Preparation and application of anti-fusobacterium nucleatum pknk and pknl protein monoclonal antibody

Abstract

The application discloses preparation and application of anti-Fusobacterium nucleatum PknK and PknL protein monoclonal antibodies and belongs to the technical field of biotechnology. The application takes two virulence proteins PknK and PknL of the Fusobacterium nucleatum as immunogens, successfully prepares monoclonal antibodies against the serine / threonine protein kinases PknK and PknL of the Fusobacterium nucleatum through hybridoma cell fusion technology, and the two monoclonal antibodies against the PknK and PknL proteins of the Fusobacterium nucleatum have good safety, thereby providing a basis for clinical drug safety. More importantly, the antibodies can not only efficiently neutralize bacterial toxins and directly eliminate the pathogenicity of the Fusobacterium nucleatum, but also can be directly combined on the bacterial surface to enhance the recognition and phagocytosis efficiency of immune cells to the bacteria, thereby clearing pathogenic bacteria, and is expected to be developed into a drug for resisting the Fusobacterium nucleatum infection and has a very good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation and application of monoclonal antibodies against PknK and PknL proteins of Cryptococcus pyogenes. Background Technology

[0002] Cryptococcus pyogenes ( Trueperella pyogenes , T. pyogenes Cryptobacterium is a Gram-positive, non-motile, non-spore-forming facultative anaerobic rod-shaped bacterium. It is the most pathogenic zoonotic pathogen in the Cryptobacterium genus, causing purulent infections in livestock, poultry, wild animals, and even humans. It not only causes serious economic losses to the livestock industry but also seriously threatens human health.

[0003] In the current technological context, the clinical diagnosis of Cryptococcus pyogenes mainly relies on nucleic acid-based molecular detection techniques (such as PCR and LAMP) and traditional bacterial culture methods. However, these methods have significant limitations: while PCR technology has a certain sensitivity, it is prone to false positives; and traditional culture methods are time-consuming and have low sensitivity, making them difficult to meet the needs of rapid diagnosis. More importantly, the existing technological system lacks highly specific recognition tools for the bacterium's key functional antigens (especially virulence proteins closely related to its pathogenicity), which not only limits the establishment of serological diagnostic methods but also hinders the development of related targeted therapy strategies.

[0004] Currently, the prevention and control of Cryptococcus pyogenes infection mainly relies on antibiotics to control and treat the related disease. However, Cryptococcus pyogenes has developed varying degrees of resistance to many antibiotics, making treatment extremely difficult. Furthermore, traditional vaccines cannot provide animals with specific protection against the bacteria. T. pyogenes Complete immune protection against infection. Therefore, research and development of [treatments / treatments] is needed. T. pyogenes Novel antibacterial therapies for infections are urgently needed. Existing research has shown that monoclonal antibodies have demonstrated good practical value in the prevention and treatment of bacterial infectious diseases. Unlike traditional antibiotics, antibacterial monoclonal antibodies typically target bacterial virulence proteins rather than proteins essential for bacterial survival. They modulate the phagocytic activity of host immune cells, promoting their recognition and engulfment of bacteria. This mechanism of action not only effectively inhibits the pathogenicity of pathogens but also reduces the risk of bacterial resistance.

[0005] Serine / threonine protein kinases are crucial signaling elements mediating transcription and regulation in bacteria, playing a vital role in stress response, metabolic regulation, and pathogenicity. Previous research in our group has shown that serine / threonine protein kinases PknK and PknL are involved in the regulation of pathogenicity in *Cryptobacillus pyogenes*. Developing monoclonal antibodies targeting PknK and PknL holds promise for reducing bacterial pathogenicity and reducing the likelihood of inducing drug resistance. Therefore, antibody therapy shows great potential as a new and promising alternative to traditional antibiotics. Currently, there are no reports on the preparation and function of monoclonal antibodies targeting *Cryptobacillus pyogenes* PknK and PknL proteins. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a method for preparing and applying monoclonal antibodies against PknK and PknL proteins of Cryptococcus pyogenes. This invention uses two virulence proteins of Cryptococcus pyogenes, PknK and PknL, as immunogens. Monoclonal antibodies against the serine / threonine protein kinases PknK and PknL of Cryptococcus pyogenes were successfully prepared through hybridoma cell fusion technology. Their in vitro functions were clarified, and an immunological detection method for Cryptococcus pyogenes based on indirect ELISA was established. This provides potential biomaterials for the diagnostic technology of Cryptococcus pyogenes infection and the development of novel anti-infection strategies.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an anti-PknK protein monoclonal antibody, wherein the anti-PknK protein monoclonal antibody is secreted by the hybridoma cell line PknK-C11 with accession number CCTCC NO:C202629.

