Anti-VIM-2 type carbapenemase monoclonal antibody and application thereof

Abstract

The invention discloses an anti-VIM-2 type carbapenemase monoclonal antibody and application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of a heavy chain of the monoclonal antibody is as shown in SEQ ID NO.9, and the amino acid sequence of a light chain of the monoclonal antibody is as shown in SEQ ID NO.11. The invention provides an anti-VIM-2 type carbapenemase (VIM-2) monoclonal antibody which can specifically recognize and combine with VIM-2 type carbapenemase (VIM-2). The monoclonal antibody prepared by the invention is strong in specificity and high in sensitivity, the titer is not lower than 1: 1.024 * 10 < 6 >, and the monoclonal antibody has no cross reaction with other carbapenemase such as IMP-1, KPC-2, OXA-23, OXA-48 and the like, can be simultaneously used for various immunological detection means such as ELISA (enzyme-linked immunosorbent assay) and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to monoclonal antibodies against VIM-2 type carbapenemase and their applications. Background Technology

[0002] Antimicrobial resistance has become a significant threat to public health and safety. The emergence of carbapenem-resistant Enterobacteriaceae (CRE) has posed a major challenge to clinical treatment. Due to their strong resistance and rapid spread, CRE consistently leads to treatment failures and increased mortality rates. More seriously, resistant bacteria can transfer to pathogens through resistance gene transmission, posing a threat of infection. Carbapenem production is a major mechanism of resistance in bacteria. Therefore, developing monoclonal antibodies against VIM-2 type carbapenemase and utilizing antigen-antibody specific binding for effective detection of carbapenemase is of great significance for clinical anti-infective treatment and hospital infection control.

[0003] Carbapenemases are a class of β-lactamases capable of hydrolyzing penicillins, cephalosporins, and monocyclic β-lactam antibiotics. According to the Amber classification, they can be divided into three categories: A (serine carbapenemases), B (metallo-β-lactamases), and D (oxacillinases). Category B carbapenemases are dominated by VIM, NDM, and IMP strains. Among these, VIM-type carbapenemases, due to their broad substrate scope, strong hydrolytic activity, and high resistance to enzyme inhibitors, have become an increasingly important determinant of clinical drug resistance in Gram-negative bacilli. VIM resistance genes can coexist with other β-lactamase genes, leading to multidrug resistance phenotypes in host bacteria, and the prevalence of these strains has become a global public health crisis. VIM-2 is the most prevalent clinical variant of VIM-type carbapenemase to date, and also the most geographically widespread variant. Therefore, studying VIM-2 carbapenemase is of great significance for the study of VIM-type carbapenemases. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody against VIM-2 carbapenemase and its applications, thereby addressing the problems existing in the prior art. This invention provides a monoclonal antibody against VIM-2 carbapenemase that specifically recognizes and binds to VIM-2 carbapenemase. The monoclonal antibody prepared by this invention exhibits high specificity, high sensitivity, and a titer of not less than 1:1.024×10⁻⁶. 6 It shows no cross-reactivity with other carbapenemases such as IMP-1, KPC-2, OXA-23, and OXA-48, and can be used simultaneously for multiple immunological detection methods such as ELISA, showing promising application prospects.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides a monoclonal antibody against VIM-2 type carbapenemase, wherein the heavy chain of the monoclonal antibody comprises CDR1 with an amino acid sequence as shown in SEQ ID NO.12, CDR2 with an amino acid sequence as shown in SEQ ID NO.13, and CDR3 with an amino acid sequence as shown in SEQ ID NO.14;

[0007] The light chain of the monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.15, CDR2 with an amino acid sequence as shown in SEQ ID NO.16, and CDR3 with an amino acid sequence as shown in SEQ ID NO.17.

[0008] Furthermore, the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.9, and the amino acid sequence of the light chain is shown in SEQ ID NO.11.

[0009] The present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of detection products for VIM-2 type carbapenemase.

