CN116239683A - A kind of anti-Clostridium difficile toxin B monoclonal antibody and its preparation method and application - Google Patents

Abstract

The present invention relates to the field of medical biological antibodies, specifically an anti-Clostridium difficile toxin B monoclonal antibody, the amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 are respectively shown in SEQ ID NO: 1-3, and the light chain CDR1, CDR1, The amino acid sequences of CDR2 and CDR3 are respectively shown in SEQ ID NO: 4‑6. The invention also provides the preparation method and application of the monoclonal antibody. The murine neutralizing antibody screened out by the present invention has good antibody activity and has the potential to be developed into a marketed product.

Description

A kind of anti-Clostridium difficile toxin B monoclonal antibody and its preparation method and application

technical field

The invention relates to the field of medical biological antibodies, in particular to a monoclonal antibody against Clostridium difficile toxin B and its preparation method and application.

Background technique

Clostridium difficile is the main pathogen of antibiotic-associated diarrhea and pseudomembranous colitis. Due to the overgrowth of pathogenic Clostridium difficile in the intestinal tract, the intestinal flora is imbalanced and a large number of virulence factors are released, which eventually induces Clostridium difficile infection ( Clostridium difficile infection, CDI).

At present, monoclonal antibody (monoclonal antibody, McAb) is widely used in the treatment of infectious diseases. In 2016, Bezlotoxumab, a fully human monoclonal antibody product against Clostridium difficile toxin B (TcdB), was approved by the US Food and Drug Administration. (Food and Drug Administration, FDA) approved for marketing, becoming the first McAb drug for the treatment of CDI patients >18 years old (Gerding D N, File T M, Jr., McDonald L C. Diagnosis and Treatment of Clostridium difficile Infection (CDI)[ J].Infect Dis Clin Pract(Baltim Md),2016,24(1):3-10.Wilcox M H,Gerding D N,Poxton I R,etal.Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection[J].N Engl J Med , 2017, 376(4):305-317.). Although it has shown good effects in reducing the recurrence rate of CDI and improving the overall cure rate, the cost of treatment is as high as 50,000 US dollars, and it is generally only suitable for severe patients with high risk of recurrence (Prabhu V S, Dubberke E R, Dorr M B, et al.Cost -effectiveness of BezlotoxumabCompared With Placebo for the Prevention of Recurrent Clostridium difficile Infection[J].Clin Infect Dis,2018,66(3):355-362.). In addition, the drug is likely to cause obvious side effects such as vomiting, abdominal pain, and diarrhea in patients. Therefore, it is urgent to develop therapeutic antibody drugs with a wider range of use, lower prices, and fewer side effects to improve the overall cure rate of CDI.

Contents of the invention

In order to solve the above problems, the object of the present invention is to provide a monoclonal antibody against Clostridium difficile toxin B and its preparation method and application.

Studies have reported that Toxin of Clostridium difficile B (TcdB) plays a key role in the pathogenesis of CDI (Aktories K, Schwan C, Jank T.Clostridium difficile Toxin Biology[J].Annu Rev Microbiol,2017,71 :281-307.), and the receptor binding domain (RBD) of TcdB is the key epitope for the toxin to enter the human body to cause disease (Tian J H, Fuhrmann S R, Kluepfel-Stahl S, et al.A novel fusion protein containing the receptor binding domains of C.difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models[J].Vaccine,2012,30(28):4249-4258.). In the present invention, the recombinant expressed TcdB RBD protein is used as the target antigen to immunize Balb/c mice, hybridoma cells are prepared, positive clones are screened by limiting dilution method, forward and reverse screening and Tcd B secondary screening are used to determine hybridoma cell lines, and mouse animal experiment To verify neutralizing activity, surface plasmon resonance (Surface Plasmon Resonance, SPR) was used to analyze antibody affinity.

The first aspect of the present invention provides an anti-clostridium difficile toxin B monoclonal antibody (2F3), the amino acid sequence of its heavy chain CDR1, CDR2, CDR3 is:

CDR1: GYTFTSYW, as shown in SEQ ID NO:1;

CDR2: IYPGNNNT, as shown in SEQ ID NO:2;

CDR3: SRGDYGSSYALDY, as shown in SEQ ID NO:3;

The amino acid sequences of light chain CDR1, CDR2, and CDR3 are:

CDR1: QDVNTA, as shown in SEQ ID NO:4;

CDR2: SASYRYTGV, as shown in SEQ ID NO:5;

CDR3: QQRHVPPRT, shown in SEQ ID NO:6.

Further, the monoclonal antibody is selected from a fragment comprising a heavy chain CDR region and a light chain CDR region, a fragment comprising a heavy chain variable region and a light chain variable region, or a fragment having a full-length heavy chain and a light chain intact antibody molecule.

Further, in the monoclonal antibody, its heavy chain contains 4 FR regions, and its light chain contains 4 FR regions; the amino acid sequences of the 4 FR regions of the heavy chain are respectively shown in SEQ ID NO: 7, SEQ ID Shown in NO:8, SEQ ID NO:9 and SEQ ID NO:10;

EVKLVESGTVLARPGASVKMSCKAS (SEQ ID NO: 7);

LHWVKQRPGQGLEWIGA (SEQ ID NO: 8);

SYSQKFKGKAKLTAVTSTSTAYMDLSSLTNDDSAVYYC (SEQ ID NO: 9);

WGQGTSVTVSSA (SEQ ID NO: 10).

