Anti-β-lactamase antibody or antigen-binding fragment thereof, biological product and application thereof

Abstract

The present invention discloses an antibody or antigen-binding fragment thereof, a biological product and its application against β-lactamase, and relates to the field of immunoassay detection technology. The antibody has a high binding affinity with β-lactamase, and can identify CTX-M-1 type β-lactamase. In addition, the present invention also provides another antibody paired with the antibody, and these two antibodies, as coating antibodies and detection antibodies, can realize the detection of β-lactamase with high sensitivity. The present invention provides core materials for the high-performance immunoassay detection method of β-lactamase, which can be used to develop reagents, test kits, test strips, antibody chips, antibody probes or detectors and other biological products. The present invention greatly facilitates the rapid detection of β-lactamase, and has good application prospects in β-lactamase residues, bacterial detection, and in vitro sample detection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay detection, and in particular to an anti-β-lactamase antibody or an antigen-binding fragment thereof, a biological product and application thereof. Background Art

[0002] Microbial drug resistance has become a global biosafety issue. β-lactam antibiotics have a long history and are widely used, but their resistance situation is more severe. The production of β-lactamases by bacteria is the main mechanism of bacterial resistance to β-lactam antibiotics. The emergence and rapid spread of extended spectrum β-lactamases (ESBLs) make clinical treatment more challenging. Among them, CTX-M-1 ESBLs spread rapidly and are widely present in Escherichia coli and Klebsiella bacteria. The cost of using traditional drug sensitivity tests and gene sequencing to study bacterial resistance is high and time-consuming. In recent years, rapid and accurate detection of β-lactamases has become an effective means of studying bacterial resistance.

[0003] At present, immunoassays have shown strong advantages in the rapid detection of β-lactamase, but most of the existing methods are based on monoclonal antibodies or polyclonal antibodies. The sensitivity and specificity of polyclonal antibodies are poor. Although monoclonal antibodies based on hybridoma technology have high sensitivity and specificity, they require animal immunization and cell culture, and it is difficult to ensure stability between batches. Recombinant antibodies use genetic engineering technology to produce antibodies from expression systems, which can effectively improve antibody performance and have higher application value.

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

[0005] The object of the present invention is to provide an anti-β-lactamase antibody or an antigen-binding fragment thereof, a biological product and application thereof to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides an anti-β-lactamase antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR region as shown in the heavy chain variable region as shown in SEQ ID NO: 1 and a light chain CDR region as shown in the light chain variable region as shown in SEQ ID NO: 2;

[0008] Or, comprising the heavy chain CDR region shown in the heavy chain variable region shown in SEQ ID NO: 17 and the light chain CDR region shown in the light chain variable region shown in SEQ ID NO: 18.

[0009] In a second aspect, the present invention provides a biological product, which includes the above-mentioned anti-β-lactamase antibody or its antigen-binding fragment, and the biological product is selected from a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0010] In a third aspect, the present invention provides the use of anti-β-lactamase antibodies and antigen-binding fragments thereof or the above-mentioned biological products in the detection of β-lactamase.

[0011] In a fourth aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned anti-β-lactamase antibody and antigen-binding fragment thereof.

[0012] In a fifth aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0013] In a sixth aspect, the present invention provides a recombinant cell, comprising: the above-mentioned recombinant vector.

[0014] The present invention has the following beneficial effects:

[0015] The present invention prepares a new recombinant antibody for β-lactamase, which has a high binding affinity with β-lactamase and can identify CTX-M-1 type β-lactamase. In addition, the present invention also provides another antibody paired with the antibody. These two antibodies, as coating antibodies and detection antibodies, can detect β-lactamase (CTX-M-1) with high sensitivity. The present invention provides core materials for high-performance immunoassay detection methods for β-lactamase (especially CTX-M-1), which can be used to develop biological products such as reagents, kits, test strips, antibody chips, antibody probes or detectors. The present invention greatly facilitates the rapid detection of β-lactamase and has good application prospects in β-lactamase residues, bacterial detection, and in vitro sample detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is the SDS-PAGE image of CTX-M-1 recombinant protein; left image: marker molecular weight; right image: lane 1: marker, lane 2: CTX-M-1, CTX-M-1 protein purification image (purity 87%);

[0018] Figure 2The SDS-PAGE images of the three antibodies (purity 95%);

[0019] Figure 3 The ELISA test results of each group of paired antibodies on protein samples with different gradients under the condition of 1:1000 dilution ratio of the detection antibody;

[0020] Figure 4 The ELISA test results of each group of paired antibodies on protein samples with different gradients under the condition of 1:4000 dilution ratio of the detection antibody;

[0021] Figure 5 This is the result of SDS-PAGE detection of 14F6E6 protein (purity 95%);

[0022] Figure 6 This is the result of SDS-PAGE detection of 18E7D5 protein (purity 95%);

[0023] Figure 7 This is the standard curve of CTX-M-1 double antibody sandwich ELISA (n=3). DETAILED DESCRIPTION

[0024] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0025] Definition

[0026] The term "antigen binding fragment" refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antigen binding fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0027] The term "antibody" herein is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, so long as they exhibit the desired biological activity, such as specific binding to respiratory syncytial virus F protein or a fragment thereof.

[0028] In the present invention, the term "complementarity determining region or complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment with its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0029] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0030] Typically, the variable region VH of the heavy chain of an antibody can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0031] The variable region VL of the light chain of an antibody can be obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0032] In a first aspect, the present invention provides an anti-β-lactamase antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR region as shown in the heavy chain variable region as shown in SEQ ID NO: 1 and a light chain CDR region as shown in the light chain variable region as shown in SEQ ID NO: 2;

[0033] Or, comprising the heavy chain CDR region shown in the heavy chain variable region shown in SEQ ID NO: 17 and the light chain CDR region shown in the light chain variable region shown in SEQ ID NO: 18.

