CN116063535B - Antibody or antigen binding fragment thereof, and preparation method and application thereof - Google Patents
Antibody or antigen binding fragment thereof, and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/65—Tetracyclines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
- A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- G01N2333/90248—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one of the donors, and incorporation of one atom of oxygen 1.14.13
- G01N2333/90251—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one of the donors, and incorporation of one atom of oxygen 1.14.13 with a definite EC number (1.14.13.-)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application provides an antibody or antigen binding fragment thereof, hybridoma cells producing the antibody or antigen binding fragment thereof, and uses thereof. The application 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
Technical Field
The application relates to the technical field of biological medicine, in particular to an antibody or an antigen binding fragment thereof, a preparation method and application thereof.
Background
Today, bacterial antibiotic resistance (Antimicrobial Resistance) has become one of the greatest threats to global health and food safety with the overuse of antibiotics. For a series of diseases caused by gram-negative bacteria such as carbapenem-resistant escherichia coli and the like, treatment is mainly dependent on colistin and tigecycline, but because colistin resistant genes (mcr) mediated by plasmids are widely spread in the global scope, clinical effects of colistin are greatly weakened, and therefore tigecycline becomes one of the 'last defense lines' for clinically treating antibiotic infection.
Tetracyclines and their derivatives (e.g., tigecycline) are a large clinical choice for the treatment of gram-negative bacterial infections and are widely used. The advent of transferable Tet (X) enzymes that destroy tetracycline antibiotics poses a significant challenge for antibacterial treatment and food/environmental safety. Unlike the efflux pump mechanism of tigecycline resistance widely studied previously, the flavin-dependent monooxygenase encoded by the Tet (X) gene takes tigecycline as a substrate and plays a certain modification role in the tigecycline so as to deactivate the tigecycline. Among them, the transmissible tigecycline resistance gene Tet (X4) gene isolated from enterobacteriaceae and acinetobacter is extremely important. Knowing its characteristics and drug resistance mechanism can provide important basis for medical and health industries.
Disclosure of Invention
In order to solve the defects in the prior art, the application provides a transferable tigecycline drug-resistant protein (Tet (X4)) which can be specifically combined, and is hopeful to block the combination of Tet (X4) and tigecycline, thereby solving the drug resistance problem.
In a first aspect of the application there is provided an antibody or 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 SEQ ID NO:1 or 10, or an amino acid sequence as set forth in SEQ ID NO:1 or 10 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more; the amino acid sequence of CDR-H2 comprises SEQ ID NO: 2. 11 or 26, or an amino acid sequence as set forth in SEQ ID NO: 2. 11 or 26 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more; the amino acid sequence of CDR-H3 comprises SEQ ID NO:3 or 12, or an amino acid sequence that hybridizes to SEQ ID NO:3 or 12 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more; the amino acid sequence of CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4. 7, 13, 27, 28 or 29, or an amino acid sequence as set forth in SEQ ID NO: 4. 7, 13, 27, 28 or 29 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more; the amino acid sequence of CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5. 8, 14 or 30, or an amino acid sequence as set forth in SEQ ID NO: 5. 8, 14 or 30 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more; the amino acid sequence of CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. 9, 15 or 31, or an amino acid sequence as set forth in SEQ ID NO: 6. 9, 15 or 31 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more.
In a specific embodiment of the present application, 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.
In a specific embodiment of the present application, 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.
In a specific embodiment of the present application, 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 or 7; the amino acid sequence of CDR-L2 comprises the amino acid sequence of SEQ ID NO:5 or 8; the amino acid sequence of CDR-L3 comprises the amino acid sequence of SEQ ID NO:6 or 9.
In a specific embodiment of the present application, 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, and a polypeptide comprising the amino acid sequence shown in seq id no; 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, an amino acid sequence shown in seq id no; 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, and a polypeptide having the amino acid sequence shown in seq id no.
In a specific embodiment of the present application, 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, and a polypeptide sequence shown in the figure; the amino acid sequence of CDR-L2 comprises the amino acid sequence of SEQ ID NO:5, and a polypeptide sequence shown in the figure; the amino acid sequence of CDR-L3 comprises the amino acid sequence of SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.
In a specific embodiment of the present application, 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: 7; the amino acid sequence of CDR-L2 comprises the amino acid sequence of SEQ ID NO:8, and a polypeptide sequence shown in the figure; the amino acid sequence of CDR-L3 comprises the amino acid sequence of SEQ ID NO: 9.
Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 are arranged in order from N-terminal to C-terminal.
Preferably, the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 are arranged in order from N-terminal to C-terminal.
Preferably, the amino acid sequence of the heavy chain variable region comprises SEQ ID NO:16 or 19, or an amino acid sequence as set forth in SEQ ID NO:16 or 19 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more.
Preferably, the amino acid sequence of the light chain variable region comprises SEQ ID NO: 17. 18 or 20, or an amino acid sequence as set forth in SEQ ID NO: 17. 18 or 20 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more.
In a specific embodiment of the application, the heavy chain variable region of the antibody or antigen binding fragment thereof comprises SEQ ID NO:16, the light chain variable region comprises SEQ ID NO:17 or 18.
