New delhi metallo-beta-lactamase binding proteins, uses and products thereof

Abstract

The application provides a binding protein of New Delhi metallo-beta-lactamase and application and products thereof, and relates to the technical field of biotechnology. The binding protein of New Delhi metallo-beta-lactamase provided by the application has the advantages of good specificity, high biological activity, strong stability, small batch difference, no influence of cell strain degradation, high affinity with New Delhi metallo-beta-lactamase, and a titer of 1:1280000 or more, and can be used for New Delhi metallo-beta-lactamase detection or preparation of purified product.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a New Delhi metallo-beta-lactamase binding protein and its application and product. BACKGROUND

[0002] New Delhi metallo-beta-lactamase (NDM) belongs to the class B metalloenzymes in carbapenemases, which can hydrolyze beta-lactam antibacterial drugs except aztreonam. Bacteria containing this enzyme are almost resistant to all antibacterial drugs, and beta-lactamase inhibitors such as sulbactam and clavulanic acid cannot effectively inhibit this type of metalloenzyme. Since NDM was reported, NDM-producing drug-resistant bacteria have been widespread in the world, and NDM is evolving rapidly. Currently, 24 NDM-1 mutant subtypes with one or more residues different from each other at different positions have been reported. The substitution of various amino acids in NDM mutant subtypes affects the stability and activity of the enzyme, and confers selective advantage in the evolution of bacterial drug resistance. Except for NDM-2 gene and NDM-13 gene located on the chromosome, the other mutant subtype encoding genes are mainly mediated by plasmid. NDM-2, 3, 4, 6, 9, 11, 14, 22, 23 and 24 differ from NDM-1 in that there is a single amino acid substitution, while the remaining mutant subtypes have multiple substitutions, and the most common substitution is that methionine at position 154 is replaced by leucine.

[0003] blaNDM is a plasmid-borne carbapenem resistance gene that can spread widely in various pathogens, bringing great difficulties to clinical anti-infection treatment. Since the first strain producing NDM-1 enzyme was discovered in 2009, strains producing NDM-type enzymes have rapidly spread to other regions and have been found and reported all over the world. The NDM-type enzyme genes with higher detection rates in the isolated bacteria prevalent in China and the world are mainly NDM-1 and NDM-5. Strains carrying blaNDM-1 gene often carry other antibacterial drug-related resistance genes such as beta-lactamases, quinolones and aminoglycosides, resulting in only a few antibacterial drugs such as polymyxin and tigecycline having certain antibacterial effect on such bacteria, and such pathogenic bacteria are therefore called "super bacteria". NDM-5 differs from NDM-1 by only 2 amino acids, with the 262nd amino acid changed from G to T and the 460th amino acid changed from A to C. Compared with NDM-1, NDM-5 has a higher resistance level to carbapenems and broad-spectrum beta-lactam antibacterial drugs.

[0004] In 2010, the Ministry of Health of China issued the 'Guidelines for the Diagnosis and Treatment of NDM-1 Producing Enterobacteriaceae Infection (Trial Version)', which requires that the laboratory diagnosis of superbug NDM-1 should include three steps of phenotype screening, phenotype confirmation and gene confirmation. At the same time, the Center for Disease Control (CDC) of the United States recommends using phenotypic screening and phenotypic confirmation methods to identify NDM-1 resistant bacteria. In addition, the M100 Antimicrobial Susceptibility Test Performance Standards (2020 edition) issued by the American Clinical and Laboratory Standardization Association (CLSI) and the Drug Resistance Mechanism Detection (2017 edition) issued by the European Union (EUCAST) also mainly use phenotypic screening for the detection and identification of carbapenem-resistant superbug NDM-1. Phenotypic screening has the advantages of accurate results and good reproducibility, but the detection process requires the cultivation of microorganisms, which takes a long time and greatly reduces the detection efficiency. With the rapid development of molecular biology and gene sequencing technology, visual rapid detection based on nucleic acid amplification technology and metagenomic sequencing have also been widely used in the emergency detection and field monitoring of NDM-resistant bacteria. These methods do not require the cultivation of pathogens, have high accuracy, but are complex to operate and require technical personnel with professional backgrounds. PCR detection requires specialized laboratory equipment, and workers need to obtain detection qualifications, which increases the cost of individual detection. The accurate detection of NDMase-producing bacteria is still a great challenge for clinical laboratory tests.

