CN116063535A - Antibody or antigen-binding fragment thereof, preparation method and application thereof - Google Patents

Abstract

The invention provides an antibody or antigen binding fragment thereof, hybridoma cells producing the antibody or antigen binding fragment thereof, and uses thereof. The invention adopts prokaryotic expression technology to successfully prepare high-purity Tet (X4) recombinant protein, uses the Tet (X4) recombinant protein as antigen to prepare hybridoma cells and generate specific antibodies or antigen binding fragments thereof, and the antibodies or antigen binding fragments thereof have strong specificity and high affinity.

Description

Antibody or antigen-binding fragment thereof, preparation method and application thereof

technical field

The invention relates to the technical field of biomedicine, in particular to an antibody or an antigen-binding fragment thereof and a preparation method and application thereof.

Background technique

Nowadays, with the overuse of antibiotics, bacterial antibiotic resistance (Antimicrobial Resistance) has become one of the biggest threats to global health and food safety. For a series of diseases caused by Gram-negative bacteria such as carbapenem-resistant Escherichia coli, etc., mainly rely on colistin and tigecycline for treatment, The gene (mcr) is widely spread around the world, and the clinical effect of colistin is greatly weakened. Therefore, tigecycline has become one of the "last line of defense" for clinical treatment of antibiotic infection.

Tetracyclines and their derivatives (such as tigecycline) are a major clinical option for the treatment of Gram-negative bacterial infections and are widely used. The emergence of transferable Tet(X) enzymes that destroy tetracycline antibiotics poses a great challenge to antimicrobial therapy and food/environmental safety. Different from the previously widely studied efflux pump mechanism of tigecycline resistance, the flavin-dependent monooxygenase encoded by the Tet(X) gene, which uses tigecycline as a substrate, plays a certain role in it. Modification, so that tigecycline loses its activity. Among them, the transferable tigecycline resistance gene Tet(X4) isolated from Enterobacteriaceae and Acinetobacter is extremely important. Understanding its characteristics and resistance mechanisms can provide important evidence for the medical and health industries.

Contents of the invention

In order to solve the defects existing in the prior art, the application provides a kind of protein that can specifically bind to transferable tigecycline resistance (Tet(X4) for short), which is expected to block the interaction between Tet(X4) and tigecycline. Combining to solve the problem of drug resistance.

The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, and/or, CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region.

Wherein, the amino acid sequence of CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 1 or 10, or has 80%, 90%, 91%, 92%, 93%, 94%, 95% with SEQ ID NO: 1 or 10 %, 96%, 97%, 98%, 99% or more identity; the amino acid sequence of CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 2, 11 or 26, or with SEQ ID NO: 2, 11 or 26 has 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity; the amino acid sequence of CDR-H3 comprises SEQ ID NO: 3 or 12 amino acid sequence, or having 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of SEQ ID NO: 3 or 12 The identity of; the amino acid sequence of CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4, 7, 13, 27, 28 or 29, or has with SEQ ID NO: 4, 7, 13, 27, 28 or 29 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity; the amino acid sequence of CDR-L2 comprises SEQ ID NO: 5, The amino acid sequence shown in 8, 14 or 30, or having 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of SEQ ID NO: 5, 8, 14 or 30 %, 98%, 99% or more identity; the amino acid sequence of CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 6, 9, 15 or 31, or has 80% with SEQ ID NO: 6, 9, 15 or 31 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

In a specific embodiment of the present invention, the amino acid sequence of CDR-H1 includes the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of CDR-H2 includes the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence of CDR-H3 The amino acid sequence comprises the amino acid sequence of SEQ ID NO: 3; the amino acid sequence of CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4, 7, 28 or 29; the amino acid sequence of CDR-L2 comprises SEQ ID NO: 5, 8 Or the amino acid sequence shown in 30; The amino acid sequence of CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 6 or 9.

In a specific embodiment of the present invention, the amino acid sequence of CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 10; The amino acid sequence of CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 11 or 26; CDR- The amino acid sequence of H3 comprises the amino acid sequence of SEQ ID NO: 12; the amino acid sequence of CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 13 or 27; the amino acid sequence of CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 14 Sequence; The amino acid sequence of CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 15 or 31.

In a specific embodiment of the present invention, the amino acid sequence of CDR-H1 includes the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of CDR-H2 includes the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence of CDR-H3 The amino acid sequence comprises the amino acid sequence of SEQ ID NO: 3; the amino acid sequence of CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4 or 7; the amino acid sequence of CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 5 or 8 Sequence; The amino acid sequence of CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 6 or 9.

In a specific embodiment of the present invention, the amino acid sequence of CDR-H1 includes the amino acid sequence shown in SEQ ID NO: 10; the amino acid sequence of CDR-H2 includes the amino acid sequence shown in SEQ ID NO: 11; the amino acid sequence of CDR-H3 The amino acid sequence comprises the amino acid sequence of SEQ ID NO: 12; the amino acid sequence of CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence of CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 14; CDR- The amino acid sequence of L3 comprises the amino acid sequence shown in SEQ ID NO: 15.

In a specific embodiment of the present invention, the amino acid sequence of CDR-H1 includes the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of CDR-H2 includes the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence of CDR-H3 The amino acid sequence comprises the amino acid sequence of SEQ ID NO: 3; the amino acid sequence of CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4; the amino acid sequence of CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 5; CDR- The amino acid sequence of L3 comprises the amino acid sequence shown in SEQ ID NO:6.

