CN116239683B

CN116239683B - Monoclonal antibody for resisting clostridium difficile toxin B, and preparation method and application thereof

Info

Publication number: CN116239683B
Application number: CN202310115787.5A
Authority: CN
Inventors: 王建锋; 杨处; 陈继军; 毛晓燕; 张超; 熊颖; 叶星
Original assignee: Lanzhou Institute Of Biological Products Co ltd
Current assignee: Lanzhou Institute Of Biological Products Co ltd
Priority date: 2023-02-15
Filing date: 2023-02-15
Publication date: 2024-12-27
Anticipated expiration: 2043-02-15
Also published as: CN116239683A

Abstract

The invention relates to the field of medical biological antibodies, in particular to a monoclonal antibody for resisting clostridium difficile toxin B, wherein the amino acid sequences of a heavy chain CDR1, a CDR2 and a CDR3 are respectively shown in SEQ ID NO. 1-3, and the amino acid sequences of a light chain CDR1, a CDR2 and a CDR3 are respectively shown in SEQ ID NO. 4-6. The invention also provides a preparation method and application of the monoclonal antibody. The screened mouse-derived neutralizing antibody has good antibody activity and potential of developing into products on the market.

Description

Monoclonal antibody for resisting clostridium difficile toxin B, and preparation method and application thereof

Technical Field

The invention relates to the field of medical biological antibodies, in particular to a monoclonal antibody for resisting clostridium difficile toxin B, and a preparation method and application thereof.

Background

Clostridium difficile is the main causative agent of antibiotic-associated diarrhea and pseudomembranous enteritis, and due to excessive proliferation of pathogenic clostridium difficile in the gut, the gut flora is deregulated, releasing a large number of virulence factors, ultimately inducing clostridium difficile infection (clostridium difficile infection, CDI).

Currently, monoclonal antibodies (monoclonal antibody, mcAb) are widely used in the treatment of infectious diseases, and in 2016, a whole humanized monoclonal antibody product Bezlotoxumab against clostridium difficile toxin B (TcdB) was approved by the U.S. food and drug administration (Food and Drug Administration, FDA) to be marketed, and as a first McAb drug (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,et al.Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection[J].N Engl J Med,2017,376(4):305-317.). for treating CDI patients of >18 years old, although it shows good effects in reducing the recurrence rate of CDI and improving the overall cure rate, the treatment cost is as high as $50000, and generally only suitable for severe patients (Prabhu V S,Dubberke E R,Dorr M B,et al.Cost-effectiveness of Bezlotoxumab Compared With Placebo for the Prevention of Recurrent Clostridium difficile Infection[J].Clin Infect Dis,2018,66(3):355-362.). with high recurrence risk, and the drug is liable to cause obvious side effects such as vomiting, abdominal pain, diarrhea, etc. in patients, so there is an urgent need to develop a therapeutic antibody drug with wider application range, lower price and less side effects to improve the overall cure rate of CDI.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a monoclonal antibody for resisting clostridium difficile toxin B, and a preparation method and application thereof.

Research reports that clostridium difficile toxin B (Toxin of Clostridium difficile B, tcdB) plays a key role (Aktories K,Schwan C,Jank T.Clostridium difficile Toxin Biology[J].Annu Rev Microbiol,2017,71:281-307.), in the pathogenic process of CDI, a receptor binding region (Receptor binding domain, RBD) of TcdB is a key antigen epitope (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.). for toxin entering human pathogens, the invention uses recombinant expression TcdB RBD protein as a target antigen to immunize Balb/c mice, hybridoma cells are prepared, positive clones are screened by a limiting dilution method, hybridoma cell strains are determined by forward and reverse screening and TcdB secondary screening, the neutralization activity is verified by a mouse animal experiment, and antibody affinity is analyzed by a surface plasmon resonance technology (Surface Plasmon Resonance, SPR).

In a first aspect of the invention there is provided an anti-clostridium difficile toxin B monoclonal antibody (2F 3) having the amino acid sequences of heavy chain CDR1, CDR2, CDR3 of:

CDR1: GYTFTSYW, shown as SEQ ID NO 1;

CDR2: IYPGNNNT as shown in SEQ ID NO 2;

CDR3: SRGDYGSSYALDY, shown as SEQ ID NO 3;

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

CDR1: QDVNTA, shown as SEQ ID NO 4;

CDR2: SASYRYTGV as shown in SEQ ID NO 5;

CDR3: QQRHVPPRT is shown as SEQ ID NO. 6.

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

Furthermore, in the monoclonal antibody, the heavy chain comprises 4 FR regions, the light chain comprises 4 FR regions, and the amino acid sequences of the 4 FR regions of the heavy chain are respectively shown as SEQ ID NO. 7, SEQ ID 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 4 FR regions of the light chain are respectively shown as 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);

PDRFTGSGSGTDFTFTITSVQAEDLAVYYC(SEQ ID NO:13);

FGGGTKVEIKRA(SEQ ID NO:14)。

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

VH:

EVKLVESGTVLARPGASVKMSCKASGYTFTSYWLHWVKQRPGQGLEWIGAIYPGNNNTSYSQKFKGKAKLTAVTSTSTAYMDLSSLTNDDSAVYYCSRGDYGSSYALDYWGQGTSVTVSSA(SEQ ID NO:15)

VL:

DIVMTQAHKFMSTSVGDRVSFTCKASQDVNTAIAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTITSVQAEDLAVYYCQQRHVPPRTFGGGTKVEIKRA(SEQ ID NO:16)

Wherein heavy chain CDR regions and light chain CDR regions are underlined.