[0005] Monoclonal antibodies are highly uniform antibodies produced by a single B cell clone, only targeting specific antigen epitopes. They are usually prepared using hybridoma cells, based on cell fusion technology, which fuses sensitized B cells with specific antibody secretion ability and myeloma cells with unlimited reproduction ability into B cell hybridomas. After culturing the cell population, specific antibodies against a specific antigen epitope, i.e. monoclonal antibodies, can be prepared. The purpose of specific antibody detection is to assist clinical diagnosis, and in some diseases, it is also an indicator for observing efficacy and prognosis. In drug resistance and infectious disease epidemiological investigations, the detection of specific antibodies also has special and important significance. Antibody immunological detection has the following advantages: high specificity, specific monoclonal antibodies can be used for single cytokine detection; simple and fast operation, no need to rely on cell lines, so no need to maintain culture, increased operability, easy to promote and convenient for screening; relatively few and easily controlled influencing factors, good reproducibility, and easy standardization of methods.

[0006] NDM can hydrolyze almost all beta-lactam antibiotics, including the most effective carbapenem antibiotics, which brings great challenges to clinical treatment and hospital infection prevention and control. The existing NDM detection methods have poor specificity and long time consumption, which delays the diagnosis and treatment of patients. CN114316056A discloses a mouse anti-NDM enzyme hybridoma cell strain, a monoclonal antibody and application thereof; CN113150137A discloses a preparation method and application of NDM-1 monoclonal antibody, but the above-mentioned monoclonal antibody is a mouse monoclonal antibody, and is a monoclonal antibody directly obtained by animal immunization. Although the mouse monoclonal antibody belongs to the most widely used antibody, there are still problems of weak affinity and poor specificity.

[0007] Therefore, the present application is proposed. SUMMARY

[0008] The first object of the present application is to provide a binding protein of New Delhi metallo-beta-lactamase, which has the characteristics of good specificity and high affinity, so as to solve the problems of mouse monoclonal antibody in practical application, and provide a new scheme for establishing detection, diagnosis, prevention and treatment of NDM enzyme.

[0009] The second object of the present application is to provide the application of the above-mentioned binding protein in preparing New Delhi metallo-beta-lactamase detection products.

[0010] The third object of the present application is to provide a colloidal gold immunochromatography test strip for detecting New Delhi metallo-beta-lactamase.

[0011] The fourth object of the present application is to provide the application of the above-mentioned binding protein in preparing New Delhi metallo-beta-lactamase purification products.

[0012] The fifth object of the present application is to provide a kit for purifying New Delhi metallo-beta-lactamase.

[0013] The sixth object of the present application is to provide a nucleic acid encoding the above-mentioned binding protein.

[0014] The seventh object of the present application is to provide a biological material.

[0015] The eighth object of the present application is to provide a preparation method of the above-mentioned binding protein.

[0016] In the first aspect, the present application provides a binding protein of New Delhi metallo-beta-lactamase, which comprises variable region A or variable region B.

[0017] The variable region A comprises: a complementarity determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO. 5, and a complementarity determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO. 6;

[0018] The variable region B comprises: a complementarity determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO. 7, a complementarity determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO. 8, a complementarity determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO. 9, a complementarity determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO. 10, a complementarity determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO. 11, and a complementarity determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO. 12.

[0019] As a further technical solution, the variable region A comprises a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 13 and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 14.

[0020] As a further technical solution, the variable region B comprises a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 15 and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 16.

[0021] In a second aspect, the application provides application of the above-mentioned binding protein in preparation of a New Delhi metallo-beta-lactamase detection product.

[0022] In a third aspect, the application provides a colloidal gold immunochromatography test strip for detecting New Delhi metallo-beta-lactamase, comprising the binding protein.

[0023] In a fourth aspect, the application provides application of the above-mentioned binding protein in preparation of a New Delhi metallo-beta-lactamase purification product.

[0024] In a fifth aspect, the application provides a kit for purifying New Delhi metallo-beta-lactamase, comprising the binding protein.

[0025] In a sixth aspect, the present application provides a nucleic acid encoding the binding protein, the nucleic acid having a nucleic acid sequence as shown in SEQ ID NO. 17 and SEQ ID NO. 18.

[0026] Alternatively, the nucleic acid has a nucleic acid sequence as shown in SEQ ID NO. 19 and SEQ ID NO. 20.

[0027] In a seventh aspect, the present application provides a biological material selected from one or more of the following (n1) to (n3):

[0028] (n1) a vector containing the nucleic acid;

[0029] (n2) a recombinant microorganism containing the nucleic acid, or a recombinant microorganism containing the vector of (n1);

[0030] (n3) a cell line containing the nucleic acid, or a cell line containing the vector of (n1).