In a specific embodiment of the present invention, the amino acid sequence of CDR-H1 includes the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of CDR-H2 includes the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence of CDR-H3 The amino acid sequence comprises the amino acid sequence of SEQ ID NO: 3; the amino acid sequence of CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence of CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 8; CDR- The amino acid sequence of L3 comprises the amino acid sequence shown in SEQ ID NO:9.

Preferably, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in order from N-terminal to C-terminal.

Preferably, the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are arranged in order from N-terminal to C-terminal.

Preferably, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16 or 19, or has 80%, 90%, 91%, 92%, 93%, 94% of SEQ ID NO: 16 or 19 %, 95%, 96%, 97%, 98%, 99% or more identity.

Preferably, the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, 18 or 20, or has 80%, 90%, 91%, 92%, More than 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

In a specific embodiment of the present invention, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 16, and the light chain variable region comprises SEQ ID NO: 17 or 18.

In a specific embodiment of the present invention, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 19, and the light chain variable region comprises SEQ ID NO: 20.

Preferably, the antibody or antigen-binding fragment thereof specifically binds to the transferable tigecycline resistance protein (Tet(X4) for short).

Preferably, the structure of the antibody or antigen-binding fragment thereof is Fab, Fab', Fab'-SH, F(ab')2, single domain antibody or single chain antibody.

The second aspect of the present invention provides a nucleic acid encoding the above-mentioned antibody or its antigen-binding fragment, the above-mentioned CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR - L3, or the above-mentioned heavy chain variable region or light chain variable region.

Preferably, the nucleotide sequence encoding the heavy chain variable region comprises SEQ ID NO: 21 or 24, or has 80%, 90%, 91%, 92%, 93%, 94% of SEQ ID NO: 21 or 24 , 95%, 96%, 97%, 98%, 99% or more identity.

Preferably, the nucleotide sequence encoding the light chain variable region comprises SEQ ID NO: 22, 23 or 25, or has 80%, 90%, 91%, 92%, 93% of SEQ ID NO: 22, 23 or 25 , 94%, 95%, 96%, 97%, 98%, 99% or more identity.

The third aspect of the present invention provides a method for preparing Tet(X4) recombinant protein.

The method includes introducing the recombinant plasmid containing the nucleotide sequence encoding Tet(X4) recombinant protein into the host cell.

Preferably, the host cell is prokaryotic, such as Escherichia coli.

The fourth aspect of the present invention provides a recombinant plasmid, which includes a nucleotide sequence encoding Tet(X4) recombinant protein.

The fifth aspect of the present invention provides a cell comprising a nucleotide sequence encoding Tet(X4) recombinant protein or comprising the above recombinant plasmid or comprising the above nucleic acid.

The sixth aspect of the present invention provides a Tet(X4) recombinant protein prepared by the above method.

The seventh aspect of the present invention provides a hybridoma cell.

Preferably, said hybridoma cells produce the above-mentioned antibodies or antigen-binding fragments thereof.

The eighth aspect of the present invention provides a method for preparing hybridoma cells.

The preparation method comprises the steps of immunizing mice with Tet(X4) recombinant protein, isolating splenocytes, and fusing with myeloma cells.

The ninth aspect of the present invention provides a medicine or a diagnostic kit, which comprises the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid or the above-mentioned hybridoma cells.

The tenth aspect of the present invention provides an application of the above-mentioned antibody or its antigen-binding fragment, the above-mentioned nucleic acid, the above-mentioned hybridoma cell, or the above-mentioned drug or diagnostic kit, and the application includes:

A) use in the preparation of products for the treatment of diseases caused by Gram-negative bacteria; or

B) Application in the manufacture of products against tigecycline resistance.

Preferably, the Gram-negative bacteria include but are not limited to Gram-negative bacteria (Escherichia coli (E. Bacteria (Enterobacter, Citrobacter, Morganella, Providencia, Proteus, etc.), Gram-negative bacteria residing in the respiratory system (Haemophilus, Moraxella, etc.) and non-glucose-fermenting Gram-negative bacteria (Pseudomonas aeruginosa, Pseudomonas, Stenotrophomonas genera (Stenotrophomonas), Burkholderia (Burkholderia), Acinetobacter (Acinetobacter, etc.).

The eleventh aspect of the present invention provides an antibody-drug conjugate, which comprises the above-mentioned antibody or antigen-binding fragment thereof covalently bound to a drug.

Preferably, the drug may be chemically synthesized drugs, antibiotics or various biological drugs. Antibiotics are preferred, such as tigecycline and the like.

The twelfth aspect of the present invention provides a method for detecting Tet(X4) protein, said detection method comprising contacting the sample to be detected with the antibody or antigen-binding fragment thereof described in this application, and then detecting the Tet(X4) protein ) complexes formed by proteins and antibodies or antigen-binding fragments thereof.

Preferably, the detection method detects the presence or content of Tet(X4) protein. Wherein, the presence means presence or absence, and the content may be expression level or protein concentration, etc.

Preferably, said antibody or antigen-binding fragment thereof may comprise a selectable marker.