The amino acid sequences of the four FR regions of the light chain are respectively shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

DIVMTQAHKFMSTSVGDRVSFTCKAS (SEQ ID NO: 11);

AWYQQKPGQSPKLLIY (SEQ ID NO: 12);

PDRFTGSGSGTDFFTITISVQAEDLAVYYC (SEQ ID NO: 13);

FGGGTKVEIKRA (SEQ ID NO: 14).

In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:15.

In a preferred embodiment of the present invention, the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:16.

VH:

EVKLVESGTVLARPGASVKMSCKAS GYTFTSYW LHWVKQRPGQGLEWIGA IYPGNNNT SYSQKFKGKAKLTAVTSTSTAYMDLSSLTNDDSAVYYC SRGDYGSSYALDY WGQGTSVTVSSA (SEQ ID NO: 15)

VL:

DIVMTQAHKFMSTSVGDRVSFTCKAS QDVNTA IAWYQQKPGQSPKLLIY SASYRYTGV PDRFTGSGSGTDFFTITISVQAEDLAVYYC QQRHVPPRT FGGGTKVEIKRA (SEQ ID NO: 16)

Wherein, underlined are heavy chain CDR region and light chain CDR region.

The second aspect of the present invention provides a polynucleotide comprising nucleotides encoding the above-mentioned monoclonal antibody.

Further, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 17, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 18 shown.

Nucleotide sequence encoding heavy chain variable region:

VH:GAGGTGAAGCTGGTGGAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGTTGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAATAATACTAGTTACAGCCAAGTTCAAGGGCAAGGCCA AACTGACTGCAGTCACATCCACCAGTACTGCCTACATGGACCTCAGCAGCCTGACAAATGATGACTCTGCGGTCTATTACTGTTCAAGAGGGGACTACGGTAGTAGTTATGCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCC (SEQ ID NO: 17)

Nucleotide sequence encoding light chain variable region:

VL:GATATTGTGATGACGCAGGCTCACAAATTCATGTCCATCAGTAGGAGACAGGGTCAGCTTCACCTGCAAGGCCAGTCAGGATGTGAATACTGCTATAGCCTGGTATCAACAGAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGCTACACTGGAGTACCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCAC TTTCACCATCACCAGTGTGCAGGCTGAAGACCTGGCAGTGTATTACTGTCAGCAACGTCATGTTCCTCCTCGGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAACGGGCT (SEQ ID NO: 18)

Further, the upstream and downstream primer sequences for amplifying the heavy chain variable region are shown in SEQ ID NO:21 and SEQ ID NO:22 respectively; the upstream and downstream primer sequences for amplifying the light chain variable region are respectively shown in SEQ ID NO:23 and shown in SEQ ID NO:24.

The third aspect of the present invention provides a recombinant expression vector comprising the above polynucleotide.

The fourth aspect of the present invention provides a host cell transformed with the above-mentioned recombinant expression vector.

The fifth aspect of the present invention provides the application of the above-mentioned monoclonal antibody in the preparation of a medicament for treating Clostridium difficile infection.

Further, the drug uses the monoclonal antibody as an active ingredient.

The sixth aspect of the present invention provides a medicament for treating Clostridium difficile infection, which includes the monoclonal antibody.

The seventh aspect of the present invention provides the application of the above monoclonal antibody in the preparation of Clostridium difficile detection reagent or detection kit.

Further, the detection reagent includes the above-mentioned monoclonal antibody that has been biomarked or chemically labeled;

The detection kit includes a labeled complex obtained by biomarking or chemically marking the above-mentioned monoclonal antibody.

The eighth aspect of the present invention provides a Clostridium difficile detection kit, which includes a labeled complex obtained by biomarking or chemically labeling the above-mentioned monoclonal antibody.

The ninth aspect of the present invention provides a method for preparing the above-mentioned monoclonal antibody, comprising: culturing the above-mentioned host cell, and inducing the expression of the above-mentioned monoclonal antibody.

Further, it specifically includes the following steps:

(A) Construct TcdB RBD recombinant expression plasmid, induce expression of TcdB RBD protein;

(B) After the three-step column chromatography, the purity of the TcdB RBD protein is more than 90% as determined by CE-SDS;

(C) BALB/c mice were immunized with TcdB RBD recombinant protein, and splenocytes were fused with SP2/0 to prepare hybridoma cells;

(D) Cloning and screening positive hybridoma cell lines by limiting dilution method, and preparing ascites animal experiments to verify antibody neutralization activity;

(E) Design primers to clone antibody heavy chain and light chain variable region genes from hybridoma cell genome, and determine antibody candidate sequences by sequencing analysis.

Advantage of the present invention and beneficial effect are:

1. Recombinantly express the TcdB RBD region as an antigen to immunize animals, which greatly improves the efficiency of neutralizing antibodies.

2. Antibody screening adopts TcdB RBD and TcdB cross-screening to further prove the reliability of the screening results.

3. The murine neutralizing antibody 2F3 screened out by the present invention has good antibody activity and has the potential to be developed into a marketed product.

Description of drawings

Figure 1. TcdB RBD gene PCR product identification results.

Figure 2. Recombinant plasmid identification; M: DL5000 DNA Marker; 1: PET-22b vector; 2: Restricted PET-22b vector; 3: Recombinant plasmid Pet-TcdB RBD; 4: Recombinant plasmid Pet-TcdB RBD .

Figure 3. PCR identification results of TcdBRBD recombinant expression plasmid; M: DL2000 DNA Marker; 1-10: positive clones of TcdBRD recombinant expression plasmid.

Figure 4. Homology comparison results of TcdB RBD recombinant expression plasmids.