[0031] In an eighth aspect, the present application provides a preparation method of the binding protein, comprising: culturing the recombinant microorganism or cell line in a culture medium, and then isolating the binding protein.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The binding protein of New Delhi metallo-beta-lactamase provided by the present application has good specificity, high biological activity, strong stability, small batch difference, is not affected by cell strain degradation, has high affinity with New Delhi metallo-beta-lactamase, and has a titer of more than 1:1280000, and can be used for New Delhi metallo-beta-lactamase detection or preparation of purified product. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0035] Figure 1 SDS-PAGE electrophoresis chart provided for Example 7;

[0036] Figure 2 NDM antibody titer detection result provided for Example 8;

[0037] Figure 3 Cross reaction result of antibody 1 provided for Example 9;

[0038] Figure 4 Cross-reactivity results for antibody 2 provided for Example 9. DETAILED DESCRIPTION

[0039] The embodiments of the present application will be described in detail based on the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts are within the scope of the present application. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0040] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The variable region of the light chain or the heavy chain is composed of framework regions interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework region of the antibody, i.e. the framework region constituting the combination of the light chain and the heavy chain, plays a role in positioning and aligning the CDRs, which are mainly responsible for binding to the antigen.

[0041] "Framework" or "FR" region means the region of an antibody variable domain that is outside the regions defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0042] Generally, the variable regions of the heavy chain and the light chain VL / VH can be obtained by connecting the CDRs and FRs numbered as follows in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0043] In the present application, CDR1-VH, CDR2-VH, and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively, and correspondingly, CDR1-VL, CDR2-VL, and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

[0044] In a first aspect, the present application provides a binding protein of New Delhi metallo-beta-lactamase, which comprises variable region A or variable region B;

[0045] The variable region A comprises: a complementarity determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO. 5, and a complementarity determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO. 6.

[0046] The sequences of the variable region A are shown in Table 1:

[0047] Table 1

[0048] CDR1-VH SYSMN SEQ ID NO. 1 CDR2-VH SISASSSYIFYADSLKG SEQ ID NO. 2 CDR3-VH ERTVYTGRYSRLYYYGLDV SEQ ID NO. 3 CDR1-VL RAGQSVGSNLA SEQ ID NO. 4 CDR2-VL GASTKAT SEQ ID NO. 5 CDR3-VL QQYDNWPPT SEQ ID NO. 6

[0049] The variable region B comprises: a complementarity determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO. 7, a complementarity determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO. 8, a complementarity determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO. 9, a complementarity determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO. 10, a complementarity determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO. 11, and a complementarity determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO. 12.

[0050] The sequences of the variable region B are shown in Table 2:

[0051] Table 2

[0052] CDR1-VH SGDYYWT SEQ ID NO. 7 CDR2-VH YISHSGGPFYNPSLKS SEQ ID NO. 8 CDR3-VH DLDYDVGHNYYYGMDV SEQ ID NO. 9 CDR1-VL RASQSVTNGLLA SEQ ID NO. 10 CDR2-VL GPSNRAT SEQ ID NO. 11 CDR3-VL QQYGSSPRT SEQ ID NO. 12

[0053] The binding protein of the New Delhi metallo-beta-lactamase provided by the application has good specificity, high biological activity, strong stability, small batch difference, is not affected by cell strain degradation, has high affinity with the New Delhi metallo-beta-lactamase, the titer reaches more than 1:1280000, and can be used for New Delhi metallo-beta-lactamase detection or preparation of purified product.

[0054] In some preferred embodiments, the variable region A comprises a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 13 and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 14.

[0055] The amino acid sequence of the heavy chain variable region VH of the variable region A is as follows: The amino acid sequence of the heavy chain variable region VH of the variable region A is as follows:

[0056] EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYSMNSVRQAPEKGLEWVASISASSSYI FYADSLKGRFTISFDNAKNTLFLQDTSLRSEDTAMYYCARERTVYTGRYSRLYY YGLDVWGQGTTVTVSS (SEQ ID NO. 13).

[0057] The amino acid sequence of the light chain variable region VL of the variable region A is as follows:

[0058] DIVMTHATASLSFSLGTTATLSCRAGQSVGSNLAWTQQKAEQVPRGLIHGASTKATG VPVRFRYSGSGTDFTLTISGLEHEDAALYYCQQYDNWPPTFGQGTKVEIK (SEQ ID NO. 14).

[0059] The variable region B comprises a heavy chain variable region VH having an amino acid sequence as set forth in SEQ ID NO. 15 and a light chain variable region VL having an amino acid sequence as set forth in SEQ ID NO. 16.

[0060] The amino acid sequence of the heavy chain variable region VH of the variable region B is as follows:

[0061] QVQVQESGPGLVKPSGSLFLVCSITGFPITSGDYYWTWIRQLPGKPLEWMGYISH SGGPFYNPSLKSPISTTREAAKNQFFLQLNSVTTQDAAMYYCAGDLDYDVGHN YYYGMDVWGQGTTVTVSS (SEQ ID NO. 15).