[0034] The present invention uses genetic engineering technology to successfully prepare a recombinant full-length antibody that can specifically identify β-lactamase (especially CTX-M-1 type), which can achieve highly sensitive, stable and rapid detection of β-lactamase (especially CTX-M-1 type). The present invention provides core materials for a high-performance immunoassay detection method for β-lactamase (CTX-M-1), which can be used to develop biological products such as reagents, kits, test strips, antibody chips, antibody probes or detectors. The present invention greatly facilitates the rapid detection of β-lactamase, and has good application prospects in β-lactamase residues, bacterial detection (especially β-lactam antibiotic-resistant bacteria), and in vitro sample detection.

[0035] Among them, the antibody numbered as 18E7D5 includes the heavy chain variable region shown in SEQ ID NO: 1 and the antibody numbered as SEQ ID NO: 2, which can specifically bind to β-lactamase. The antibody numbered as 14F6E6 includes the heavy chain variable region shown in SEQ ID NO: 17 and the light chain variable region shown in SEQ ID NO: 18, which can specifically bind to β-lactamase. These two antibodies can be paired for high-sensitivity detection of β-lactamase, such as 18E7D5 as a coating antibody and 14F6E6 as a detection antibody; or 14F6E6 as a coating antibody and 18E7D5 as a detection antibody.

[0036] By inputting the above sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 17-18 into the CDR labeling system, the corresponding CDR sequences can be obtained.

[0037] In a preferred embodiment of the present invention, the heavy chain CDR region includes CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown in SEQ ID NOs: 3-5, and the light chain CDR region includes CDR-L1, CDR-L2 and CDR-L3, and their amino acid sequences are shown in SEQ ID NOs: 6-8 or 19-21.

[0038] Antibody 18E7D5, heavy chain VH amino acid sequence length is 118aa, and the sequence information of the antibody is as follows:

[0039] HFR1: EVKLVESGGGLVQPGGSLRLSCATSGFTFS (SEQ ID NO: 13);

[0040] CDR-H1:DYYMS (SEQ ID NO: 3);

[0041] HFR2:WVRQPPGKALEWLG (SEQ ID NO:14);

[0042] CDR-H2: FIRNKVNGYTTEYSASVKG (SEQ ID NO: 4);

[0043] HFR3: RFTISRDDSQSILYLQMNTLRTEDSATYYCAR (SEQ ID NO: 15);

[0044] CDR-H3: DWDGFAY (SEQ ID NO: 5);

[0045] HFR4: WGQGTLVTVSA (SEQ ID NO:16);

[0046] Light chain VL amino acid sequence: 112aa.

[0047] LFR1: DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO:9);

[0048] CDR-L1:RSSQSLVYSNGNTYLQ (SEQ ID NO:6);

[0049] LFR2: WYLQKPGQSPKLLIY (SEQ ID NO:10);

[0050] CDR-L2:KVSNRFS (SEQ ID NO:7);

[0051] LFR3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO: 11);

[0052] CDR-L3: SQSTHVPLT (SEQ ID NO:8);

[0053] LFR4: FGAGTKLELK (SEQ ID NO: 12).

[0054] Antibody 14F6E6, heavy chain VH amino acid sequence length is 118aa, and its amino acid information is as follows:

[0055] HFR1: EVKLVESGGGLVQPGGSLRLSCATSGFTFT (SEQ ID NO: 25);

[0056] CDR-H1:DYYMS (SEQ ID NO: 3);

[0057] HFR2:WVRQPPGKALEWLG (SEQ ID NO:14);

[0058] CDR-H2: FIRNKVNGYTTEYSASVKG (SEQ ID NO: 4);

[0059] HFR3: RFTISRDNSQSILYLQMNTLRAEDSATYYCAR (SEQ ID NO: 26);

[0060] CDR-H3: DWDGFAY (SEQ ID NO: 5);

[0061] HFR4:WGQGTLVTVSA (SEQ ID NO:16);

[0062] The length of the light chain VL amino acid sequence of antibody 14F6E6 is 112aa, and its amino acid sequence is as follows:

[0063] LFR1: DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO:9);

[0064] CDR-L1: RSSQSLVHSNGNNYLH (SEQ ID NO:19);

[0065] LFR2: WYLQRPGQSPKLLIY (SEQ ID NO:22);

[0066] CDR-L2: KVSKRLS (SEQ ID NO:20);

[0067] LFR3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYF (SEQ ID NO: 23);

[0068] CDR-L3: CSQSTHVPLT (SEQ ID NO:21);

[0069] LFR4: FGAGTKLEVK (SEQ ID NO: 24).

[0070] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region, and / or a light chain framework region;

[0071] In a preferred embodiment of the present invention, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 85% homologous to the amino acid sequences shown in SEQ ID NOs: 9-12, or LFR1, LFR2, LFR3 and LFR4 that are at least 85% homologous to the amino acid sequences shown in SEQ ID NOs: 9, 22, 23, and 24, for example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 10 ... The amino acid sequences shown in 9, 22, 23, and 24 have LFR1, LFR2, LFR3, and LFR4 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous.

[0072] The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that have at least 85% homology to the amino acid sequences shown in SEQ ID NOs: 13-16, or HFR1, HFR2, HFR3 and HFR4 that have at least 85% homology to the amino acid sequences shown in SEQ ID NOs: 25, SEQ ID NO: 14, SEQ ID NO: 26, and SEQ ID NO: 16, respectively. For example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs: 13-16, or HFR1, HFR2, HFR3 and HFR4 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 14, SEQ ID NO: 26, and SEQ ID NO: 16.