Figure 5. Purification results of protein expression products by chromatography.

Figure 6. SDS-PAGE analysis of TcdB RBD recombinant protein expression and purification results; M: protein maker; 1: non-induced bacterial supernatant; 2: non-induced bacterial precipitation; 3: induced bacterial supernatant; 4: induced bacterial supernatant Bacterial precipitation; 5: Ni column chromatography flow through; 6: Ni column chromatography 20mmol/L imidazole elution; 7: Ni column chromatography 60mmol/L imidazole elution; 8: Hydrophobic chromatography flow through; 9: Hydrophobic layer analysis sample peak; 10: anion exchange chromatography sample peak.

Figure 7. Western Blot detection of TcdB RBD recombinant protein expression and purification results; M: Western Blotmaker; 1: Uninduced bacterial supernatant; 2: Uninduced bacterial precipitation; 3: Induced bacterial supernatant; 4: Induced bacterial supernatant Precipitation; 5: Ni column chromatography flow through; 6: Ni column chromatography 20mmol/L imidazole elution; 7: Ni column chromatography 60mmol/L imidazole elution; 8: Hydrophobic chromatography flow through; 9: Hydrophobic chromatography Sample peak; 10: Anion exchange chromatography sample peak.

Figure 8. CE-SDS results of TcdB RBD recombinant protein after three-step chromatography purification

Figure 9. Western Blot analysis of TcdB RBD recombinant protein specificity results; M: Western Blot maker; 1: TcdB RBD protein; 2: Clostridium difficile pure TcdB.

Figure 10. Cell fusion process.

Figure 11. Purification of monoclonal antibody and SDS-PAGE analysis results; M: protein maker; 1: non-reduced IgG; 2: reduced IgG.

Figure 12. The specificity analysis results of monoclonal antibodies; M1: protein maker; M2: Western Blot maker; 1: non-reduced TcdB; 2: reduced TcdB.

Figure 13. Clostridium difficile toxin B toxin virulence assay results.

Figure 14. Affinity analysis results of neutralizing active antibody 2F3 and TcdB.

Figure 15. Affinity analysis results of Bezlotoxumab and TcdB.

Figure 16. PCR product identification results of VH, Vκ, Vλ chains of murine antibodies; M: DL5000 DNA Marker; 1-3: VH; 4-5: Vκ; 6: Vλ.

Figure 17. Analysis results of antibody gene structure.

Detailed ways

The specific implementation modes provided by the present invention will be described in detail below in conjunction with the examples.

Example 1:

1. Materials and methods

1.1 Test equipment and reagents

1.1.1 Virus species and cells

Clostridium difficile strain LCL084 was provided by the Clostridium Laboratory of Lanzhou Institute of Biological Products. SP2/0 myeloma cells were provided by the Fifth Laboratory of Lanzhou Institute of Biological Products.

1.1.2 Carrier

E.coil BL21 (DE3) competent cells were prepared by the Fifth Laboratory of Lanzhou Institute of Biological Products Co., Ltd. The PET-22b vector was provided by the Fifth Laboratory of Lanzhou Institute of Biological Products.

1.2.3 Experimental animals

Both female BALB/c mice and female Kunming mice were provided by the SPF grade animal room of Lanzhou Institute of Biological Products.

1.2 Main reagents and equipment

Primers were synthesized by Shanghai Sangon Bioengineering Company; high-fidelity KOD DNA polymerase was purchased from Japan TOYOBO Company; LB medium was purchased from American Invitrogen Company; BamHI and XhoI restriction endonucleases, DL2000 DNA Marker, nucleic acid dyes, IPTG, ECL Hypersensitive chemiluminescence kit, IPTG, goat anti-rabbit monoclonal antibody, HRP-labeled goat anti-mouse IgG were purchased from Shanghai Sangon Bioengineering Company; Ni Sepharose TM High Performance, DEAE Sepharose FastFlow, Phenyl Sepharose Fast Flow, Protein G Fillers were purchased from GE Company in the United States; Clostridium difficile pure toxin B and anti-Clostridium difficile toxin B monoclonal antibody were purchased from Abcam Company in the United Kingdom; gel recovery kits, PCR purification kits, and plasmid mini-prep kits were purchased from QIAGEN Company in Germany ; PVDF membrane, protein pre-stained Marker, and cell culture plates were purchased from Thermo Company of the United States; newborn fetal bovine serum, RPMI 1640 medium, HT additive, and HAT supplement were purchased from Gibco Company of the United States; Freund's complete adjuvant, Freund's incomplete Adjuvant was purchased from SIGMA Company of the United States; PEG 1450 was purchased from Bioss Company; 96-well ELISA plate was purchased from Costar Company of the United States; Envision microplate reader was purchased from PerkinElmer Company; PCR amplification instruments were purchased from Bio-Rad Company; Purchased from Beijing Liuyi Instrument Factory; fluorescence microscope was purchased from Olympus IX71; capillary electrophoresis instrument, high-speed centrifuge and PA 800plus SDS-MW Analysis Kit were purchased from Beckman Coulter; NanoVue micro-spectrophotometer, chromatography The system AKTAAVANT, Biacore T200, and ImageQuant gel imaging system were purchased from GE; the CO ₂ incubator was purchased from SANYO; the cell analyzer was purchased from Count star.

