Use of gonococcal ferritin in the preparation of products against gonococci

Abstract

This invention provides the application of gonococcal ferritin in the preparation of anti-gonococcal products, relating to the field of antibody technology. The application of gonococcal ferritin subunits in the preparation of antibodies against gonococcal ferritin inhibits gonococcal infection by intervening in the iron metabolism pathway of gonococci, without inducing classic drug resistance mutations. This overcomes the limitations of existing technologies that rely on antibiotics, achieving broad-spectrum inhibition of drug resistance lineages. It solves the technical problem in existing technologies where multidrug / pan-drug resistance of gonococci leads to complete failure of antibiotic treatment, and the lack of therapeutic targets targeting its core survival mechanisms.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to the application of gonococcal ferritin in the preparation of anti-gonococcal products. Background Technology

[0002] Gonorrhea is caused by the Gram-negative diplococcus Neisseria gonorrhoeae (gonococcus) Neisseria gonorrhoeae Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrhoeae. It can infect the urogenital tract, rectum, oropharyngeal mucosa, and conjunctiva. Urogenital infections most commonly cause purulent urethritis in men and cervicitis in women. However, approximately 50% of infected women and a small percentage of infected men are asymptomatic. Without timely and effective treatment, it can lead to a series of serious short-term or long-term complications.

[0003] There is currently no effective vaccine for gonorrhea; antibiotics are the only truly effective treatment. Since sulfonamide antibiotics were first used to treat gonorrhea in the mid-1930s, Neisseria gonorrhoeae has demonstrated a strong ability to develop drug resistance. It not only develops resistance through selective mutations but also acquires resistance genes from other bacterial species, especially non-gonococcal Neisseria gonorrhoeae. Because Neisseria gonorrhoeae has developed or acquired resistance to all first-line empirical treatments, the only currently effective empirical single-agent treatment option is the third-generation broad-spectrum cephalosporin ceftriaxone. However, in 2009, the highly ceftriaxone-resistant strain H041 was first discovered. And in 2015, the "super-drug-resistant Neisseria gonorrhoeae" strain FC428 was discovered. penA The strain (60.001, ceftriaxone MIC = 0.5 mg / L) has rapidly spread and become prevalent globally. The emergence of highly drug-resistant gonococci has made gonorrhea treatment a major global challenge.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide the application of the gonococcal ferritin subunit as a target in the preparation of anti-gonococcal products, in order to solve the technical problem in the prior art where gonococcal multidrug / pan-drug resistance leads to the complete failure of antibiotic treatment and the lack of a therapeutic target that targets its core survival mechanism.

[0006] A second objective of this invention is to provide the application of the gonococcal ferritin subunit in the preparation of antibodies against gonococcal ferritin.

[0007] The third objective of this invention is to provide a method for preparing an antibody against gonococcal ferritin.

[0008] The fourth objective of this invention is to provide an antibody against gonococcal ferritin.

[0009] The fifth objective of this invention is to provide the application of the antibody against gonococcal ferritin prepared by the above-described method in the preparation of drugs for treating gonococcal infection.

[0010] The sixth objective of this invention is to provide a drug.

[0011] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the application of the bacterial iron protein subunit as a target in the preparation of products against gonococci.

[0012] Furthermore, the product has the function of inhibiting the growth of gonococci; and / or, has the function of preventing or treating gonococcal infection; Preferably, the product includes at least one of antibody, mRNA vaccine, DNA vaccine, vector vaccine, live attenuated vaccine, or inactivated vaccine.

[0013] Secondly, the present invention provides the application of the gonococcal ferritin subunit in the preparation of antibodies against gonococcal ferritin.

[0014] Furthermore, the gonococcal ferritin subunit includes BfrA protein or BfrB protein.

[0015] Thirdly, the present invention provides a method for preparing an antibody against gonococcal ferritin, comprising immunizing animals with gonococcal ferritin subunits as immunogens, separating serum from the immunized animals, and purifying the antibody against gonococcal ferritin.

[0016] Furthermore, the gonococcal ferritin subunit includes BfrA protein or BfrB protein.

[0017] Furthermore, the immunization includes immunizing each animal three times, with the initial immunization dose of gonococcal ferritin subunit being 0.2~1 mg / animal, preferably 0.5 mg / animal, and each subsequent immunization dose being independently half of the initial immunization dose; Preferably, the second immunization is administered between the 10th and 20th day after the first immunization, and more preferably on the 14th day. Preferably, the three immunizations are administered between the 7th and 14th day after the second immunization, with the 7th day being the most preferred. Preferably, the immunization method is subcutaneous injection at multiple sites; Preferably, the preparation method further includes enhancing immunity; Preferably, the booster immunization is administered between the 7th and 14th day after three successful immunizations, with the 7th day being the most preferred. Preferably, the animal includes at least one of chicken, rabbit, mouse, sheep or horse, with rabbit being the most preferred.