The "antigen-binding fragment" of the present invention is a part of the antibody that retains the specific binding activity of the whole antibody, that is, any part of the antibody can specifically bind to the epitope on the target molecule of the whole antibody. It includes eg Fab, Fab', F(ab')2 and variants of these fragments. For example, the heavy and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, the full length heavy or light chain of an intact antibody, or a heavy or light chain from an intact antibody single CDR.

The "CDR" mentioned in the present invention represents a complementarity-determining region (complementarity-determining region), which can be determined by the Kabat numbering, Chothia numbering, IMGT numbering, AHo numbering (Honegger numbering) schemes commonly used in the prior art.

When "comprising" in the present invention is used to describe the sequence of a protein or nucleic acid in this application, the protein or nucleic acid may consist of the sequence, or may have additional Amino acids or nucleotides, but still have the activity described in the present invention.

The "identity" mentioned in the present invention means that in terms of using amino acid sequence or nucleotide sequence, those skilled in the art can adjust the sequence according to actual work needs without changing the structure or activity of the original sequence, so that Compared with the specific sequence described in the present invention, the used sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27% , 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44 %, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% identity.

The recombinant plasmid was obtained by prokaryotic expression technology, Tet (X4) recombinant protein was obtained by mass expression in Escherichia coli, and high-purity protein was obtained by using a protein purifier. Induced by different temperatures, it was concluded that the soluble expression level of Tet(X4) protein had no significant difference between 20°C and 37°C, and the purified protein had good biological activity. During the subsequent monoclonal antibody preparation and verification process, a total of 4 mice were immunized. After the immunization was completed, the "left" mouse was selected for fusion experiment according to the detection titer. After fusion, 34 positive clones were retained for subcloning through antigen screening and re-examination, and 30 subcloning supernatants were collected for affinity sorting. Finally, 5D3D3 was selected for antibody preparation in ascites, and the obtained antibodies were detected by SDS-PAGE and WB of recombinant antigens, showing clear bands. Monoclonal antibody is an antibody secreted by lymphocytes that only targets a specific epitope, and has good specificity and strong affinity. In this study, a high-purity Tet(X4) protein was prepared using a prokaryotic expression system, and its monoclonal antibody was obtained, which can provide a basis for further explaining the drug resistance mechanism of Tet(X4) drug-resistant strains and controlling its prevalence worldwide .

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail in conjunction with the accompanying drawings, wherein:

Figure 1: PCR identification results of the Tet(X4) recombinant plasmid.

Figure 2: The expression level of Tet(X4) protein in different processes under the induction condition of 20°C.

Figure 3: The expression level of Tet(X4) protein in different processes under the induction condition of 37°C.

Figure 4: SDS-PAGE image of antibodies.

Figure 5: Antibody Western-Blot diagram.

Figure 6: Tet(X4) protein denaturation curve and aggregation curve overlay.

Figure 7: Overlay of antibody denaturation and aggregation curves.

Figure 8: Chemiluminescence Imaging.

Figure 9: Quantitative analysis results, where Ckneg represents tet (X4) negative bacteria, CKpos represents tet (X4) positive bacteria without drug addition, T2 represents the concentration of tigecycline 2 μg/mL, T4 represents the concentration of tigecycline 4 μg/mL mL, T8 represents the concentration of tigecycline 8μg/mL.

Figure 10: Antibody standard curve.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Reagent and instrument source used in the embodiment:

Escherichia coli [Escherichia coli BL21(DE3)] was purchased from Shanghai Weidi Biotechnology Co., Ltd., and the prokaryotic expression plasmid pET-28a was provided by Beijing Liuhe Huada Gene Technology Co., Ltd.

Nucleic acid restriction endonuclease rTaq, DNA T7, T7t ligase, DL2000 nucleic acid marker were all purchased from Yubao Bioengineering (Dalian) Co., Ltd.;

SDS-PAGE protein loading buffer and protein molecular weight standards (10-150kD, not pre-stained) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.;

SDS-PAGE gel kit (Beiyuntian Biotechnology Co., Ltd.);

Freund's adjuvant, Balb/C mice, PBS, DMEM medium (Hyclone, SH30809.01B), fetal bovine serum (no phage), double antibody (Hyclone, SY30010), L-glutamate (Amresc, DH144 -1), PEG4000 (SIGMA, p7181), human lymphocyte separation medium (LTS1077), HAT (SIGNA, H0262-IVL), 200-mesh filter, semi-solid medium, 96-well fine cell culture plate (Corning Incroporated Costar, 3699), cell hemocytometer, RPMI-1640 medium, HRP-labeled goat anti-mouse IgG, FC (Jacksan, 115-035-071);

Isopropyl-β-D-1-thiogalactopyranoside (isopropyl-β-D-1-thiogalactopyranoside, IPTG), imidazole and other reagents were purchased from Sigma-aldrich;

Ultrasound: Ultrasonic cell pulverizer SCIENTZ-IID was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.;

The protein purification system was purchased from Bio-Rad Life Medical Products (Shanghai) Co., Ltd.