In a second aspect of the invention, there is provided a polynucleotide comprising nucleotides encoding the monoclonal antibody described above.

Further, the nucleotide sequence for coding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 17, and the nucleotide sequence for coding the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 18.

Nucleotide sequence encoding the heavy chain variable region:

VH:GAGGTGAAGCTGGTGGAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGTTGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAATAATACTAGTTACAGCCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCCACCAGTACTGCCTACATGGACCTCAGCAGCCTGACAAATGATGACTCTGCGGTCTATTACTGTTCAAGAGGGGACTACGGTAGTAGTTATGCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGT CTCCTCAGCC(SEQ ID NO:17)

nucleotide sequence encoding a light chain variable region:

VL:GATATTGTGATGACGCAGGCTCACAAATTCATGTCCACATCAGTAGGAGACAG GGTCAGCTTCACCTGCAAGGCCAGTCAGGATGTGAATACTGCTATAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGCTACACTGGAGTACCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCACCAGTGTGCAGGCTGAAGACCTGGCAGTGTATTACTGTCAGCAACGTCATGTTCCTCCTCGGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAACGGGCT(SEQ ID NO:18)

Further, the upstream and downstream primer sequences of the amplified heavy chain variable region are shown as SEQ ID NO. 21 and SEQ ID NO. 22, respectively, and the upstream and downstream primer sequences of the amplified light chain variable region are shown as SEQ ID NO. 23 and SEQ ID NO. 24, respectively.

In a third aspect of the present invention there is provided a recombinant expression vector comprising a polynucleotide as described above.

In a fourth aspect of the invention, there is provided a host cell transformed with the recombinant expression vector described above.

In a fifth aspect of the invention there is provided the use of a monoclonal antibody as described above in the manufacture of a medicament for the treatment of clostridium difficile infection.

Furthermore, the medicine takes the monoclonal antibody as an active ingredient.

In a sixth aspect of the invention there is provided a medicament for the treatment of clostridium difficile infection comprising said monoclonal antibody.

In a seventh aspect of the invention there is provided the use of a monoclonal antibody as described above in the preparation of a clostridium difficile detection reagent or kit.

Further, the detection reagent comprises the monoclonal antibody marked by the biomarker or the chemical marker;

the detection kit comprises a labeled compound obtained by the monoclonal antibody through biological labeling or chemical labeling.

According to an eighth aspect of the present invention, there is provided a clostridium difficile detection kit comprising a labelled complex of the monoclonal antibody described above, the labelled complex being labelled with a biomarker or chemical label.

In a ninth aspect of the present invention, there is provided a method for producing the above monoclonal antibody, comprising culturing the above host cell and inducing expression of the monoclonal antibody.

Further, the method specifically comprises the following steps:

(A) Constructing a TcdB RBD recombinant expression plasmid, and inducing expression of a TcdB RBD protein;

(B) The purity of the TcdB RBD protein after three steps of column chromatography is more than 90 percent through CE-SDS verification;

(C) Immunizing a BALB/c mouse by using a TcdB RBD recombinant protein, and fusing spleen cells and SP2/0 to prepare hybridoma cells;

(D) Cloning and screening positive hybridoma cell strains by a limiting dilution method, and preparing an ascites animal experiment method to verify the neutralizing activity of the antibody;

(E) Primers were designed to clone antibody heavy and light chain variable region genes from the hybridoma cell genome and sequencing analysis was performed to determine antibody candidate sequences.

The invention has the advantages and beneficial effects that:

1. The recombinant expression TcdB RBD region is used as antigen to immunize animal to raise the neutralizing antibody obtaining efficiency greatly.

2. The antibody screening adopts TcdB RBD and TcdB cross screening, further proving the reliability of the screening result.

3. The screened murine neutralizing antibody 2F3 has good antibody activity and has the potential of being developed into a product on the market.

Drawings

FIG. 1. Identification of TcdB RBD gene PCR product.

FIG. 2 shows the identification of recombinant plasmid, M is DL5000 DNA MARKER, 1 is PET-22b vector, 2 is PET-22b vector, 3 is recombinant plasmid PET-TcdB RBD, and 4 is recombinant plasmid PET-TcdB RBD.

FIG. 3 shows the results of PCR identification of TcdB RBD recombinant expression plasmid, M: DL2000 DNA MARKER, and 1-10: tcdB RBD recombinant expression plasmid positive clone.

FIG. 4 shows the homology comparison result of TcdB RBD recombinant expression plasmid.

FIG. 5 shows the results of respective chromatographic purifications of the protein expression products.

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 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.

FIG. 7 shows the results of Western Blot detection of TcdB RBD recombinant protein expression and purification, M: western Blot maker, 1, no induced bacterial supernatant, 2, no induced bacterial precipitation, 3, induced bacterial supernatant, 4, induced 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 chromatography sample peaks, 10, anion exchange chromatography sample peaks.