In a specific embodiment of the application, the heavy chain variable region of the antibody or antigen binding fragment thereof comprises SEQ ID NO:19, the light chain variable region comprises SEQ ID NO:20.
preferably, the antibody or antigen binding fragment thereof specifically binds to a transferable tigecycline resistance protein (abbreviated as Tet (X4)).
Preferably, the antibody or antigen binding fragment thereof has the structure of a Fab, fab '-SH, F (ab') 2, single domain antibody or single chain antibody.
In a second aspect of the application, there is provided a nucleic acid encoding an antibody or antigen binding fragment thereof as defined above, a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 or a heavy chain variable region or light chain variable region as defined above.
Preferably, the nucleotide sequence encoding the heavy chain variable region comprises SEQ ID NO:21 or 24, or a sequence corresponding to SEQ ID NO:21 or 24 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more.
Preferably, the nucleotide sequence encoding the light chain variable region comprises SEQ ID NO: 22. 23 or 25, or a sequence corresponding to SEQ ID NO: 22. 23 or 25 has an identity of 80%, 90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or more.
In a third aspect of the application, a method for preparing a Tet (X4) recombinant protein is provided.
The method comprises introducing a recombinant plasmid comprising a nucleotide sequence encoding a Tet (X4) recombinant protein into a host cell.
Preferably, the host cell is prokaryotic, such as E.coli.
In a fourth aspect of the present application, there is provided a recombinant plasmid comprising a nucleotide sequence encoding a Tet (X4) recombinant protein.
In a fifth aspect of the application, there is provided a cell comprising a nucleotide sequence encoding a Tet (X4) recombinant protein or comprising the recombinant plasmid described above or comprising the nucleic acid described above.
In a sixth aspect of the present application, there is provided a Tet (X4) recombinant protein prepared by the above method.
In a seventh aspect of the application, there is provided a hybridoma cell.
Preferably, the hybridoma produces an antibody or antigen-binding fragment thereof as described above.
In an eighth aspect of the present application, there is provided a method for producing a hybridoma cell.
The preparation method comprises the steps of immunizing mice by using a Tet (X4) recombinant protein, separating spleen cells and fusing the spleen cells with myeloma cells.
In a ninth aspect of the application, there is provided a pharmaceutical or diagnostic kit comprising an antibody or antigen binding fragment thereof as defined above, a nucleic acid as defined above or a hybridoma cell as defined above.
In a tenth aspect, the present application provides the use of an antibody or antigen binding fragment thereof as defined above, a nucleic acid as defined above, a hybridoma cell as defined above or a pharmaceutical or diagnostic kit as defined above, said use comprising:
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.
Preferably, the gram-negative bacteria include, but are not limited to, gram-negative bacteria of enterobacteria (e.coli), klebsiella (Klebsiella), ganoderma (Serratia), enterobacteria (Enterobacter), citric acid bacteria (Citrobacter), morganella (Morganella), providencia (Providencia), proteus (Proteus), etc., gram-negative bacteria (Haemophilus), moraxella (Moraxella), etc.), gram-negative bacteria (glucose non-fermented), pseudomonas (Pseudomonas aeruginosa), oligotrophic bacteria (stephromonas), burkholderia (Burkholderia), acinetobacter (Acinetobacter), etc., which are colonized in the respiratory system.
In an eleventh aspect of the application, there is provided an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof as described above covalently bound to a drug.
Preferably, the drug may be a chemical synthetic drug, an antibiotic or various biological drugs. Antibiotics, such as tigecycline, and the like, are preferred.
In a twelfth aspect of the present application, there is provided a method for detecting a Tet (X4) protein, which comprises contacting a sample to be detected with an antibody or antigen-binding fragment thereof of the present application, and then detecting a complex formed by the Tet (X4) protein and the antibody or antigen-binding fragment thereof.
Preferably, the detection method detects the presence or amount of the Tet (X4) protein. Wherein the presence indicates the presence or absence of the protein, and the content may be an expression level, a protein concentration, or the like.
Preferably, the antibody or antigen binding fragment thereof may comprise a selectable marker.
An "antigen binding fragment" as used herein is a portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of the antibody is capable of specifically binding to an epitope on the target molecule of the intact antibody. It includes, for example, 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 individual CDRs from the heavy or light chain of an intact antibody.
"CDR" as described herein represents complementarity determining region (complementarity-determining region) which can be determined by the Kabat numbering, chothia numbering, IMGT numbering, AHo numbering (Honyger numbering) schemes commonly used in the art.
The term "comprising" as used herein to describe the sequence of a protein or nucleic acid may consist of the sequence or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.
"identity" as used herein means that a person skilled in the art can adjust the sequence according to actual working needs without changing the structure or activity of the original sequence, and that the sequence used is (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%, 96%,97%, 99%, 99.99.99%, 99.5%, 99.99.99% and the like.