[0073] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region, and the constant region comprises a heavy chain constant region and / or a light chain constant region;

[0074] In a preferred embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0075] In a preferred embodiment of the present invention, the heavy chain constant region is selected from at least one of IgG2a and IgG2b, and the light chain constant region is selected from a κ-type light chain constant region.

[0076] In a preferred embodiment of the present invention, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, rabbit or human;

[0077] In a preferred embodiment of the present invention, the species of origin of the constant region is mouse or human.

[0078] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of an antibody. The antigen-binding fragment of the above-mentioned antibody usually has the same binding specificity as the antibody from which it is derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragment of the above-mentioned antibody can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds.

[0079] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0080] In a preferred embodiment of the present invention, the β-lactamase is a CTX-M family β-lactamase.

[0081] In a preferred embodiment of the present invention, the β-lactamase is CTX-M-1 type β-lactamase or CTX-M-10 type β-lactamase.

[0082] In a second aspect, the present invention provides a biological product, which includes the above-mentioned anti-β-lactamase antibody or its antigen-binding fragment, and the biological product is selected from a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0083] In one embodiment, the above reagent is a product of an anti-β-lactamase antibody or an antigen-binding fragment thereof, and may also include functional components such as a protein stabilizer and a protective agent. The protein stabilizer is selected from: sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20. The protective agent is selected from a cryoprotectant, such as a polyol and a sugar. The polyol is selected from, for example, sorbitol, mannitol or a mixture thereof.

[0084] In a preferred embodiment of the present invention, the reagent includes: an anti-β-lactamase antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment thereof is labeled with a detectable marker; the form of the reagent includes but is not limited to solid, liquid, and semi-solid.

[0085] The antibody chip refers to a chip formed by immobilizing the above-mentioned anti-β-lactamase antibody or its antigen-binding fragment on a carrier.

[0086] Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0087] In a preferred embodiment of the present invention, the detectable marker is selected from fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent agents and nanoparticle markers.

[0088] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0089] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0090] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0091] In an alternative embodiment, the radioactive isotopes include but are not limited to212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0092] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0093] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; the nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0094] In a preferred embodiment of the present invention, the kit includes a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase; for example, the antibody or antigen-binding fragment thereof is connected to the solid phase by chemical coupling.

[0095] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates and membranes;

[0096] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon and nitrocellulose membranes;

[0097] In a preferred embodiment of the present invention, when the solid phase is a microplate, the microplate is coated with the above-mentioned anti-β-lactamase antibody or its antigen-binding fragment, and the kit further comprises a detection antibody, the heavy chain variable region of the detection antibody is shown in SEQ ID NO: 17, and the light chain variable region of the detection antibody is shown in SEQ ID NO: 18;

[0098] Or, when the solid phase is a microwell plate, the microwell plate is coated with a coating antibody, the heavy chain variable region of the coating antibody is shown as SEQ ID NO: 17, and the light chain variable region of the antibody is shown as SEQ ID NO: 18; the kit also includes a detection antibody, which is the above-mentioned anti-β-lactamase antibody or its antigen-binding fragment.

[0099] In a preferred embodiment of the present invention, the detection antibody is labeled with a detectable marker, such as horseradish peroxidase or alkaline phosphatase.

[0100] In a third aspect, the present invention provides the use of anti-β-lactamase antibodies and antigen-binding fragments thereof or the above-mentioned biological products in the detection of β-lactamase.

[0101] In a preferred embodiment of the present invention, the application includes at least one of the following application modes:

[0102] (1) Detection of bacteria producing β-lactamases; such as detection of bacteria resistant to β-lactam antibiotics, including but not limited to Escherichia coli and Klebsiella;

[0103] (2) Detection of β-lactamase residues in dairy products; dairy products include but are not limited to cow's milk, goat's milk and other dairy products.

[0104] (3) Detection of β-lactamase resistance genes in test samples or environmental samples, without the purpose of disease diagnosis;

[0105] In a preferred embodiment of the present invention, the subject sample is selected from at least one of the subject's skin, serum, blood or culture thereof, urine, intestinal tract and respiratory tract.

[0106] In addition, the present invention also provides the use of anti-β-lactamase antibodies and antigen-binding fragments thereof or the above biological products in the preparation of β-lactamase detection products.

[0107] In a fourth aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned anti-β-lactamase antibody and antigen-binding fragment thereof.

[0108] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0109] In a fifth aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0110] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can introduce the target DNA fragment directly or indirectly (such as packaging into a virus) into a host cell by transformation, transfection or transduction to express the target gene.

[0111] One type of vector is a plasmid, which is a circular double-stranded DNA molecule into which the target DNA fragment can be connected. Another type of vector is a viral vector, which can package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0112] A person skilled in the art can also transcribe the nucleic acid sequence of the present invention into RNA by in vitro transcription, and further transfect, transduce or transform the RNA into a host cell to express the antibody of the present invention or its functional fragment to exert the biological efficacy of the present invention.

[0113] In a sixth aspect, the present invention provides a recombinant cell, comprising: the above-mentioned recombinant vector.

[0114] In a preferred embodiment of the present invention, the recombinant cell is a bacterium, a fungus or a 293 cell, a 293T cell, a 293FT cell, a CHO cell, a COS cell, or a Per6 cell. 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those skilled in the art.

[0115] In a preferred embodiment of the present invention, the bacteria is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis.

[0116] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0117] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parental cells in nucleic acid content, but may contain mutations.