[0018] Fourthly, the present invention provides an antibody against gonococcal ferritin, which is prepared by the above-described preparation method.

[0019] Fifthly, the present invention provides the application of the antibody against gonococcal ferritin prepared by the above-described preparation method in the preparation of products for anti-gonococcal infection or detection of gonococcal pathogens; Preferably, the product includes at least one of a reagent, a kit, or a drug.

[0020] In a sixth aspect, the present invention provides a medicine comprising an antibody against gonococcal ferritin prepared by the above-described preparation method; Preferably, it also includes a pharmaceutically acceptable carrier.

[0021] The application of the gonococcal ferritin subunit provided in this invention in the preparation of antibodies against gonococcal ferritin inhibits gonococcal infection by intervening in the iron metabolism pathway of gonococci, without inducing classical drug resistance mutations. This overcomes the limitations of existing technologies that rely on antibiotics, achieving broad-spectrum inhibition of drug resistance lineages. Simultaneously, it can be used as a vaccine target in gonococcal vaccine research. This solves the technical problem in existing technologies where multidrug / pan-drug resistance in gonococci leads to complete failure of antibiotic treatment, and there is a lack of therapeutic targets targeting its core survival mechanisms. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 The recombinant plasmid pET-28a(+)- provided in Example 1 of this invention bfrA The results were verified by double enzyme digestion and sequencing. Lane M in A contains a 10000bp DNA marker, and lane 1 contains pET-28a(+)- bfrA Plasmid, lane 2 is pET-28a(+)- bfrA Plasmid double enzyme digestion product; B is pET-28a(+)- bfrA Partial sequence alignment results from plasmid sequencing; Figure 2 The recombinant plasmid pET-28a(+)- provided in Example 1 of this invention bfrB The results were verified by double enzyme digestion and sequencing. Lane M in A contains a 10000bp DNA marker, and lane 1 contains pET-28a(+)- bfrB Plasmid, lane 2 is pET-28a(+)-bfrB Plasmid double enzyme digestion product; B is pET-28a(+)- bfrB Partial sequence alignment results from plasmid sequencing; Figure 3 The above are the SDS-PAGE detection results of BfrA and BfrB-induced proteins provided in Example 2 of this invention, where lane M in A is the protein marker; lane 1 is pET-28a(+)- bfrA Pre-inducing cells; Lane 2 is pET-28a(+)- bfrA Post-induction bacterial cells; lane 3 is pET-28a(+)- bfrA Supernatant protein after induction; lane 4 is the flow medium during purification; lanes 5 and 6 are purified BfrA protein; lane M in B is the protein marker; lane 1 is pET-28a(+)- bfrB Pre-inducing cells; Lane 2 is pET-28a(+)- bfrB Post-induction bacterial cells; lanes 3 and 4 were pET-28a(+)- bfrB Inclusion body protein after induction; lanes 5 and 6 contain purified BfrB protein; Figure 4 The results of the titer and reactivity detection of BfrA and BfrB polyclonal antibodies provided in Example 3 of the present invention are shown. In this example, A and C are the titer detection results of BfrA and BfrB polyclonal antibodies, respectively, and B and D are the reactivity detection results of BfrA and BfrB polyclonal antibodies by Western blot. Detailed Implementation

[0024] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0025] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Antibodies against gonococcal ferritin were prepared using the gonococcal ferritin subunit as an immunogen. These antibodies inhibit gonococcal infection by intervening in the gonococcal iron metabolism pathway without inducing classical resistance mutations. This demonstrates that the gonococcal ferritin subunit can serve as a target for anti-gonococcal activity, overcoming the limitations of existing technologies that rely on antibiotics and achieving broad-spectrum inhibition of resistance lineages. Furthermore, it can be used as a vaccine target in gonococcal vaccine research. This solves the technical problem in existing technologies where multidrug / pan-drug resistance in gonococci leads to complete failure of antibiotic treatment, and there is a lack of therapeutic targets targeting its core survival mechanisms.

[0028] Accordingly, the present invention provides, in one aspect, the application of the gonococcal ferritin subunit as a target in the preparation of anti-gonococcal products.

[0029] In some specific embodiments, the product has the function of inhibiting the growth of gonococci; and / or, has the function of preventing or treating gonococcal infection.

[0030] The products include at least one of antibody, mRNA vaccine, DNA vaccine, vector vaccine, live attenuated vaccine, or inactivated vaccine.

[0031] According to another aspect of the invention, the use of the gonococcal ferritin subunit in the preparation of antibodies against gonococcal ferritin is also provided.