[0062] The amino acid sequence of the light chain variable region VL of the variable region B is as follows:

[0063] DISLTQAPASLSFSLGETATLPCRASQSVTNGLLAWVQQKAEQVPRLLIHGPSN RATGFPVRFSGTGSGTDFTLTISSLEPEDAAVYFCQQYGSSPRTFGQGTKVEIK (SEQ ID NO. 16).

[0064] In some preferred embodiments, the binding protein further comprises a light chain constant region and a heavy chain constant region. The constant region, in combination with the variable region, results in a complete antibody.

[0065] In a second aspect, the present application provides use of the above binding protein in the preparation of a New Delhi metallo-beta-lactamase detection product.

[0066] The binding protein provided by the application has high specificity, high biological activity, high stability, and high affinity with New Delhi metallo-beta-lactamase, and can be used for preparing New Delhi metallo-beta-lactamase detection products.

[0067] In a third aspect, the application provides a colloidal gold immunochromatography test strip for detecting New Delhi metallo-beta-lactamase, comprising the binding protein.

[0068] The colloidal gold immunochromatography test strip may be, for example, a double-antibody sandwich colloidal gold immunochromatography test strip in which a colloidal gold-labeled anti-NDMase monoclonal antibody I is embedded on a sample binding pad, an anti-NDMase monoclonal antibody II and a goat anti-mouse antibody are coated on a test line (T) and a control line (C), respectively. If the detection sample is positive, the NDMase in the sample binds with the fluorescent microsphere-labeled NDMase antibody I to form a complex, which moves along the test strip under chromatography, and is captured by the pre-coated NDMase antibody II on the test line (T) to form an immune complex and present a red band. If the detection sample is negative, no immune complex is formed, and no band appears on the test line. The colloidal gold-labeled NDMase antibody I passes through the control line (C) and is captured, and a band should appear. A quantitative dropper is used to add the bacterial suspension to the sample well of the test strip, and the detection result is judged by naked eye after 15 minutes, and the drug resistance of NDMase can be output.

[0069] In a fourth aspect, the application provides use of the binding protein described above in preparing a New Delhi metallo-beta-lactamase purification product.

[0070] The binding protein provided by the application has high specificity, high biological activity, high stability, and high affinity with New Delhi metallo-beta-lactamase, and can be used for preparing a New Delhi metallo-beta-lactamase purification product.

[0071] In a fifth aspect, the application provides a kit for purifying New Delhi metallo-beta-lactamase, comprising the binding protein.

[0072] In a sixth aspect, the application provides a nucleic acid encoding the binding protein, which has a nucleic acid sequence as shown in SEQ ID NO. 17 and SEQ ID NO. 18, and can express a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 13 and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 14.

[0073] The nucleotide sequence is as follows:

[0074] GAGGTGCAGCTGGTGGAGTCTTCCGGCGGATTAGTGAAGCCTGGCGGCTCCCTGAAACTCTCCTGTGCCGCATCTGGGTTCACTTTCAGTAGCTATAGCATGAACTCTGTTCGTCAGGCTCCAGAGAAAGGCCTGGAGTGGGTTGCATCCATTAGTGCTAGTAGTAGTTACATATTCTACGCAGACTCACTGAAGGGCCGATTCACCATCTCCTTCGACAATGCCAAGAACACCCTGTTCCTGCAAGACACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGAGCGGACGGTATATACTGGGCGCTACTCCCGACTCTACTACTACGGTTTGGACGTCTGGGGACAAGGAACGACGGTCACCGTCTCATCA (SEQ ID NO. 17).

[0075] GATATTGTGATGACACACGCTACAGCTAGTCTGAGTTTTTCTCTTGGAACAACAGCAACACTGTCATGCAGGGCCGGTCAGAGTGTTGGCAGCAACTTAGCCTGGACACAGCAGAAAGCAGAGCAAGTTCCCCGGGGTCTTATCCATGGTGCGTCCACCAAGGCCACTGGAGTCCCAGTCCGGTTCAGATACTCTGGCTCTGGAACAGACTTCACTCTCACCATCAGCGGTCTAGAACATGAAGATGCTGCACTTTACTACTGTCAGCAGTATGATAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO. 18).

[0076] Alternatively, the nucleic acid has a nucleic acid sequence as shown in SEQ ID NO. 19 and SEQ ID NO. 20, and is capable of expressing a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 15 and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 16.