FIG. 8 shows the result of CE-SDS after three-step chromatographic purification of TcdB RBD recombinant protein

FIG. 9 Western Blot analysis of TcdB RBD recombinant protein specificity results, M: western Blot maker, 1: tcdB RBD protein, 2: clostridium difficile pure TcdB.

FIG. 10. Cell fusion process.

FIG. 11 shows the results of purification and SDS-PAGE analysis of monoclonal antibodies, M: protein maker, 1: non-reducing IgG, and 2: reducing IgG.

FIG. 12 shows the results of a monoclonal antibody specificity assay, M1: protein maker, M2: western Blot maker, 1: non-reduced TcdB, 2: reduced TcdB.

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

FIG. 14 shows the results of affinity analysis of neutralizing active antibody 2F3 with TcdB.

Fig. 15. Affinity analysis results of bezlotoxumab with TcdB.

FIG. 16 shows the results of PCR product identification of VH, Vκ, and Vλ chains of murine antibodies, M: DL5000 DNA MARKER, 1-3: VH, 4-5:V κ, 6:V λ.

FIG. 17 shows the results of analysis of antibody gene structure.

Detailed Description

The following provides a detailed description of embodiments of the present invention with reference to examples.

Example 1:

1. Materials and methods

1.1 Test apparatus and reagents

1.1.1 Toxic species, cells

Clostridium difficile strain LCL084 is supplied by clostridium perfringens institute of biological products, clostridium difficile laboratory. SP2/0 myeloma cells were supplied by the fifth research laboratory of the Lanzhou Biotechnology institute.

1.1.2 Vectors

E.coil BL21 (DE 3) competent cells were prepared by the fifth laboratory of all responsibility-limited company of Lanzhou Biotechnology research. The PET-22b vector was supplied by the fifth research laboratory of the Lanzhou Biotechnology institute.

1.2.3 Laboratory animals

Female BALB/c mice and female Kunming mice are both supplied by SPF class animal chambers of the Lanzhou Biotechnology institute.

1.2 Main reagents and apparatus

The primer is synthesized by Shanghai biological engineering company; high-fidelity KOD DNA polymerase was purchased from TOYOBO Co., japan; LB media was purchased from Invitrogen corporation, USA; bamHI and XhoI restriction endonucleases, DL2000 DNA MARKER, nucleic acid dyes, IPTG, ECL super-sensitized chemiluminescent kits, IPTG, goat anti-rabbit monoclonal antibodies, HRP-labeled goat anti-mouse IgG were purchased from Shanghai Biotechnology, ni Sepharose TM High Performance, DEAE Sepharose Fast Flow, phenyl Sepharose Fast Flow, protein G filler was purchased from GE, clostridium difficile pure toxin B, anti-Clostridium difficile toxin B monoclonal antibodies were purchased from UK Abcam, gel recovery kits, PCR purification kits, plasmid miniprep kits were purchased from QIAGEN, PVDF membranes, protein pre-dye markers, cell culture plates were purchased from Thermo, fresh fetal calf serum, RPMI1640 medium, HT additives, HAT supplements were purchased from Gibco, freund's complete adjuvant, freund's incomplete adjuvant was purchased from Sigma, PEG was purchased from Bioss, 96-well ELISA plates were purchased from Cor, engin, elkin Elmer, auger-Stran, fluorescent dye-60, fluorescent dye-transfer systems, fluorescent dye-60, fluorescent dye-probe-60, fluorescent dye-probe-35, fluorescent dye-measuring systems, fluorescent dye-transfer dye-set-60, fluorescent dye-transfer-station dye-set dye set dye set.

1.2 Experimental methods

1.2.1 Construction of TcdB RBD prokaryotic expression plasmid

According to the clostridium difficile toxin B gene sequence (sequence number: CP 046327.1) provided by NCBI database, genome DNA of LCL084 toxigenic strain is used as a template, bamHI and XhoI are selected as enzyme cutting sites, primer is designed by using Primer5.0 software, and TcdB RBD gene sequence is amplified. The primer is represented by tcdB-RBD, the TcdB RBD consists of 533 amino acids, 1834-2366aa in TcdB protein, and PCR primer synthesized by Shanghai Bioengineering company.

TABLE 1 Clostridium difficile toxin B RBD PCR primers

The TcdB RBD gene is amplified by PCR by taking genomic DNA of clostridium difficile LCL084 toxigenic strain as a template. The PCR products and the XhoI are respectively digested, and the PET-22b vector is identified by 1% agarose gel electrophoresis and cut into gel for recovery. The size of TcdB RBD after enzyme digestion is about 1600bp. E.coil BL21 (DE 3) was transformed using T4 DNA ligase to ligate the vector and sequences, ampicillin (AMP) -resistant LB agar plates were plated, positive clones were picked for sequencing, and 100% identical strains were retained.