[0032] In some specific embodiments, the gonococcal ferritin subunit includes BfrA protein or BfrB protein.

[0033] The antibody includes at least one of monoclonal antibody, humanized antibody, fully human antibody, bispecific antibody, or antibody fragment.

[0034] According to another aspect of the present invention, a method for preparing an antibody against gonococcal ferritin is also provided, comprising immunizing animals with gonococcal ferritin subunits as an immunogen, separating serum from the immunized animals, and purifying the antibody against gonococcal ferritin.

[0035] Antibodies against gonococcal ferritin were prepared by animal immunization to ensure efficient antibody induction and obtain antibodies with high titers, high affinity and low cross-reactivity.

[0036] BfrA and BfrB proteins can be used as immunogens alone or in combination. Data shows that both have good antibacterial activity in the preparation of antibodies against gonococcal infection. When the two are used in combination as immunogens, the antibacterial effect of the obtained antibodies is significantly improved.

[0037] In some specific embodiments, the gonococcal ferritin subunit includes BfrA protein or BfrB protein.

[0038] In some specific embodiments, the immunization includes three immunizations per animal. The initial immunization dose of gonococcal ferritin subunit is 0.2-1 mg / animal, preferably 0.5 mg / animal, and each subsequent immunization dose is independently half of the initial immunization dose. In some specific embodiments, the second immunization is administered from day 10 to day 20 after the first immunization, preferably day 14; in some specific embodiments, the third immunization is administered from day 7 to day 14 after the second immunization, preferably day 7; in some specific embodiments, a booster immunization is also included; in some specific embodiments, the booster immunization is administered from day 7 to day 14 after the third immunization, preferably day 7. This three-step immunization process involves the first antigen exposure initiating the initial response, the second promoting IgG class switching and high-frequency somatic mutations, and the third consolidating the memory B cell pool. A booster immunization is administered at the peak antibody titer to obtain high-titer antibodies.

[0039] In some specific implementations, immunization is performed via subcutaneous multi-point injection. This creates multiple local immune foci, significantly improving antigen utilization.

[0040] In some specific embodiments, the animal includes at least one of chicken, rabbit, mouse, sheep or horse, preferably rabbit.

[0041] According to another aspect of the present invention, an antibody against gonococcal ferritin is also provided, which is prepared by the above-described preparation method.

[0042] According to another aspect of the present invention, the use of the antibody against gonococcal ferritin prepared by the above-described preparation method in the preparation of products for anti-gonococcal infection or detection of gonococcal pathogens is also provided.

[0043] In some specific embodiments, the product includes at least one of a reagent, a kit, or a drug.

[0044] According to another aspect of the present invention, a medicament is also provided comprising an antibody against gonococcal ferritin prepared by the above-described preparation method; In some specific implementations, a pharmaceutically acceptable carrier is also included.

[0045] Specifically, the pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, solubilizers, diluents, wetting agents, disintegrants, lubricants, fillers, binders, penetration enhancers, pH adjusters, stabilizers, surfactants, absorption enhancers, thickeners, antioxidants, plasticizers, propellants, atomizing agents, suspending agents, dispersants, colorants, and flavoring agents. Those skilled in the art will recognize that a carrier often has multiple functions; for example, starch can act as both a disintegrant and a binder. Those skilled in the art can routinely select the above-mentioned carriers based on the properties of the drug and the route of administration.

[0046] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0047] The gonococci WHO P, WHO K, WHO L, WHO J, and ATCC49226 used in the following examples are standard strains provided by the National Center for STD Control and Prevention, Chinese Center for Disease Control and Prevention; FA19 and FA1090 were provided by the Dermatology Hospital of Southern Medical University; gonococci G001, G002, P001, P004, P006, P008, P010, P011, P016, Z006, Z007, Z009, M001, M004, M005, M006, and G015 are clinical gonococcal isolates provided by the Dermatology Hospital of Southern Medical University; Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Staphylococcus aureus, Shigella flexneri, and Salmonella enteritidis are quality control strains provided by the Dermatology Hospital of Southern Medical University.

[0048] Example 1: Construction of Prokaryotic Expression Recombinant Plasmid 1. Based on the ferritin-encoding gene of Neisseria gonorrhoeae in GenBank bfrA (LT591901.1, Region: 1013853..1014317) and bfrBThe sequence of the gene (LT591901.1, region: 1014345..1014818) was introduced at both ends. Nco I and Xho I Enzyme cleavage sites are retained, and the C-terminal 6×His tag is preserved for protein purification. Proteins containing enzyme cleavage sites are then purified. bfrA and bfrB The gene sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0049] 2. Enzyme digestion: using restriction endonucleases Nco I and Xho I The synthesized gene fragment and pET-28a(+) plasmid were double-digested and purified.