The recombinant plasmid is obtained by utilizing a prokaryotic expression technology, a large amount of Tet (X4) recombinant protein is obtained by expression in escherichia coli, and high-purity protein is obtained by using a protein purifier. The soluble expression level of Tet (X4) protein is obtained through different temperature induction, no obvious difference exists between 20 ℃ and 37 ℃, and the purified protein has good biological activity. In the subsequent preparation and verification process of the monoclonal antibody, 4 mice are immunized in total, and after immunization, a 'left' mouse is selected for impact according to the detection titer condition to carry out a fusion experiment. And finally reserving 34 positive clone strains for subcloning through antigen screening and rechecking after fusion, collecting 30 subcloning supernatant strains, and carrying out affinity sequencing. Finally, 5D3D3 is selected for ascites beating to prepare antibodies, and the obtained antibodies respectively carry out SDS-PAGE and WB detection of recombinant antigens, and all show clear bands. The monoclonal antibody is an antibody secreted by lymphocytes and only aims at a specific antigen epitope, and has good specificity and strong affinity. The research utilizes a prokaryotic expression system to prepare high-purity Tet (X4) protein and obtain monoclonal antibodies thereof, and can provide a basis for further explaining the drug resistance mechanism of Tet (X4) drug-resistant strains and controlling the prevalence of Tet (X4) drug-resistant strains in the global scope.
Drawings
Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: PCR identification result of Tet (X4) recombinant plasmid.
Fig. 2: expression levels of Tet (X4) protein in different processes under induction conditions of 20 ℃.
Fig. 3: expression levels of Tet (X4) protein in different processes under induction conditions of 37 ℃.
Fig. 4: SDS-PAGE of antibodies.
Fig. 5: western-Blot image of antibody.
Fig. 6: tet (X4) protein denaturation curve and aggregation curve.
Fig. 7: antibody denaturation curve and aggregation curve.
Fig. 8: chemiluminescent imaging.
Fig. 9: and (3) quantitatively analyzing the result, wherein Ckneg represents tet (X4) negative bacteria, CKpos represents tet (X4) positive bacteria without adding medicine, T2 represents tigecycline concentration of 2 mug/mL, T4 represents tigecycline concentration of 4 mug/mL, and T8 represents tigecycline concentration of 8 mug/mL.
Fig. 10: antibody standard curve.
Detailed Description
The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Reagents and instrumental sources used in the examples:
coli [ Escherichia coli BL (DE 3) ] was purchased from Shanghai-Viedi Biotechnology Inc., and the prokaryotic expression plasmid pET-28a was supplied by Beijing Liuhua macrogene technologies Co.
Nucleic acid restriction endonucleases rTaq, DNA T7, T7T ligase, DL2000 nucleic acid Marker were all purchased from Takara Bio-engineering (Dalian);
SDS-PAGE protein loading buffer, protein molecular weight standard (10-150 kD, non-pre-dye) were purchased from Shanghai Biyun biotechnology Co;
SDS-PAGE gel kit (Biyun biotechnology Co., ltd.);
freund's adjuvant, balb/C mice, PBS, DMEM medium (Hyclone, SH30809.01B), fetal bovine serum (phage-free), diabody (Hyclone, SY 30010), L-glutamate (Amresc, DH 144-1), PEG4000 (SIGMA, p 7181), human lymphocyte isolates (LTS 1077), HAT (SIGNA, H0262-IVL), 200 mesh filters, semi-solid medium, 96 Kong Xi howl plates (Corning Incroporated Costar, 3699), cell count plates, RPMI-1640 medium, HRP-labeled sheep anti-mouse 1gG, FC (Jacksan, 115-035-071);
isopropyl- β -D-thiogalactoside (IPTG), imidazole, and other reagents were purchased from Sigma-aldrich company;
ultrasonic: the ultrasonic cell mill SCIENTZ-IID was purchased from Ningbo New Zhi Biotech Co., ltd;
protein purification systems were purchased from berle life medicine products (Shanghai) Inc.
Example 1: expression and purification of Tet (X4) recombinant proteins
1. Experimental method
1. Transformation of Tet (X4) recombinant plasmid
50. Mu.L of competent cells BL21 (DE 3) are taken, 10. Mu.L of prepared recombinant plasmid pET28a-tet (X4) is added under the semi-molten state of the cells, the mixture is flicked and mixed uniformly, the mixture is transferred into a precooled electrorotating cup for reaction for 10min, the electrorotating cup is placed into an electrorotating instrument (180V, 200 omega) for transformation, 500. Mu.L of SOC culture medium is added into the electrorotating cup after transformation, the culture is carried out for 1h at 37 ℃ at 180rpm, and bacteria are coated on LB agar culture medium containing kanamycin (50. Mu.g/mL) by a glass rod and are cultured at a constant temperature of 37 ℃. The single colony grown on the next day was picked for PCR positive verification and sequencing.
pET28a-tet (X4) sequence:
the target gene synthesis manufacturer: bioengineering (Shanghai) Co., ltd.