[0077] The nucleotide sequence is as follows:

[0078] CAGGTCCAGGTGCAGGAGTCAGGCCCTGGCCTGGTGAAACCCTCAGGGTCACTCTTCCTCGTCTGCTCTATTACTGGATTCCCCATCACCAGTGGCGATTACTACTGGACCTGGATCCGTCAGCTCCCTGGGAAACCACTAGAATGGATGGGCTACATCTCTCACAGTGGGGGACCCTTCTATAATCCGTCCCTCAAGAGCCCCATCTCCACTACTAGAGAAGCAGCCAAGAACCAGTTCTTTCTGCAATTGAACTCTGTGACCACACAGGACGCAGCCATGTATTACTGTGCAGGAGATCTCGACTATGATGTTGGTCACAACTACTACTACGGCATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCATCA (SEQ ID NO. 19).

[0079] GATATTAGTCTAACACAGGCTCCAGCTTCTCTGAGTTTTTCTCTTGGTGAAACAGCAACACTGCCCTGCAGGGCCAGTCAGAGTGTTACCAACGGTTTGTTAGCCTGGGTCCAGCAGAAAGCAGAGCAAGTTCCCCGGCTCCTTATCCATGGTCCGTCCAACAGGGCCACTGGTTTCCCAGTCCGGTTCAGTGGCACTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTAGAACCTGAAGATGCTGCAGTTTACTTCTGTCAGCAATATGGGAGCTCACCCCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO. 20).

[0080] In a seventh aspect, the present application provides a biological material containing a nucleic acid, which includes, but is not limited to, a vector, a recombinant microorganism, and a cell line. The "vector" refers to a substance capable of achieving replication and / or expression of a target gene, and introducing the target gene into a prokaryotic or eukaryotic cell, which includes, but is not limited to, a plasmid, a bacteriophage, a viral genome, or a virus, etc. The "recombinant microorganism" refers to a microorganism that, after being manipulated, exhibits a genotype or phenotype different from that of the wild type of the microorganism.

[0081] Specifically, the biological material is selected from the following: (n1) a vector containing the nucleic acid; (n2) a recombinant microorganism containing the nucleic acid, or a recombinant microorganism containing the vector of (n1); (n3) a cell line containing the nucleic acid, or a cell line containing the vector of (n1).

[0082] In an eighth aspect, the present application provides a preparation method of the above-mentioned binding protein, comprising: culturing the recombinant microorganism or cell line in a culture medium, and then isolating the binding protein.

[0083] Since the recombinant microorganism or cell line can express the above-mentioned binding protein, culturing the recombinant microorganism or cell line can obtain a fermentation broth rich in the binding protein, and the binding protein can be obtained after isolation. The preparation method is simple and convenient.

[0084] The present application will be further described below through specific examples and comparative examples, but it should be understood that these examples are only for more detailed description and should not be understood as limiting the present application in any form.

[0085] Example 1: Preparation of antigen

[0086] The present application obtains the conserved sequence of NDM enzyme (NDM1-15, NDM17-31) through NCBI (National Center for Biotechnology Information, National Center for Biotechnology Information) sequence alignment. The conventional enzyme cutting and ligation technology in the field of molecular biology is used to construct the expression plasmid pET-28a(+)-PM, and the CaCl2 heat shock method is used to transform the recombinant vector into the E. coli DH5a competent cells. The positive clones are selected by using LB medium containing 100 μg / mL ampicillin. The E. coli is cultured conventionally, the plasmid is extracted for PCR identification to determine the presence of the target gene. After the extracted expression plasmid pET-28a(+)-PMAA is transformed into the E. coli BL21(DE3) competent cells, it is cultured on the selective medium, the single colony resistant to 100 μg / mL ampicillin is selected, and then liquid culture is carried out overnight. 1 mL of the overnight culture is inoculated into 200 mL of LB medium containing 100 μg / mL ampicillin, and shaken and cultured until the logarithmic phase (OD600 is 0.5-0.6). IPTG (1 mmol / L) is added, and the culture is induced at 16°C for 3 h. The fermentation broth is purified by nickel column, and high-purity protein is obtained by prokaryotic gene expression.

[0087] Example 2: Animal immunization

[0088] Select the age, weight of about 1.5 kg New Zealand white rabbits, in standard animal house for 3 days, if no abnormal conditions, then start the immune: 100 μg NDM enzyme antigen added to 0.5 mL autoclaved saline, with a mini vortex mixer mix well, add 0.5 mL Freund's complete adjuvant, with a syringe each other push and pull for emulsification, subcutaneous injection of New Zealand white rabbits back multiple point immunization; two weeks later, booster immunization, then every other week booster immunization, a total of six times, and from the third immunization, immune one week after taking the white rabbit ear vein blood 200-500 μL, determine the titer and affinity; after the last immunization, take the spleen for cell fusion, for the preparation of hybridoma cells.

