CN115724980A

CN115724980A - Binding protein of new Delhi metallo beta-lactamase and application and product thereof

Info

Publication number: CN115724980A
Application number: CN202211186237.4A
Authority: CN
Inventors: 刘春龙; 付成华; 粟艳; 周泽奇
Original assignee: Dana Hunan Biotechnology Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dana Hunan Biotechnology Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-03-03

Abstract

The invention provides a binding protein of new Delhi metallo beta-lactamase and application and a product thereof, relating to the technical field of biology. The binding protein of the new Delhi metallo-beta-lactamase provided by the invention has the advantages of good specificity, high biological activity, strong stability, small batch-to-batch difference, no influence by cell strain degradation, high affinity with the new Delhi metallo-beta-lactamase, titer reaching more than 1.

Description

Binding protein of new Delhi metallo beta-lactamase and application and product thereof

Technical Field

The invention relates to the technical field of biology, in particular to a binding protein of new Delhi metallo beta-lactamase and application and a product thereof.

Background

New Delhi metallo beta-lactamase (NDM) belongs to B-type metalloenzyme in carbapenemase, and can be used for removing beta-lactam antibacterial drugs except aztreonam by water. Bacteria containing the enzyme are resistant to almost all antibacterial drugs, and beta-lactamase inhibitors such as sulbactam, clavulanic acid and the like can not effectively inhibit the metalloenzyme. NDM has been reported since, NDM-producing drug-resistant bacteria have been widely prevalent around the world, NDM is rapidly evolving, and 24 NDM-1 mutant subtypes that differ from each other at one or more residues at different positions have been reported. The substitution of various amino acids in the mutant subtype of NDM affects the stability and activity of the enzyme, giving it a selective advantage in the evolution of resistance in bacteria. In addition to the location of the NDM-2 gene and the NDM-13 gene on the chromosome, other mutant subtype-encoding genes are mainly mediated by plasmids. NDM-2, 3, 4, 6, 9, 11, 14, 22, 23 and 24 differ from NDM-1 by a single amino acid substitution, while the remaining mutant subtypes have multiple substitutions, with the most common substitution being a substitution of methionine at position 154 with leucine.

The bla NDM is a carbapenem drug-resistant gene carried by a plasmid, can be widely spread in various pathogens and brings great difficulty to clinical anti-infective treatment. Since the first discovery of NDM-1 enzyme-producing strains in 2009, NDM-type enzyme-producing strains have rapidly spread to other regions, and have been discovered and reported all over the world. The NDM type enzyme genes with high detection rate in the separating bacteria popular in China and the world are mainly NDM-1 and NDM-5. Strains carrying blaNDM-1 genes often carry drug-resistant genes related to other antibacterial drugs such as beta lactamases, quinolones, aminoglycosides and the like, so that only a few antibacterial drugs such as polymyxin, tigecycline and the like have certain antibacterial effects on the bacteria, and the pathogenic bacteria are also called as 'super bacteria'. NDM-5 differs from NDM-1 by only 2 amino acids, the amino acid at position 262 being changed from G to T and the amino acid at position 460 being changed from A to C. Compared with NDM-1, NDM-5, the antibacterial composition has higher drug resistance level to carbapenems and broad-spectrum beta-lactam antibacterial drugs.

The clinical guidelines for infection diagnosis and treatment of bacteria of the family of drug-resistant Enterobacteriaceae, NDM-1 produced by Ministry of health in China (trial edition) were published in 2010, and laboratory diagnosis of the superbacteria NDM-1 should include 3 steps of phenotype screening, phenotype confirmation and gene confirmation. Meanwhile, the Center for Disease Control (CDC) recommends the identification of NDM-1 resistant bacteria using phenotypic screening and phenotypic confirmation. In addition, the detection and identification of carbapenem-resistant superbacterial NDM-1 by the M100 antimicrobial drug susceptibility test execution Standard (2020 edition) issued by the American clinical and laboratory standardization institute CLSI and the drug resistance mechanism test (2017 edition) issued by the EUCAST of the European Union are also dominated by phenotypic screening. Phenotype screening has the advantages of accurate result, good repeatability and the like, but the detection process needs to culture microorganisms, the time consumption is quite long, and the detection efficiency is greatly reduced. With the rapid development of molecular biology and gene sequencing technologies, visual rapid detection and metagenome sequencing based on a nucleic acid amplification technology are also widely applied to emergency detection and field monitoring of NDM drug-resistant bacteria, the methods do not need to culture pathogens, the result accuracy is high, but the operation is complex, technical personnel with professional background are needed, PCR detection needs to be provided with special experimental instruments, workers need to obtain detection qualification, and the single detection cost is high. Accurate detection of NDM enzyme-producing bacteria remains a significant challenge for clinical laboratory examination.