1.2.2 Recombinant expression and purification of TcdB RBD protein

Inoculating recombinant bacteria activated overnight in advance into LB (Amp) culture medium according to the proportion of 1:100, culturing at 37 ℃ and 220rpm until OD600 = 0.6-0.8, adding a certain amount of IPTG with the final concentration of 1mM, at 25 ℃, inducing expression at 220rpm for 12h, centrifuging at 9500rpm for 30min, dissolving bacterial precipitate in PBS buffer, and crushing bacterial precipitate by a pressure crusher. 10000g is centrifuged for 15min, and the bacterial supernatant and bacterial precipitate are collected, and the expression condition of TcdB RBD protein is analyzed by SDS-PAGE and Western Blot. If the detection was successful, the broken supernatant was purified manually using Ni Sepharose ^TM High Performance medium, washed with 20mM imidazole followed by gradient elution with sequentially increasing imidazole concentration, the collected Ni affinity chromatography eluate was adjusted to 120mS/cm in conductivity, a Phenyl Sepharose Fast Flow hydrophobic chromatography column was equilibrated with 1mol/L (NH ₄)₂SO₄ in 0.05M PB buffer (pH 7.0, conductivity 5 mS/cm), the collected sample peak eluate was adjusted to 5mS/cm in conductivity, a DEAE anion chromatography column was equilibrated with 0.05M PB buffer (pH 7.0, conductivity 5 mS/cm) containing 1mol/L NaCl in conductivity 85mS/cm, and the sample peak eluate was collected by linear elution with 0.05M PB buffer (pH 7.0, conductivity 85 mS/cm) in TCRBD protein purity and activity after three-step Western-chromatography using CE-SDS, western-dB analysis.

1.2.3 Preparation of anti-TcdB monoclonal antibodies

BALB/c mice were immunized with high purity TcdB RBD recombinant protein, blood was collected from the tail vein 7 days after the third immunization, ELISA was used to determine serum titers, and the spleen cells of the mice with the highest antibody titers were selected for hybridoma preparation. After 3 days of boost immunization with the antigen, the mouse immune spleen cells 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 are cloned to more than 1/4 of the whole-well visual field, ELISA cross screening is carried out on the hybridoma cells by using the TcdB RBD recombinant protein and pure TcdB. According to the indirect ELISA detection result, selecting positive hybridoma cells with high absorbance OD ₄₅₀ reading value, small cell cloning number and good cell state, and carrying out cloning culture on the cells by adopting a limiting dilution method. Until all monoclonal cell lines are positive in indirect ELISA detection result, and cloning is terminated. Positive monoclonal hybridoma cells are subjected to expansion culture and passage, indirect ELISA detection is further carried out during passage to detect the passage stability of the cells, and cell freezing is carried out. BALB/C female mice were taken, 0.2mL of paraffin oil was injected into each abdominal cavity for sensitization of the mice, about 1X 10 ⁶ positive hybridoma cells were injected into each abdominal cavity after one week, when the abdomen of the mice was significantly distended and mobility was inconvenient, the abdominal skin of the mice was cut off with dissecting scissors, the peritoneum was exposed, a sterile syringe needle was inserted into the abdomen of the mice, ascites were collected and centrifuged at 4 ℃ (12000 rpm X30 min), fat and paraffin oil in the abdominal cavity of the mice were removed, and the intermediate layer ascites was collected and purified.

1.2.4 Purification of ascites from mice

The ascites was first salted out and crude extracted by saturated ammonium sulfate precipitation, centrifuged at 4℃for 10000rpm X30 min, the supernatant was discarded, the precipitate was resuspended in PBS buffer, filtered through a 0.45 μm filter membrane and purified by Protein G agarose gel. 5mL of Protein G pre-packed column was connected to the corresponding column site of AKTA AVANT Protein purification system, and 5 column volumes were washed with purified water, 0.1mol/L NaOH, and purified water, respectively, and then 10 column volumes were equilibrated with PBS. After UV 280 zeroing, loading cell culture supernatant and ascites crude pure samples according to the loading of not more than 30mg/mL, washing 5 column volumes with impurity removal buffer (0.5M NaCl+10mM PB,pH 7.0-7.4), PB rebalancing, eluting with 0.03M NaAC (pH=3.7), collecting elution peak of UV 280>50mAU, adding 1mol/L Tris-HCl (pH=10.2), and adjusting pH to about 7.0. The 5 column volumes were rinsed with 0.1mol/L NaOH and pure water, respectively, and the pre-packed column was kept in 20% ethanol. NanoVue ultra micro spectrophotometry Protein A280 method for measuring the concentration of purified antibody.

1.2.5 Analysis of specificity of monoclonal antibodies

The specificity of monoclonal antibodies is identified by Western-Blot, clostridium difficile pure TcdB (positive control) and TcdB RBD proteins to be detected are separated by SDS-PAGE electrophoresis, after membrane transfer, the membrane is closed at room temperature for 2 hours, primary antibodies are used for resisting the TcdB monoclonal antibodies after purification, the primary antibodies are incubated at room temperature for 1 hour, PBS-T membranes are washed for 3-5 times and 10 minutes each time, secondary antibodies are used for marking goat anti-mouse monoclonal antibodies by HRP, the primary antibodies are combined at room temperature for 1 hour, PBS-T membranes are washed for 3-5 times and 10 minutes each time, substrates are added for color development, and the membrane is exposed for 30 seconds, and the results are observed.