2. Inducible expression and purification of Tet (X4) recombinant protein
Single colonies were picked and inoculated in LB liquid medium containing kanamycin (50. Mu.g/mL) and shaken overnight at 37 ℃; the culture was inoculated at a ratio of 1:100 into 200mL of LB liquid medium containing kanamycin (50. Mu.g/mL), and expanded to OD 600 Adding IPTG to a final concentration of 0.5mmol/L at a final concentration of 0.6-0.8, shaking and culturing for 16h at 20 ℃, centrifuging for 10min at 9000g at 4 ℃ to collect thalli, washing three times by PBS, adding 5mL His Bind Columns Buffer (20 mmol/L Tris-HCl,500mmol/L NaCl,10mmol/L imidazole, pH 8.0) for ultrasonic treatment for 10min in an ice bath, centrifuging for 10min at 10000g at 4 ℃, and respectively retaining supernatant and precipitate.
The resulting supernatant was purified on a protein purifier using a Ni-NTA column with His Bind Elution Buffer (20 mmol/L Tris-HCl,500mmol/LNaCl,500mmol/L imidazole), and the eluate was collected and identified by SDS-PAGE.
3. Western Blot analysis of Tet (X4) recombinant proteins
Taking supernatant after induction, after induction and pyrolysis, precipitation after pyrolysis, purification of a flowing liquid, purification of a leaching solution and purification of a leaching solution, performing 10% SDS-PAGE (constant voltage 80V electrophoresis for 20min, and constant voltage 120V electrophoresis for 60 min), taking one gel to perform Coomassie brilliant blue staining, transferring the other gel to a PVDF membrane by constant current 200V electrophoresis according to a wet transfer method of Bio-Rad company for 120min, and transferring proteins on the gel. PVDF membrane is incubated with 5% skimmed milk powder at 37deg.C for 1h, then washed with TBST 3 times for 10min each time, incubated overnight at 4deg.C with murine His primary antibody (1:2000 dilution), washed with TBST 3 times the next day, then incubated with horseradish peroxidase (1:2000 dilution) at 4deg.C for 1h, washed with TBST 5 times, and developed by chemiluminescence.
2. Experimental results
1. Identification of recombinant plasmid transformation results
After the single colony obtained after successful transcription is subjected to PCR amplification, the product is subjected to 1% agarose gel electrophoresis, a Tet (X4) recombinant plasmid is used as a positive control, the obtained band is about 1500bp, the size of the band is consistent with the length of a target fragment, and the result is shown in figure 1. Positive clones were sequenced and aligned with the Tet (X4) standard sequence (NG_ 065852.1) in NCBI, and the sequencing results were consistent with the results in the database without mutations.
2. Expression, purification, SDS-PAGE and Western-Blot of Tet (X4) protein
The positive bacteria with correct sequence are subjected to prokaryotic expression and purification, and the Tet (X4) recombinant protein is respectively subjected to induced expression at two different temperatures of 20 ℃ and 37 ℃ in order to obtain more soluble expression proteins. SDS-PAGE verification is carried out on samples of supernatant, sediment, purified flow through liquid, purified eluent and purified eluent before induction, after induction and after pyrolysis, and Western-Blot results and Coomassie brilliant blue results are shown in figures 2-3. It can be seen that the Tet (X4) protein has a considerable amount of soluble expressed protein under two different induction conditions of 20℃and 37 ℃. The size of the target protein band eluted by the protein purifier is about 45kd, which is consistent with the size obtained by inquiry on NCBI.
3. Stability verification of prepared Tet (X4) protein
1. The stability of the Tet (X4) protein prepared was determined using a Uncle, and the experimental parameters were:
heating interval: 25-95 DEG C
Rate of temperature rise: 0.5 ℃/min
Detection of particle size and polydispersity by DLS before warming
2. The results are shown in tables 1, 2 and FIG. 6
TABLE 1
| Sample of | Conc.(mg/ml) | Tm1(℃) | Tm2(℃) | Tagg266(℃) |
| 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. Tet (X4) monoclonal antibody preparation and verification
1) Antiserum preparation and potency detection
0.05mg of the Tet (X4) recombinant protein prepared in example 1 was diluted with PBS, mixed with Freund's adjuvant in a volume ratio of 1:1, and emulsified at 4℃for 3min to 5min. The first immunization was with Freund's complete adjuvant, followed by booster immunization with Freund's incomplete adjuvant. Transferring the emulsified Tet (X4) recombinant protein into a 1mL syringe, and removing bubbles in the syringe. The Balb/C mice to be protected are taken out of the cage and placed in a special holder and injected subcutaneously on the back in multiple spots. Each immunization was separated by 2 weeks. Total immunization was 3-5 times. Blood is taken and detected after three-day immunization, and if the titer reaches the threshold, the eyes are taken, the blood is taken, the neck is pulled, and the spleen is taken. A total of 4 mice were immunized.
Tet (X4) recombinant protein was diluted to 1. Mu.g/mL with coating solution CB, 100. Mu.L/well and added to the ELISA plate at 4℃overnight. Taking out the liquid in the ELISA plate dry-beating hole, closing by 5% skimmed milk, adding 200 mu L/hole of the ELISA plate, incubating for 2h at 37 ℃, and washing the plate 3 times by TBS. The immune sera were diluted in duplicate at 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000 … … with dilution, 100 μl per well, incubated for 1h at 37 ℃. The plates were washed 3 times with TBS. HRP-labeled goat anti-mouse IgG (1:10000 dilution with enzyme) was added, 100. Mu.L/well incubated at 37℃for 40min, and the plates were washed 5 times with TBS. TMB substrate is added, 90 mu L/hole is reserved, and the temperature is 37 ℃ and protected from light for 5-20 min. Adding stop solution, 50 mu L/hole, and reading by an enzyme label instrument (wavelength 450 nm), wherein the maximum dilution of positive reaction is the serum titer of the immunized mice.