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone and directed only to a specific epitope, and are generally prepared by using hybridoma cells, and after sensitized B cells having the ability to secrete specific antibodies and myeloma cells having an unlimited reproductive ability are fused into B cell hybridomas based on a cell fusion technique, and cultured into a cell population, specific antibodies directed to one 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 index for observing curative effect and prognosis, and in drug resistance and epidemiological investigation of infectious diseases, the detection of specific antibody also has special and important significance. The immunological detection of antibodies has the following advantages: the specificity is high, and the specific monoclonal antibody is used, so that the kit can be used for detecting a single cytokine; the operation is simple, convenient and quick, and does not depend on cell strains, so that the maintenance culture is not needed, the operability is increased, the popularization is easy, and the general investigation is convenient; the influence factors are relatively few and are easy to control, the repeatability is good, and the method is easy to standardize.

NDM hydrolyzes almost all β -lactam antibiotics, including the most potent carbapenem antibiotics at present, presenting significant challenges for clinical therapy and prevention and control of nosocomial infections. The existing NDM detection method has poor specificity and long time consumption, and delays the diagnosis and treatment of patients. CN114316056A discloses a mouse anti-NDM enzyme hybridoma cell strain, a monoclonal antibody and application; CN113150137A discloses a preparation method and application of NDM-1 monoclonal antibody, but the monoclonal antibody is murine monoclonal antibody and is directly obtained by animal immunization. Although murine monoclonal antibodies are among the most widely used antibodies, they still suffer from poor affinity and poor specificity.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention 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 problem of practical application of mouse-derived monoclonal antibodies and provide a new scheme for establishing detection, diagnosis, prevention and treatment of NDM enzyme.

The second purpose of the invention is to provide the application of the binding protein in preparing a new Delhi metallo beta-lactamase detection product.

The third purpose of the invention is to provide a colloidal gold immunochromatographic test strip for detecting new Delhi metallo-beta-lactamase.

The fourth purpose of the invention is to provide the application of the binding protein in preparing a new Delhi metallo beta-lactamase purified product.

The fifth object of the present invention is to provide a kit for purifying neodrimetal beta-lactamase.

The sixth object of the present invention is to provide a nucleic acid encoding the above-mentioned binding protein.

A seventh object of the present invention is to provide a biomaterial.

The eighth object of the present invention is to provide a method for producing the above-mentioned binding protein.

In a first aspect, the present invention provides a binding protein for a new delrin metallo beta-lactamase, said binding protein comprising a variable region a or a variable region B;

the variable region a includes: a complementarity determining region CDR1-VH having an amino acid sequence shown in SEQ ID NO.1, a complementarity determining region CDR2-VH having an amino acid sequence shown in SEQ ID NO.2, a complementarity determining region CDR3-VH having an amino acid sequence shown in SEQ ID NO.3, a complementarity determining region CDR1-VL having an amino acid sequence shown in SEQ ID NO.4, a complementarity determining region CDR2-VL having an amino acid sequence shown in SEQ ID NO.5, and a complementarity determining region CDR3-VL having an amino acid sequence shown in SEQ ID NO. 6;

the variable region B includes: CDR1-VH having the amino acid sequence shown in SEQ ID NO.7, CDR2-VH having the amino acid sequence shown in SEQ ID NO.8, CDR3-VH having the amino acid sequence shown in SEQ ID NO.9, CDR1-VL having the amino acid sequence shown in SEQ ID NO.10, CDR2-VL having the amino acid sequence shown in SEQ ID NO.11, and CDR3-VL having the amino acid sequence shown in SEQ ID NO. 12.