1.2 Experimental method

1.2.1 Construction of TcdB RBD prokaryotic expression plasmid

According to the Clostridium difficile toxin B gene sequence (sequence number: CP046327.1) provided by the NCBI database, the genomic DNA of the toxin-producing strain LCL084 was used as a template, BamHI and XhoI were selected as restriction sites, and primers were designed using Primer5.0 software. The TcdB RBD gene sequence was amplified. The primer is represented by tcdB-RBD. TcdB RBD consists of 533 amino acids and is located at 1834-2366aa of TcdB protein. PCR primers were synthesized by Shanghai Sangong Bioengineering Company.

Table 1. Clostridium difficile toxin B RBD PCR primers

The TcdB RBD gene was amplified by PCR using the genomic DNA of the Clostridium difficile LCL084 toxin-producing strain as a template. The PCR product and PET-22b vector were digested by BamHI and XhoⅠ respectively, identified by 1% agarose gel electrophoresis and recovered by cutting the gel. The size of TcdBRD after digestion is about 1600bp. Use T4 DNA ligase to connect the vector and the sequence, transform E.coil BL21(DE3), smear the LB agar plate containing ampicillin (AMP) resistance, pick positive clones for sequencing, and keep the strains with 100% identical sequences.

1.2.2 Recombinant expression and purification of TcdB RBD protein

Introduce the recombinant bacteria activated overnight into LB (Amp) medium at a ratio of 1:100, culture at 37°C, 220rpm to OD600=0.6-0.8, add a certain amount of IPTG with a final concentration of 1mM, induce at 25°C, 220rpm Express for 12 hours; centrifuge at 9500rpm for 30 minutes, dissolve the cell pellet in PBS buffer, and crush the cell with a pressure breaker. Centrifuge at 10,000 g for 15 minutes, collect the supernatant and precipitate of the broken bacteria, and analyze the expression of TcdB RBD protein by SDS-PAGE and Western Blot. If the expression is detected successfully, use Ni Sepharose ^TM High Performance medium to manually purify the bacterial supernatant. After washing with 20mM imidazole, increase the concentration of imidazole in sequence for gradient elution, and adjust the conductivity of the collected Ni affinity chromatography eluate to 120mS/cm, equilibrate the Phenyl Sepharose Fast Flow hydrophobic chromatography column with 0.05M PB buffer containing 1mol/L (NH ₄ ) ₂ SO ₄ , and perform linearity with 0.05M PB buffer (pH 7.0, conductivity 5mS/cm) For elution, adjust the conductivity of the collected sample peak eluent to 5mS/cm, equilibrate the DEAE anion chromatography column with 0.05M PB buffer (pH 7.0, conductivity 5mS/cm), and use 0.05 MPB buffer (pH 7.0, conductivity 85mS/cm) was used for linear elution, and the sample peak eluate was collected. The purity and activity of TcdB RBD protein purified by three-step chromatography were analyzed by CE-SDS and Western-Blot.

1.2.3 Preparation of anti-TcdB monoclonal antibody

BALB/c mice were immunized with high-purity TcdB RBD recombinant protein. Seven days after the third immunization, blood was collected from the tail vein, and the serum titer was determined by ELISA. The mouse splenocytes with the highest antibody titer were selected for the preparation of hybridomas . Three days after the pure antigen booster immunization, the immune spleen cells of the mice were fused with SP2/0 myeloma cells under the action of PEG1450, and the cells were screened under pressure using HAT selection medium. When the hybridoma cells in the 96-well cell culture plate were cloned to more than 1/4 of the field of view of the whole well, the hybridoma cells were cross-screened by ELISA using TcdB RBD recombinant protein and pure TcdB. According to the results of indirect ELISA detection, the positive hybridoma cells with high absorbance OD ₄₅₀ reading value, small number of cell clones and good cell state were selected, and the cells were cloned and cultured by limiting dilution method. Cloning was terminated until the indirect ELISA test results of all monoclonal cell lines were positive. The positive monoclonal hybridoma cells were expanded and cultured and passaged. During the passage, an indirect ELISA test was performed to detect the passage stability of the cells, and the cells were cryopreserved. Take BALB/c female mice, inject 0.2mL paraffin oil into each mouse for mouse sensitization, and inject about 1×10 ⁶ positive hybridoma cells into each mouse after one week, and wait until the abdomen of the mouse is obviously enlarged and moves. When it is inconvenient, use dissecting scissors to cut the abdominal skin of the mouse to expose the peritoneum, insert a sterile syringe needle into the abdomen of the mouse, collect the ascites and centrifuge at 4°C (12000rpm×30min), remove the fat and paraffin oil in the abdominal cavity of the mouse, and collect the middle layer of ascites, to be purified.

1.2.4 Purification of mouse ascites

The ascites was first crudely extracted by saturated ammonium sulfate precipitation, salted out, centrifuged at 4°C (10000rpm × 30min), the supernatant was discarded, the precipitate was resuspended in PBS buffer, filtered through a 0.45μm filter membrane, and then purified by Protein G agarose gel. Connect 5 mL of Protein G prepacked column to the corresponding column position of AKTA Avant protein purification system, wash 5 column volumes with purified water, 0.1mol/L NaOH, and purified water respectively, and then equilibrate the packing with PBS for 10 column volumes. After UV 280 zero calibration, load the cell culture supernatant and crude ascitic fluid samples at a load of no more than 30 mg/mL, and wash 5 columns with impurity removal buffer (0.5M NaCl+10mM PB, pH 7.0-7.4) Volume, PB re-equilibrated, eluted with 0.03M NaAC (pH=3.7), collected the eluted peak with UV 280>50mAU, added 1mol/L Tris-HCl (pH=10.2) to adjust the pH to about 7.0. Wash 5 column volumes with 0.1mol/L NaOH and pure water respectively and store the prepacked column in 20% ethanol. The NanoVue ultra-micro spectrophotometer Protein A280 method was used to determine the concentration of purified antibodies.