2. Preparation of trophoblasts
1) Preparation of mouse abdominal cavity macrophage
Taking 8-10 weeks Balb/C mice, taking eyeballs, bleeding, pulling necks, killing the mice, and soaking the mice in 75% alcohol for sterilization for 5min. The mice were transferred to an ultra clean bench and were held supine with sterilized needles on foam with sterilized newspaper pads, with scissors forceps opening the abdominal skin and blunt dissection fully exposing the peritoneum. 3-5mL DMEM was aspirated with a 5mL syringe and injected into the intraperitoneal rinse, and the medium was aspirated and transferred to a 50mL centrifuge tube.
2) Preparation of mouse spleen cells
Aseptically cutting off the peritoneum, exposing the abdominal cavity, cutting off the connective tissue to separate spleen, dividing the spleen into four sections, placing the four sections in the center of a 200-mesh separation net, folding the separation net twice, clamping the open end of the separation net by using a hemostatic forceps, adding 3-6mL of DMEM, grinding, blowing and mixing uniformly, and sucking out and transferring to a 50mL centrifuge tube. 3-6mL DMEM was added again, and the mixture was blown and mixed well to form a single spleen cell suspension, which was aspirated and transferred to a 50mL centrifuge tube.
Macrophages and spleen cells were collected and centrifuged at 1500rpm for 5min, the supernatant was discarded, and the cells were suspended in 1mL of fetal bovine serum from one mouse and stored at 4 ℃.
3. Cell fusion
1) Activation and preparation of SP2/0
Resuscitates SP2/0 cells before fusion, subcultures the cells in DMEM medium containing 10% FBS for one week, and performs aseptic operation on the DMEM medium for 2 times and 1×10 when the cell growth state is good 6 Cells/injections were subcutaneously injected in the back of mice. The tumor can be obviously seen about 8-10 days, and after the tumor grows to the diameter of 1-3cm, the immunized qualified mice are subjected to impact immunization, and the mice are fused after 3 days. The back skin is cut off aseptically, the tumor mass is cut off and cut into small pieces, the small pieces are moved to the center of a 200-mesh filter screen and placed in a 10cm dish, 5-10mL DMEM is added, the small pieces are ground into single cell suspensions, and all the single cell suspensions are sucked into a 50mL centrifuge tube. Then 5mL of DMEM was added, the mixture was blown and mixed well, and all were aspirated into a 50mL centrifuge tube. Then, 5mL of DMEM was added, the mixture was blown and mixed uniformly, and all the mixture was sucked into a 50mL centrifuge tube. 20mL of myeloma cell suspension is gently picked up along the tube wall into a 50mL centrifuge tube with 20mL of lymphocyte separation liquid added in advance, the centrifugation is carried out for 15min at 2000r/min, after the upper cell suspension is discarded, the middle white myeloma cell suspension is transferred into another 50mL centrifuge tube, the myeloma cell suspension is suspended by 10mL of DMEM culture medium, the centrifugation is carried out for 5min at 1500r/min, and the cells are washed twice. The supernatant was discarded, and myeloma cells were resuspended in 10mL of DMEM medium and counted for later use.
2) Preparation of immune spleen cells
One of the Balb/C mice with qualified immunity is taken, the eyeball is removed for bloodletting, the whole neck is pulled for sacrifice, blood is collected and separated, and the serum is used as positive control serum in antibody detection. The immune spleen cell suspension was isolated and prepared in the same manner as the method for preparing the trophoblast from the spleen.
3) Cell fusion
1mL PEG and 40mL DMEM medium were placed at 37℃with 5% CO 2 The incubator is preheated. Individual of myeloma cells and spleen cellsThe mixture was mixed in a ratio of 1:3 to 1:10, centrifuged at 1500rpm for 5min, the supernatant was discarded, the residual liquid was blotted dry, and 1mL of PEG preheated at 37℃was added. 40mL of DMEM medium was added to the fusion system to terminate the PEG effect. After the first 10mL was added, DMEM medium was then added to 40mL along the vessel wall. Centrifuge at 800rpm for 5min and discard supernatant. The mixed cells after fusion were suspended with 2-4mL of fetal bovine serum, and the amount of the fused cells was added to a semi-solid medium containing 25% fetal bovine serum of feeder cells, glutamate, diabody and HAT. Pouring the mixed semi-solid into 3.5cm plates, placing all the plates in sterilized wet box, placing at 37deg.C, 5% CO 2 Culturing in an incubator.
4) Selective cultivation
Cells were grown to day ten, colonies were picked into 96 well plates according to cell colony size, cultured for 2-3 days, and cell culture supernatants were taken and screened simultaneously by indirect ELISA and indirect competition ELISA.