[0009] Based on the above technical solution, the anti-PknK protein monoclonal antibody is further described as an antibody of IgG1 subtype and κ light chain.

[0010] Secondly, the present invention provides an anti-PknL protein monoclonal antibody, wherein the anti-PknL protein monoclonal antibody is secreted by the hybridoma cell line PknL-B5 with accession number CCTCC NO:C202632.

[0011] Based on the above technical solution, the anti-PknL protein monoclonal antibody is further described as an antibody of IgG1 subtype and κ light chain.

[0012] Thirdly, the present invention provides a hybridoma cell line PknK-C11 that secretes a monoclonal antibody against PknK protein, which was deposited at the China Center for Type Culture Collection on February 3, 2026, with accession number CCTCC NO:C202629.

[0013] Fourthly, the present invention provides a hybridoma cell line PknL-B5 that secretes monoclonal antibodies against PknL protein, which was deposited at the China Center for Type Culture Collection on February 3, 2026, with accession number CCTCC NO:C202632.

[0014] Fifthly, the present invention provides the use of the above-mentioned anti-PknK protein monoclonal antibody, anti-PknL protein monoclonal antibody, hybridoma cell line PknK-C11 secreting anti-PknK protein monoclonal antibody, and hybridoma cell line PknL-B5 secreting anti-PknL protein monoclonal antibody in the preparation of drugs for the prevention or treatment of Cryptococcus pyogenes.

[0015] Based on the above technical solution, the drug further includes pharmaceutically acceptable excipients.

[0016] Fifthly, the present invention provides the use of the above-mentioned anti-PknK protein monoclonal antibody, anti-PknL protein monoclonal antibody, hybridoma cell line PknK-C11 secreting anti-PknK protein monoclonal antibody, and hybridoma cell line PknL-B5 secreting anti-PknL protein monoclonal antibody in the preparation of reagents or kits for detecting Cryptococcus pyogenes.

[0017] In a sixth aspect, the present invention provides a kit for detecting Cryptococcus pyogenes, wherein the kit contains the aforementioned anti-PknK protein monoclonal antibody or anti-PknL protein monoclonal antibody.

[0018] Based on the above technical solution, the coating protein is further defined as recombinant protein PknK or recombinant protein PknL, and HRP-labeled goat anti-mouse IgG is used as the secondary antibody.

[0019] Based on the above technical solution, the kit further includes a colorimetric reagent, a termination reagent, a blocking solution, and a washing solution.

[0020] Based on the above technical solution, the colorimetric reagent is TMB colorimetric solution; the termination reagent is 1~3M sulfuric acid solution.

[0021] Based on the above technical solution, the blocking solution is a 5% BSA solution; the washing solution is a PBST buffer.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention has for the first time obtained two monoclonal antibodies that are respectively anti-PknK and PknL proteins of Cryptococcus pyogenes. Both of these monoclonal antibodies are IgG1 subtype antibodies with κ light chain, which are the most common and effective antibody types involved in humoral immunity. They provide candidate molecules for the development of antibody drugs for prevention or treatment of Cryptococcus pyogenes infection.

[0023] (2) The monoclonal antibody obtained in this invention can effectively bind to the PknK and PknL proteins of Cryptococcus pyogenes and can be used for immunological detection such as ELISA and Western Blot. Through the binding reaction of antigen and antibody, the diagnosis of Cryptococcus pyogenes infection can be achieved.