1.2.5 Specificity analysis of monoclonal antibodies

Western-Blot was used to identify the specificity of the monoclonal antibody. The pure TcdB of Clostridium difficile (positive control) and the TcdB RBD protein to be detected were separated by SDS-PAGE electrophoresis. Monoclonal antibody, incubate at room temperature for 1 hour; wash membrane with PBS-T 3-5 times, 10 minutes each time; use HRP-labeled goat anti-mouse monoclonal antibody as secondary antibody, combine at room temperature for 1 hour; wash membrane with PBS-T 3-5 times, each time 10min; add substrate to develop color, expose for 30s, and observe the results.

1.2.6 Titer detection of monoclonal antibody

Coat the 96-well ELISA plate with Clostridium difficile pure toxin B, block overnight at 4°C, wash the plate and add the monoclonal antibody after washing in order from the first row, incubate at 37°C for 1 hour, add peroxide after washing the plate The goat anti-mouse secondary antibody labeled with enzyme was used to incubate at 37°C for 45 minutes. After washing the plate, add substrate solution A and solution B, incubate for 15 minutes, add stop solution, and detect its OD ₄₅₀ reading value with a microplate reader. The positive control is the mouse serum after antigen immunization, and the negative control is the non-immunized mouse serum. The maximum dilution corresponding to the OD ₄₅₀ reading > 2.1 times and greater than 0.2 of the negative control is taken as the antibody titer.

1.2.7 Monoclonal antibody neutralizing activity analysis

1.2.7.1 Clostridium difficile toxin B toxin toxicity assay

The toxin toxicity of TcdB was measured with reference to the 2015 edition of Pharmacopoeia Botulinum Toxin Potency Determination Method, and the toxin toxicity of TcdB was measured with the half lethal dose (50% lethal dose, LD ₅₀ ) in mice as the detection standard. Refer to the literature that the LD ₅₀ of TcdB in CD1 mice is 1×10 ^-3 mg/kg. Toxin B was serially diluted with reference to this dose, and a total of 5 toxin dose dilutions were set up, and the toxin doses were 0.25×10 ^-3 mg/kg, 0.5×10 ^-3 mg/kg, 1×10 ^-3 mg/kg, 2×10 ^-3 mg/kg and 4×10 ^-3 mg/kg, each dilution is 2.5mL. 14-16g female Kunming mice (SPF grade), inject 4 for each dilution. Each mouse was intraperitoneally injected with 0.5 mL, observed continuously for 96 hours after injection, recorded the death of mice, and counted the results.

The LD ₅₀ is calculated by the improved Cole's method for statistical results, and the calculation method is as follows:

LgLD ₅₀ = logarithmic value of maximum dose - difference between logarithmic values of two adjacent dose groups × (sum of mortality in each dose group - 0.5).

1.2.7.2 Neutralization test

Adopt mouse test to carry out the qualitative analysis of neutralizing activity of monoclonal antibody, test is set up sample group, toxin group, toxin inactivation control group and blank group respectively; In the sample group, quantitatively draw the antibody solution to be tested of different dilutions (respectively 2F3 sample group, 4H7 sample group, 7C2 sample group, 8C10 sample group, 9C9 sample group, 9A12 sample group, 9H7 sample group, 10F7 sample group). Then it was mixed with the toxin dose diluent corresponding to 5LD ₅₀ , the mixture was incubated at 37° C. for 1 hour, and immediately injected intraperitoneally into Kunming mice weighing 14-16 g. Four mice were injected with each dilution, and each was injected intraperitoneally with 0.5 mL. The antibody solution in the sample group was divided into 3 different dilutions (low dilution B1; medium dilution B2; high dilution B3;); B1, B2, The IgG concentrations corresponding to the three dilutions of B3 were 10 μg/mL, 1 μg/mL, and 0.1 μg/mL, respectively. In the toxin group, Kunming mice were only injected with diluted toxin solution; in the toxin inactivation control group, they were injected with toxin solution that had been heat-inactivated at 100°C; in the blank group, Kunming mice were only injected with PBS buffer solution; mice were observed continuously for 4 days after injection Survival, statistical results.

1.2.8 Surface plasmon resonance analysis of monoclonal antibody affinity

Coupling ligand: Mouse monoclonal antibody and marketed anti-TcdB McAbBezlotoxumab were coated on the CM5 chip by amino coupling method, the concentration of the conjugated antibody was 5 μg/ml, and the coating solution was 10 mM sodium acetate with pH=5.0. The running buffer is HBS-EP+. First, the chip was activated with EDC and NHS, and then the mouse antibody was bound to the chip at a flow rate of 10 μL/min for 7 minutes, and the unbound epitope was blocked with ethanolamine. Use the other channel of unconjugated antibody after activation as the reference channel for detection.

Affinity measurement: the running buffer is HBS-EP+, and the flow rate is 30 μL/min. After the baseline is stable, the TcdB is serially diluted with the running buffer and slowly flows over the surface of the chip as an analyte, and the affinity is measured by a single cycle method. The flow rate was 30 μL/min, binding for 120 s, dissociation for 600 s, and the medium was regenerated with pH 1.5 Gly-HCl buffer for 30 s. The resulting sensorgrams were fitted using its analysis software to obtain affinity data between antibodies and antigens.