4. Subclone and subclone detection
The cells to be subcloned are gently blown to prepare single cell suspension, and the single cell suspension is added into a counting plate for counting, so as to calculate the cell density. The antigen was diluted to 2. Mu.g/ml with coating solution CB, 100. Mu.L/well and the ELISA plate was added at 4℃overnight. Taking out the liquid in the ELISA plate dry-beating hole, closing by 5% skimmed milk, adding 200 mu L/hole of the ELISA plate, incubating for 2h at 37 ℃, and washing the plate 3 times by TBS. The plates were washed 3 times with 100. Mu.L/well cell supernatant, incubated for 1h at 37 ℃. HRP-labeled goat anti-mouse IgG (1:10000 dilution with enzyme) was added, 100. Mu.L/well incubated at 37℃for 40min, and the plates were washed 5 times with TBS. TMB substrate, 90. Mu.L/well, was added and protected from light at 37℃for 20-30min. Adding a stop solution, 50 mu L/hole, and reading by an enzyme-labeled instrument (wavelength is 450 nm), wherein the corresponding dilution value is the cell titer when the OD value is 2.0.
5. Expansion culture of frozen and resuscitated cells
Cells larger than 1x10 6 Freezing and storing at each time, and centrifuging at 1500rpm for 5min. The cells were cryopreserved in a 1:9 ratio of DMSO to fetal bovine serum. The cell cryopreservation tube was placed in a gradient cryopreservation box at room temperature and then directly placed at-80 ℃ overnight. And taking out the cells in the gradient freezing box, and placing the cells in a liquid nitrogen tank for long-term storage.
And (5) rapidly taking the cell cryopreservation tube out of the liquid nitrogen tank, and rapidly shaking the cell cryopreservation tube in a constant-temperature water bath kettle at 37 ℃ until the cell melts. Adding 5mL of DMEM medium, mixing, centrifuging at 1200rpm for 5min, discarding supernatant, adding 20% complete medium, 37 deg.C, and 5% CO 2 Culturing in an incubator, and passaging.
6. Antibody titer detection
The Tet (X4) recombinant protein prepared in example 1 was diluted to 2. Mu.g/ml with coating solution CB, 100. Mu.L/well was added to the ELISA plate, and the temperature was 4℃overnight. Taking out the liquid in the ELISA plate dry-beating hole, closing by 5% skimmed milk, adding 200 mu L/hole of the ELISA plate, incubating for 2h at 37 ℃, and washing the plate 3 times by TBS. The antibody was diluted in a doubling ratio of 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL, 0.0625. Mu.g/mL, 0.03125. Mu.g/mL, 0.015625. Mu.g/mL, 100. Mu.L per well and incubated at 37℃for 1h. The plates were washed 3 times with TBS. HRP-labeled goat anti-mouse IgG (1:10000 dilution with enzyme) was added, 100. Mu.L/well incubated at 37℃for 40min, and the plates were washed 5 times with TBS. TMB substrate is added, 90 mu L/hole is reserved, and the temperature is 37 ℃ and protected from light for 5-20 min. Adding stop solution, 50 mu L/hole, and reading by an enzyme label instrument (wavelength 450 nm), wherein the maximum dilution of positive reaction is the titer of the antibody.
2. Experimental results
1. Antiserum preparation and potency detection
According to the serum titer, the mouse numbered "left" had the highest titer, and in theory the specificity of both mice was also stronger, so the mice were selected for the fusion experiment. Specific antisera potency data are presented in table 3 and table 4.
Table 3: tri-exemption potency detection
Table 4: four-way potency detection
2. Cell fusion and subclone detection
1 mouse is selected for fusion after fusion screening, and 34 strains are selected for subcloning after the fusion and rechecking once by using immune antigens. And then, subcloning screening to obtain 30 positive clones, collecting cell supernatant and carrying out affinity sequencing on the positive clones. Finally, 5D3D3 is selected according to the supernatant gradient to beat ascites to prepare the antibody.
3. Monoclonal antibody purification and antibody titer detection
The subtype of the cells is identified after the cells are frozen and resuscitated, and the cell strain 5D3D3 is identified as igG (see table 5), so that Protein G columns are selected for purification, the obtained antibodies are respectively named an A antibody and a B antibody, and CDR sequences of the antibodies are shown in table 6.
Table 5: cell strain subtype identification table
Table 6: CDR sequences of antibodies
Wherein, the heavy chain variable region of the A antibody is shown in SEQ ID NO:16, the nucleotide sequence of the code is shown as SEQ ID NO:21, the light chain variable region is as set forth in SEQ ID NO:17 or 18, the nucleotide sequences encoded by which are shown in SEQ ID NOs: 22 or 23, specifically as follows:
the heavy chain variable region of the B antibody is shown in SEQ ID NO:19, the encoded nucleotide sequence is shown as SEQ ID NO:24, the light chain variable region is as set forth in SEQ ID NO:20, and the coded nucleotide sequence is shown as SEQ ID NO: 25. The method comprises the following steps:
then detecting antibody titer, performing SDS-PAGE and WB detection of recombinant antigen (Tet (X4) recombinant protein) to obtain target bands, and specifically, the target bands can be seen in FIG. 4 and FIG. 5. It was shown that the hybridoma cells prepared in the examples produced specific antibodies that specifically bound to the Tet (X4) recombinant protein.