[0089] Example 3: preparation and screening of hybridoma cells

[0090] The prepared rabbit antiserum was tested for titer, and if qualified, the rabbit spleen was used for cell fusion to prepare a monoclonal hybridoma cell line, as follows: the immunized New Zealand white rabbits were sacrificed, the spleen was removed under sterile conditions, washed once with cell culture solution, then ground, the obtained cells were centrifuged and washed twice with cell culture solution; logarithmic growth phase SP2 / 0 myeloma cells were mixed with spleen cells, washed once with cell culture solution without fetal bovine serum, centrifuged, and the supernatant was discarded, polyethylene glycol solution was added, and the mixture was treated at 37°C for about 90 seconds; the reaction was terminated with cell culture solution without fetal bovine serum, centrifuged, and the cells were resuspended in HAT selection medium containing 20% fetal bovine serum, then added to a 96-well plate and cultured at 37°C, 5.0% CO2; the cells in the 96-well plate that grew well were diluted to 1-3 cells / mL with cell culture solution, added to a 96-well plate, and cultured at 37°C, 5.0% CO2; each cell strain was numbered, and the cell strain with positive supernatant was selected for expansion culture, and finally a hybridoma cell strain was obtained. The obtained hybridoma cells were screened by ELISA, the growth of the cells was observed on the 5th day after fusion, the titer of the cell culture supernatant was detected by indirect ELISA on the 10th to 14th day, the positive hybridoma cells with the strongest titer were expanded to a cell positive rate of 100%, the hybridoma cell strain was obtained, and stored in liquid nitrogen for use.

[0091] Example 4: isolation of antibody variable region genes from hybridoma cells by RT-PCR

[0092] After the hybridoma cells are homogenized, cell lysate is added for RNA extraction, and isopropanol is added to precipitate RNA from the water phase layer. After centrifugation, the precipitated RNA is washed to remove impurities, resuspended, and then reverse transcribed to obtain cDNA. The specific primers of New Zealand rabbits are used for PCR, with the cDNA of the hybridoma cells as the template, to amplify the heavy and light chain variable region genes of the antibody. The 50 μL system contains 5 μL cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM specific primers. The PCR amplification is performed according to the following conditions: pre-denaturation at 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 50 s, 35 cycles; 72°C for 7 min. The obtained PCR product is identified by 1% agarose gel electrophoresis, the target fragment is recovered, and sequencing is performed. The sequencing results are compared with the IMGT database (http: / / www.imgt.org / IMGT_vquest / vquest) to obtain the antibody variable region gene fragment.

[0093] Example 5: Construction, expression, and purification of the monoclonal antibody

[0094] A signal peptide is introduced at the N-terminus of the antibody heavy chain variable region gene and the N-terminus of the light chain variable region gene. Then, homologous recombination primers are used to add homologous recombination arms at both ends of the antibody heavy chain variable region gene and both ends of the light chain variable region gene, respectively. Double enzymes are used to linearize the expression plasmid containing the rabbit antibody heavy and light chain IgG1 constant region to generate homologous recombination arms. The variable region gene fragment with the added homologous recombination arms and the linearized plasmid are connected by homologous recombination to form a complete expression vector. The recombination product is transformed into TOP10 E. coli competent cells, and the plasmid is amplified.

[0095] The obtained monoclonal antibody heavy and light chain expression plasmids were added into Opti-Mem transfection medium at a ratio of 1:1, mixed well, then 4 times the mass of transfection reagent PEI was added, mixed, and then placed at room temperature for 30 min in the dark, and then added into 293T cells; after incubation for 6 h, the transfection system was removed, FreeStyleTM293 expression medium was added, and the AKTA protein purification system was used to purify the expressed antibody supernatant by affinity purification (Protein A) to obtain a monoclonal antibody against NDM enzyme. The specific steps are as follows: (1) centrifuge the expressed antibody supernatant at 2500 x g at room temperature for 10 min to remove the precipitate; (2) wash the affinity purification column containing Protein A with 10 times the volume of binding buffer (Binding Buffer) to wash thoroughly; (3) pass the expression supernatant through the purification column at a flow rate of 5 mL / min; (4) wash the purification column with 20 times the volume of the purification column with binding buffer (Binding Buffer); (5) elute the purification column with 0.1 M citric acid buffer at pH 3.0-3.5 until the elution peak drops to the equilibrium state, and adjust the pH to 7.0 with 1 M Tris-HCl buffer at pH 9.0; (6) use a concentration centrifugal column to concentrate the purified monoclonal antibody, use PBS as the antibody storage buffer, and finally use the BSA protein concentration detection method to determine the concentration of the concentrated antibody.