1.2.9 Retrieval of antibody variable region sequences

The total RNA of hybridoma cells was extracted by the Trizol method, and the first strand of cDNA was synthesized by reverse transcription. The antibody variable region genes of hybridoma cells were obtained by PCR cloning, and the genes were cloned into T vectors, and positive clones were selected for sequencing. According to the principle of independent amplification of VH, Vκ and Vλ chains in mice, primers were designed, and the primer sequences are shown in Table 2.

Table 2. Primers for PCR amplification of VH, Vκ, Vλ chains of murine antibodies

2. Experimental results

2.1 Amplification of the target gene and product identification results

Using the genomic DNA of Clostridium difficile strain LCL084 as a template, the PCR product of TcdB RBD was amplified, and analyzed by 1% agarose gel electrophoresis, the target band with a size of about 1600bp was seen, which was consistent with the expected size, as shown in the figure 1.

2.2 Results of plasmid construction

The amplified product of TcdB RBD sequence in 2.1 and the carrier PET-22b were double-digested with BamHI and XhoI, and analyzed by 1% agarose gel electrophoresis. It can be seen that the bands are about 1600bp and 5500bp respectively, and the sizes are consistent with the expectations. The agarose gel electrophoresis identification results of the TcdBRBD sequence amplification product and the vector PET-22b digestion product are shown in Figure 2.

The recombinant expression plasmid was transformed into E.coil BL21(DE3), and positive clones were picked after overnight culture. A total of 10 single clones were picked and cultured separately. After PCR, 5 μL of the transformed single culture product was verified by 1% agarose gel electrophoresis analysis, and a positive band appeared at 1600 bp, which was consistent with the expectation. The identification results of the recombinant plasmids are shown in Figure 3.

The verified positive bacteria were sent for sequencing, and the sequencing results were compared with the main board sequence by Megalign. The homology of the TcdBRD gene sequence inserted in the plasmid and the main board sequence was 100%, that is, there was no mutation in the gene, indicating that the recombinant plasmid was constructed correctly. The results of the homology comparison are shown in Figure 4.

2.3 Expression and purification results of TcdB RBD recombinant protein

The induced TcdB RBD recombinant protein was analyzed by Western Blot technology, and the HRP-labeled mouse anti-His tag monoclonal antibody was used to detect whether the recombinant protein carried the tag. After detection, the His tag was positive, which proved that the TcdBRD recombinant protein was successfully expressed. Afterwards, the protein expression product was separated and purified by affinity chromatography, hydrophobic chromatography and anion exchange chromatography in sequence, and the purification results of each chromatography of the protein expression product are shown in FIG. 5 .

The eluate from each peak was collected separately, and the eluate was separated by SDS-PAGE electrophoresis, stained, and decolorized. A protein band of about 60KD could be seen in the eluate of the highest peak, which may be the target protein, and the protein purity increased sequentially. The SDS-PAGE and Western-Blot results of the purified TcdB RBD recombinant protein samples are shown in Figure 6 and Figure 7 .

2.4 Analysis results of TcdB RBD recombinant protein properties

2.4.1 CE-SDS analysis of protein purity results

The protein purity of TcdB RBD purified by three-step chromatography was detected by non-reducing CE-SDS method, and the target protein peak was integrated by 32Karat software. The test report is shown in Figure 8, and the results show that the purity of TcdB RBD protein is about 91%, indicating that the purification effect is good.

2.4.2 TcdB RBD recombinant protein specificity analysis results

The specificity of TcdB RBD recombinant protein was detected by Western-Blot method. Firstly, the pure TcdB of Clostridium difficile (positive control) and the TcdB RBD protein to be detected were fixed on PVDF membrane, and rabbit-derived toxin B monoclonal antibody was used as the primary antibody binding protein. HRP-labeled goat anti-rabbit monoclonal antibody was used as the secondary antibody to analyze the binding of Clostridium difficile pure TcdB and TcdB RBD recombinant protein to rabbit-derived toxin B monoclonal antibody. It was found that both showed positive reactions, indicating that the TcdB RBD recombinant protein was successfully expressed. The result is shown in Figure 9.

2.5 BALB/c mouse serum anti-TcdB protein antibody titer results

The titer of mouse serum was detected by indirect ELISA method, and the serum of unimmunized mice was used as a negative control. The results are shown in Table 3. Mice with a serum dilution factor greater than 20,000 when the OD ₄₅₀ reading value is about 1.0 can be used for the preparation of hybridoma cells. It can be seen from the table that the above 5 mice can all be used for the preparation of monoclonal antibodies, among which No. 2 mouse has the highest antibody titer (>160000), and was selected to prepare anti-TcdB RBD monoclonal antibody.

Table 3. BALB/c mouse serum anti-TcdB RBD protein antibody titer results

2.6 Cell Fusion Results

Three days after the booster immunization of BALB/c mice, mouse splenocytes and SP2/0 myeloma cells were taken for cell fusion under the action of PEG1450 fusion agent. After fusion, the cell fusion was observed with an inverted microscope. The results are shown in Figure 10 The red circle indicates that the fused cells began to divide on the second day after fusion, and slowly divided into two from one. With the prolongation of the fusion time, the cells gradually divided into cell clusters, which can be observed under the microscope on the twelfth day Clear and bright clusters of cells indicate successful cell fusion.

2.7 Screening and cloning results of positive hybridoma cells

Positive hybridoma cells were screened by indirect ELISA method. Firstly, TcdB RBD recombinant protein was used for positive screening, and then pure TcdB was used for secondary screening. Positive hybridoma cells with high absorbance OD ₄₅₀ readings, few cell clones, and good cell state were selected for cloning culture. After 3 rounds of cloning and screening by limiting dilution method, a total of 8 positive hybridoma cell lines were obtained, named respectively: 2F3, 4H7, 7C2, 8C10, 9C9, 9A12, 9H7, 10F7. The results are shown in Table 4. Indirect ELISA was performed on them during passage, and it was determined that they were all positive hybridoma cell lines stably secreting specific monoclonal antibodies.