4. Monoclonal antibody stability and potency
Stability of antibody a (heavy chain variable region shown as SEQ ID NO:16, light chain variable region shown as SEQ ID NO: 17), b (heavy chain variable region shown as SEQ ID NO:16, light chain variable region shown as SEQ ID NO: 18), c (heavy chain variable region shown as SEQ ID NO:19, light chain variable region shown as SEQ ID NO: 20) was examined.
1) The stability of the prepared antibodies was determined using a Uncle, and experimental parameters were:
heating interval: 25-95 DEG C
Rate of temperature rise: 0.5 ℃/min
Detection of particle size and polydispersity by DLS before warming
2) Stability results
The results show that the stability of the a, b and c antibodies is better, and the results of the exemplary a antibodies are shown in tables 7, 8 and fig. 7.
TABLE 7
| Sample of | Conc.(mg/ml) | Tm1(℃) | Tm2(℃) | Tagg266(℃) |
| a antibody | 1 | 72.79 | - | 65.7 |
TABLE 8
Example 3 Western-Blot verification
1. Materials and methods
1) Material
Preparing the obtained Tet (X4) mouse monoclonal antibody; tet (X4) positive wild-type bacteria and tet (X4) negative wild-type bacteria; western and IP cell lysates, PMSF, horseradish peroxidase-labeled goat anti-mouse IgG (H+L) were all purchased from Shanghai Biyun biotechnology Co., ltd, and chemiluminescent fluids were purchased from Semer Feishmania technology Co., ltd.
2) Method of
Bacterial culture: streaking tet (X4) positive wild bacteria and tet (X4) negative wild bacteria on LB agar medium, culturing overnight at 37 ℃, then picking single colony into 1mL LB broth medium, culturing at 37 ℃ at 180rpm for 6 hours, respectively transferring bacterial liquid into 5mL LB medium according to the proportion of 1:100, adding tigecycline with different concentrations, and culturing overnight at 37 ℃ at 180rpm, wherein the final concentration of tigecycline is 0 mug/mL, 2 mug/mL, 4 mug/mL, 8 mug/mL.
Protein extraction: centrifuging at 10000rpm for 5min, removing supernatant, washing thallus with precooled PBS for three times, re-suspending thallus with 1mLPBS, adding lysozyme to reach final concentration of 0.25mg/mL, mixing, standing on ice for 30min, incubating at 37deg.C for 5min, and standing on ice for 30min. PMSF with a final concentration of 1mM is added into the lysate, 200 mu L of lysate is added into the bacterial cells treated by lysozyme, the bacterial cells are left stand for 10min after flicking, 5min and 50w of the bacterial cells are lysed by an ultrasonic cytoclasis instrument, the operation is carried out for 3s, the operation is stopped for 2s, and finally the supernatant is obtained by centrifugation.
Protein concentration homogenization: protein concentration was measured using the Bradford method, protein sample buffer was added, and then the concentration of each protein sample was adjusted to be uniform to 0.8 μg/μl with lysate.
Western-Blot validation: protein samples were boiled and centrifuged at 10000rpm for 5min, and SDS-PAGE was performed using 10% gel, each sample being loaded with 16. Mu.g, under 80V 30min,120V 90min conditions. And (3) placing the PVDF film into a methanol solution for activation for 1min, soaking the film, glue, sponge, filter paper and the like in a film transferring buffer solution, clamping the film, the glue, the sponge, the filter paper and the like in the order of white surface, white sponge, two layers of filter paper, film, glue, two layers of filter paper, black sponge and black surface, and transferring the film by a wet transfer method for 300mA 2h. After the transfer, the membrane was blocked with 5% skim milk, 180rpm shaker at 37℃for 1h, washed 3 times with TBST, tet (X4) mouse monoclonal antibody and primary anti-dilution solution were added at 1:1000, incubated overnight at 4℃for 3 times with TBST the next day, then horseradish peroxidase and 5% skim milk were added at 1:1000, incubated at 4℃for 1h, washed 3 times with TBST, imaged with chemiluminescent solution, and grey scale analyzed with imageJ software.
2. Results and analysis
As can be seen from fig. 8, the loading sequence is: channels 1 and 2 are tet (X4) negative bacteria, channels 3 and 4 are tet (X4) positive bacteria under non-medicated culture conditions, channels 5 and 6 are tet (X4) positive bacteria under culture conditions with a tigecycline concentration of 2 mug/mL, channels 7 and 8 are tet (X4) positive bacteria under culture conditions with a tigecycline concentration of 4 mug/mL, and channels 9 and 10 are tet (X4) positive bacteria under culture conditions with a tigecycline concentration of 8 mug/mL. The Tet (X4) negative bacteria loading channel has no protein band, while the Tet (X4) positive bacteria channels all show bands with different degrees, so that the Tet (X4) mouse monoclonal antibody has good binding capacity with Tet (X4) protein. FIG. 9 shows a quantitative analysis chart of the results of which the test (X4) was negative, the test (X4) positive bacteria were not medicated, and the tigecycline concentration was 2. Mu.g/mL, the tigecycline concentration was 4. Mu.g/mL, and the tigecycline concentration was 8. Mu.g/mL, respectively. the Tet (X4) positive bacteria and the negative bacteria show a significant difference in the expression of Tet (X4), and in addition, under the condition of dosing, the expression of the Tet (X4) protein is significantly improved, and the expression quantity is stable.
