

[0128] (1) A549 cells were prepared at a ratio of 1×10 5 The cells were seeded at a density of 1 cell / ml in 24-well plates and incubated overnight at 37°C to allow for full cell adhesion.

[0129] (2) The treatment group was pretreated with 20 μg / ml and 100 μg / ml SpaF recombinant protein (37℃, 1h).

[0130] (3) Preparation of 10 8 CFU / ml bacterial suspension was used to infect cells at a 100:1 infection ratio (37℃, 2h). The control group consisted of bacterial cells infected with cells without SpaF protein, and the blank group consisted of an equal volume of complete culture medium. Each experiment was conducted in parallel with 3 wells.

[0131] (4) Discard the culture medium 2 hours after infection and wash the wells 3 times with PBS.

[0132] (5) Adhesion determination: After lysing cells with 1% Triton X-100, the lysate was spread on blood agar plates and incubated at 37°C for 24-48 hours. Colony forming units (CFU) were counted.

[0133] 1.3 SpaF polyclonal antibody inhibition of Corynebacterium striatum cell adhesion assay

[0134] (1) 24 hours before the infection experiment, 1×10 5 A549 cells were seeded into 24-well culture plates at a density of cells / well and incubated overnight at 37°C in a 5% CO2 cell culture incubator.

[0135] (2) Pick a single colony of Corynebacterium banding and culture it in a 15ml centrifuge tube containing 5ml TSB until the logarithmic growth phase.

[0136] (3) Prepare the bacterial suspension and adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / ml. Bacterial-to-cell infection ratio (MOI) is 100 / 1.

[0137] (4) Split SpaF antibody at titers of 1:20, 1:50, and 1:100 into 100 μl of bacterial suspension (1×10⁻⁶). 7 CFU was pre-incubated at 37°C for 1 hour.

[0138] (5) After incubation, 100 μl of each bacterial suspension pre-incubated with the antibody was added to a 24-well plate containing A549 monolayer cells and infected at 37°C for 2 hours. Each concentration was set up in triplicate.

[0139] (6) After the infection is terminated, the culture medium is aspirated and the cell monolayer is gently washed three times with PBS to remove unattached bacteria.

[0140] (7) Add 1% Triton X-100 cell lysis buffer to each well for lysis, inoculate the diluted lysate onto blood agar plates, and incubate at 37°C for 24-48 h before counting colonies.

[0141] 2. Experimental Results:

[0142] 2.1 SpaF protein adhesion inhibition effect

[0143] like Figure 10 As shown, the adhesion ability of bacteria to A549 cells was significantly weakened after pretreatment with SpaF recombinant protein. Compared with the positive control group, the inhibition rate of bacterial adhesion in the 20 μg / ml treatment group was 44.1% (P < 0.05), and the inhibition rate of bacterial adhesion in the 100 μg / ml treatment group was 62.7% (P < 0.05). The epithelial cell adhesion inhibition effect was protein concentration dependent.

[0144] 2.2 SpaF polyclonal antibody adhesion inhibition effect

[0145] like Figure 11 As shown, the adhesion ability of *Corynebacterium striatum* to A549 cells was weakened after pretreatment with SpaF polyclonal antibody. Compared with the positive control group, for strain CS-1, the inhibition rate of bacterial adhesion was 5.16% in the 1:100 antibody titer treatment group, 14.76% in the 1:50 antibody titer treatment group, and 33% in the 1:20 antibody titer treatment group (P < 0.05). The epithelial cell adhesion inhibition effect was antibody dose-dependent, and the inhibition effect was most significant in the 1:20 antibody concentration treatment group.

[0146] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A SpaF polyclonal antibody, characterized in that, The SpaF polyclonal antibody is prepared by using SpaF protein as an antigen, mixing the antigen with an adjuvant to obtain an immunogen, then immunizing animals, collecting positive serum, and separating and purifying it.

2. The SpaF polyclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the SpaF protein is shown in SEQ ID NO.

2.

3. The SpaF polyclonal antibody as described in claim 1, characterized in that, The antigen and physiological saline were diluted at a mass-to-volume ratio of 4 μg:(2-5) μl, and then mixed with an equal volume of the adjuvant to obtain the immunogen.

4. The SpaF polyclonal antibody as described in claim 1, characterized in that, The separation and purification were performed using a HiTraprProtein AFF affinity chromatography column.

5. Application of SpaF protein in any of the following: (1) Application in the preparation of drugs that inhibit the formation of biofilms of Corynebacterium styloides; (2) Application in the preparation of drugs for treating Corynebacterium tumefaciens infection.

6. Application of SpaF protein in the preparation of drugs that inhibit cell adhesion.

7. The application as described in claim 5 or 6, characterized in that, The amino acid sequence of the SpaF protein is shown in SEQ ID NO.

2.

8. The use of the SpaF polyclonal antibody as described in any one of claims 1-4 in any of the following: (1) Application in the preparation of drugs that inhibit the formation of biofilms of Corynebacterium styloides; (2) Application in the preparation of drugs for treating Corynebacterium tumefaciens infection.

9. The use of the SpaF polyclonal antibody as described in any one of claims 1-4 in the preparation of a medicament for inhibiting cell adhesion.

10. A drug, characterized in that, The polyclonal antibody described in any one of claims 1-4 is the main active ingredient.

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