[0024] (3) The two monoclonal antibodies against PknK and PknL proteins of Cryptococcus pyogenes obtained in this invention both exhibit good safety profiles, providing a foundation for safe clinical use. More importantly, these antibodies not only efficiently neutralize bacterial toxins and directly eliminate the pathogenicity of Cryptococcus pyogenes, but also directly bind to the bacterial surface, enhancing the recognition and phagocytic efficiency of immune cells and clearing pathogens. They hold promise for development into a novel therapy against Cryptococcus pyogenes infection. Attached Figure Description

[0025] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0026] Figure 1 The figure shows the serum titer results of mice immunized with PknK (A) and PknL (B) proteins.

[0027] Figure 2 The results show the titer of anti-PknK protein monoclonal antibody (A) and anti-PknL protein monoclonal antibody (B) in mouse ascites fluid.

[0028] Figure 3 The purification and identification results of anti-PknK protein monoclonal antibody (A) and anti-PknL protein monoclonal antibody (B) in mouse ascites fluid.

[0029] Figure 4 The results show the stability assay of anti-PknK protein monoclonal antibodies and anti-PknL protein monoclonal antibodies secreted by positive hybridoma cells.

[0030] Figure 5 The results show the specificity identification of anti-PknK protein monoclonal antibodies and anti-PknL protein monoclonal antibodies, where A: anti-PknK protein monoclonal antibody; B: anti-PknL protein monoclonal antibody; M: 8~195 and 10~180 kDa Markers; 1: Cryptococcus pyogenes; 2: Staphylococcus aureus; 3: Streptococcus pneumoniae; 4: Escherichia coli; 5: Pseudomonas aeruginosa.

[0031] Figure 6 The results show the subtype identification of anti-PknK protein monoclonal antibody (A) and anti-PknL protein monoclonal antibody (B).

[0032] Figure 7The results show the binding ability of anti-PknK protein monoclonal antibodies and anti-PknL protein monoclonal antibodies to Cryptorchidica pyogenes.

[0033] Figure 8 To investigate the effects of anti-PknK protein monoclonal antibody (A) and anti-PknL protein monoclonal antibody (B) on the number of Cryptorchidica colonies phagocytosed by macrophages, compared with the control group, The difference was highly significant (P < 0.01). The difference was significant (0.01 < P < 0.05), n = 3.

[0034] Figure 9 The results show the inhibitory effects of anti-PknK protein monoclonal antibody (A) and anti-PknL protein monoclonal antibody (B) on bacterial hemolysis. This indicates a highly significant difference (P < 0.01). The difference is considered significant (0.01 < P < 0.05), n = 3.

[0035] Figure 10 The results show the hemolytic activity of anti-PknK protein monoclonal antibodies and anti-PknL protein monoclonal antibodies.

[0036] Figure 11 The effects of anti-PknK protein monoclonal antibody (A) and anti-PknL protein monoclonal antibody (B) on the viability of RAW264.7 cells were investigated. This indicates a highly significant difference (P < 0.01). The difference is considered significant (0.01 < P < 0.05), n = 3. Detailed Implementation