[0118] In a preferred embodiment of the present invention, the yeast is selected from at least one of the following genera: Dekkera, Brettanomyces, Hansenula, Kluyveromyces, Pichia, Candida, Kluyveromyces Kluyveromyces ), Debaryomyces spp. Debaryomyces ), Kazachstania , Wickerhamomyces , Lindnera , Zygotorulaspora , Zygosaccharomyces ( Zygosaccharomyces ), Rhodosporidium Rhodosporidium ) and Schizosaccharomyces spp. ( Schizosaccharomyces ).

[0119] The above-mentioned recombinant cells are prepared by transforming the recombinant expression vector into a host cell (such as a microorganism) by conventional methods in the art; the host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate itself and the foreign gene carried can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0120] In a preferred embodiment of the present invention, the recombinant cell refers to at least one of a resting cell of a recombinant bacterium, a living cell of a recombinant bacterium, a dead cell of a recombinant bacterium, and a cell fragment of a recombinant bacterium.

[0121] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0122] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0123] The present invention utilizes β-lactamase CTX-M-1 recombinant protein to immunize mice to produce sensitized B lymphocytes, removes mouse spleen cells and fuses them with syngeneic myeloma cells to prepare hybridoma cells, obtains a large number of monoclonal antibodies by in vivo induction, and selects monoclonal antibodies with higher titers after separation, purification and identification. The monoclonal antibodies obtained by screening are further subjected to pairing experiments to obtain antibody pairs that can simultaneously recognize CTX-M-1 recombinant proteins, and the corresponding hybridoma cells are subjected to genome sequencing. The antibody gene sequence obtained by sequencing is introduced into an expression system for full-length expression to obtain recombinant antibodies, and the recombinant antibodies are verified by ELISA.

[0124] Example 1

[0125] Antigen expression.

[0126] Antigen information: Beta-lactamase CTX-M-1 (29-291aa)

[0127] Expression region: 29-291aa, full length of mature protein.

[0128] Tags: N-terminal 6xHis-KSI-tagged

[0129] Destination sequence:

[0130] QTADVQQKLAELERQSGGRLGVALINTADNSQILYRADERFAMCSTSKVMAVAAVLKKSESEPNLLNQRVEIKKSDLVNYNPIAEKHVDGTMSLAELSAAALQYSDNVAMNKLISHVGGPASVTAFARQLGD ETFRLDRTEPTLNTAIPGDPRDTTSPRAMAQTLRNLTLGKALGDSQRAQLVTWMKGNTTGAASIQAGLPASWVVGDKTGSGDYGTTNDIAVIWPKDRAPLILVTYFTQPQPKAESRRDVLASAAKIVTNGL.

[0131] Reference link: https: / / www.uniprot.org / uniprotkb / P28585 / entry

[0132] Part I: Gene cloning encoding the target sequence:

[0133] A. PCR amplification, 200 µl system, PCR amplification of target fragment, product recovery.

[0134] 200 µl PCR system was prepared by amplifying the following system:

[0135]

[0136] After mixing, amplify according to the following PCR conditions:

[0137] 94℃, 4min; 94℃ 45sec, 52℃ 60sec, 72℃ 5min, cycle 30 times; 72℃ 10min.

[0138] B. Connection and conversion

[0139] The PCR product and vector pET28a were connected for 2 hours under the action of ligase; transformed into TOP10 host cells; the plate was inverted and cultured in a constant temperature incubator at 37°C, and colonies appeared after 12 to 16 hours.

[0140] C. Recombinant screening and identification

[0141] Aliquot 40ul LB medium into PCR tubes, pick 8 single colonies of each clone into PCR tubes, mix well, and place in a 37℃ shaker at 220 rpm for 2 hours. Take 2 µl from each tube as a template for bacterial solution PCR, and those with positive results can be sequenced for identification.

[0142] D. Sequencing and identification

[0143] The plasmid was analyzed and compared with the target amino acid and no mutation was found.

[0144] Part II: Protein expression and purification

[0145] 1. Conversion:

[0146] 1.1. Take out competent E. coli BL21 (DE3) cells from the ultra-low temperature refrigerator and thaw them on ice;

[0147] 1.2. Add plasmid (0.5-1 μg), pipette gently to mix thoroughly, and place on ice for 30 min;

[0148] 1.3. Heat in a water bath at 42°C for 90 seconds and place on ice for 1-2 minutes;

[0149] 1.4. Add 800 μL pre-warmed LB liquid medium and culture at 37°C and 158 rpm for 50 min.

[0150] 1.5. Centrifuge at 6000 rpm for 4 min, remove part of the supernatant (650 μL), resuspend and mix the remaining bacterial solution (150 μL), and then apply it to the resistance LB plate;

[0151] 1.6. Invert the plate and incubate at 37°C for 12-16 hours until a single colony appears.

[0152] 1.7. Select a single colony containing the recombinant plasmid and place it in 3 mL LB liquid medium. Culture it at 37°C overnight and store it at -20°C.

[0153] 2. Small-scale expression and identification

[0154] 2.1. Select a single colony containing the recombinant plasmid and place it in 3 mL LB liquid medium containing antibiotics. Cultivate at 37°C with shaking until the OD600 is about 0.6.

[0155] 2.2 Take part of the bacterial solution as the control group, add IPTG inducer (final concentration 1 mM) to the remaining bacterial solution, and culture at 37 °C with shaking for 3 h;

[0156] 2.3 Take 0.15 mL of bacterial suspension from each group, centrifuge at 12000×g for 2 min, resuspend and lyse the bacterial pellet with 40 μL 1× loading buffer, boil in boiling water for 5 min, and take 10 μL for protein electrophoresis.