Embodiment 1: Expression and purification of Tet (X4) recombinant protein

1. Experimental method

1. Transformation of Tet(X4) recombinant plasmid

Take 50 μL of competent cells BL21(DE3), add 10 μL of the prepared recombinant plasmid pET28a-tet(X4) in the semi-thawed state of the cells, flick and mix well, transfer to a pre-cooled electroporation cup to react for 10 minutes, and place in the electroporation apparatus (1800V, 200Ω) for transformation, add 500 μL SOC medium to the transformed electroporation cup, incubate at 180 rpm at 37°C for 1 hour, spread the bacteria on LB agar medium containing kanamycin (50 μg/mL) with a glass rod On, 37 ℃ constant temperature culture. The next day, the single colony that grew out was picked for PCR positive verification and sequencing.

pET28a-tet(X4) sequence:

Target gene synthesis manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd.

2. Induced expression and purification of Tet(X4) recombinant protein

Pick a single colony, inoculate in LB liquid medium containing kanamycin (50 μg/mL), shake overnight at 37°C; inoculate the culture into 200 mL of kanamycin (50 μg/mL) ) in LB liquid medium, expand the culture until the OD ₆₀₀ value is 0.6-0.8, add IPTG to make the final concentration 0.5mmol/L, culture with shaking at 20°C for 16h, centrifuge at 9000g at 4°C for 10min to collect the bacteria, and wash with PBS three times After washing, add 5mL His Bind Columns Buffer (20mmol/L Tris-HCL, 500mmol/L NaCl, 10mmol/L imidazole, pH 8.0), sonicate in an ice bath for 10min, centrifuge at 10000g 4°C for 10min, and save the supernatant and precipitate respectively.

The obtained supernatant was purified by Ni-NTA column using His Bind Elution Buffer (20mmol/L Tris-HCL, 500mmol/LNaCl, 500mmol/L imidazole) on the protein purifier, and the eluate was collected and identified by SDS-PAGE .

3. Western Blot analysis of Tet(X4) recombinant protein

Take the pre-induction, post-induction, lysed supernatant, lysed precipitate, purified flow-through, purified eluate, and purified eluent samples for 10% SDS-PAGE (constant voltage 80V electrophoresis for 20min, switch to constant voltage 120V electrophoresis 60 min), one piece of gel was stained with Coomassie Brilliant Blue, and the other gel was electrophoretically transferred at a constant current of 200V for 120 min according to the wet transfer method of Bio-Rad Company, and the protein on the gel was transferred to the PVDF membrane. PVDF membrane was incubated with 5% skimmed milk powder at 37°C for 1 hour, washed 3 times with TBST, 10 minutes each time, added mouse His primary antibody (1:2000 dilution) and incubated overnight at 4°C, washed 3 times with TBST the next day Add horseradish peroxidase (diluted 1:2000) and incubate at 4°C for 1 h, wash the membrane 5 times with TBST, and develop by chemiluminescence.

2. Experimental results

1. Identification of recombinant plasmid transformation results

After the single colony obtained from successful transcription was amplified by PCR, the product was subjected to 1% agarose gel electrophoresis, and the Tet(X4) recombinant plasmid was used as a positive control. The obtained band was about 1500bp, and the size was consistent with the length of the target fragment. The result is shown in the figure 1. The positive clones were sequenced and compared with the standard sequence (NG_065852.1) of Tet(X4) in NCBI. The sequencing results were consistent with those in the database and there was no mutation.

2. Expression, purification and SDS-PAGE and Western-Blot of Tet(X4) protein

Prokaryotic expression and purification of positive bacteria with correct sequencing were carried out. In order to obtain more soluble expressed proteins, Tet (X4) recombinant protein was induced and expressed at two different temperatures of 20°C and 37°C. Take samples of pre-induction, post-induction, post-lysed supernatant, post-lysed precipitate, purified flow-through, purified eluate, and purified eluent for SDS-PAGE verification. The results of Western-Blot and Coomassie Brilliant Blue are shown in Figure 2- 3. It can be seen that Tet(X4) protein has a considerable amount of soluble expressed protein under two different induction conditions of 20°C and 37°C. The band size of the target protein after elution by the protein purifier is about 45kd, which is consistent with the size obtained from the query on NCBI.

3. Stability verification of the prepared Tet(X4) protein

1. Use Uncle to measure the stability of the prepared Tet(X4) protein, and its experimental parameters are:

Heating range: 25-95°C

Heating rate: 0.5°C/min

DLS detection of particle size and polydispersity before heating

2. See Table 1, 2 and Figure 6 for the results

Table 1

sample Conc.(mg/ml) Tm1(°C) Tm2(°C) Tagg266 (°C) Tet(X4) 0.46 45 52.65 34.51

Table 2

Example 2: Preparation of hybridoma cells and production of monoclonal antibodies

1. Experimental method

1. Preparation and verification of Tet(X4) monoclonal antibody

1) Antiserum preparation and titer detection

Dilute 0.05 mg of Tet(X4) recombinant protein prepared in Example 1 with PBS, mix it with Freund's adjuvant at a volume ratio of 1:1, and emulsify at 4°C for 3 min-5 min. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations. Transfer the emulsified Tet(X4) recombinant protein into a 1mL syringe, and remove the air bubbles in the syringe. The Balb/C mice to be immunized were taken out of the cage and placed in a special fixed frame, and multi-point subcutaneous injections were performed on the back. Each immunization was separated by 2 weeks. A total of 3-5 times of immunization. One week after the third immunization, blood was taken for testing. If the titer reached the impact immunization, the eyeballs were removed, the blood was taken, and the neck was pulled to kill, and the spleen was taken. A total of 4 mice were immunized.