[0050] 3. Ligation: The purified... bfrA and bfrB The gene fragments were mixed with the pET-28a(+) vector, and the two gene fragments were ligated to pET-28a(+) using T4 DNA ligase.

[0051] 4. Transformation: The ligation products were transformed into DH5α competent cells and plated onto LB solid culture plates containing kanamycin (50 μg / ml), and incubated overnight at 37 ℃.

[0052] 5. Plasmid extraction: Select single clones into LB liquid medium containing kanamycin, and after scale-up culture, extract plasmids using a plasmid extraction kit.

[0053] 6. Identification: Take 1 μl of the extracted plasmid for preliminary identification by 1% agarose gel electrophoresis. Simultaneously, take 1 μl of... Nco I and Xho I After double enzyme digestion, electrophoresis was performed again for verification.

[0054] 7. Sequencing verification: The recombinant plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing sequence was compared with the reference sequence to confirm the correctness of the construction.

[0055] 8. The constructed recombinant plasmids were named pET-28a(+)-bfrA and pET-28a(+)-bfrB, respectively.

[0056] Example 2 Protein Expression and Purification 1. Transformation: Remove competent cells from a -70 ℃ ultra-low temperature freezer, incubate on ice for 5 min, add 100 ng of the successfully constructed recombinant plasmid, mix well, and incubate on ice for 30 min. Heat shock at 42 ℃ for 90 s, then incubate on ice for 5 min. Add 900 μl of antibiotic-free LB medium, and incubate at 37 ℃ and 220 rpm for 1 h. After centrifugation, retain approximately 100 μl of bacterial culture, spread 50 μl onto an LB agar plate containing kanamycin (50 μg / ml), and incubate overnight at 37 ℃.

[0057] 2. Inoculation: Pick a single clone and inoculate it into LB medium containing kanamycin. Shake overnight at 37°C and 220 rpm.

[0058] 3. Induction of expression: Inoculate fresh LB medium containing kanamycin at a ratio of 1:20 and culture until the OD600 is 0.4~0.6 (0.6 in this example). Add IPTG to a final concentration of 1.0 mM and induce at 37℃ and 220 rpm for 4 h. Collect the bacterial culture before and after induction, prepare protein samples, and perform SDS-PAGE electrophoresis analysis for identification.

[0059] 4. BfrA protein purification 1) Cell treatment. Protein expression was induced in large quantities, and the cells were collected by centrifugation. The cells were resuspended in 30 ml of 1×PBS, and sonicated on ice for 10-15 min (3 s intervals, 3 s pause, 15 min in this example). The cells were then centrifuged at 12000 rpm for 15 min at 4°C, and the precipitate and supernatant were collected separately. SDS-PAGE analysis showed that BfrA protein was mainly found in the supernatant.

[0060] 2) Nickel column purification. Equilibrate the nickel column sequentially with ddH2O and non-denaturing equilibration buffer. Load the supernatant sample onto the column and repeat once. Wash and elute sequentially with 1×PBS, non-denaturing wash buffer, and non-denaturing elution buffer containing 60 mM and 500 mM imidazole. Collect the eluents from each stage and perform electrophoresis analysis.

[0061] 3) Dialysis: The eluent containing the target protein is placed into a dialysis bag and dialyzed in 1×PBS buffer.

[0062] 4) Concentration and quantification: The protein after dialysis was concentrated by ultrafiltration, the protein concentration was measured, and SDS-PAGE was performed for verification.

[0063] 5. BfrB protein purification 1) Cell treatment. Protein expression was induced in large quantities, and the cells were collected by centrifugation. The cells were resuspended in 30 ml of 1×PBS, and 30 μl of β-mercaptoethanol and 300 μl of PMSF were added. The cells were then sonicated under ice. The cells were centrifuged at 12000 rpm for 15 min at 4°C, and the precipitate (inclusion bodies) was collected. The precipitate was washed three times with 1×PBS, then resuspended in PBS containing 8 M urea and 30 μl of β-mercaptoethanol. After standing for 30–60 min, the supernatant was collected by centrifugation (60 min in this example) to obtain denatured and dissolved BfrB protein.

[0064] 2) Nickel column purification. Proteins were purified under denaturing conditions by sequentially using equilibration buffer (20 mM PB, 8 M urea, 500 mM NaCl, pH 7.4), washing buffer (containing 10 mM imidazole), and elution buffer (containing 100 mM imidazole).

[0065] 3) Protein dialysis: Collect the eluted protein, perform stepwise dialysis by gradually decreasing the urea concentration, and finally store it in 1×PBS containing 4 M urea and 10% glycerol.

[0066] 4) The concentration and quantification methods are the same as above.