As a further technical scheme, the variable region A comprises a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO.13 and a light chain variable region VL with an amino acid sequence shown as SEQ ID NO. 14;

as a further technical scheme, the variable region B comprises a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO.15 and a light chain variable region VL with an amino acid sequence shown as SEQ ID NO. 16.

In a second aspect, the invention provides the use of the binding protein in the preparation of a new Dreher metallo beta-lactamase detection product.

In a third aspect, the invention provides a colloidal gold immunochromatographic test strip for detecting neodril metallo-beta-lactamase, which comprises the binding protein.

In a fourth aspect, the invention provides the use of the above binding protein in the preparation of a purified product of neodrime beta-lactamase.

In a fifth aspect, the invention provides a kit for purifying neodrimetallo beta-lactamase, the kit comprising the binding protein.

In a sixth aspect, the invention 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;

or, the nucleic acid has a nucleic acid sequence shown as SEQ ID NO.19 and SEQ ID NO. 20.

In a seventh aspect, the present invention provides a biomaterial selected from one or more of the following (n 1) to (n 3):

(n 1) a vector containing the nucleic acid;

(n 2) a recombinant microorganism containing the nucleic acid, or a recombinant microorganism containing the vector of (n 1);

(n 3) a cell line containing the nucleic acid, or a cell line containing the vector of (n 1).

In an eighth aspect, the present invention provides a method for preparing the above binding protein, comprising: culturing said recombinant microorganism or cell line in a culture medium and isolating said binding protein.

Compared with the prior art, the invention has the following beneficial effects:

the binding protein of the new Delhi metallo-beta-lactamase provided by the invention has the advantages of good specificity, high biological activity, strong stability, small batch difference, no influence from cell strain degradation, high affinity with the new Delhi metallo-beta-lactamase, and high titer reaching more than 1.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an SDS-PAGE electrophoresis image provided in example 7;

FIG. 2 shows the results of the detection of the NDM antibody titer provided in example 8;

FIG. 3 shows the cross-reaction results of antibody 1 provided in example 9;

figure 4 is the cross-reaction results for antibody 2 provided in example 9.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

It is 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 may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region is made up of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

By "framework" or "FR" regions is meant regions of the antibody variable domain that are excluded from those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR 1, FR2, FR3 and FR 4) separated by CDRs.

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

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

the variable region a includes: a complementarity determining region CDR1-VH having an amino acid sequence shown in SEQ ID NO.1, a complementarity determining region CDR2-VH having an amino acid sequence shown in SEQ ID NO.2, a complementarity determining region CDR3-VH having an amino acid sequence shown in SEQ ID NO.3, a complementarity determining region CDR1-VL having an amino acid sequence shown in SEQ ID NO.4, a complementarity determining region CDR2-VL having an amino acid sequence shown in SEQ ID NO.5, and a complementarity determining region CDR3-VL having an amino acid sequence shown in SEQ ID NO. 6.

The respective sequences of the above variable region A are shown in Table 1:

TABLE 1

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

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

The respective sequences of the above variable region B are shown in Table 2:

TABLE 2

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

The binding protein of the new Delhi metallo-beta-lactamase provided by the invention has the advantages of good specificity, high biological activity, strong stability, small batch-to-batch difference, no influence by cell strain degradation, high affinity with the new Delhi metallo-beta-lactamase, titer reaching more than 1.

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

The amino acid sequence of the heavy chain variable region VH of variable region a is as follows:

EVQLVESSGGLVKPGGSLKLSCAASGFTFSSYSMNSVRQAPEKGLEWVASISASSSYIFYADSLKGRFTISFDNAKNTLFLQDTSLRSEDTAMYYCARERTVYTGRYSRLYYYGLDVWGQGTTVTVSS(SEQ ID NO.13)。

the amino acid sequence of the light chain variable region VL of variable region a is as follows:

DIVMTHATASLSFSLGTTATLSCRAGQSVGSNLAWTQQKAEQVPRGLIHGASTKATGVPVRFRYSGSGTDFTLTISGLEHEDAALYYCQQYDNWPPTFGQGTKVEIK(SEQ ID NO.14)。

the variable region B comprises a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO.15 and a light chain variable region VL with an amino acid sequence shown as SEQ ID NO. 16.