Dilute the Tet(X4) recombinant protein to 1 μg/mL with coating solution CB, add 100 μL/well to the microtiter plate, and leave overnight at 4°C. Take out the ELISA plate and pat dry the liquid in the well, block with 5% skimmed milk, add 200 μL/well to the ELISA plate, incubate at 37°C for 2 hours, and wash the plate three times with TBS. Dilute the immune serum according to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000... with sample dilution, 100μL per well, 37 Incubate at ℃ for 1h. Wash the plate 3 times with TBS. Add HRP-labeled goat anti-mouse IgG (1:10000 enzyme dilution), 100 μL/well, incubate at 37°C for 40 min, and wash the plate 5 times with TBS. Add TMB substrate, 90 μL/well, and protect from light at 37°C for 5-20 minutes. Add stop solution, 50 μL/well, read with microplate reader (wavelength 450nm), the maximum dilution of positive reaction is the serum titer of immunized mice.

2. Preparation of trophoblasts

1) Preparation of mouse peritoneal macrophages

Take 8-10 week old Balb/C mice, kill them by removing eyeballs, bloodletting, and neck pulling, and immerse the mice in 75% alcohol for 5 minutes to disinfect. The mice were moved into the ultra-clean workbench, and the mice were fixed supine on the foam lined with sterilized newspapers with sterilized needles. The abdominal skin was opened with scissors and forceps, and the peritoneum was fully exposed by blunt peeling. Use a 5mL syringe to draw 3-5mL DMEM and inject it into the peritoneal cavity for flushing, then suck out the culture medium and transfer it to a 50mL centrifuge tube.

2) Preparation of mouse spleen cells

Aseptically cut the peritoneum to expose the abdominal cavity, cut off the connective tissue to separate the spleen, divide it into four sections and place them in the center of a 200-mesh separation net, fold the separation net twice, clamp the opening end of the separation net with hemostatic forceps, and add 3-6mL DMEM , grind, mix by pipetting, aspirate and transfer to a 50mL centrifuge tube. Add 3-6mL DMEM again, pipette and mix to form a single splenocyte suspension, aspirate and transfer to a 50mL centrifuge tube.

Collect macrophages and spleen cells and centrifuge at 1500rpm for 5min to discard the supernatant, suspend the cells according to the amount of 1mL fetal bovine serum for one mouse, and store at 4°C.

3. Cell Fusion

1) Activation and preparation of SP2/0

Resuscitate SP2/0 cells before fusion, and subculture them in 10% FBS DMEM medium for one week. When the cells grow well, take the cells and wash them twice in aseptically operated DMEM medium, and inject ^1x106 cells/only in small Subcutaneously on the back of the mouse. The tumor can be clearly seen in about 8-10 days. After the tumor grows to a diameter of 1-3cm, the immune-qualified mice are subjected to shock immunization, and they fuse after 3 days. Cut the back skin aseptically, cut off the tumor mass and cut it into small pieces, move it to the center of a 200-mesh filter and place it in a 10cm dish, add 5-10mL DMEM, grind it into a single-cell suspension, and suck it all into a 50mL centrifuge tube . Then add 5mL DMEM, mix by pipetting, and suck it all out into a 50mL centrifuge tube. Add 5 mL of DMEM, mix by pipetting, and suck it all out into a 50 mL centrifuge tube. Gently pick 20mL of myeloma cell suspension along the tube wall into a 50mL centrifuge tube pre-added with 20mL of lymphocyte separation medium, centrifuge at 2000r/min for 15min, discard the upper layer of cell suspension, and remove the white myeloma cells in the middle layer Transfer the suspension to another 50mL centrifuge tube, suspend it with 10mL DMEM medium, centrifuge at 1500r/min for 5min, and wash the cells twice. Discard the supernatant, resuspend the myeloma cells in 10 mL DMEM medium, count and set aside.

2) Preparation of immune splenocytes

Take a Balb/C mouse qualified for immunization, remove the eyeballs and bleed completely, and kill it by pulling the neck completely. The blood is collected and separated, and the serum is used as the positive control serum for antibody detection. Immune splenocyte suspension was isolated and prepared in the same way as the preparation of trophoblasts to obtain the spleen.

3) cell fusion

Place 1 mL of PEG and 40 mL of DMEM medium in a 37 °C, 5% _CO2 incubator pre-warmed. Mix the number of myeloma cells and splenocytes at a ratio of 1:3-1:10, centrifuge at 1500rpm for 5min, discard the supernatant, blot the residual liquid, and add 1mL PEG preheated at 37°C. Add 40 mL of DMEM medium to the fusion system to terminate the effect of PEG. After adding the first 10mL, add DMEM medium along the tube wall to 40mL. Centrifuge at 800rpm for 5min and discard the supernatant. Suspend the fused mixed cells with 2-4mL fetal bovine serum, and add them into semi-solid medium containing 25% fetal bovine serum containing trophoblasts, glutamine, double antibody and HAT according to the amount of fused cells. Pour the mixed semi-solid into a 3.5cm petri dish, and then place all the petri dishes in a sterilized wet box, and place them in a 37°C, 5% CO ₂ incubator for cultivation.