[0157] 3. Expand cultivation:

[0158] 3.1 Take 10 μL of the bacteria stored at -20 ℃ and inoculate it into 100 ml of LB liquid medium and culture it overnight with shaking;

[0159] 3.2 Take 50 mL of bacterial solution and inoculate it into 1 L of LB liquid medium, and culture it at 37 °C until the OD600 is about 0.6;

[0160] 3.3 Add IPTG inducer to a final concentration of 1 mM and continue shaking culture at 37 °C for 2.5-3 h;

[0161] 3.4 Centrifuge at 8000 rpm for 3 min to collect the cells, resuspend in 50 mL pre-cooled STET buffer and place on ice for 30 min;

[0162] 3.5 Ultrasonic disruption of bacteria, centrifugation at 16000 rpm at 4 °C for 50 min, collection of supernatant and precipitate;

[0163] 3.6 Take a small amount of supernatant and precipitate for SDS-PAGE detection, and store the remaining supernatant and precipitate at 4 ℃ for later use.

[0164] 4. Protein Purification

[0165] A Ni column was used for protein purification. After the column was balanced, the sample was loaded at a flow rate of 1 ml / min. The column was washed with 20 mMTris-HCl, 0.5 M NaCl, pH 8.0, plus 20 mM, 60 mM, 200 mM and 500 mM imidazole (until the G250 detection did not turn blue). The effluent was collected and the protein purity was analyzed by gel running. The theoretical size of CTX-M-1 (target fragment 29-291aa) was predicted to be 43.5 kDa, and the SDS-PAGE observed size was about 43 kDa ( Figure 1 ), the theoretical value and the observed value are consistent, and the protein is successfully expressed.

[0166] Example 2

[0167] Preparation of antibodies.

[0168] 1. Mouse Immunization

[0169] Antigen emulsification: Take 0.05 mg of antigen, dilute it with PBS, mix it with Freund's adjuvant in a volume ratio of 1:1 (total volume 0.8 mL), and emulsify it at 4°C for 3-5 minutes.

[0170] Antigen injection: transfer the emulsified antigen into a 1 mL syringe, remove the bubbles in the syringe, place the immunized mice (4 mice) in a special holder, and perform multiple subcutaneous injections on the back.

[0171] Immunization cycle: Each immunization is 2 weeks apart. A total of 4 immunizations. Blood is collected one week after the third immunization for titer testing. When the titer reaches the requirement, the eyeballs are removed to collect blood, the animals are killed by pulling the neck, and the spleen is removed.

[0172] 2. Hybridoma Cell Preparation

[0173] Activation and preparation of SP2 / 0

[0174] 1). Resuscitate SP2 / 0 cells before fusion and subculture in 10% FBS DMEM medium for one week. When the cells are in good growth condition, take 1x10 6 Each cell was injected subcutaneously into the back of the mouse (injected at 5-10 points). Tumors were clearly visible after about 8-10 days. When the tumor grew to a diameter of 1-3 cm, the immune-qualified mice were subjected to shock immunization, and fused after 3 days.

[0175] 2). Aseptically cut the back skin, 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 10 cm dish, add 5-10 ml DMEM, grind it into a single cell suspension, and aspirate all of it into a 50 ml centrifuge tube.

[0176] 3). Gently add 20 ml of myeloma cell suspension along the tube wall to a 50 ml centrifuge tube pre-added with 20 ml of lymphocyte separation solution, centrifuge at 2000 r / min for 15 min, discard the upper layer of cell suspension, transfer the middle layer of white myeloma cell suspension to another 50 ml centrifuge tube, suspend with 10 ml of DMEM medium, centrifuge at 1500 r / min for 5 min, wash the cells twice. Discard the supernatant, resuspend the myeloma cells with 10 ml of DMEM medium, count and set aside.

[0177] Preparation of immune splenocytes

[0178] Take a Balb / C mouse that has passed the immunization, remove the eyeball, bleed, and kill it by pulling the neck completely, collect the blood and separate it, and use the serum as the positive control serum for antibody detection. Separate and prepare the immune spleen cell suspension.

[0179] Cell fusion

[0180] 1). Myeloma cells and spleen cells were mixed in a ratio of 1:3-1:10, centrifuged at 1500rpm for 5min, discarded the supernatant, aspirated the residual liquid, and gently tapped the centrifuge tube to loosen the cells.

[0181] 2). Slowly add 1 ml PEG preheated at 37°C within 60 seconds, and let stand for 60 seconds.

[0182] 3). Add 40 ml of DMEM medium to the fusion system to terminate the effect of PEG, slowly at first and then quickly, gently shaking the centrifuge tube while adding, mix well. Centrifuge at 800 rpm for 5 minutes and discard the supernatant.

[0183] 4). Use 2-4 ml of fetal bovine serum to suspend the mixed cells after fusion. Add the semi-solid medium containing 25% fetal bovine serum containing trophoblasts, glutamine, double antibody and HAT according to the amount of fused cells, and mix gently. Pour the mixed semi-solid into a 3.5 cm plate (2-3 ml), and then place all the plates in a sterilized wet box at 37°C 5% CO 2 Culture in an incubator.

[0184] Selective training

[0185] 0 day is counted from the day of fusion. On the 10th day of cell growth, colonies can be picked into 96-well plates (200 μl HT complete medium containing nutrient cells in each well) according to the size of cell colonies. After culturing for 2-3 days (the medium should be replaced with HT complete medium once during this period), the cell culture supernatant can be taken and positive cell lines can be screened by indirect ELISA and indirect competitive ELISA methods.

[0186] Subclone screening

[0187] The positive cells were selected for subclone screening. 1 subclone / well, 50 μl was taken at a concentration of 1x10 3 Add the cell suspension of 100 μl / ml to 4.95 ml culture medium and mix well (add 100 μl to a 96-well plate), and spread 6 strips of 100 μl / well to a 96-well plate containing trophoblast cells.