1.2.6 Detection of the titre of monoclonal antibodies

Coating a 96-well ELISA plate with clostridium difficile pure toxin B, closing the plate at 4 ℃ overnight, sequentially adding a double-ratio diluted monoclonal antibody from the first row after washing the plate, incubating for 1h at 37 ℃, adding a peroxidase-labeled goat anti-mouse secondary antibody after washing the plate, incubating for 45min at 37 ℃, adding substrate A solution and substrate B solution after washing the plate, incubating for 15min, adding a stop solution, and detecting the OD ₄₅₀ reading value by using an ELISA reader. The positive control is the serum of the mice immunized by the antigen, the negative control is the serum of the mice not immunized, and the maximum dilution corresponding to the OD ₄₅₀ reading value > 2.1 times and more than 0.2 of the negative control is taken as the antibody titer.

1.2.7 Analysis of neutralizing Activity of monoclonal antibodies

1.2.7.1 Clostridium difficile toxin B toxin virulence assay

The toxin virulence of TcdB was determined by reference to the 2015 edition pharmacopoeia type a botulinum toxin potency assay, with the toxin virulence of TcdB taking the half lethal dose (50%lethal dose,LD ₅₀) of mice as the detection standard. See literature TcdB in CD1 mice LD ₅₀ is 1X 10 ^-3 mg/kg, with reference to this dose to serial dilutions of toxin B, setting 5 toxin dose dilutions, toxin dose 0.25X10 ^-3mg/kg、0.5×10^-3mg/kg、1×10^-3mg/kg、2×10^-3 mg/kg and 4X 10 ^-3 mg/kg, each dilution 2.5mL, selecting female Kunming mice (SPF grade) weighing about 14-16g, each dilution injected 4. Each abdominal cavity is injected with 0.5mL, the mice are continuously observed for 96 hours after injection, and the death condition of the mice is recorded and the statistical result is recorded.

The statistical result is calculated by the modified koehne method to obtain LD ₅₀, and the calculation method is as follows:

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

1.2.7.2 Neutralization test

Qualitative analysis of neutralizing activity of monoclonal antibodies was performed by using a mouse test, which was set up with a sample group, a toxin inactivation control group, and a blank group, respectively, in which different dilutions of the antibody solutions to be tested (2F 3 sample group, 4H7 sample group, 7C2 sample group, 8C10 sample group, 9C9 sample group, 9a12 sample group, 9H7 sample group, and 10F7 sample group) were quantitatively aspirated. Then, the mixture was mixed with a toxin dose diluent corresponding to 5LD ₅₀, and the mixture was incubated at 37℃for 1 hour, and immediately, it was intraperitoneally injected into a Kunming mouse weighing 14-16 g. 4 mice were injected with each dilution, 0.5mL of each intraperitoneal injection, and the antibody solution in the sample group was divided into 3 different dilutions (low dilution B1; medium dilution B2; high dilution B3; respectively), and IgG concentrations corresponding to the three dilutions B1, B2, and B3 were 10. Mu.g/mL, 1. Mu.g/mL, and 0.1. Mu.g/mL, respectively. The toxin group of Kunming mice is injected with diluted toxin solution, the toxin inactivation control group is injected with toxin solution subjected to heat inactivation treatment at 100 ℃, the blank group of Kunming mice is injected with PBS buffer solution, and the survival condition of the mice is observed for 4 days after injection, and the statistical result is obtained.

1.2.8 Analysis of monoclonal antibody affinity by surface plasmon resonance

Coupling ligand, namely coating a mouse monoclonal antibody and a marketed antibody TcdB McAb Bezlotoxumab on a CM5 chip by an amino coupling method, wherein the concentration of the coupling antibody is 5 mug/ml, the coating solution is 10mM sodium acetate with pH=5.0, and the running buffer is HBS-EP+. The chip was first activated with EDC, NHS, after which murine antibodies were bound to the chip at a flow rate of 10 μl/min for 7 minutes, and the unbound epitopes were blocked with ethanolamine. The other channel, to which the antibody was not coupled after activation, was used as a reference channel for detection.

Affinity measurement, namely, running buffer is HBS-EP+, the flow rate is 30 mu L/min, tcdB is used as an analyte to slowly flow through the surface of the chip after gradient dilution by the running buffer after the baseline is stable, and the affinity measurement is carried out by adopting a single-cycle method. The flow rate was 30. Mu.L/min, combined with 120s, dissociated for 600s and the medium was regenerated with Gly-HCl buffer pH 1.5 for 30s. The obtained sensorgram is fitted by using analysis software to obtain affinity data of the antibody and the antigen.

1.2.9 Modulation of antibody variable region sequences

Extracting total RNA of the hybridoma cells by adopting a Trizol method, synthesizing a first cDNA chain by reverse transcription, cloning by PCR to obtain a variable region gene of the antibody of the hybridoma cells, cloning the gene to a T vector, picking positive clones, and sequencing. Primers were designed according to the principle of independent amplification of VH, vκ and vλ chains in mice, and the primer sequences are shown in table 2.