4) Selective cultivation

After the cells grew to the tenth day, the colonies were picked and cultured in 96-well plates according to the size of the cell colonies. After 2-3 days, the cell culture supernatant was collected and screened by indirect ELISA method and indirect competition ELISA method at the same time.

4. Subcloning and subcloning detection

Take the cells to be subcloned by blowing gently to make a single-cell suspension, count them on a counting plate, and calculate the cell density. Dilute the antigen to 2 μg/ml with coating solution CB, add 100 μL/well to the microtiter plate, and overnight at 4°C. Take out the ELISA plate and pat dry the liquid in the well, block with 5% skimmed milk, add 200 μL/well to the ELISA plate, incubate at 37°C for 2 hours, and wash the plate three times with TBS. Add 100 μL/well cell supernatant, incubate at 37°C for 1 h, and wash the plate 3 times with TBS. Add HRP-labeled goat anti-mouse IgG (1:10000 enzyme dilution), 100 μL/well, incubate at 37°C for 40 min, and wash the plate 5 times with TBS. Add TMB substrate, 90 μL/well, at 37°C, protected from light for 20-30min. Add stop solution, 50 μL/well, read with a microplate reader (wavelength 450 nm), and the corresponding dilution value when the OD value is 2.0 is the titer of the cells.

5. Cell cryopreservation and recovery expansion culture

When the cells are larger than 1x10 ⁶ , freeze them and centrifuge at 1500rpm for 5min. DMSO to fetal bovine serum ratio of 1:9 cryopreservation medium to freeze cells. The cell cryopreservation tubes were placed in a gradient freezer box at room temperature, and then placed directly at -80°C overnight. Take out the cells in the gradient freezing box and store them in a liquid nitrogen tank for long-term storage.

Quickly take the cell cryopreservation tube out of the liquid nitrogen tank, and place it in a constant temperature water bath at 37°C and shake it quickly until the cells thaw. Add 5 mL of DMEM medium and mix well, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 20% complete medium, culture in a 37°C, 5% CO ₂ incubator, and pass.

6. Antibody titer detection

Dilute the Tet(X4) recombinant protein prepared in Example 1 to 2 μg/ml with coating solution CB, add 100 μL/well to the microtiter plate, and overnight at 4°C. Take out the ELISA plate and pat dry the liquid in the well, block with 5% skimmed milk, add 200 μL/well to the ELISA plate, incubate at 37°C for 2 hours, and wash the plate three times with TBS. Dilute the antibody according to 2μg/mL, 1μg/mL, 0.5μg/mL, 0.25μg/mL, 0.125μg/mL, 0.0625μg/mL, 0.03125μg/mL, 0.015625μg/mL with sample dilution, each well 100μL, incubate at 37°C for 1h. Wash the plate 3 times with TBS. Add HRP-labeled goat anti-mouse IgG (1:10000 enzyme dilution), 100 μL/well, incubate at 37°C for 40 min, and wash the plate 5 times with TBS. Add TMB substrate, 90 μL/well, at 37°C, protected from light for 5-20 minutes. Add stop solution, 50 μL/well, read with microplate reader (wavelength 450nm), the maximum dilution of positive reaction is the titer of antibody.

2. Experimental results

1. Antiserum preparation and titer detection

According to the serum titer, the mouse numbered "left" has the highest titer. Theoretically, the specificity of these two mice will be stronger, so this mouse was selected for the fusion experiment. See Table 3 and Table 4 for specific antiserum titer data.

Table 3: Three free titer detection

Table 4: Potency detection of four vaccines

2. Cell fusion and subcloning detection

After fusion screening, one mouse was selected for fusion, and after fusion, it was re-examined with immune antigen, and 34 strains were selected for subcloning. Afterwards, 30 positive strains were obtained by subcloning screening, and the cell supernatant was collected and affinity sorted for the positive clones. Finally, 5D3D3 was selected according to the supernatant gradient to infuse ascitic fluid to prepare antibodies.

3. Monoclonal antibody purification and antibody titer detection

After the cells were cryopreserved and resuscitated, their subtypes were identified, and the cell line 5D3D3 was identified as igG1 (see Table 5), so they were purified using Protein G columns, and the obtained antibodies were named A antibody and B antibody, respectively. See Table 6 for CDR sequences.

Table 5: Cell line subtype identification table

Table 6: CDR sequences of antibodies

Wherein, the heavy chain variable region of antibody A is shown in SEQ ID NO: 16, the encoded nucleotide sequence is shown in SEQ ID NO: 21, and the light chain variable region is shown in SEQ ID NO: 17 or 18, The encoded nucleotide sequence is shown in SEQ ID NO: 22 or 23, specifically as follows:

The heavy chain variable region of antibody B is shown in SEQ ID NO: 19, the encoded nucleotide sequence is shown in SEQ ID NO: 24, the light chain variable region is shown in SEQ ID NO: 20, the encoded nucleotide sequence The acid sequence is shown in SEQ ID NO:25. details as follows:

Then the antibody titer was detected by SDS-PAGE and WB detection of the recombinant antigen (Tet(X4) recombinant protein), and the target bands were obtained, as shown in Figure 4 and Figure 5 for details. It was shown that the hybridoma cells prepared in the examples produced specific antibodies specifically binding to the Tet(X4) recombinant protein.