Table 4. Positive screening results of hybridoma cells

2.8 Mass preparation and purification results of monoclonal antibodies

A large amount of monoclonal antibodies were prepared by the mouse ascites method. Taking one of the antibodies as an example, the ascites after crude extraction was purified using the AKTA Avant protein chromatography system and Protein G prepacked column, and the sample peak eluate was purified by NanoVue ultra- The protein concentration measured by a micro-spectrophotometer was 1500 μg/ml. Because the molecular weight of the monoclonal antibody was relatively large (150-180KD), it was subjected to β-mercaptoethanol reduction treatment, and the purified monoclonal antibody was analyzed by reducing and non-reducing SDS-PAGE electrophoresis. Antibodies were analyzed for purity. The results are shown in Figure 11. It can be seen from the figure that the monoclonal antibody is reduced to 55KD (heavy chain) and 25KD (light chain) by the reducing agent β-mercaptoethanol, and the molecular weight of the non-reduced monoclonal antibody is about 150KD, which is consistent with the molecular weight of IgG . Moreover, high-purity monoclonal antibodies were obtained by using the protein G medium purification method, and the purity of the antibodies reached more than 90%.

2.9 Specific analysis results of monoclonal antibodies

The Western-Blot method was used to analyze and identify the specificity of monoclonal antibodies. Taking one of the antibodies as an example, the results are shown in Figure 12. The left picture is the TcdB reduced and non-reduced SDS-PAGE electrophoresis, and the right picture is the monoclonal antibody. Western-Blot results of the binding of the cloned antibody to TcdB. The results show that the monoclonal antibody secreted by the hybridoma cell line can specifically recognize the reduced and non-reduced whole TcdB toxin molecule protein (molecular weight 270KD).

2.10 Titer test results of monoclonal antibodies

The titer of the purified monoclonal antibody was determined by the indirect ELISA method. Taking one of the antibodies as an example, the results are shown in Table 5. The titer of the antibody was 1:3.2×10 ⁶ .

Table 5. Monoclonal antibody titer test results

2.11 Analysis results of monoclonal antibody neutralizing activity

2.11.1 Clostridium difficile toxin B toxin toxicity test results

Clostridium difficile toxin B toxin toxicity assay results are shown in Figure 13, as can be seen from the table, the mortality rate of mice in the 0.25 μg/kg and 0.5 μg/kg toxin dosage groups are all below 50%, and the 1 μg/kg toxin dosage group has 50 % of the mice died, and all the mice in the 2 μg/kg and 4 μg/kg toxin dose groups died. Therefore, it is calculated that the toxin toxicity of TcdB is LD ₅₀ =1 μg/kg. The experimental results are consistent with the LD ₅₀ values of TcdB in CD1 mice referenced in the literature.

2.11.2 Neutralization test results

The neutralizing activity of the monoclonal antibodies was determined using a mouse assay. The test set up the sample group, the toxin group, the toxin inactivation control group and the blank group respectively; the survival of the mice after injection is shown in Table 6, and the mice in the toxin inactivation control group and the blank group survived 96 hours after injection without symptoms . The mice in the toxin group and 4H7, 7C2, 9C9, 9A12, and 9H7 sample groups at each dilution all died 24 hours after injection, and the mice in the 2F3, 8C10, and 10F7 sample groups all survived 24 hours after injection, but the 8C10 and 10F7 sample groups Only one mouse survived in the low dilution B1, and the mice in the B2 and B3 dilution groups all died. Since only one mouse in the B1 group survived, it cannot be determined that they are neutralizing active antibodies. After 24 hours of injection, the mice in 2F3-B3 were obviously sick, and 2F3-B1, 2F3-B2, and 2F3-B3 survived 3, 2, and 1 respectively. From the results, it can be seen that the antibody dilution varies from low to high. Rat survival also showed a certain gradient. After 48 hours, 2 mice and 1 mouse survived in the 2F3-B1 and 2F3-B2 groups respectively, and all the mice in the 2F3-B3 group died. It was judged to be a neutralizing antibody.

Table 6. Analysis results of neutralizing activity of monoclonal antibodies

2.12 Neutralizing antibody affinity analysis results

The affinity of neutralizing antibody 2F3 and marketed anti-TcdB McAb Bezlotoxumab to TcdB was analyzed by Biacore T200. Antibody affinity was determined by the single-cycle method. The coupling amount of antibody 2F3 to the chip was 3413.5RU, and the coupling amount of Bezlotoxumab to the chip was 4412.6RU. Make two-fold serial dilutions of TcdB in HBS-EP Buffer, a total of 5 dilutions. Each step on the curve in the figure represents the response value of the antibody binding to different dilutions of TcdB, and the change of the response value can reflect the process of the antibody interacting with TcdB. Equation fitting was performed with a one-to-one binding model. The affinity results are shown in Figure 14 and Figure 15. The ka and kd values in the figure represent the association rate constant and dissociation rate constant of the antibody 2F3 and TcdB respectively. The equilibrium solution of the interaction between the two can be obtained by calculating the ratio of kd/ka The distance constant KD, generally the smaller the KD value, the higher the affinity between the two. The equilibrium dissociation constants KD of the neutralizing antibody 2F3 and Bezlotoxumab and TcdB calculated by the software were 1.188E-8M and 4.985E-9M, respectively.