[0037] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0038] Both PknK and PknL proteins were expressed, purified, and prepared in our laboratory (the amino acid sequences of PknK and PknL proteins are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively). Mouse myeloma cells Sp2 / 0-Ag14[SP2 / 0](CL-0445) were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0039] SEQ ID NO:1 MQESTLGAYTLRWSMGGNYATSVASSPIFLLTDPASDPGARLYARRLCDGMGALARTGVIPEPEQVRRVWADALAAAESDLATITLMASDHDIPDIGLRAALLAAVSVEGNMRWLVHSIGGLGVIHDDGTRHTTITSSTELPMRARDRFVLLSDAVIRGHSSGRTNNVISQLPRAQRAAAVLVSSVPAAGRDAATAIVVDVHDADSLAALDFPETLMSAEVEAVSEPSLVSDWAMQVLGVQGRARVDESPLTAPDLDDFVFQSYLGSGGFADVFLYEEQTPKRLVAIKVLKSDGRAGRHFRSEIDVMGQLGAHPSIVSIYDAALAPTGQPYIVMQYCPGPSLSDQLSRGPLPVADALRMGIQLSGATHTAHMLGIAHYDIKPSNVLTTAFGRYALSDFGIATLVGNASTDVLGMSLPWAAPEVLRGEECGCPADIYSVGATLYTALYGHAPFAVAGLGKRAYIEHVHTGDIAYPPIDGISEPVNAAIVDLLSAALERDVAARTSSVEMIGRGLQEIQQAMGLAATELEIPQTETWNTPPSGIVPL SEQ ID NO:2 VKPDPLLGAVIDHRYAISARIARGGMATVYHATDRRLERKVAIKVIHDHLAEQPDFVRRFISEARAAASLSSPHIVAVHDQGVASLPSGERPYLVMELMSGPDLRSELAAYGSFSLGVSLEIVRQVLSGLAVAHEADIIHRDIKPENVLLTAALEPTSIDPKFQ AKLTDFGLARAASDATSTHTNSMLGTVGYVAPEFITDGATSKASDIYSIGIMLYELIAGQLPFQGESGMNVAFKHVNDTMPRLADQADWMPPAVDALISLFTAKSPAKRPVNAQAALDALTDVVASLPEETLIRRIPVFPTEQAPTQAIAAGTSVLPISPTQVI GDGSNFSDLIQPALAGRQDTQVSRHNTQVEDLRRRRRRWPILLVLLIALLGGGSYGTWWYFTQGPGLRINVPNVINLPQADAESALTNAGLVFDVVKDYSDDIVAGHVLAIDPAVGVPIHPSTPVKLVVSEGVEHVNVPDVIGKPQDEASSILQSGRLGVEVTQ AYSEDIAEGAVISQTPASGSFVPHSTSVKLIISLGKEPLPVPDLTKDTLESANRRLDEIGMKGAVTEEFSDTVPAGVVIRQDPVPGEHRHRGDTINMVVSKGPELVKVPNVFGLQEAQATKILKEAGFGVTYNRFLGGYFGTVRAQSPGAGEMVKPGTLITITVV Example 1 1. Preparation of monoclonal antibodies against PknK and PknL proteins 1.1 Animal immunization program and cell fusion Female BALB / c mice aged 5-6 weeks were immunized with PknK and PknL proteins as immunogens according to the standard animal immunization program (PknK and PknL proteins were administered at a dose of 50 μg / mouse; immunization was repeated every two weeks), with 5 mice in each group. After three booster immunizations, blood was collected from the orbital sinus of the mice (the collected serum was used to obtain polyclonal antibodies against PknK and PknL proteins). The antibody titers against PknK or PknL proteins in the serum were detected by indirect ELISA (see 1.2 Screening and Subclonal Culture of Positive Hybridoma Cells). The results of the mouse serum titer determination are shown below. Figure 1 As shown.

[0040] Then, select mice with the highest serum titers and inject them intraperitoneally with recombinant PknK or PknL protein, 100 μg / mouse. Three days later, spleen cells from mice immunized with PknK and PknL protein were mixed with mouse myeloma cells Sp2 / 0-Ag14 [SP2 / 0] (CL-0445) at a ratio of 10:1 in a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min at room temperature and discard the supernatant. Gently tap the bottom of the centrifuge tube with your finger to disperse the precipitated cells. Place the centrifuge tube in a 37°C water bath and slowly add 1 mL of preheated PEG (37°C) to the centrifuge tube over 1 min, rotating the centrifuge tube as you add. Then, incubate the tube in a 37°C water bath for 1 min. Add 1 mL of incomplete DMEM medium over 1 min while continuously and slowly stirring the cells. Add another 1 mL of incomplete DMEM medium over 1 min while continuously and slowly stirring the cells. Finally, add 10 mL of DMEM medium over 3 min. Prepare DMEM incomplete medium and stir the cells slowly and continuously. Remove the centrifuge tubes from the water bath, seal them with sealing film, and gently place them in an incubator to stand for 10 minutes. Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, aspirate the remaining medium, resuspend the cell pellet in 3 mL of incomplete medium, add the prepared HAT complete medium, and mix gently. Add the cell suspension to 0.1 mL of feeder cell layer to each well of a 96-well plate. Incubate the plate at 37°C in a 5% CO2 incubator for 5 days without shaking the plate. After 7 days of fusion, observe and record the wells where fused hybridoma cells appear.