4. Monoclonal antibody stability and potency

Detect antibody a (the heavy chain variable region is shown in SEQ ID NO: 16, the light chain variable region is shown in SEQ ID NO: 17), b (the heavy chain variable region is shown in SEQ ID NO: 16, the light chain variable region is shown in SEQ ID NO: 16, and the light chain variable region is shown in SEQ ID NO: 16) Chain variable region as shown in SEQ ID NO: 18), c (heavy chain variable region as shown in SEQ ID NO: 19, light chain variable region as shown in SEQ ID NO: 20) stability.

1) Use Uncle to measure the stability of the prepared antibody, and its experimental parameters are:

Heating range: 25-95°C

Heating rate: 0.5°C/min

DLS detection of particle size and polydispersity before heating

2) Stability results

The results showed that the a, b, and c antibodies all had good stability, and the exemplary a antibody results are shown in Table 7, 8 and Figure 7.

Table 7

sample Conc.(mg/ml) Tm1(°C) Tm2(°C) Tagg266 (°C) a antibody 1 72.79 - 65.7

Table 8

Embodiment 3 Western-Blot verification

1. Materials and methods

1) Material

Prepared Tet(X4) mouse monoclonal antibody; tet(X4) positive wild bacteria and tet(X4) negative wild bacteria; Western and IP cell lysate, PMSF, horseradish peroxidase labeled goat anti-mouse IgG (H+L) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and the chemiluminescence liquid was purchased from Thermo Fisher Scientific (China) Co., Ltd.

2) method

Bacterial culture: streak tet(X4) positive wild bacteria and tet(X4) negative wild bacteria on LB agar medium, culture overnight at 37°C, then pick a single colony into 1mL LB broth medium, 37°C 180rpm Cultivate for 6 hours, transfer the bacterial solution to 5 mL LB medium at a ratio of 1:100, add different concentrations of tigecycline to make the final concentration of tigecycline 0 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg /mL, culture overnight at 37°C 180rpm.

Protein extraction: Centrifuge at 10,000rpm for 5min, remove the supernatant, wash the cells with pre-cooled PBS three times, then resuspend the cells with 1mL of PBS, add lysozyme to make the final concentration 0.25mg/mL, mix them upside down and keep on ice After 30 minutes, incubate at 37°C for 5 minutes, and put it on ice again for 30 minutes. Add PMSF with a final concentration of 1mM to the lysate, add 200 μL of lysate to the cells treated with lysozyme, let it stand for 10 minutes after flicking, use an ultrasonic cell disruptor to lyse the cells for 5 minutes, 50w, work for 3s, stop for 2s, Finally, centrifuge to get the supernatant.

Protein concentration homogenization: use Bradford method to measure protein concentration, add protein sample buffer, and then use lysate to adjust the concentration of each protein sample to make it uniform to 0.8 μg/μL.

Western-Blot verification: After boiling the protein sample, centrifuge at 10,000rpm for 5min, use 10% gel for SDS-PAGE electrophoresis, load 16μg of each sample, and the conditions are 80V for 30min and 120V for 90min. Place the PVDF membrane in methanol solution for 1min activation, soak the membrane, glue, sponge, filter paper, etc. in the transfer buffer, follow the procedure of white flour-white sponge-two-layer filter paper-membrane-glue-two-layer filter paper-black sponge-black surface Clamped in sequence, and then transferred to the membrane by wet transfer method, 300mA for 2h. After transferring the membrane, use 5% skimmed milk to block the membrane, 180rpm shaker at 37°C for 1 hour, wash the membrane 3 times with TBST, add Tet(X4) mouse monoclonal antibody and primary antibody diluent at a ratio of 1:1000 , incubate overnight at 4°C, wash the membrane 3 times with TBST the next day, then add horseradish peroxidase and 5% skimmed milk at a ratio of 1:1000, incubate at 4°C for 1 hour, wash the membrane 3 times with TBST, and add chemiluminescence The liquid was imaged, and the grayscale was analyzed with ImageJ software.

2. Results and analysis

The chemiluminescence imaging image can be seen from Figure 8. The order of loading samples is as follows: channels 1 and 2 are tet(X4) negative bacteria, channels 3 and 4 are tet(X4) positive bacteria in culture conditions without adding drugs, and channels 5 and 6 are tet(X4) positive bacteria. The tet(X4) positive bacteria under the culture condition of 2 μg/mL tigecycline concentration, channel 7 and 8 are the tet(X4) positive bacteria under the culture condition of 4 μg/mL tigecycline concentration, channel 9 and 10 It is the tet(X4) positive bacteria under the culture condition that the concentration of tigecycline is 8 μg/mL. There is no protein band in the channel for loading tet(X4)-negative bacteria, while the channels for tet(X4)-positive bacteria show different bands. It can be seen that the Tet(X4) mouse monoclonal antibody and Tet(X4) protein have Good binding ability. The quantitative analysis chart is shown in Figure 9, which are respectively tet(X4) negative bacteria, tet(X4) positive bacteria without drug addition, tigecycline concentration 2 μg/mL, tigecycline concentration 4 μg/mL, tigecycline The concentration is 8 μg/mL. Tet(X4) positive bacteria and negative bacteria showed significant differences in the expression of Tet(X4). In addition, in the case of drug addition, the expression of Tet(X4) protein was significantly increased, and the expression level was stable.