[0010] Optionally, the detection product may include reagents, kits, or chips.

[0011] The present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of products for identifying whether drug-resistant bacteria produce VIM-2 type carbapenemase.

[0012] Optionally, the detection product may include reagents, kits, or chips.

[0013] The present invention also provides a kit for detecting carbapenemase, the kit comprising the above-mentioned monoclonal antibody.

[0014] Optionally, the kit may include an ELISA kit.

[0015] The present invention also provides the use of the above-mentioned monoclonal antibody in the preparation of medicaments for the prevention and / or treatment of infections caused by VIM-2 type carbapenemase-resistant bacteria.

[0016] The present invention discloses the following technical effects:

[0017] This invention provides a monoclonal antibody against VIM-2 carbapenemase. It is based on the gene sequence encoding VIM-2 carbapenemase as the target gene. Through gene amplification, transformation, induced expression, and purification, a recombinant protein with VIM-2 carbapenemase activity is obtained and used as an immunogen. This protein is prepared by immunizing BALB / c mice using immunological methods and can specifically recognize and bind to VIM-2 carbapenemase. The monoclonal antibody prepared by this invention has high specificity and sensitivity, with a heavy chain form of IgG2a and a light chain form of Kappa, and a titer of not less than 1:1.024×10⁻⁶. 6 It shows no cross-reactivity with other carbapenemases such as imipenemase (IMP-1), Klebsiella pneumoniae carbapenemase-2 (KPC-2), oxacillinase-23 (OXA-23), and oxacillinase-48 (OXA-48), and can be used simultaneously for multiple immunological detection methods such as ELISA, showing good application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 SDS-PAGE was used to identify the expression of recombinant VIM-2 protein; where M is the marker; lanes 1-4 are whole cells before induction, whole cells after induction, sonicated supernatant after induction, and sonicated precipitate after induction, respectively.

[0020] Figure 2 The results of Carba NP assay for VIM-2 recombinant protein are shown; where A is the control group and B is the experimental group (VIM-2 recombinant protein).

[0021] Figure 3 The results are the serum titer test results of immunized mice;

[0022] Figure 4 This is a diagram of ascites fluid purification of monoclonal antibody 5D11; where M is the marker; lanes 1-2 are the ascites fluid of monoclonal antibody and the purified monoclonal antibody 5D11, respectively.

[0023] Figure 5 The image shows the results of indirect ELISA identification of the titer of monoclonal antibody 5D11.

[0024] Figure 6 The image shows the affinity identification results for the monoclonal antibody 5D11.

[0025] Figure 7 The image shows the Western blotting results of the monoclonal antibody 5D11, where M is the marker and lane 1 is the recombinant VIM-2 protein.

[0026] Figure 8 The image shows the results of indirect ELISA identification of the cross-reactivity of the monoclonal antibody 5D11. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0033] Example 1: Selection and Preparation of Immunogens

[0034] The preparation process of the immunogen includes the following steps:

[0035] The recombinant prokaryotic expression vector pET-30a-Vim-2-BL21(DE3) was constructed as follows: The VIM-2 gene sequence was obtained from GeneBank (Accession Number: NZ_CP063457.1). The VIM-2 target gene sequence was sent to Sangon Biotech for optimization. Based on the optimized VIM-2 target gene sequence (pUC57-VIM-2), specific primers were designed: VIM-2-upstream primer P: CGCGGATCCATGGTTGATAGCAGCGGCGAATACC (SEQ ID NO.1), and VIM-2-downstream primer R: CCGCTCG AGTTATTCAACAACGCTACGGTTGG (SEQ ID NO.2). Using the optimized VIM-2 target gene sequence (pUC57-VIM-2) as a template, the VIM-2 target gene was amplified by PCR. The VIM-2 target gene and pET-30a vector plasmid were digested with BamHI and XhoI and then digested with T4. DNA ligase was used for ligation, and the cells were transformed into competent DH5α cells to construct a recombinant plasmid. The bacterial culture was sent to Sangon Biotech for sequencing. The sequenced sequence was compared with the target gene sequence of VIM-2. After expanding the culture of DH5α-pET-30a-VIM-2 cells with the correct sequence, the recombinant plasmid was extracted and transformed into BL21 competent cells, successfully constructing the expression strain pET-30a-VIM-2-BL21. The results of the sonicated supernatant obtained after IPTG-induced expression are as follows: Figure 1 As shown in the figure. Recombinant VIM-2 protein with a purity greater than 90% was obtained after ultrasonic disruption. The recombinant protein was identified using the Carba NP method, and the results are shown in the figure. Figure 2 As shown.