TABLE 2 primers for PCR amplification of VH, Vκ, and Vλ chains of murine antibodies

2. Experimental results

2.1 Amplification of the Gene of interest and product identification results

The PCR product of TcdB RBD is amplified by taking genomic DNA of clostridium difficile strain LCL084 as a template, and the target band with the size of about 1600bp is found by 1% agarose gel electrophoresis analysis, and the size is consistent with the expected size, as shown in figure 1.

2.2 Plasmid construction results

After the amplified product of the TcdB RBD sequence in 2.1 and the vector PET-22b are digested with BamHI and XhoI, bands of about 1600bp and 5500bp are respectively seen to be consistent with expectations by 1% agarose gel electrophoresis analysis. The result of agarose gel electrophoresis identification of the amplified product of the TcdB RBD sequence and the vector PET-22b enzyme digestion product is shown in figure 2.

The recombinant expression plasmid is transformed into E.coil BL21 (DE 3), positive clones are picked up after overnight culture, and 10 single clones are picked up for culture respectively. After taking 5 mu L of single culture product after transformation, the analysis and verification of 1% agarose gel electrophoresis prove that positive bands appear at 1600bp, which is consistent with expectations. The recombinant plasmid identification results are shown in FIG. 3.

And (3) verifying that positive bacteria are sent to sequence, comparing the sequencing result with a main board sequence through Megalign, wherein the homology between a TcdB RBD gene sequence inserted in the plasmid and the main board sequence is 100%, namely, the genes have no mutation, so that the recombinant plasmid is correctly constructed. The homology alignment results are shown in FIG. 4.

2.3 Expression and purification results of TcdB RBD recombinant protein

The TcdB RBD recombinant protein after induced expression is analyzed by Western Blot technology, whether the recombinant protein carries the tag or not is detected by using an HRP-marked mouse anti-His tag monoclonal antibody, and the TcdB RBD recombinant protein is proved to be successfully expressed by detecting the His tag positive. And then subjecting the protein expression product to affinity chromatography, hydrophobic chromatography and anion exchange chromatography in sequence, wherein the purification results of the protein expression product are shown in figure 5.

And respectively collecting the eluates of each peak, separating the eluates by SDS-PAGE electrophoresis, dyeing, decolorizing, and then observing about 60KD protein bands in the eluate of the highest peak, wherein the protein bands can be target proteins, and the protein purity is sequentially increased. SDS-PAGE and Western-Blot results of purified samples of TcdB RBD recombinant protein are shown in FIGS. 6 and 7.

2.4TcdB RBD recombinant protein property analysis result

2.4.1CE-SDS analysis of protein purity results

The protein purity of the TcdB RBD after three-step chromatography purification is detected by using a non-reducing CE-SDS method, and the target protein peak is integrated by using 32Karat software. The detection report is shown in FIG. 8, and the result shows that the purity of the TcdB RBD protein is about 91%, which shows that the purification effect is good.

2.4.2TcdB RBD recombinant protein specificity analysis results

The specificity of TcdB RBD recombinant protein is detected by a Western-Blot method, namely, the pure TcdB (positive control) of clostridium difficile and the TcdB RBD protein to be detected are fixed on a PVDF membrane, a rabbit-source toxin B monoclonal antibody is used as a primary antibody binding protein, an HRP-marked goat anti-rabbit monoclonal antibody is used as a secondary antibody, and the binding condition of the pure TcdB and the TcdB RBD recombinant protein of clostridium difficile and the rabbit-source toxin B monoclonal antibody is analyzed. Both were found to exhibit a positive response, indicating successful expression of the TcdB RBD recombinant protein. The results are shown in FIG. 9.

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

Mouse serum titers were detected by indirect ELISA using non-immunized mouse serum as a negative control. The results are shown in Table 3, and mice with serum dilutions greater than 20000 at OD ₄₅₀ reading of approximately 1.0 can be used for hybridoma cell preparation. As can be seen from the table, the above 5 mice can be used for preparing monoclonal antibodies, wherein the mouse number 2 has the highest antibody titer (> 160000) and is selected to prepare the anti-TcdB RBD monoclonal antibody.

TABLE 3 BALB/c mouse serum anti-TcdB RBD protein antibody titre results

2.6 Cell fusion results

Three days after BALB/c mice are boosted, spleen cells of the mice and SP2/0 myeloma cells are taken to be subjected to cell fusion under the action of PEG1450 fusion agent, and after the cell fusion is observed by an inverted microscope, the result is shown in figure 10, the fused cells begin to divide in red circles after the fusion on the next day, are gradually divided into two by one, and gradually divide into cell clusters along with the extension of the fusion time, and clear and bright cell clusters can be observed under the scope on the twelfth day, so that the cell fusion is successful.

2.7 Screening and cloning results of Positive hybridoma cells

Screening positive hybridoma cells by adopting an indirect ELISA method, carrying out positive preliminary screening by using TcdB RBD recombinant protein, and then carrying out secondary screening by using pure TcdB. Positive hybridoma cells with high absorbance OD ₄₅₀ reading value, small cell cloning number and good cell state are selected for cloning culture. After 3 rounds of cloning and screening by limiting dilution method, 8 positive hybridoma cell strains are obtained, which are respectively named as 2F3, 4H7, 7C2, 8C10, 9C9, 9A12, 9H7 and 10F7. The results are shown in Table 4, which were tested by indirect ELISA during passage and were determined to be positive hybridoma cell lines stably secreting specific monoclonal antibodies.