In the Western-Blot verification, there was no band in the tet(X4)-negative bacterial protein sample that did not express Tet(X4) protein, but it appeared in the tet(X4)-positive bacterial protein sample that expressed Tet(X4) protein Clear bands, and the gray value of the bands increase with the increase of protein expression, which reflects that the Tet(X4) mouse monoclonal antibody has a good binding ability to Tet(X4) protein.

Example 4

Detect antibody a (the heavy chain variable region is shown in SEQ ID NO: 16, the light chain variable region is shown in SEQ ID NO: 17), b (the heavy chain variable region is shown in SEQ ID NO: 16, the light chain variable region is shown in SEQ ID NO: 16, and the light chain variable region is shown in SEQ ID NO: 16) Chain variable region as shown in SEQ ID NO: 18), c (heavy chain variable region as shown in SEQ ID NO: 19, light chain variable region as shown in SEQ ID NO: 20) stability.

1. Experimental method

(1) Dilute the tetx4 protein 1000 times with the coating solution, add to the microtiter plate, 100 μL/well, and coat overnight at 4°C;

(2) Pour off the liquid in the well, wash once with washing solution, add 150 μL of blocking solution to each well, block at a constant temperature of 37°C for 1 hour, then pour off the blocking solution and shake dry;

(3) Add the tetx4 antibody diluted in gradient concentration to the microtiter plate for reaction, the antibody concentrations are 1ug/mL, 0.67ug/mL, 0.50ug/mL, 0.40ug/mL, 0.33ug/mL, 0.11ug/mL, 0.037ug/mL, 0.012ug/mL, each concentration has 3 repetitions, 50μL/well, react at 37°C for 30min, wash with washing solution for 3 times, and then dry the liquid in the well;

(4) Add 100 μL of HRP-goat anti-mouse IgG to each well, react at 37°C for 30 minutes, and wash as above;

(5) Add freshly prepared TMB solution, 100 μL/well, and develop color at 37°C for 15 minutes in the dark;

(6) Add 2mol/L H ₂ SO ₄ , 50 μL/well;

(7) read each hole OD _450nm with microplate reader;

(8) Taking the concentration of the tetx4 antibody as the abscissa and the OD value corresponding to each concentration of the tetx4 antibody as the ordinate, use the GraphPad prism9 software to draw a standard curve according to four-parameter fitting, as shown in Figure 10.

2. Experimental results

The results showed that all three antibodies had high ability to bind to tetx4 protein, and their EC50 values were all between 0.4566 and 0.5775.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

Claims (10)

1. An antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region, and/or CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region; wherein,,

the amino acid sequence of CDR-H1 comprises the amino acid sequence of SEQ ID NO:1 or 10;

the amino acid sequence of CDR-H2 comprises SEQ ID NO: 2. 11 or 26;

the amino acid sequence of CDR-H3 comprises SEQ ID NO:3 or 12;

the amino acid sequence of CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4. 7, 13, 27, 28 or 29;

the amino acid sequence of CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5. 8, 14 or 30;

the amino acid sequence of CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. 9, 15 or 31.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein,

the amino acid sequence of CDR-H1 comprises the amino acid sequence of SEQ ID NO:1, and a polypeptide sequence shown in the specification;

the amino acid sequence of CDR-H2 comprises SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);

the amino acid sequence of CDR-H3 comprises SEQ ID NO:3, an amino acid sequence of seq id no;

the amino acid sequence of CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4. 7, 28 or 29;

the amino acid sequence of CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5. 8 or 30;

the amino acid sequence of CDR-L3 comprises the amino acid sequence of SEQ ID NO:6 or 9.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein,

the amino acid sequence of CDR-H1 comprises the amino acid sequence of SEQ ID NO:10, and a polypeptide comprising the amino acid sequence shown in seq id no;

the amino acid sequence of CDR-H2 comprises SEQ ID NO:11 or 26;

the amino acid sequence of CDR-H3 comprises SEQ ID NO:12, an amino acid sequence of seq id no;

the amino acid sequence of CDR-L1 comprises the amino acid sequence of SEQ ID NO:13 or 27;

the amino acid sequence of CDR-L2 comprises the amino acid sequence of SEQ ID NO:14, an amino acid sequence shown in seq id no;

the amino acid sequence of CDR-L3 comprises the amino acid sequence of SEQ ID NO:15 or 31.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region comprises SEQ ID NO:16 or 19;

the amino acid sequence of the light chain variable region comprises SEQ ID NO: 17. 18 or 20.

5. The antibody or antigen-binding fragment thereof of claim, wherein the antibody or antigen-binding fragment thereof specifically binds to a transferrin-resistant protein.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof has the structure of Fab, fab '-SH, F (ab') 2, single domain antibody, or single chain antibody.

7. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. A hybridoma cell producing the antibody or antigen-binding fragment thereof of any one of claims 1-6.

9. A pharmaceutical or diagnostic kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid of claim 7, or the hybridoma cell of claim 8.

10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-6, a nucleic acid according to claim 7, a hybridoma cell according to claim 8 or a pharmaceutical or diagnostic kit according to claim 9, wherein said use comprises:

a) The application in preparing products for treating diseases caused by gram-negative bacteria; or (b)

B) Use in the preparation of a product resistant to tigecycline.

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