[0036] The optimized pUC57-VIM-2 gene sequence based on the codon bias of E. coli is as follows:

[0037] ATGTTCAAACTGCTGAGCAAACTGCTGGTTTACCTGACCGCAAGCATCATGGCGATC

[0038] GCTTCTCCGCTGGCGTTCTCTGTTGATAGCAGCGGCGAATACCCGACCGTTTCTGAAATC

[0039] CCGGTTGGCGAAGTTCGTCTGTACCAGATTGCAGACGGCGTTTGGAGCCACATCGCGAC

[0040] CCAGAGCTTCGACGGTGCGGTTTACCCGTCTAACGGTCTGATCGTTCGTGACGGTGATG

[0041] AACTGCTGCTGATCGACACCGCATGGGGTGCAAAAAACACCGCGGCGCTGCTGGCGGA

[0042] AATCGAAAAACAGATCGGCCTGCCGGTGACCCGTGCGGTTTCCACCCACTTCCACGATG

[0043] ATCGTGTTGGTGGTGTAGATGTTCTGCGCGCGGCGGGCGTGGCGACCTACGCGAGCCCG

[0044] AGCACCCGTCGTCTGGCGGAAGTTGAAGGCAACGAAATCCCGACCCACTCCCTGGAAG

[0045] GCCTGTCTAGCAGCGGCGATGCTGTTCGTTTCGGTCCGGTTGAACTGTTCTACCCAGGC

[0046] GCGGCGCACAGCACCGATAACCTGGTTGTTTACGTTCCGTCTGCTTCTGTTCTGTACGGC

[0047] GGCTGCGCGATCTACGAACTGAGCCGCACCTCTGCGGGTAACGTTGCGGATGCGGACCT

[0048] GGCGGAATGGCCGACCTCTATTGAACGTATCCAGCAGCACTACCCGGAAGCGCAGTTCG

[0049] TTATTCCGGGTCACGGCCTGCCGGGCGGCCTGGATCTGCTGAAACACACCACCAACGTT

[0050] GTTAAAGCGCACACCAACCGTAGCGTTGTTGAATAA(SEQ ID NO.3)。

[0051] Recombinant proteins of IMP-1 (Accession Number: AP012280), KPC-2 (GenBank Accession Number: KJ151293.1), OXA-23 (Accession Number: KF740470.1), and OXA-48 (Accession Number: KT175900.1) were prepared using the same method and used for subsequent specificity identification of monoclonal antibodies.

[0052] Example 2: Preparation of Monoclonal Antibodies

[0053] 1. Animal immunization

[0054] (1) Freund's complete adjuvant was added to the recombinant immunogen VIM-2 protein and emulsified for the first immunization. Two female BALB / c mice aged 4-8 weeks were immunized by subcutaneous injection at multiple points on the back, with an immunization dose of 10 μg / mouse.

[0055] (2) Three weeks later, BALB / c mice were boosted with the same method and dosage after emulsification of Freund's incomplete adjuvant with the immunogen.

[0056] (3) Three weeks later, blood was collected from the tail vein to determine serum titer, and mice with higher titers were selected. Figure 3 Three to four days before cell fusion, BALB / c mice were given a super-immunization with an adjuvant-free immunogen via tail vein injection at a dose of 50 μg per mouse.