[0067] Example 3: Preparation and purification of rabbit-derived polyclonal antibodies Using the purified BfrA and BfrB proteins from Example 2 as antigens, two New Zealand white rabbits were immunized to prepare polyclonal antibodies.

[0068] 1. Animals: Purchase New Zealand White rabbits weighing approximately 2.5 kg, pre-feed them for 2 weeks, and tag them.

[0069] 2. Preparation of Immunogens: Take the purified proteins BfrA and BfrB obtained in Example 2, and allow them to return to room temperature. Emulsify the antigens with an equal volume of Freund's adjuvant (using complete adjuvant for the initial immunization and incomplete adjuvant for subsequent immunizations) using a dual-syringe, repeatedly pushing and drawing until the emulsion does not disperse when dropped into 37°C water. The initial antigen concentration is 1 mg / ml, with subsequent immunization doses halved.

[0070] 3. Immunization: Multiple subcutaneous injections are used, 0.5 ml per rabbit, with 0.1~0.2 ml injected at each point (0.1 ml injected at each point in this example).

[0071] 4. Immunization schedule: The second immunization is given 14 days after the first immunization, and the third immunization is given 7 days after the second immunization. On the 7th day after the third immunization, blood is collected from the marginal ear artery to test the antibody titer. Those with a normal titer will receive a booster immunization 7 days later, and then a whole blood sample will be collected 7 days after that.

[0072] 5. Serum preparation: After collecting whole blood, incubate in a 37°C water bath for 15-30 min (30 min in this example), cool, and then allow to stand at 4°C for separation. Collect the supernatant, centrifuge at 12000 rpm for 2 min, add 0.02% sodium thimerosal to the supernatant, and store at -20°C.

[0073] 6. Antibody purification: 1) Cleaning the chromatography column: Wash the affinity chromatography column with 20 ml of pure water and 20 ml of 1×PBS in sequence.

[0074] 2) Filter the serum: Take 10 ml of serum and filter it through a 0.45 μm filter.

[0075] 3) Sample loading: Load the filtrate onto the affinity chromatography column, and repeat the loading once with the percolation solution.

[0076] 4) Wash the column: Wash the column with 20 ml of 1×PBS.

[0077] 5) Elution of antibodies: Elution was performed using 0.2 M glycine solution (pH 2.7), the elution curve was monitored, and the antibody peaks were collected.

[0078] 6) Antibody preservation: Adjust the pH to 7.0 with 1M sodium bicarbonate, mix the antibody preservation solution (containing glycerol, BSA and preservative sodium thimerosal) and antibody at a volume ratio of 3:2, dispense into containers and store at -70 ℃.

[0079] Example 4: Preparation and Concentration Determination of Neisseria gonorrhoeae Protein Samples 1. Cell preparation: Take 300 μl of logarithmic growth phase bacterial culture, centrifuge at 8000 rpm for 3 min at 4℃, remove the supernatant, add 1 ml of 1×PBS to wash once, centrifuge again to remove the supernatant.

[0080] 2. Preparation of lysis buffer: Use a bacterial protein lysis kit (manufacturer: Sangon Biotech (Shanghai) Co., Ltd., product number: C500003-0010) to prepare lysis working buffer at a ratio of 1 ml of solution A, 1 μl of solution B and 10 μl of solution C.

[0081] 3. Protein lysis: Add 300 μl of lysis working solution to the bacterial cell pellet, mix thoroughly by pipetting, vortex for 30 s, and shake on a shaker at room temperature for 10 min. Centrifuge at 12000 rpm for 5 min at 4℃, and collect the supernatant for subsequent experiments.

[0082] 4. Protein concentration determination (BCA method) 1) Preparation of standards: Dilute the protein standards with PBS to 0.5 mg / ml.

[0083] 2) Preparation of BCA working solution: Mix 50 volumes of solution A with 1 volume of solution B.

[0084] 3) Sample addition: Add 0 μl, 1 μl, 2 μl, 4 μl, 8 μl, 12 μl, 16 μl, and 20 μl of standard to the 96-well plate sequentially, and bring the volume to 20 μl with standard diluent. The corresponding concentrations are 0 mg / ml, 0.025 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, and 0.5 mg / ml (0~0.5 mg / ml). Add 20 μl of sample to the sample well.

[0085] 4) Reaction solution: Add 200 μl of BCA working solution to each well and incubate at 37 °C for 20-30 min (30 min in this example).

[0086] 5) Detection: The absorbance at 562 nm was measured using a multi-functional microplate reader, a standard curve was plotted, and the protein concentration of the sample was calculated.