[0096] Through the above method, monoclonal antibody 1 (referred to as antibody 1) and monoclonal antibody 2 (referred to as antibody 2) were finally obtained. The amino acid sequences of the heavy chain variable region and the light chain variable region of antibody 1 are as follows:

[0097] Signal peptide-VH:

[0098] MDWTWRFLFVVAAATGVQSEVQLVESSGGLVKPGGSLKLSCAASGFTFSSYSMNSVRQAPEKGLEWVASISASSSYIFYADSLKGRFTISFDNAKNTLFLQDTSLRSEDTAMYYCARERTVYTGRYSRLYYYGLDVWGQGTTVTVSS (SEQ ID NO. 21).

[0099] Signal peptide-VL:

[0100] MDMRVPAQLLGLLLLWLSGARCDIVMTHATASLSFSLGTTATLSCRAGQSVGSNLAWTQQKAEQVPRGLIHGASTKATGVPVRFRYSGSGTDFTLTISGLEHEDAALYYCQQYDNWPPTFGQGTKVEIK (SEQ ID NO. 22).

[0101] The amino acid sequences of the heavy chain variable region and the light chain variable region of antibody 2 are as follows:

[0102] Signal peptide-VH:

[0103] MDWTWRFLFVVAAATGVQSQVQVQESGPGLVKPSGSLFLVCSITGFPITSGDYYWTWIRQLPGKPLEWMGYISHSGP FYNPSLKSPISTTREAAKNQFFLQLNSVTTQDAAMYYCAGDLDYDVGHNYYYGMDVWGQGTTVTVSS (SEQ ID NO. 23).

[0104] Signal peptide-VL:

[0105] MDMRVPAQLLGLLLLWLSGARCDISLTQAPASLSFSLGETATLPCRASQSVTNGLLAWVQQKAEQVPRLLIHGPSNRATGFPVRFSGTGSGTDFTLTISSLEPEDAAVYFCQQYGSSPRTFGQGTKVEIK (SEQ ID NO. 24).

[0106] Example 6: Determination of molecular weight

[0107] The molecular weight of the monoclonal antibody was identified by SDS-PAGE electrophoresis, 5 μg of sample was added to each electrophoresis lane, and a known molecular weight standard series was used as a reference, first electrophoresis at 90 V for 20 min, and then electrophoresis at 140 V until the indicator was completely run out, the gel was taken out, stained with coomassie brilliant blue, and the molecular weight of the biological raw material was analyzed after staining. The SDS-PAGE electrophoresis map is shown in Figure 1 (Figure, the first column is marker, the second column is antibody 1, and the third column is antibody 2).

[0108] Example 7: Determination of titer

[0109] The main steps are as follows: (1) the NDM enzyme antigen is diluted with PBS to 1 ng / μL, and 100 μL per well is added to the 96-well enzyme-labeled plate, and coated at 37°C for 2 h; (2) the supernatant is discarded, and the plate is washed with 0.01M PBST for 3 times, and 100 μL of PBST containing 3% BSA is added to each well, and blocked at 37°C for 2 h; (3) the supernatant is discarded, and the plate is washed with PBST for 5 times, and the purified and concentrated antibody is gradiently diluted, and the concentration is diluted from 1:1000 times to 1:2560000, and 100 μL per well is added, and incubated at 37°C for 1 h; (4) the antibody diluent is discarded, and the plate is washed with PBST for 6 times, and the goat anti-rabbit IgG-HRP is diluted with 1:5000 blocking solution, and 100 μL per well is added, and incubated at 37°C for 1 h; (5) the secondary antibody diluent is discarded, and the plate is washed with PBST for 6 times, and TMB is added, 100 μL per well, and incubated at 37°C for 15 min in the dark; (6) 50 μL of 1M dilute sulfuric acid is added to each well to terminate the reaction, and the absorbance is measured at 450 nm. The results are shown in Figure 2 As shown in (1# is antibody 1, and 2# is antibody 2), the screened monoclonal antibody has strong binding capacity to NDM enzyme, and the titer of NDM enzyme antigen reaches 1:1280000 (OD value>0.5).

[0110] Example 8: Comparison with existing antibodies

[0111] The affinity activity (titer) of the antibodies obtained by the application, the natural antibodies and the published mouse monoclonal antibodies (purchased from Zhuhai Bomei Biotechnology Co., Ltd.) to NDM enzyme is detected by ELISA method, and the specific steps are as in Example 7. The results are shown in Table 3, which shows that the monoclonal antibody in the application has increased affinity and enhanced biological activity compared with the prior art.