[0057] 2. Cell fusion and monoclonal antibody preparation

[0058] Using polyethylene glycol, spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 at a ratio of 8:1. The fused cells were then screened using HAT selective medium. Twelve days post-fusion, positive hybridoma cells were initially screened using indirect ELISA with recombinant VIM-2 protein as the coating antigen. The steps of the indirect ELISA method are as follows:

[0059] (1) Dilute the uncoupled VIM-2 recombinant protein with CBS solution to a coating solution with a concentration of 2 μg / mL and coat the microplate with 100 μl / well, and block overnight at 4℃.

[0060] (2) Dilute the hybridoma supernatant (primary antibody) twice with 5% skim milk and add it to the ELISA plate in sequence, 50 μl / well. The positive control is ASFV positive serum from pigs. Incubate at 37°C for 30 min.

[0061] (3) Discard the primary antibody, wash the plate with PBST, wash it clean, and pat it dry;

[0062] (4) Add 50 μl of diluted HRP-labeled goat anti-mouse IgG (secondary antibody) to each well. Incubate at 37°C for 30 min.

[0063] (5) Discard the secondary antibody, rinse thoroughly with PBST, and pat dry;

[0064] (6) Add 100 μl of freshly prepared TMB colorimetric solution to each well and react in the dark for 15 min.

[0065] (7) Add 50 μl of 2M H2SO4 to each well to terminate the reaction;

[0066] (8) Read the OD of each well using an ELISA reader. 450 value.

[0067] 3. Subcloning of hybridoma cells using limiting dilution method

[0068] Dilute the above-mentioned positive hybridoma cells to approximately 1.5 cells / ml using 1640 / 10 complete medium. Add 100 μl of each cell to a 96-well plate pre-coated with 100 μl of feeder cells and incubate at 37°C in a 5% CO2 incubator for 6-8 days. Further screen for positive hybridoma cells using indirect ELISA. Perform 2-3 subcloning cycles until a hybridoma cell line stably secreting anti-P54 protein monoclonal antibody is obtained. The selected positive monoclonal cells are then expanded into larger cultures at a cell number of 1-2 × 10⁻⁶ cells / ml. 6 / The tubes are frozen.

[0069] 4. Stability identification of monoclonal hybridoma cell lines

[0070] The established monoclonal hybridoma cell line was cultured continuously for 3 months and repeatedly frozen and thawed in liquid nitrogen to identify the stability of the hybridoma cells; the results showed that the monoclonal hybridoma cell line had good stability.

[0071] 5. Preparation of monoclonal antibodies using in vivo ascites induction method

[0072] Multiparous female Balb / c mice were selected and injected intraperitoneally with 500 μl of sterile paraffin. One week later, the obtained monoclonal hybridoma cells were injected intraperitoneally again at a dose of 2 × 10⁻⁶. 5 One cell was collected, and after a week, once the mouse abdomen was swollen, ascites fluid was extracted, centrifuged, and the supernatant was collected. The ascites fluid was then purified using the ammonium octanoate sulfate method.

[0073] Example 3: Purification and Identification of Antibodies

[0074] 1. Antibody purification using saturated ammonium sulfate precipitation method, the procedure is as follows:

[0075] (1) Take 5 ml of monoclonal antibody ascites fluid, add 5 ml of PBS buffer, and then add 2.5 ml of saturated ammonium sulfate solution dropwise to make it a final concentration of 20% ammonium sulfate solution. Stir while adding, mix thoroughly, and let stand for 30 min.

[0076] (2) Centrifuge at 8000 r / min for 20 min and discard the precipitate to remove fibrin.

[0077] (3) Add 12.5 ml of saturated ammonium sulfate solution to the supernatant, mix thoroughly, and let stand for 30 min.

[0078] (4) Centrifuge at 8000 r / min for 20 min and discard the supernatant.

[0079] (5) Add 10 ml of PBS buffer to the precipitate to dissolve it, then add 5 ml of saturated ammonium sulfate solution to make it a 33% ammonium sulfate solution. Mix well and let stand for 30 min.