[0188] Subclone detection

[0189] 1). Coating: Dilute the antigen to 2 μg / ml with coating solution, add 100 μl / well to the ELISA plate, and incubate overnight at 4°C.

[0190] 2). Blocking: Take out the ELISA plate and pat dry the liquid in the wells, block with 5% skim milk (dissolved in PBS), add 200 μl / well of the ELISA plate, incubate at 37℃ for 2h, and wash the plate 3 times with TBS.

[0191] 3). Sample addition: add 100 μl / well of cell supernatant, incubate at 37°C for 1 hour, and wash the plate 3 times with TBS.

[0192] 4). Add the second antibody: Add HRP-labeled goat anti-mouse IgG (1:10000 enzyme dilution), 100ul / well, incubate at 37℃ for 40min, and wash the plate 5 times with TBS.

[0193] 5). Color development: Add TMB substrate, 90 μl / well, incubate at 37°C in the dark for 20-30 min.

[0194] 6). Stop: Add stop solution, 50 μl / well, read with microplate reader (wavelength 450 nm), when the OD value is greater than 1, the corresponding dilution value is the cell titer.

[0195] The absorbance of ELISA test of 30 hybridoma cells is shown in the following table:

[0196]

[0197] Thirty positive clones were screened through subcloning, and based on the titer test results of the cell supernatant, the cell lines 14F6E6, 18H3G3, and 18E7D5 hybridoma cells were selected to prepare antibodies for later verification.

[0198] 3. Monoclonal antibody preparation:

[0199] Hybridoma cells were injected into the peritoneal cavity of Balb / C mice, and the ascites was collected and the supernatant was set aside. The ascites supernatant was purified using affinity chromatography column A. After balancing the column, the supernatant was mixed with sodium acetate buffer at a ratio of 1:4, loaded at a rate of 0.5 ml / min, and the penetration solution was collected (for subsequent SDS-PAGE detection). After loading, continue to rinse with sodium acetate buffer until the G250 detection is colorless. Rinse the column bed with glacial acetic acid eluent, collect the elution peak, and quickly adjust the pH of the elution peak to neutral with saturated sodium carbonate. Ultrafilter and concentrate the elution peak to the same volume as the ascites supernatant, and load it into a dialysis bag. Centrifuge at 4°C, 12,000 rpm for 10 minutes, collect the supernatant, and store it at 4°C for SDS-PAGE detection. Figure 2 The purified antibodies were stored at -20°C.

[0200] Example 3

[0201] Antibody pairing

[0202] Antibody Matrix Pairing

[0203] 1). Coating: Dilute the antibody to 2 µg / ml with coating solution, add 100 µl / well to the ELISA plate, and incubate at 4°C overnight or at 37°C for 2 hours.

[0204] 2). Blocking: Take out the ELISA plate and pat dry the liquid in the wells, block with 5% skim milk (dissolved in PBS), add 200ul / well to the ELISA plate, incubate at 37℃ for 2h, and wash the plate 3 times with TBS.

[0205] 3). Sample addition: Use sample diluent to dilute the protein at 2 μg / ml, and then dilute it in two-fold gradient to 1, 0.5, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.001953125,0.0009765625, 0.00048828125, 0.000244140625, 0.0001220703125,0.00006103515625 μg / ml (using PBS as negative control), 100ul per well, incubate at 37℃ for 2h.

[0206] 4). Add labeled antibody: Add Biotin-labeled antibody (1:1000 or 1:4000 dilution), 100ul / well, incubate at 37℃ for 60min, wash the plate 3 times with TBS.

[0207] 5). Add enzyme conjugate: Add avidin-HRP linked antibody (1:4000 enzyme dilution), 100ul / well, incubate at 37℃ for 60min, wash the plate 5 times with TBS.

[0208] 6). Color development: Add TMB substrate, 90 μl / well, incubate at 37°C in the dark for 20 min.

[0209] 7). Stop: Add stop solution, 50 μl / well, read with a microplate reader (dual wavelengths of 450 nm and 630 nm), the maximum dilution with a positive reaction (OD value greater than 1) is the antibody pairing titer.

[0210] The pairing results of the three antibodies after dilution 1000 times and 4000 times are as follows: Figure 3 and Figure 4 According to the experimental results, 14F6E6 and 18E7D5 were selected as the antibody pair for subsequent recombinant expression. When the target protein concentration was as low as about 3 ng / mL, a high OD value was still maintained.

[0211] Example 4

[0212] Antibody recombinant expression and ELISA verification.

[0213] 1. Antibody variable region sequencing: Total RNA was extracted from 14F6E6 and 18E7D5 hybridoma cell lines, and the antibody heavy chain and light chain genes were obtained by RT-PCR. After purification, they were connected to the heavy chain and light chain vectors pSecTag2A respectively and transferred into competent cells HEK 293F for culture. After the colonies were grown on the plates, sequencing was performed. The results of antibody variable region sequencing are as follows:

[0214] 14F6E6 (subtype IgG2b, Igκ), the heavy chain VH amino acid sequence length is 118 aa, and the light chain VL amino acid sequence length of antibody 14F6E6 is 112 aa.

[0215] Antibody 18E7D5 (subtype IgG2a, Igκ)

[0216] The amino acid sequence length of the heavy chain VH is 118aa, and the amino acid sequence of the light chain VL is 112aa.