[0112] Table 3

[0113]

[0114] Note: 1# is antibody 1, and 2# is antibody 2.

[0115] Example 9: Cross reaction

[0116] KPC, NDM, VIM, IMP and OXA-48 enzymes are used to coat the enzyme-labeled plate, and the coating amount per well is 50 ng; the monoclonal antibody is diluted to 10 ng / mL, and added to each enzyme-labeled plate, 100 μL per well, and incubated at 37°C for 1 h; after washing, HRP-labeled goat anti-rabbit secondary antibody is added, 100 μL per well, and incubated at 37°C for 0.5 h; after washing, TMB is added, and incubated at 37°C for 15 min, and the reading is terminated. The results are shown in Figure 3 and Figure 4As shown, it is indicated that the monoclonal antibody does not cross-react with other types of carbapenamases, and is highly specific.

[0117] Example 10: Antibody Pairing Verification

[0118] Two antibodies, one as a capture antibody and the other as a labeled antibody (HRP enzyme labeled), and the capture antibody is also HRP enzyme labeled as a control group. The capture antibody is coated on the antigen plate, and then the antigen is added in a multiple dilution, and after incubation, the unbound antigen is washed away. Then the labeled antibody is added for incubation, and after incubation, the unbound labeled antibody is washed away. Finally, the color developing solution is added for color development. If color development can be achieved, it indicates that the labeled antibody is specifically combined with the antigen, and the capture antibody and the labeled antibody are a pair of matched antibodies. If color development cannot be achieved, it indicates that the labeled antibody cannot be combined with the antigen, and thus is washed away. The capture antibody and the labeled antibody are not matched antibodies. The results are shown in Table 4, indicating that the two antibodies have the best ability to combine with the antigen.

[0119] Table 4

[0120]

[0121] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An antibody to a New Delhi metallo-β-lactamase, characterized in that, The antibody comprises a variable region A or a variable region B; The variable region A comprises a complementarity determining region CDR1-VH of an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH of an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH of an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL of an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL of an amino acid sequence as shown in SEQ ID NO. 5, and a complementarity determining region CDR3-VL of an amino acid sequence as shown in SEQ ID NO. 6; The variable region B comprises a complementarity determining region CDR1-VH of an amino acid sequence as shown in SEQ ID NO. 7, a complementarity determining region CDR2-VH of an amino acid sequence as shown in SEQ ID NO. 8, a complementarity determining region CDR3-VH of an amino acid sequence as shown in SEQ ID NO. 9, a complementarity determining region CDR1-VL of an amino acid sequence as shown in SEQ ID NO. 10, a complementarity determining region CDR2-VL of an amino acid sequence as shown in SEQ ID NO. 11, and a complementarity determining region CDR3-VL of an amino acid sequence as shown in SEQ ID NO.

12.

2. The antibody according to claim 1, characterized in that, The variable region A comprises a heavy chain variable region VH of an amino acid sequence as shown in SEQ ID NO. 13 and a light chain variable region VL of an amino acid sequence as shown in SEQ ID NO.

14.

3. The antibody according to claim 1, characterized in that, The variable region B comprises a heavy chain variable region VH of an amino acid sequence as shown in SEQ ID NO. 15 and a light chain variable region VL of an amino acid sequence as shown in SEQ ID NO.

16.

4. Use of the antibody of any one of claims 1-3 in the preparation of a New Delhi metallo-beta-lactamase detection product.

5. A colloidal gold immunochromatographic test strip for detecting New Delhi metallo-β-lactamase, characterized in that, The antibody of any one of claims 1-3.

6. Use of the antibody of any one of claims 1-3 in the preparation of a New Delhi metallo-beta-lactamase purification product.

7. A kit for purifying New Delhi metallo-β-lactamase, characterized by, The kit comprises the antibody of any one of claims 1-3.

8. A nucleic acid encoding the antibody of any one of claims 1-3. The nucleic acid has a nucleic acid sequence as shown in SEQ ID NO. 17 and SEQ ID NO. 18; Alternatively, the nucleic acid has a nucleic acid sequence as shown in SEQ ID NO. 19 and SEQ ID NO.

20.

9. A biomaterial, characterized in that, selected from one or more of the following (n1)~(n3): (n1) a vector containing the nucleic acid of claim 8; (n2) a recombinant microorganism containing the nucleic acid of claim 8, or a recombinant microorganism containing the vector of (n1); (n3) a cell line containing the nucleic acid of claim 8, or a cell line containing the vector of (n1).

10. A method of producing the antibody as claimed in any one of claims 1 to 3, characterized in that, comprises: culturing the recombinant microorganism or cell line described in claim 9 in a culture medium, and then isolating the antibody.

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