[0080] (6) Centrifuge at 8000 r / min for 20 min and discard the supernatant to remove albumin.

[0081] (7) Repeat step (5) 2-3 times.

[0082] (8) Dissolve the precipitate with 5 ml of PBS buffer, put it into a dialysis bag, dialyze with PBS buffer at 4°C, and change the buffer 4 times.

[0083] (9) Centrifuge at 8000 r / min for 20 min, discard the precipitate, and the supernatant is the purified antibody. Figure 4 The antibody was named 5D11, its concentration was measured, and after aliquoting, it was stored at -20℃.

[0084] 2. Monoclonal antibody titer determination

[0085] The indirect ELISA assay was performed according to Example 2, with a slight difference in the primary antibody: the purified monoclonal antibody was serially diluted 1:1000 with 5% skim milk and added sequentially to the ELISA plate at 50 μl / well. The positive control was positive serum from mice immunized with VIM-2 protein. The plate was incubated at 37°C for 30 min. Other steps were performed according to Example 2. The ELISA results showed that the monoclonal antibody titer was 1:1.024×10⁻⁶. 6 ( Figure 5 ).

[0086] 3. Subtype identification

[0087] The monoclonal antibody was identified using a mouse monoclonal antibody isotype identification kit (Sigma). The results showed that the monoclonal antibody belonged to IgG2a and the light chain type was Kappa.

[0088] 4. Affinity assessment

[0089] (1) Using CBS as the coating solution, the VIM-2 recombinant protein was diluted to concentrations of 1 μg / mL and 2 μg / mL for coating;

[0090] (2) Different concentrations of monoclonal antibody were added to the coated wells, and the OD values ​​of different wells were analyzed using i-ELISA. 450nm Perform the measurement;

[0091] (3) Using 1 / CmAb as the x-axis and 1 / OD as the y-axis 450nm Plot a standard curve with 50% OD as the ordinate to obtain a linear equation. 450nm Substituting the reciprocal of the product into the formula yields the corresponding antibody concentration, which is then converted to molar concentration and substituted into the following formula to obtain the affinity constant Kaff:

[0092] Kaff=(n-1) / 2(n[Ab′]t-[Ab]t).

[0093] In the formula, n = [Ag]t / [Ag′]t; [Ag]t represents the concentration of the coating antigen, [Ab]t represents the molar concentration of the monoclonal antibody, [Ab′]t represents the concentration of the antibody bound to the antigen at equilibrium, and [Ag′]t represents the concentration of the antigen bound to the antibody at equilibrium.

[0094] See results Figure 6 The regression curves for this monoclonal antibody are as follows: y = 0.008057*x + 0.1021 (R²) 2 =0.9971), y=0.003402*x+0.2004(R 2 =0.9952), and the calculated affinity constant of the monoclonal antibody 5D11 is 1.81 × 10⁻⁶. 9 L / mol.

[0095] 5. Monoclonal antibody specificity identification (cross-reactivity identification with other carbapenemases (IMP-1, KPC-2, OXA-23 and OXA-48))

[0096] Western blotting results of the binding of monoclonal antibody 5D11 to recombinant VIM-2 protein expressed in prokaryotes (prepared in Example 1) are as follows: Figure 7As shown, the monoclonal antibody specifically reacts with the VIM-2 protein. Monoclonal antibody ascites fluid was added to reaction wells coated with recombinant proteins of VIM-2, IMP-1, KPC-2, OXA-23, and OXA-48 at concentrations of 1 μg / mL, respectively. Cross-reactivity between the monoclonal antibody and IMP-1, KPC-2, OXA-23, and OXA-48 was determined using an indirect ELISA method. The results are shown below. Figure 8 As shown, the monoclonal antibody only reacted with VIM-2, and the reaction results with other carbapenemases (IMP-1, KPC-2, OXA-23 and OXA-48) were all negative, proving that the monoclonal antibody has good specificity for reacting with VIM-2 and no cross-reaction with several other common carbapenemases.