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

QVQVQESGPGLVKPSGSLFLVCSITGFPITSGDYYWTWIRQLPGKPLEWMGYISHSGGPFYNPSLKSPISTTREAAKNQFFLQLNSVTTQDAAMYYCAGDLDYDVGHNYYYGMDVWGQGTTVTVSS(SEQ ID NO.15)。

the amino acid sequence of the light chain variable region VL of variable region B is as follows:

DISLTQAPASLSFSLGETATLPCRASQSVTNGLLAWVQQKAEQVPRLLIHGPSNRATGFPVRFSGTGSGTDFTLTISSLEPEDAAVYFCQQYGSSPRTFGQGTKVEIK(SEQ ID NO.16)。

in some preferred embodiments, the binding protein further comprises a light chain constant region and a heavy chain constant region. The constant region is combined with the variable region to give a complete antibody.

The binding protein provided by the invention has the advantages of good specificity, high biological activity, strong stability and high affinity with the new Delhi metallo beta-lactamase, and can be used for preparing new Delhi metallo beta-lactamase detection products.

The colloidal gold immunochromatographic test strip can be, for example: a double-antibody sandwich colloidal gold immunochromatography method is adopted, a colloidal gold-labeled anti-NDM enzyme monoclonal antibody I is embedded on a sample combination pad, and an anti-NDM enzyme monoclonal antibody II and a goat anti-mouse antibody are respectively coated on a detection line (T) and a quality control line (C). If the detected sample is positive, NDM enzyme in the kit is combined with NDM enzyme antibody I marked by fluorescent microspheres to form a complex, the complex moves forwards along the paper strip under the action of chromatography, and is captured by precoated NDM enzyme antibody II when passing through a detection line (T), so that an immune complex is formed and a red strip is presented. If the detection sample is negative, no immune complex is formed, and no strip appears at the detection line. When the colloidal gold-labeled NDM enzyme antibody I passes through the quality control line (C), the antibody I is captured, and a band should appear. And (3) taking the bacterial suspension by using a quantitative dropper, dripping the bacterial suspension into a test strip sample adding hole, and judging the detection result by naked eyes after 15min, so that the drug resistance condition of the NDM enzyme can be output.

The binding protein provided by the invention has the advantages of good specificity, high biological activity, strong stability and high affinity with the new Delhi metallo beta-lactamase, and can be used for preparing a new Delhi metallo beta-lactamase purified product.

In a sixth aspect, the invention provides a nucleic acid encoding the binding protein, the nucleic acid having the nucleic acid sequences shown as SEQ ID No.17 and SEQ ID No.18, capable of expressing a heavy chain variable region VH having the amino acid sequence shown as SEQ ID No.13 and a light chain variable region VL having the amino acid sequence shown as SEQ ID No. 14.

The nucleotide sequence is as follows:

GAGGTGCAGCTGGTGGAGTCTTCCGGCGGATTAGTGAAGCCTGGCGGCTCCCTGAAACTCTCCTGTGCCGCATCTGGGTTCACTTTCAGTAGCTATAGCATGAACTCTGTTCGTCAGGCTCCAGAGAAAGGCCTGGAGTGGGTTGCATCCATTAGTGCTAGTAGTAGTTACATATTCTACGCAGACTCACTGAAGGGCCGATTCACCATCTCCTTCGACAATGCCAAGAACACCCTGTTCCTGCAAGACACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGAGCGGACGGTATATACTGGGCGCTACTCCCGACTCTACTACTACGGTTTGGACGTCTGGGGACAAGGAACGACGGTCACCGTCTCATCA(SEQ ID NO.17)。

GATATTGTGATGACACACGCTACAGCTAGTCTGAGTTTTTCTCTTGGAACAACAGCAACACTGTCATGCAGGGCCGGTCAGAGTGTTGGCAGCAACTTAGCCTGGACACAGCAGAAAGCAGAGCAAGTTCCCCGGGGTCTTATCCATGGTGCGTCCACCAAGGCCACTGGAGTCCCAGTCCGGTTCAGATACTCTGGCTCTGGAACAGACTTCACTCTCACCATCAGCGGTCTAGAACATGAAGATGCTGCACTTTACTACTGTCAGCAGTATGATAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA(SEQ ID NO.18)。

or the nucleic acid has the nucleic acid sequences shown as SEQ ID NO.19 and SEQ ID NO.20, and can express the heavy chain variable region VH with the amino acid sequence shown as SEQ ID NO.15 and the light chain variable region VL with the amino acid sequence shown as SEQ ID NO. 16.