[0041] 1.2 Screening and subclonal culture of positive hybridoma cells Recombinant proteins PknK and PknL (10 μg / mL) were coated onto ELISA plates and incubated at 4°C for 12 h. The supernatant of positive hybridoma cells was detected using an indirect ELISA method. The specific steps were as follows: wash the plate with PBST buffer, shaking for 3 min each time, then blot dry, repeating 3 times. Then, add 100 μL of 5% BSA blocking buffer (PBST as solvent) to each well, incubate at 37°C for 2 h, repeat the washing process, add 200 μL of primary antibody (collected hybridoma cell supernatant), and incubate at 37°C for 2 h. Blank and negative control wells were also prepared (blank control: PBST as primary antibody; negative control: mouse serum not immunized with recombinant protein as primary antibody), and the plate was washed repeatedly. Finally, 100 μL of secondary antibody (HRP-labeled goat anti-mouse IgG) was added at a ratio of 1:5000, and the plate was incubated at 37°C for 2 h. The plate was washed repeatedly, and finally, 50 μL of secondary antibody (HRP-labeled goat anti-mouse IgG) was added to each well. μL of single-component TMB chromogenic solution was incubated at 37°C in the dark for 30 min, and then stop solution (2 M sulfuric acid solution) was added immediately. OD was then measured using a microplate reader. 450The ratio of sample wells to negative wells is used as the criterion for determining a positive result. A ratio greater than 2.1 is considered positive, and a ratio less than 2.1 is considered negative. Cells that test positive and show good growth are transferred to 24-well cell culture plates for expansion culture and then frozen.

[0042] Subcloning of positive hybridoma cells was performed using a limiting dilution method. First, viable cells in positive wells were accurately counted and diluted to 100 cells / mL with complete culture medium. After culturing at 37°C and 5% CO2 for 5 days, cell cloning was observed under a microscope. Only single clones were recorded as growing. Cell clones were visible to the naked eye after 8-9 days, and antibody detection was performed promptly. Positive cell wells were selected for further subcloning and passaged at least three times. Cell cloning was considered complete when the positivity rate of the supernatant containing all single cell colonies in a detection plate reached 100%. After three subcloning processes, strongly positive hybridoma cells were selected.

[0043] The positive hybridoma cell line secreting monoclonal antibody against PknK protein was named PknK-C11 and deposited at the China Center for Type Culture Collection (CCTCC) on February 3, 2026, with accession number CCTCC NO: C202629; the positive hybridoma cell line secreting monoclonal antibody against PknL protein was named PknL-B5 and deposited at the China Center for Type Culture Collection (CCTCC) on February 3, 2026, with accession number CCTCC NO: C202632.

[0044] 1.3 Preparation, purification, and antibody concentration detection of ascites fluid Six 8-week-old BALB / c mice were selected, and each mouse was injected intraperitoneally with 0.5 mL of liquid paraffin. Seven to ten days later, cells were inoculated intraperitoneally at a density of 5 × 10⁻⁶. 5 The mice were inoculated with positive hybridoma cells at a concentration of [number] cells / mL. Seven days after inoculation, ascites production was observed. Ascites fluid was collected when the abdomen was significantly distended. The fluid was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. Antibodies were purified using rProtein G chromatography. The antibody purification efficiency was assessed by SDS-PAGE, and antibody concentration was determined using the BCA method. The titers of anti-PknK and PknL monoclonal antibodies in mouse ascites fluid are shown below. Figure 2 As shown, the purification effect is as follows. Figure 3 .