TABLE 4 Positive screening results for hybridoma cells

2.8 Mass production and purification results of monoclonal antibodies

A large amount of monoclonal antibodies are prepared by adopting a mouse ascites method, taking one strain of the monoclonal antibodies as an example, purifying the ascites after crude extraction by using a AKTA AVANT Protein chromatography system and a Protein G pre-packed column, measuring the Protein concentration of a sample peak eluent by using a NanoVue ultra-micro spectrophotometer to be 1500 mug/ml, carrying out beta-mercaptoethanol reduction treatment on the monoclonal antibodies due to the large molecular weight (150-180 KD), and carrying out purity analysis on the purified monoclonal antibodies by utilizing reduction and non-reduction SDS-PAGE electrophoresis. As a result, as shown in FIG. 11, it was revealed that the monoclonal antibody was reduced to 55KD (heavy chain) and 25KD (light chain) by the reducing agent beta-mercaptoethanol, and the molecular weight of the non-reducing monoclonal antibody was about 150KD, which corresponds to the molecular weight of IgG. And the high-purity monoclonal antibody is obtained by using a Protein G medium purification method, and the purity of the antibody reaches more than 90 percent.

2.9 Results of monoclonal antibody specificity analysis

The specificity of the monoclonal antibody is analyzed and identified by adopting a Western-Blot method, taking one antibody as an example, the result is shown in FIG. 12, the left image is a TCdB reduction and non-reduction SDS-PAGE electrophoresis diagram, and the right image is a Western-Blot result diagram of the combination of the monoclonal antibody and the TcdB. The result shows that the monoclonal antibody secreted by the hybridoma cell strain can specifically recognize the full TcdB toxin molecular protein (molecular weight 270 KD) subjected to reduction and non-reduction treatment.

2.10 Results of detection of the titers of monoclonal antibodies

The titers of the purified monoclonal antibodies were determined by indirect ELISA, and the results are shown in Table 5, with one of the antibodies as an example, and the titers of the antibodies were 1:3.2X10 ⁶.

TABLE 5 monoclonal antibody titre detection results

2.11 Results of monoclonal antibody neutralization Activity analysis

2.11.1 Clostridium difficile toxin B toxin virulence determination results

As shown in FIG. 13, it was found that the mortality rate of mice in the toxin dose groups of 0.25. Mu.g/kg and 0.5. Mu.g/kg was 50% or less, and that the mice in the toxin dose groups of 1. Mu.g/kg were 50% dead, and that the mice in the toxin dose groups of 2. Mu.g/kg and 4. Mu.g/kg were all dead. The toxin virulence of TcdB was calculated to be LD ₅₀ = 1 μg/kg. The experimental results are in accordance with the literature-referenced LD ₅₀ values of TcdB in CD1 mice.

2.11.2 Neutralization test results

The neutralizing activity of the monoclonal antibodies was determined using a mouse assay. The test is provided with a sample group, a toxin inactivation control group and a blank group respectively, the survival conditions of mice after injection are shown in table 6, and the mice in the toxin inactivation control group and the mice in the blank group both survive after 96 hours of injection without disease symptoms. Mice in each of the toxin and 4H7, 7C2, 9C9, 9a12, 9H7 sample groups all died after 24H of injection, mice in each of the 2F3, 8C10, 10F7 sample groups survived 24H of injection, but 1 mouse in each of the 8C10 and 10F7 sample groups survived only at the low dilution B1, and mice in both the B2 and B3 dilution groups died, and since only 1 mouse in the B1 group survived, it was not possible to determine that they were neutralizing active antibodies. After 24h of injection, the incidence of mice in 2F3-B3 was evident, and 3, 2F3-B1, 2F3-B2, and 2F3-B3 survived 3, 2, and 1 respectively, and from this result, it was seen that a gradient was also present as the dilution of the antibody was low to high. After 48h, 2 mice in groups 2F3-B1 and 2F3-B2 still survived, respectively, and all mice in groups 2F3-B3 died. And judging the antibody to be a neutralizing antibody.

TABLE 6 monoclonal antibody neutralization Activity analysis results

2.12 Neutralizing antibody affinity assay results

The affinity of neutralizing antibody 2F3 and marketed antibody TcdB McAb Bezlotoxumab for TcdB was analyzed by Biacore T200. The antibody affinity was determined by a 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. TcdB was diluted in HBS-EP Buffer in a two-fold gradient for a total of 5 dilutions. Each step on the graph represents the response of the antibody to TcdB at different dilutions, and the course of the antibody interaction with TcdB can be reflected by the change in response. Equation fitting was performed with a one-to-one joint model. The affinity results are shown in FIG. 14 and FIG. 15, wherein ka and KD values represent the binding rate constant and dissociation rate constant of the antibody 2F3 and TcdB, respectively, and the equilibrium dissociation constant KD of the interaction between the two can be obtained by calculating the ratio of KD/ka, and generally, the smaller the KD value, the higher the affinity between the two. The equilibrium dissociation constants KD of the neutralizing antibodies 2F3 and Bezlotoxumab and TcdB were calculated by software to be 1.188E-8M and 4.985E-9M, respectively.