2.13 Results of PCR amplification of VH, Vκ, Vλ chains of murine antibodies

Using the mouse hybridoma cell cDNA as a template, the VH, Vκ, Vλ chains of the mouse antibody were amplified, and analyzed by 1% agarose gel electrophoresis. The results can be seen from Figure 16, and the VH and Vκ chains were amplified by PCR. , the Vλ chain was not amplified, indicating that the light chain of the mouse antibody is of the κ type. The VH electrophoresis band is about 360bp, and the Vκ electrophoresis band is about 340bp, and the band size is in line with the expectation.

2.14 Results of antibody variable region sequence analysis

The verified positive bacteria were sent for sequencing, and the sequencing results were compared with the NCBI database by BLAST software. After comparison, it was found that the amplified VH and Vκ sequences were the variable region sequences of murine antibodies. Further use the V-BASE2 database to analyze the gene structure of the variable region gene sequence of the murine antibody obtained by sequencing, and locate the complementarity determining region CDR and framework region FR on the VH and VL of the antibody. The results of antibody gene structure analysis are shown in FIG. 17 .

3 Discussion

Clostridium difficile LCL084 strain is a strictly obligate anaerobic bacterium with harsh culture conditions. The strain has low toxin production, large molecular weights of toxins TcdA and TcdB, and similar physical and chemical properties, which bring great challenges to the antigen preparation work of the present invention. According to literature reports, it is known that TcdB RBD is the key epitope for the toxin to enter the human body and cause disease. And the prokaryotic expression system can be used to realize the large-scale preparation of the protein, so the present invention chooses to clone the TcdB RBD gene into the PET-22b prokaryotic expression vector, and transform Escherichia coli for recombinant expression. After three-step chromatography purification, CE -SDS results show that the purity of the recombinant protein is over 90%, which fully meets the antigen immunity requirements for McAb prepared by hybridoma technology, greatly reduces the antibody screening strength of the present invention, and lays the foundation for the successful screening of positive McAb cell lines established a solid foundation.

The mouse in vivo test is a commonly used standard method for measuring the neutralizing activity of antitoxin McAb. The present invention adopts the animal test to analyze the antibody neutralizing activity of the 8 positive hybridoma cell lines screened, by mixing the neutralized pure TcdB with the McAb solution Intraperitoneal injection into the mice, according to the death of the mice, preliminary screening of antibodies that have inhibitory effects on the toxin. The results showed that only the 2F3 antibody strain could neutralize 5 LD ₅₀ toxins TcdB, while the other 7 antibody strains could not neutralize and protect mice. A McAb with neutralizing activity was successfully screened by this method.

The present invention adopts SPR technology to evaluate the affinity of McAb through a single cycle method. The mouse-derived neutralizing antibody 2F3 and the fully human Bezlotoxumab (anti-TcdB McAb already on the market) were respectively immobilized on the surface of the CM5 chip by the amino coupling method, and the pure TcdB antigen molecule after gradient dilution was used as the mobile phase slowly through the microfluidic chuck. Transmitted to the surface of the sensor chip, according to the change of the response value, it reflects the whole process of the interaction between the antibody and TcdB. Using this technique, the equilibrium dissociation constant KD between mouse neutralizing antibody 2F3 and TcdB is 1.188E-8M; while the equilibrium dissociation constant KD between Bezlotoxumab and TcdB is 4.985E-9M; there is no significant difference in the affinity data between the two, indicating that The murine neutralizing antibody 2F3 screened out by the present invention has good antibody activity and has the potential to be developed into a marketed product.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. These equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

Claims (10)

1. an anti-clostridium difficile toxin B monoclonal antibody is characterized in that the amino acid sequence of its heavy chain CDR1, CDR2, CDR3 is: CDR1: GYTFTSYW, as shown in SEQ ID NO:1; CDR2: IYPGNNNT, as shown in SEQ ID NO:2; CDR3: SRGDYGSSYALDY, as shown in SEQ ID NO:3; The amino acid sequences of light chain CDR1, CDR2, and CDR3 are: CDR1: QDVNTA, as shown in SEQ ID NO:4; CDR2: SASYRYTGV, as shown in SEQ ID NO:5; CDR3: QQRHVPPRT, shown in SEQ ID NO:6. 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is selected from fragments comprising heavy chain CDR regions and light chain CDR regions, fragments comprising heavy chain variable regions and light chain variable regions. Fragments of variable regions, or complete antibody molecules with full-length heavy and light chains. 3. A polynucleotide comprising nucleotides encoding the monoclonal antibody according to claim 1 or 2. 4. A recombinant expression vector, characterized in that it comprises the polynucleotide according to claim 3. 5. A host cell transformed with the recombinant expression vector according to claim 4. 6. The application of the monoclonal antibody described in claim 1 or 2 in the preparation of a medicament for the treatment of Clostridium difficile infection. 7. A medicine for treating Clostridium difficile infection, characterized in that it comprises the monoclonal antibody according to claim 1 or 2. 8. The application of the monoclonal antibody described in claim 1 or 2 in the preparation of Clostridium difficile detection reagent or detection kit. 9. A Clostridium difficile detection kit, characterized in that the detection kit comprises a labeled complex obtained by biomarking or chemically marking the monoclonal antibody according to claim 1 or 2. 10. The preparation method of the monoclonal antibody according to claim 1 or 2, comprising: culturing the host cell according to claim 5, and inducing the expression of the monoclonal antibody.

Images