[0087] 5. Protein sample preparation: Take an appropriate amount of protein supernatant, mix it with 5× Loading buffer at a ratio of 4:1, centrifuge briefly, boil at 100℃ for 5 min, centrifuge again, and take the supernatant for later use.

[0088] Detection methods 1. Coomassie brilliant blue staining 1) Electrophoresis: Use a 12% or 4-20% gradient Hepes precast gel, loading 5 μl of protein sample and protein marker into each well. After loading according to the experimental design, electrophoresis at a constant voltage of 120 V until the minimum protein reaches the bottom of the gel.

[0089] 2) Staining and destaining: After electrophoresis, remove the gel and place it in Coomassie Brilliant Blue rapid staining solution. Stain on a shaker at room temperature for 30 min. Then destain with distilled water until the bands are clear.

[0090] 3) Imaging: Images are acquired using a gel imaging system.

[0091] 2. Western blot detection 1) Electrophoresis: 1. SDS-PAGE electrophoresis was performed using the Coomassie brilliant blue staining electrophoresis method.

[0092] 2) Transfer: A semi-dry transfer method was used, with the transfer buffer prepared using 5× rapid semi-dry transfer buffer: anhydrous ethanol: deionized water = 1:1:3. After activation with methanol, the PVDF membrane was assembled with the gel to form a transfer "sandwich" structure (filter paper-PVDF membrane-gel-filter paper).

[0093] 3) Blocking: After transfer, block with 5% skim milk at room temperature for 1 hour.

[0094] 4) Antibody incubation: The primary antibody diluted 1:1000 (incubated overnight at 4°C or 1 h at 37°C) and the secondary antibody diluted 1:5000 (incubated at room temperature for 1 h) were incubated sequentially. After each incubation, the antibody was washed 3 times with TBST for 10 min each time.

[0095] 5) Development: Mix ECL developer solution A and solution B in a 1:1 ratio, drop the mixture onto the PVDF membrane strip, and use a gel imaging system to image and photograph the membrane.

[0096] 3. ELISA testing 1) Coating: Dilute the protein to 1 μg / ml with coating buffer, add 50 μl to a polystyrene coating plate, and coat overnight at 4°C.

[0097] 2) Blocking: Wash the plate once with PBST, add 150 μl of 1% BSA to each well, and block at 37℃ for 1 h.

[0098] 3) Primary antibody incubation: Add 50 μl of serially diluted test antibody to each well, and set up a negative control (rabbit serum that has not been immunized with any protein). Incubate at 37°C for 30 min, and wash 3 times with PBST.

[0099] 4) Secondary antibody incubation: Add 50 μl of HRP-labeled goat anti-rabbit secondary antibody diluted 1:50000 to each well, incubate at 37℃ for 45 min, and wash 3 times with PBST.

[0100] 5) Color development and termination: Add 50 μl of TMB substrate to each well, incubate at 37°C for 10 min, and then add 50 μl of 1 M sulfuric acid to terminate the reaction.

[0101] 6) Detection: OD was measured using a multifunctional microplate reader. 450 Save the data and analyze the results.

[0102] 4. Detection of gonococcal immunofluorescence 1) Smearing and fixation: Spread the bacterial culture onto a detachable glass slide, allow it to air dry, fix it with immunostaining permeabilization buffer at room temperature, and punch holes for 10 min. Wash 3 times with PBS.

[0103] 2) Blocking and antibody incubation: Block with immunostaining blocking solution at room temperature for 30 min; add 1:200 diluted primary antibody and 1:100 diluted FITC-labeled secondary antibody successively, incubate for 30 min each, and wash 3 times with PBS.

[0104] 3) Mounting and observation: Mount the slide with an anti-fluorescence quenching mounting medium containing DAPI, and observe and acquire images under a confocal microscope.

[0105] 5. Cell immunofluorescence detection 1) Cell culture and infection: HeLa cells were cultured at a concentration of 1×10⁻⁶ cells / mL. 5 / wells were inoculated into 12-well plates and infected with Neisseria gonorrhoeae at an MOI of 100:1 during the logarithmic growth phase, and incubated for 4 h.

[0106] 2) Fixation and permeabilization: Wash 3 times with PBS, add 500 μl of immunostaining permeabilization buffer (containing 1% Triton X-100), treat at room temperature for 15 min, and wash with PBS.

[0107] 3) Blocking: Block with immunostaining blocking solution at room temperature for 30 min.

[0108] 4) Primary antibody: Dilute rabbit primary antibody 1:200, incubate at room temperature for 30 min, and wash 3 times with PBS.

[0109] 5) Secondary antibody: Dilute FITC-labeled anti-rabbit secondary antibody 1:1000, incubate at 4℃ in the dark for 30 min, wash 3 times with PBST, 500 μl / well, shake gently for 5 min each time.