[0045] 2. Detection of secretion stability and specificity of monoclonal antibodies against PknK and PknL proteins Positive hybridoma cells were cryopreserved in liquid nitrogen, thawed, and continuously passaged in vitro. Cell supernatant was collected every 5 passages, and titers were determined using indirect ELISA to assess antibody secretion by the positive hybridoma cells. The results of the secretory stability of anti-PknK and PknL protein monoclonal antibodies are as follows: Figure 4 As shown.

[0046] Cryptococcus pyogenes (BM-H06-3), Staphylococcus aureus (ATCC 29213), Streptococcus pneumoniae (ATCC 49619), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 15692) were used as test bacteria. Total bacterial protein was extracted from these test bacteria, and the protein samples from each strain were subjected to SDS-PAGE, transferred to PVDF membranes, and then blocked overnight with 5% BSA solution. The specificity of the prepared monoclonal antibodies was detected using the prepared anti-PknK and anti-PknL protein monoclonal antibodies as primary antibodies and HRP-labeled goat anti-mouse IgG as secondary antibodies. The specificity identification results of the anti-PknK and anti-PknL protein monoclonal antibodies are as follows: Figure 5 As shown in the figure (1: Cryptococcus pyogenes; 2: Staphylococcus aureus; 3: Streptococcus pneumoniae; 4: Escherichia coli; 5: Pseudomonas aeruginosa).

[0047] 3. Identification of monoclonal antibody subtypes against PknK and PknL proteins Identification was performed according to the instructions on the mouse monoclonal antibody subtype identification kit. The specific steps were followed exactly as instructed, and the OD values ​​were finally read using a microplate reader. 450 OD 450 The well with the highest value corresponds to the corresponding subtype; the subtype identification results of anti-PknK and PknL protein monoclonal antibodies are as follows: Figure 6 As shown.

[0048] 4. Establishment of a detection method for Cryptobacterium pyogenes based on indirect ELISA Cryptobacterium pyogenes BM-H06-3, isolated and preserved in our laboratory, was inoculated into BHI medium and cultured at 37°C with shaking until the logarithmic growth phase (OD200). 600 =0.6). Take 3 mL of bacterial culture, centrifuge at 12000 rpm for 5 min, and discard the supernatant. Gently resuspend the bacterial cells in pre-cooled PBS (pH 7.4), and repeat the centrifugation and washing twice. Resuspend the washed bacterial pellet in 4% paraformaldehyde solution, fix at 4℃ for 2 h, and dilute with PBS (pH 7.4) to OD. 600=0.15, the diluted bacterial suspension was coated. Then, washing and blocking were performed according to the indirect ELISA method. Next, serially diluted anti-PknK or PknL protein monoclonal antibodies (initial concentration 400 μg / mL, 2-fold serial dilution) were added to the wells. After incubation and washing, horseradish peroxidase-labeled secondary antibodies were added. Finally, TMB substrate solution was added for colorimetric reaction, and absorbance was measured at 450 nm. The judgment criterion for this method is: if the OD value of the corrected monoclonal antibody well is ≥ 2.1 times the OD value of the negative control (i.e., P / N ratio ≥ 2.1), it is considered a positive binding. The results of the binding ability of anti-PknK and PknL protein monoclonal antibodies to *Cryptobacillus pyogenes* are as follows. Figure 7 As shown, a positive result indicates that the detected bacteria is Cryptococcus pyogenes, marking the successful establishment of this detection method.