In Western-Blot verification, no band is displayed in a Tet (X4) negative bacterial protein sample which does not express the Tet (X4) protein, clear bands are displayed in a Tet (X4) positive bacterial protein sample which expresses the Tet (X4) protein, and the gray value of the bands increases with the increase of the protein expression quantity, so that the good binding capability of the Tet (X4) mouse monoclonal antibody and the Tet (X4) protein is reflected.
Example 4
Stability of antibody a (heavy chain variable region shown as SEQ ID NO:16, light chain variable region shown as SEQ ID NO: 17), b (heavy chain variable region shown as SEQ ID NO:16, light chain variable region shown as SEQ ID NO: 18), c (heavy chain variable region shown as SEQ ID NO:19, light chain variable region shown as SEQ ID NO: 20) was examined.
1. Experimental method
(1) Diluting tetx4 protein 1000 times with coating solution, adding to an ELISA plate, 100 mu L/hole, and coating at 4deg.C overnight;
(2) Pouring out the liquid in the holes, washing 1 time with a washing liquid, adding 150 mu L of sealing liquid into each hole, sealing for 1 hour at the constant temperature of 37 ℃, pouring out the sealing liquid, and spin-drying;
(3) Adding the tetx4 antibody with concentration gradient dilution into an ELISA plate for reaction, wherein the concentration of the antibody is 1ug/mL,0.67ug/mL,0.50ug/mL,0.40ug/mL,0.33ug/mL,0.11ug/mL,0.037ug/mL and 0.012ug/mL respectively, 3 repeats are carried out on each concentration, 50 mu L/hole is reacted for 30min at 37 ℃, and washing liquid is washed for 3 times and then liquid in the hole is dried;
(4) 100 mu L of HRP-goat anti-mouse IgG is added into each hole, the mixture is reacted for 30min at 37 ℃, and the mixture is washed as above;
(5) Adding a freshly prepared TMB solution, 100 mu L/hole and developing for 15min at 37 ℃ in dark;
(6) By adding 2mol/L H 2 SO 4 50. Mu.L/well;
(7) Reading OD of each well with an ELISA reader 450nm ；
(8) Standard curves were plotted as four parameter fits using GraphPad prism9 software with tetx4 antibody concentrations on the abscissa and OD values corresponding to each tetx4 antibody concentration on the ordinate, see fig. 10.
2. Experimental results
The results show that all three antibodies have a high capacity to bind to the tetx4 protein, with EC50 values between 0.4566 and 0.5775.
The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (5)
1. An antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof specifically binds to a transferrin-resistant protein, said antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region, and CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region; wherein, the liquid crystal display device comprises a liquid crystal display device,
the amino acid sequence of CDR-H1 is SEQ ID NO:1, cdr-H2 has the amino acid sequence of SEQ ID NO:2, cdr-H3 has the amino acid sequence of SEQ ID NO:3, cdr-L1 has the amino acid sequence of SEQ ID NO:4, cdr-L2 has the amino acid sequence of SEQ ID NO:5, and CDR-L3 is SEQ ID NO:6, preparing a base material;
or alternatively, the process may be performed,
the amino acid sequence of CDR-H1 is SEQ ID NO:1, cdr-H2 has the amino acid sequence of SEQ ID NO:2, cdr-H3 has the amino acid sequence of SEQ ID NO:3, cdr-L1 has the amino acid sequence of SEQ ID NO:7, cdr-L2 has the amino acid sequence of SEQ ID NO:8, and CDR-L3 is SEQ ID NO:9, a step of performing the process;
or alternatively, the process may be performed,
the amino acid sequence of CDR-H1 is SEQ ID NO:10, cdr-H2 has the amino acid sequence of SEQ ID NO:11, cdr-H3 has the amino acid sequence of SEQ ID NO:12, cdr-L1 has the amino acid sequence of SEQ ID NO:13, cdr-L2 has the amino acid sequence of SEQ ID NO:14, and CDR-L3 is SEQ ID NO:15.
2. the antibody or antigen-binding fragment thereof according to claim 1, wherein,
the amino acid sequence of the heavy chain variable region is SEQ ID NO:16, and the light chain variable region is SEQ ID NO:17;
or alternatively, the process may be performed,
the amino acid sequence of the heavy chain variable region is SEQ ID NO:16, and the light chain variable region is SEQ ID NO:18;
or alternatively, the process may be performed,
the amino acid sequence of the heavy chain variable region is SEQ ID NO:19, and the light chain variable region is SEQ ID NO:20.
3. 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, or a single chain antibody.
4. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3.
5. A pharmaceutical or diagnostic kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3 or the nucleic acid of claim 4.
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