The nucleotide sequence is as follows:

CAGGTCCAGGTGCAGGAGTCAGGCCCTGGCCTGGTGAAACCCTCAGGGTCACTCTTCCTCGTCTGCTCTATTACTGGATTCCCCATCACCAGTGGCGATTACTACTGGACCTGGATCCGTCAGCTCCCTGGGAAACCACTAGAATGGATGGGCTACATCTCTCACAGTGGGGGACCCTTCTATAATCCGTCCCTCAAGAGCCCCATCTCCACTACTAGAGAAGCAGCCAAGAACCAGTTCTTTCTGCAATTGAACTCTGTGACCACACAGGACGCAGCCATGTATTACTGTGCAGGAGATCTCGACTATGATGTTGGTCACAACTACTACTACGGCATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCATCA(SEQ ID NO.19)。

GATATTAGTCTAACACAGGCTCCAGCTTCTCTGAGTTTTTCTCTTGGTGAAACAGCAACACTGCCCTGCAGGGCCAGTCAGAGTGTTACCAACGGTTTGTTAGCCTGGGTCCAGCAGAAAGCAGAGCAAGTTCCCCGGCTCCTTATCCATGGTCCGTCCAACAGGGCCACTGGTTTCCCAGTCCGGTTCAGTGGCACTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTAGAACCTGAAGATGCTGCAGTTTACTTCTGTCAGCAATATGGGAGCTCACCCCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA(SEQ ID NO.20)。

in a seventh aspect, the present invention provides biomaterials comprising nucleic acids, including but not limited to vectors, recombinant microorganisms and cell lines. The "vector" refers to a substance capable of achieving replication and/or expression of a target gene and introduction of the target gene into prokaryotic or eukaryotic cells, and includes, but is not limited to, plasmids, phages, viral genomes or viruses, and the like. The term "recombinant microorganism" refers to a microorganism which, when manipulated, is not identical in genotype or phenotype to that exhibited by the wild-type of the microorganism.

In particular, the biological material is selected from the following: (n 1) a vector containing the nucleic acid; (n 2) a recombinant microorganism containing the nucleic acid, or a recombinant microorganism containing the vector of (n 1); (n 3) a cell line containing the nucleic acid, or a cell line containing the vector of (n 1).

Since the recombinant microorganism or cell line can express the binding protein, the fermentation broth rich in the binding protein can be obtained by culturing the recombinant microorganism or cell line, and the binding protein is obtained after separation. The preparation method is simple and convenient.

The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.

Example 1: preparation of antigens

The present invention obtains the conserved sequence of NDM enzymes (NDM 1-15, NDM17-31) by sequence alignment of NCBI (National Center for Biotechnology Information ). The expression plasmid pET-28a (+) -PM is constructed by adopting the conventional enzyme digestion and connection technology in the field of molecular biology, caCl is adopted ₂ Heat shock method recombinant vectors were transformed into E.coli DH5a competence. Positive clones were selected using LB medium containing 100. Mu.g/mL ampicillin. Conventionally culturing Escherichia coli, extracting plasmid, performing PCR identification, and determining the existence of target gene. The extracted expression plasmid pET-28a (+) -PMAA was transformed into E.coli BL21 (DE 3) -competent cells, which were then spread and cultured on a selective medium, and single colonies resistant to 100. Mu.g/mL ampicillin were selected and cultured overnight in a liquid medium. 1mL of the overnight culture was inoculated into 200mL of LB medium containing 100. Mu.g/mL ampicillin, and shake-cultured until logarithmic phase(OD 600 is 0.5-0.6), adding IPTG (1 mmol/L), inducing and culturing at 16 ℃ for 3h, purifying the fermentation liquor by a nickel column, and obtaining the high-purity protein by prokaryotic gene expression.