[0049] 5. Functional identification of monoclonal antibodies against PknK and PknL proteins 5.1 Assessment of the phagocytic capacity of immune cells promoted by monoclonal antibodies against PknK and PknL proteins Collect the cultured RAW264.7 cell suspension and adjust its cell density to 5 × 10⁻⁶ cells / mL. 4 RAW264.7 cell suspension was seeded into 24-well plates and incubated at 37°C (5% CO2) for 18 h. Four treatment groups were set up: PBS (pH 7.4) control group, anti-PknK (or PknL) polyclonal antibody group, anti-PknK (or PknL) monoclonal antibody group, and anti-PknK (or PknL) monoclonal antibody and polyclonal antibody mixed group (mixed at 1:1). The antibodies of each treatment group were diluted with PBS (pH 7.4) and added to the cell wells. The cells were incubated at 37°C, 5% CO2 for 4 h. Each group was repeated in triplicate, and the amount of antibody in each group was 20 μg / well.

[0050] Single colonies of *Cryptobacter pyogenes* BM-H06-3 were picked and cultured in BHI medium at 37°C with shaking at 180 rpm until the logarithmic growth phase. The bacterial culture was then washed three times with PBS (pH 7.4) and adjusted to OD200. 600 The value was approximately 0.2. After antibody incubation, 200 μL of bacterial culture was added to each cell well and gently shaken to ensure adequate bacterial contact with the cells. The mixture was then incubated at 37°C in a 5% CO2 incubator for 2 hours. After incubation, gentamicin (100 μg / mL) was added to each well, and the mixture was incubated at 37°C in a 5% CO2 incubator for 2 hours to kill any unphagocytosed extracellular bacteria. After sterilization, the supernatant was discarded, and the cells were washed three times with sterile PBS (pH 7.4). 1 mL of sterile water was added to lyse the cells, and the cell lysis buffer was diluted 10 times. 1 ~106 Two 10 μL dilutions were taken and dropped onto MHA blood agar plates, incubated at 37°C in a 5% CO2 bacterial incubator for 36 h, and colony counts were performed to analyze the effects of the two monoclonal antibodies on macrophage phagocytic function and clarify the role of the two antibodies in enhancing the host immune response. The results are as follows: Figure 8 As shown, the results indicate that the two monoclonal antibodies can promote the phagocytic capacity of macrophages and enhance the host immune response.

[0051] 5.2 Evaluation of the neutralizing effect of anti-PknK and PknL protein monoclonal antibodies on bacterial hemolysin Cryptobacterium pyogenes BM-H06-3 was cultured to OD200. 600 =0.3, anti-PknK and anti-PknL monoclonal antibodies were added to achieve concentrations of 10, 20, and 40 μg / mL, respectively, and cultured for 36 h to reach the stationary phase. After centrifugation (5000 rpm, 5 min), 100 μL of bacterial supernatant was added to 850 μL PBS (pH 7.4), followed by 50 μL of 10% erythrocyte suspension. The mixture was incubated at 37℃ for 30 min, centrifuged (5000 rpm, 5 min), and 100 μL of supernatant was transferred to a 96-well plate. The absorbance was measured at 543 nm. The group without anti-PknK or anti-PknL monoclonal antibodies (BM-H06-3 group) was considered 100% hemolysis. The hemolysis rate was calculated to analyze the neutralizing effect of the two monoclonal antibodies on bacterial hemolysins, clarifying the role of the two antibodies in reducing bacterial pathogenicity. Results are as follows. Figure 9 As shown, the results indicate that the two monoclonal antibodies can neutralize the hemolysin secreted by bacteria, inhibit bacterial hemolysis, and reduce bacterial pathogenicity.

[0052] 6. In vitro safety assessment of monoclonal antibodies against PknK and PknL proteins 6.1 Assessment of the hemolytic activity of monoclonal antibodies against PknK and PknL proteins Fresh rabbit blood was collected and defibrinated. One mL of defibrinated rabbit blood was mixed with 10 times its volume of physiological saline, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The precipitated red blood cells were washed three times with physiological saline until the supernatant was no longer red. The obtained red blood cells were suspended in a 2% saline solution. Anti-PknK and PknL protein monoclonal antibodies were serially diluted with PBS (pH 7.4) (e.g., 1:10, 1:100, 1:1000). Samples were added to 1.5 mL centrifuge tubes according to different groups and incubated at 37°C for 1 h. Three replicates were used for each group. After incubation, the samples were centrifuged at 2000 rpm for 5 min, and the supernatant was transferred to 96 wells. The OD value (hemoglobin release) of the supernatant at 540 nm was measured using a microplate reader. The in vitro biosafety of the anti-PknK and PknL protein monoclonal antibodies was analyzed by calculating the hemolysis rate. Results are as follows: Figure 10 As shown, the results indicate that both monoclonal antibodies are safe.