2.13PCR amplification of VH, vkappa, vlambda chain results of murine antibodies

The mouse hybridoma cDNA was used as a template to amplify the VH, V.kappa.and V.lambda.chains of the murine antibody, and the results of analysis by 1% agarose gel electrophoresis revealed that the VH and V.kappa.chains were amplified by PCR and the V.lambda.chains were not amplified, indicating that the light chain of the murine antibody was kappa type, as shown in FIG. 16. The VH electrophoresis band is about 360bp, the Vκ electrophoresis band is about 340bp, and the band size is consistent with the expectations.

2.14 Results of antibody variable region sequence analysis

And (3) sequencing the verified positive bacteria, and comparing the sequencing result with NCBI database by BLAST software, wherein the amplified VH and V kappa sequences are variable region sequences of the murine antibody. The variable region gene sequence of the murine antibody obtained by sequencing is further subjected to gene structure analysis by utilizing a V-BASE2 database, and complementarity determining region CDR and framework region FR on antibody VH and VL are positioned. The results of the analysis of the antibody gene structure are shown in FIG. 17.

3. Discussion of the invention

Clostridium difficile LCL084 strain is a strict obligate anaerobic bacterium, and has strict culture conditions. The strain has low toxin yield, large molecular weight of toxins TcdA and TcdB and similar physicochemical properties, and brings great challenges to the antigen preparation work of the invention. According to literature reports, tcdB RBD is known to be a key epitope for toxin entering human body to cause diseases. The protein can be prepared in a large quantity by utilizing a prokaryotic expression system, so that the TcdB RBD gene is directionally cloned to a PET-22b prokaryotic expression vector and converted into escherichia coli for recombinant expression, and after three-step chromatography purification, a CE-SDS result shows that the purity of the recombinant protein reaches more than 90 percent, the purity of the protein completely meets the antigen immunization requirement of preparing McAb by a hybridoma technology, the antibody screening strength of the invention is greatly reduced, and a solid foundation is laid for successful screening of positive McAb cell strains.

The invention adopts animal test to analyze the neutralizing activity of the antibody of 8 positive hybridoma cell strains, and the pure TcdB after neutralization and McAb mixed solution are injected into the body of the mouse in the abdominal cavity, thus the antibody with inhibiting effect on toxin is primarily screened according to the death condition of the mouse. The results showed that only 2F3 this strain was able to neutralize 5 toxins TcdB of LD ₅₀, while none of the other 7 strains had a neutralizing protective effect on mice. 1 McAb with neutralizing activity was successfully screened by this method.

The invention adopts SPR technology to evaluate the affinity of McAb by a single cycle method. The method comprises the steps of selecting a murine neutralizing antibody 2F3 and a fully human source Bezlotoxumab (anti-TcdB McAb marketed) to be respectively fixed on the surface of a CM5 chip through an amino coupling method, slowly transmitting pure TcdB antigen molecules subjected to gradient dilution to the surface of a sensor chip through a microfluidic chuck as a mobile phase, and reflecting the whole process of interaction of the antibody and the TcdB according to the change of a response value. The equilibrium dissociation constant KD of the murine neutralizing antibody 2F3 and the TcdB is 1.188E-8M, the equilibrium dissociation constant KD of Bezlotoxumab and the TcdB is 4.985E-9M, and the affinity data of the two are not significantly different, so that the screened murine neutralizing antibody 2F3 has good antibody activity and the potential of developing into a product on the market.

While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.

Claims

1. A monoclonal antibody against clostridium difficile toxin B, characterized in that the amino acid sequences of heavy chain CDR1, CDR2, CDR3 are:

CDR1: GYTFTSYW, shown as SEQ ID NO 1;

CDR2: IYPGNNNT as shown in SEQ ID NO 2;

CDR3: SRGDYGSSYALDY, shown as SEQ ID NO 3;

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

CDR1: QDVNTA, shown as SEQ ID NO 4;

CDR2: SASYRYTGV as shown in SEQ ID NO 5;

CDR3: QQRHVPPRT is shown as SEQ ID NO. 6.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody is selected from the group consisting of 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, and a complete antibody molecule having a full length heavy chain and a light chain.

3. A polynucleotide comprising nucleotides encoding the monoclonal antibody of claim 1 or 2.

4. A recombinant expression vector comprising the polynucleotide of claim 3.

5. A host cell transformed with the recombinant expression vector of claim 4.

6. Use of a monoclonal antibody according to claim 1 or 2 in the manufacture of a medicament for the treatment of clostridium difficile infection.

7. A medicament for the treatment of clostridium difficile infection, comprising a monoclonal antibody according to claim 1 or 2.

8. Use of a monoclonal antibody according to claim 1 or 2 for the preparation of a clostridium difficile detection reagent or detection kit.

9. A clostridium difficile detection kit comprising a labelled complex of the monoclonal antibody of claim 1 or 2, labelled by a biomarker or chemical.

10. A method for producing 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.