[0217] 2. Recombinant Expression of Antibodies

[0218] First, the variable region genes of the heavy and light chains of the sequenced antibodies were amplified by PCR. After amplification, the target gene was verified by running on a 1% agarose gel. The target gene was then connected to the plasmid pSecTag2A and transferred into the host cell HEK 293F for recombinant antibody expression. The specific experimental steps for transfection and expression are as follows:

[0219] 2.1 Preparation of cells for transfection

[0220] 2.1.1 Before transient transfection of cells, the cell density and viability must be determined. To ensure the transfection effect, it is recommended to use cells in the exponential growth phase (density of about 4~6×10 6 / ml), and the cell transfection with a survival rate greater than 98%;

[0221] 2.1.2 The cells were directly diluted into KOP293 culture medium without centrifugation to a cell density of 2×10 6 pcs / ml;

[0222] 2.1.3 Shake flasks in 5% CO 2 Incubate the cells in a constant temperature shaker at 37°C and 120 rpm for 10 min before starting transfection.

[0223] 2.2 Transient transfection (taking transfection of 200ml cell suspension as an example)

[0224] 2.2.1 Prepare two 50ml sterile centrifuge tubes, add 10ml KPM and 200 μg sterile plasmids (Ab-VH-pSecTag plasmid 66 μg, Ab-VL-pSecTag2A plasmid 134 μg) to one of them, and gently pipette to mix; take another centrifuge tube, add 10ml KPM and 1ml TA-293 transfection reagent, and gently pipette to mix;

[0225] 2.2.2 Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid and mix gently by pipetting;

[0226] 2.2.3 Let stand at room temperature for 10 minutes to prepare the plasmid-vector complex;

[0227] 2.2.4 Remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO 2 Incubate the culture in a constant temperature shaker. Add appropriate amount of antibiotics as needed after 3 hours.

[0228] 2.2.5 24 hours after transfection, add 1.2 ml of 293 cell protein expression enhancer (KE-293) to increase product expression;

[0229] 2.2.6 Transient transfection nutrient additives (KT-Feed 50×) can increase the expression of the product and can be added once 24 hours after transfection;

[0230] 2.2.7 Measure the product expression level on the 4th day after transfection.

[0231] 2.3 Product expression and purification

[0232] 2.3.1 Sampling of secreted proteins

[0233] Collect the cell supernatant into a 50 ml centrifuge tube, centrifuge at 3500 rpm for 15 min, separate the supernatant into a new 50 ml centrifuge tube and mark it accordingly.

[0234] 2.3.2 Loading of secreted proteins

[0235] After collecting the cell supernatant, pour the supernatant into the balanced column (Protein A filler) and let gravity flow through the sample (repeat this process twice or more).

[0236] 2.3.3 Cleaning of secreted proteins

[0237] After loading, wash with 50 mL Wash Buffer (20 mM PB), and then let the Wash buffer flow out by gravity.

[0238] 2.3.3 Elution of secreted proteins

[0239] After washing, elute with glycine eluent until the G250 detection solution does not change color, dialyze overnight into 1*PBS buffer, and concentrate the protein by ultrafiltration the next day.

[0240] 3.114F6E6 antibody SDS test results

[0241] The ultrafiltration concentrated protein was subjected to SDS-PAGE detection. Figure 5 Ab-VH-pSecTag2A / Ab-VL-pSecTag2A, tagged with C-terminal mFc2b-tagged, with theoretical sizes of 50 / 27 kDa.

[0242] SDS test results refer to Figure 5 As shown, lane 1: Marker (14.4, 18.4, 25.0, 35.0, 45.0, 66.2, 116.0 kDa), lane 2: cell secretion supernatant stock solution, lane 3: cell secretion supernatant flow-through, lane 4: 1*PBS buffer sample.

[0243] 3.2 18E7D5 antibody SDS test results

[0244] The ultrafiltration concentrated protein was subjected to SDS-PAGE detection. Figure 6 Ab-VH-pSecTag2A / Ab-VL-pSecTag2A, tagged with C-terminal mFc2a, theoretical size is 50 / 27 kDa.

[0245] SDS test results refer to Figure 6 As shown, lane 1: Marker (14.4, 18.4, 25.0, 35.0, 45.0, 66.2, 116.0 kDa), lane 2: cell secretion supernatant stock solution, lane 3: cell secretion supernatant flow-through, lane 4: 1*PBS buffer sample.

[0246] According to the SDS-PAGE test results, it can be concluded that the recombinant antibody was successfully prepared.

[0247] 3.ELISA Validation

[0248] The recombinantly expressed 14F6E6 and 18E7D5 antibodies were validated by ELISA, and the standard curves were as follows: Figure 7 The test steps are as follows:

[0249] 1). Coating: Dilute 18E7D5 antibody to 2 μg / ml with coating solution, add 100 μl / well to ELISA plate, and incubate at 4℃ overnight or 37℃ for 2h.

[0250] 2). Blocking: Take out the ELISA plate and pat dry the liquid in the wells, block with 5% skim milk (dissolved in PBS), add 200ul / well to the ELISA plate, incubate at 37℃ for 2h, and wash the plate 3 times with TBS.

[0251] 3) Sample addition: dilute CTX-M-1 and CTX-M-2 in series with sample diluent (PBS is used as negative control), 100 μl per well, incubate at 37℃ for 2h.

[0252] 4). Add labeled antibody: Add Biotin-labeled 14F6E6 antibody (0.25 mg / ml, 1:4000 dilution), 100 μl / well, incubate at 37°C for 60 min, and wash the plate 3 times with TBS.

[0253] 5). Add enzyme conjugate: add avidin-HRP (0.25 mg / ml, 1:4000 dilution), 100 ul / well, incubate at 37°C for 60 min, wash the plate 5 times with TBS.

[0254] 6). Color development: add TMB substrate, 90ul / well, incubate at 37℃ in dark for 20min.