[0110] 6) Cytoskeleton staining: Rhodamine-labeled phalloidin was diluted 1:200 with PBS, 500 μl / well, incubated at room temperature in the dark for 30 min, and washed 3 times with PBST.

[0111] 7) Nuclear staining: DAPI (5 mg / ml) was diluted 1:3000 with 1×PBS, 200 μl / well, and incubated at room temperature in the dark for 10 min.

[0112] 8) Image acquisition: Observe and capture multi-channel fluorescence images under a fluorescence microscope.

[0113] 6. In vitro antibacterial activity of BfrA and BfrB polyclonal antibodies against Neisseria gonorrhoeae 1) Strains selection: Select drug-resistant gonococcal strains and gonococcal susceptible strains, as shown in Table 1.

[0114] 2) Resuscitation: Remove the strain from the ultra-low temperature freezer and immediately place it in a 37°C incubator for thawing. In a biosafety cabinet, take 3-5 loops and inoculate onto a preheated TM plate, streak the plate in sections, and incubate overnight in a 37°C, 5% CO2 incubator.

[0115] 3) Activation of strain: After the strain to be revived grows to the logarithmic growth phase, select clones, streak them, and transfer them to blood agar plates. Incubate at 37°C and 5% CO2 for 18-20 h (18 h in this example).

[0116] 4) Preparation of bacterial suspension: Remove the culture plate, scrape colonies into sterile physiological saline, adjust the bacterial suspension to 0.5 McFarland point using a turbidimeter, and then dilute it 10-fold with sterile physiological saline to a final concentration of 10-fold. 7CFU / ml concentration.

[0117] 5) Inoculation: Add 150 μl of the prepared 0.5 McFarland spot bacterial suspension to 10.5 ml of MH broth diluent and mix thoroughly. Using a multichannel pipette, add 100 μl of the diluted bacterial suspension to each well of a 96-well microplate containing different concentrations of BfrA and BfrB polyclonal antibodies. The antibody concentrations were set as follows: 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.13 μg / ml, 1.56 μg / ml, 0.78 μg / ml, 0.39 μg / ml, and 0.2 μg / ml. Wells without polyclonal antibodies were used as growth controls, and MH broth without bacteria was used as a blank control. A total of 12 groups were prepared.

[0118] 6) Incubation: Cover the inoculated 96-well microplate with the plate lid and place it in a humidified box to prevent moisture evaporation. Incubate at 37°C and 5% CO2 for 20-24 h (24 h in this example) and observe the results.

[0119] 7) MIC value interpretation: Observe the blank control wells without bacterial strain to confirm that the liquid is clear and there is no sterile growth. At the same time, observe the growth control wells without antibodies to confirm that the bacteria are growing normally and will become turbid. Observe the growth of bacteria one by one from low concentration to high concentration, and use the sterile growth wells observed by the naked eye as the MIC value.

[0120] 8) Criteria for interpreting drug sensitivity results: MIC (mg / L or μg / ml) is the lowest concentration at which polyclonal antibodies inhibit the growth of Neisseria gonorrhoeae.

[0121] Test results 1. Expression and purification of ferritin BfrA and BfrB proteins 1) pET-28a(+)- bfrA and pET-28a(+)- bfrB Double enzyme digestion and sequencing identification of plasmids Recombinant plasmid pET-28a(+)- bfrA and pET-28a(+)- bfrB After double digestion with Nco I and Xho I, the fragments were analyzed by 1% agarose gel electrophoresis. The results showed that the insert size was 473 bp (…). Figure 1 (A) and 482bp ( Figure 2 The size of the inserted plasmid (A) is consistent with the expected size. Sequencing alignment confirmed that the plasmid insertion sequence is a perfect match for the expected sequence. Figure 1 China B and Figure 2 The result (B) indicates that the two recombinant plasmids were successfully constructed.

[0122] 2) Expression and purification of BfrA and BfrB proteins The successfully constructed recombinant plasmids were transformed into BL21 strain, and after IPTG induction, the proteins were purified by nickel affinity chromatography. The bacterial cells before and after induction, as well as the purified proteins, were collected for SDS-PAGE electrophoresis analysis. The results showed that BfrA protein was mainly expressed in the supernatant, while BfrB protein was expressed in inclusion bodies. At approximately 18 kDa (… Figure 3 (A) and 22 kDa ( Figure 3 Clear target protein bands were observed at (B) and (B) positions, consistent with the expected molecular weights of BfrA and BfrB. After purification, prokaryotic expression proteins of high purity BfrA and BfrB were successfully obtained.