[0053] 6.2 Cytotoxicity assessment of monoclonal antibodies against PknK and PknL proteins The cultured RAW264.7 cells were transferred to a clean bench, the cell culture medium was discarded, and the cells were resuspended in 2 mL of cell culture medium. 10 μL of the resuspended cells were then counted on a cell counting chamber, and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 4 / well. Diluted cells were seeded into 96-well plates, 100 μL of cell suspension per well, and incubated at 37°C, 5% CO2 for 1 h to allow cell adhesion. 100 μL of different concentrations (25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) of the target anti-PknK or PknL protein monoclonal antibody (with blank and negative controls) were added to each well, and the plates were incubated at 37°C, 5% CO2 for 18 h. Medium containing 10% CCK-8 was prepared, and the original medium in each well was discarded. 100 μL of medium containing 10% CCK-8 was added to each well, and the plates were incubated at 37°C, 5% CO2 for 4 h. Cell viability was calculated by measuring absorbance at 450 nm, and the effect on cell activity in vitro was analyzed. Results are as follows: Figure 11 As shown, the results indicate that both monoclonal antibodies are non-toxic to cells.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against PknK protein, characterized in that, The anti-PknK protein monoclonal antibody was secreted by the hybridoma cell line PknK-C11, which has the accession number CCTCC NO:C202629.

2. A monoclonal antibody against PknL protein, characterized in that, The anti-PknL protein monoclonal antibody was secreted by the hybridoma cell line PknL-B5 with accession number CCTCC NO:C202632.

3. A hybridoma cell line, PknK-C11, that secretes a monoclonal antibody against the PknK protein, characterized in that, It was deposited at the China Center for Type Culture Collection on February 3, 2026, with accession number CCTCC NO:C202629.

4. A hybridoma cell line, PknL-B5, that secretes a monoclonal antibody against the PknL protein, characterized in that, It was deposited at the China Center for Type Culture Collection on February 3, 2026, with accession number CCTCC NO:C202632.

5. The use of the anti-PknK protein monoclonal antibody of claim 1, the anti-PknL protein monoclonal antibody of claim 2, the hybridoma cell line PknK-C11 secreting anti-PknK protein monoclonal antibody of claim 3, and the hybridoma cell line PknL-B5 secreting anti-PknL protein monoclonal antibody of claim 4 in the preparation of drugs for the prevention or treatment of Cryptococcus pyogenes.

6. The use of the anti-PknK protein monoclonal antibody of claim 1, the anti-PknL protein monoclonal antibody of claim 2, the hybridoma cell line PknK-C11 secreting anti-PknK protein monoclonal antibody of claim 3, and the hybridoma cell line PknL-B5 secreting anti-PknL protein monoclonal antibody of claim 4 in the preparation of reagents or kits for detecting Cryptococcus pyogenes.

7. A kit for detecting Cryptococcus pyogenes, characterized in that, The kit contains the anti-PknK protein monoclonal antibody of claim 1 or the anti-PknL protein monoclonal antibody of claim 2.

8. The reagent kit according to claim 7, characterized in that, The coating protein was recombinant protein PknK or recombinant protein PknL, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody.

9. The reagent kit according to claim 7, characterized in that, The kit also includes a colorimetric reagent, a termination reagent, a blocking solution, and a washing solution.

10. The kit according to claim 9, characterized in that, The colorimetric reagent was TMB colorimetric solution; the stop reagent was 1-3M sulfuric acid solution; the blocking solution was 5% BSA solution; and the washing solution was PBST buffer.

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