[0255] 7). Stop: Add stop solution, 50 μl / well, and read with a microplate reader (wavelength 450 nm).

[0256] For CTX-M-1, the samples were diluted with PBS (pH = 7.4) to 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95, 0.97, 0.48, 0.24, and 0.12 ng / mL, and the standard curve was drawn with the logarithm of the concentration of CTX-M-1 as the abscissa and the detection OD value as the ordinate. CTX-M-1 showed a good linear relationship in the range of 0.97 ng / mL to 15.625 ng / mL, and the linear regression equation was: y = 1.991*x-5.647, the correlation coefficient R2 was 0.9803, and the limit of detection (LOD) was defined as the protein concentration corresponding to 3 times the OD value of the negative control, which was 0.71 ng / mL.

[0257] For CTX-M-2, the sample was diluted with PBS (pH = 7.4) to 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95, 0.97, 0.48, 0.24, 0.12 ng / mL, and the logarithm of the concentration of CTX-M-2 was used as the abscissa and the OD value was used as the ordinate to draw a standard curve. CTX-M-2 and CTX-M-1 have a high homology (80%), but this method has a very low response to CTX-M-2 signals, indicating that the synthesized antibody has a high specificity.

[0258] In summary, the present invention successfully prepared a pair of recombinant full-length antibodies that can specifically recognize β-lactamase (CTX-M-1 type) by using genetic engineering technology, which is of great significance for achieving highly sensitive, stable and rapid detection of β-lactamase (CTX-M-1 type), and can provide core materials for the identification of β-lactam antibiotic-resistant bacteria.

[0259] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-β-lactamase antibody or an antigen-binding fragment thereof, characterized in that: The antibody or its antigen-binding fragment comprises a heavy chain CDR region and a light chain CDR region, wherein the heavy chain CDR region comprises CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NOs: 3-5, respectively; the light chain CDR region comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NOs: 6-8, respectively.

2. The anti-β-lactamase antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region and a light chain framework region; the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 which have at least 85% homology to the amino acid sequences shown in SEQ ID NOs: 9-12, in sequence; The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 85% homology to the amino acid sequences shown in SEQ ID NOs: 13-16, respectively.

3. The anti-β-lactamase antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:

2.

4. An anti-β-lactamase antibody or an antigen-binding fragment thereof, characterized in that: The antibody or its antigen-binding fragment comprises a heavy chain CDR region and a light chain CDR region, wherein the heavy chain CDR region comprises CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs: 3-5, respectively; and the light chain CDR region comprises CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs: 19-21, respectively.

5. The anti-β-lactamase antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region and a light chain framework region; the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 having at least 85% homology with the amino acid sequences shown in SEQ ID NOs: 9, 22, 23 and 24, respectively; The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 85% homology to the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:16, respectively.

6. The anti-β-lactamase antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:

18.

7. The anti-β-lactamase antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region, wherein the constant region comprises a heavy chain constant region and / or a light chain constant region; The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

8. The anti-β-lactamase antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region is selected from at least one of IgG2a and IgG2b, and the light chain constant region is selected from a κ-type light chain constant region.

9. The anti-β-lactamase antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

10. The anti-β-lactamase antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The β-lactamase is a CTX-M family β-lactamase.

11. A biological product, characterized in that It comprises the anti-β-lactamase antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and the biological product is selected from a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

12. The biological product according to claim 11, characterized in that The reagent comprises: the anti-β-lactamase antibody or its antigen-binding fragment, and the antibody or its antigen-binding fragment is marked with a detectable marker; The detectable marker is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents or nanoparticle markers.

13. The biological product according to claim 11, characterized in that The kit comprises a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase; The solid phase is selected from the group consisting of microspheres, plates and membranes.

14. The biological product according to claim 13, characterized in that The solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon or nitrocellulose membranes.

15. The biological product according to claim 14, characterized in that When the solid phase is a microwell plate, the heavy chain variable region sequence of the antibody coated on the microwell plate is as shown in SEQ ID NO: 1, and the light chain variable region sequence is as shown in SEQ ID NO:

2. The kit further comprises a detection antibody, and the heavy chain variable region sequence of the detection antibody is as shown in SEQ ID NO: 17, and the light chain variable region sequence of the detection antibody is as shown in SEQ ID NO: 18; Or, when the solid phase is a microwell plate, the microwell plate is coated with a coating antibody, the heavy chain variable region of the coating antibody is shown in SEQ ID NO: 17, and the light chain variable region of the antibody is shown in SEQ ID NO: 18; the kit further comprises a detection antibody, the heavy chain variable region sequence of the detection antibody is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 2; The detection antibody is labeled with a detectable marker.

16. Use of the anti-β-lactamase antibody and antigen-binding fragment thereof according to any one of claims 1 to 10 or the biological product according to any one of claims 11 to 15 in β-lactamase detection; The application is selected from at least one of the following application modes: (1) The purpose of detecting bacteria that produce β-lactamase is not for the purpose of disease diagnosis; (2) Use of the test for detecting β-lactamase residues in dairy products; (3) Detection of β-lactamase resistance genes in test samples or environmental samples, without the purpose of disease diagnosis; The subject sample is selected from at least one of the subject's skin, serum, blood or culture thereof, urine, intestinal tract and respiratory tract.

17. An isolated nucleic acid molecule, characterized in that It encodes the anti-β-lactamase antibody and antigen-binding fragment thereof according to any one of claims 1 to 10.

18. A recombinant vector, characterized in that: It comprises the nucleic acid molecule according to claim 17.

19. A recombinant cell, characterized in that It includes: The recombinant vector according to claim 18.

Images