[0097] Example 4: Monoclonal Antibody Variable Region Gene Amplification and Sequencing

[0098] 1. Primer design

[0099] Based on the sequence characteristics of murine monoclonal antibodies, primer sequences for the heavy chain variable region were designed:

[0100] P1: 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC-3' (SEQ ID NO. 4);

[0101] P2: 5'-GGCTCGAGGAGAGTCCAGCTTGCAGCAGTCAGG-3' (SEQ ID NO. 5).

[0102] Design primer sequences for the light chain variable region:

[0103] P3: 5'-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3' (SEQ ID NO. 6);

[0104] P4: 5'-GGGAATTCATGGAGACAGACACACTCCTGCTAT-3' (SEQ ID NO. 7).

[0105] 2. PCR amplification

[0106] The variable region sequences of the monoclonal antibodies were obtained using molecular cloning technology and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0107] The sequencing results are as follows: The gene sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 5D11 are shown in SEQ ID NO. 8 and SEQ ID NO. 10, respectively; the amino acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 5D11 are shown in SEQ ID NO. 9 and SEQ ID NO. 11, respectively. The CDR regions of the heavy chain and light chain are shown in SEQ ID NO. 12-17.

[0108] SEQ ID NO.8:

[0109] GGAGTCCAGCTTGCAGCAGTCAGGGGCAGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAATATAAAAGACTACTATATGCACTGGGTGAAACAGAGGCCTCAACAGGGCCTGGAGTGGATTGGATGGATAGATCCAGAGAATG GAAATACTATATAAGACCCGAAGTTCCAGGGCAAGGCCTTTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTCAGGACACTGCCGTCTATTACTGTGCTATGATTACGACGAGGGCCTTCTGGGGCCAAGGGACCACGGT.

[0110] SEQ ID NO.9:

[0111] GVQLAAVRGRACEARGLSQVVLQSFWLQYKRLLYALGETEASTGPGVDWMDRSREWKYYIRPEVPGQGLYNSRHILQHSLPAAQQPDISGHCRLLLCYDYDEGLLGPRDHG.

[0112] SEQ ID NO.10:

[0113] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA。

[0114] SEQ ID NO.11:

[0115] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK。

[0116] SEQ ID NO.12:

[0117] WLQYKRLL(CDRH1)。

[0118] SEQ ID NO.13:

[0119] DRSREWKY(CDRH2)。

[0120] SEQ ID NO.14:

[0121] YDYDEGL(CDRH3)。

[0122] SEQ ID NO.15:

[0123] RASKSVSTSGYSYMH(CDRL1)。

[0124] SEQ ID NO.16:

[0125] LVSNLES(CDRL2)。

[0126] SEQ ID NO.17:

[0127] QHIRELTR(CDRL3)。

[0128] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody against VIM-2 type carbapenemase, characterized in that, The heavy chain of the monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.12, CDR2 with an amino acid sequence as shown in SEQ ID NO.13, and CDR3 with an amino acid sequence as shown in SEQ ID NO.14; The light chain of the monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.15, CDR2 with an amino acid sequence as shown in SEQ ID NO.16, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

17.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.9, and the amino acid sequence of the light chain is shown in SEQ ID NO.

11.

3. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of detection products for VIM-2 type carbapenemase.

4. The application according to claim 3, characterized in that, The testing products include reagents, kits, or chips.

5. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a product for identifying whether drug-resistant bacteria produce VIM-2 type carbapenemase.

6. The application according to claim 5, characterized in that, The testing products include reagents, kits, or chips.

7. A kit for detecting carbapenemase, characterized in that, The kit contains the monoclonal antibody as described in claim 1 or 2.

8. The reagent kit according to claim 7, characterized in that, The kit includes an ELISA kit.

9. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of infections caused by VIM-2 type carbapenemase-resistant bacteria.

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