[0123] 2. Identification of the specificity and sensitivity of BfrA and BfrB polyclonal antibodies 1) Determination of the titer and reactivity of polyclonal antibodies Using the antibody prepared in Example 3 as a sample, the antibody titer and EC were detected by ELISA. 50 (Half-maximal effect dilution factor). Results showed that the BfrA antibody titer was 1:1,024,000, and the purified antibody EC... 50 The ratio is 1:210,711 ( Figure 4 (A); BfrB antibody titer was 1:1,024,000, purified antibody EC 50 The ratio is 1:1,030,289 ( Figure 4 (C). Both antibodies exhibited high titers and binding efficiency.

[0124] 2) Specificity assessment Western blot analysis of antibody specificity showed that the BfrA and BfrB polyclonal antibodies specifically recognized the corresponding proteins in gonococcal standard strains and clinical isolates at approximately 18 kDa and 22 kDa, respectively. Figure 4 Lanes B and D are used for the following strains: lanes 1-7 contain WHO L, WHO K, WHO G, WHO E, WHO D, WHO P, and WHO J, lanes 8-9 contain clinically isolated gonococcal strains G001 and G002, lane 10 contains WHO L, and lanes 11-18 contain Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Staphylococcus aureus, Shigella flexneri, and Salmonella enteritidis, respectively. Data show that neither strain nor strain exhibits cross-reactivity with a variety of non-gonococcal bacteria (including Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Staphylococcus aureus, Shigella flexneri, Salmonella enteritidis, and Staphylococcus epidermidis). This indicates that the prepared antibodies have good specificity.

[0125] 3. Polyclonal antibody activity against drug-resistant Neisseria gonorrhoeae The results of the polyclonal antibody activity against Neisseria gonorrhoeae are shown in Table 1. The data show that the purified BfrA and BfrB polyclonal antibodies have good antibacterial activity against a variety of multidrug-resistant or superdrug-resistant Neisseria gonorrhoeae within a certain concentration range, with MICs ranging from 3.125 to 12.5 μg / ml. In contrast, the purified serum from the control rabbit serum did not show any antibacterial activity.

[0126] Table 1

[0127] The criteria for determining resistance, sensitivity, and intermediate sensitivity were based on CLS1. Performance Standards for Antimicrobial Susceptibility Testing. 36th ed. CLSI supplement M100. Clinical and Laboratory Standards Institute; 2026. R indicates resistance, I indicates intermediate sensitivity, and S indicates sensitivity.

[0128] 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. Application of gonococcal ferritin subunit as a target in the preparation of anti-gonococcal products.

2. The application according to claim 1, characterized in that, The product has the function of inhibiting the growth of gonococci; and / or has the function of preventing or treating gonococcal infection; Preferably, the product includes at least one of antibody, mRNA vaccine, DNA vaccine, vector vaccine, live attenuated vaccine, or inactivated vaccine.

3. Application of gonococcal ferritin subunit in the preparation of antibodies against gonococcal ferritin.

4. The application according to any one of claims 1 to 3, characterized in that, The gonococcal ferritin subunit includes BfrA protein or BfrB protein.

5. A method for preparing an antibody against gonococcal ferritin, characterized in that, This includes immunizing animals with gonococcal ferritin subunits as immunogens, separating serum from the immunized animals, and purifying the serum to obtain antibodies against gonococcal ferritin.

6. The preparation method according to claim 5, characterized in that, The gonococcal ferritin subunit includes BfrA protein or BfrB protein.

7. The preparation method according to claim 5, characterized in that, The immunization includes three immunizations per animal. The initial immunization dose of gonococcal ferritin subunit is 0.2-1 mg / animal, preferably 0.5 mg / animal, and each subsequent immunization dose is independently half of the initial immunization dose. Preferably, the second immunization is administered between the 10th and 20th day after the first immunization, and more preferably on the 14th day. Preferably, the three immunizations are administered between the 7th and 14th day after the second immunization, with the 7th day being the most preferred. Preferably, the immunization method is subcutaneous injection at multiple sites; Preferably, the preparation method further includes enhancing immunity; Preferably, the booster immunization is administered between the 7th and 14th day after three successful immunizations, with the 7th day being the most preferred. Preferably, the animal includes at least one of chicken, rabbit, mouse, sheep or horse, with rabbit being the most preferred.

8. An antibody against gonococcal ferritin, characterized in that, It is prepared by the preparation method according to any one of claims 3 to 7.

9. The use of the antibody against gonococcal ferritin prepared by the preparation method according to any one of claims 5 to 7 in the preparation of products for anti-gonococcal infection or detection of gonococcal pathogens; Preferably, the product includes at least one of a reagent, a kit, or a drug.

10. A drug, characterized in that, The antibody prepared by the method according to any one of claims 5 to 7 contains an antibody against gonococcal ferritin. Preferably, it also includes a pharmaceutically acceptable carrier.

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