Example 2: animal immunization

Selecting a new zealand big ear white rabbit with the age being suitable and the weight being about 1.5 kg, feeding the new zealand big ear white rabbit in a standard animal house for 3 days, and starting to immunize if no abnormal condition exists: adding 100 mu g of NDM enzyme antigen into 0.5mL of autoclaved physiological saline, fully and uniformly mixing by using a micro vortex oscillator, adding 0.5mL of Freund's complete adjuvant, fully mixing and emulsifying by mutually pushing and pulling an injector, and carrying out back subcutaneous multi-point injection immunization on the New Zealand big ear rabbits; after two weeks, boosting is carried out, then boosting is carried out once every other week for six times, and from the third time of immunization, 200-500 mu L of auricle venous blood of white rabbits is taken after one week of immunization, and the titer and the affinity are measured; after the last immunization, spleen was taken for cell fusion for preparation of hybridoma cells.

Example 3: preparation and screening of hybridoma cells

Performing titer detection on the prepared rabbit antiserum, and performing cell fusion on rabbit spleen under the qualified condition to prepare a monoclonal hybridoma cell strain by the following method: killing immunized New Zealand big ear rabbit, taking out spleen under aseptic condition, washing with cell culture solution for 1 time, grinding, sieving with stainless steel sieve, centrifuging the obtained cell, and washing with cell culture solution for 2 times; mixing SP2/0 myeloma cells in logarithmic phase with spleen cells, washing with cell culture solution without fetal calf serum, centrifuging, removing supernatant, adding polyethylene glycol solution, and treating at 37 deg.C for about 90 s; terminating the reaction with cell culture medium without fetal bovine serum, centrifuging, resuspending cells with HAT selection medium containing 20% fetal bovine serum, adding the cells to a 96-well plate, 37 deg.C, 5.0% CO ₂ Medium culture; diluting cells with cell culture solution in 96-well plate to 1-3 cells/mL, adding into 96-well plate, placing into cell culture box, and adding CO at 37 deg.C and 5.0% ₂ Culturing under the condition, numbering each cell strain, selecting the cell strain with positive culture solution supernatant, performing amplification culture, and finallyObtaining the hybridoma cell strain. Screening the obtained hybridoma cells by adopting an ELISA method, observing the growth condition of the cells on the 5 th day after fusion, detecting the titer of cell culture supernatant by adopting an indirect ELISA method on the 10 th to 14 th days, expanding and culturing the positive hybridoma cells with the strongest titer until the cell positive rate reaches 100%, obtaining a hybridoma cell strain by strain determination, and freezing and storing the hybridoma cell strain in liquid nitrogen for later use.

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

Homogenizing hybridoma cells, adding cell lysate to perform RNA extraction, precipitating RNA from an aqueous phase layer by using isopropanol, washing the precipitated RNA after centrifugation, removing impurities, and performing reverse transcription after heavy suspension to obtain cDNA; PCR was carried out using specific primers from New Zealand big ear rabbits, heavy and light chain variable region genes of the antibody were amplified using hybridoma cDNA as a template, and 50. Mu.L of the system contained 5. Mu.L of cDNA, hotStarTaq Plus enzyme, dNTPs and 0.5. Mu.M specific primers, and PCR amplification was carried out under the following conditions: pre-denaturation at 94 ℃ for 5min; 30s at 94 ℃, 30s at 55 ℃, 50s at 72 ℃ and 35 cycles; 7min at 72 ℃; the obtained PCR product is identified by 1% agarose gel electrophoresis, the target fragment is recovered, the sample is sent for sequencing, and the sequencing result is compared with an IMGT database (http:// www.imgt.org/IMGT _ vquest/vquest) to obtain the antibody variable region gene fragment.

Example 5: construction, expression and purification of monoclonal antibodies

Introducing signal peptides into the N end of the heavy chain variable region gene and the N end of the light chain variable region gene of the antibody, then respectively adding homologous recombination arms into the two ends of the heavy chain variable region gene and the two ends of the light chain variable region gene of the antibody by utilizing homologous recombination primers, and linearizing expression plasmids containing rabbit antibody heavy and light chain IgG1 constant regions by using double enzymes to generate homologous recombination arms; the variable region gene segment added with the homologous recombination arm is connected with the linearized plasmid in a homologous recombination mode to form a complete expression vector, the recombinant product is transformed into TOP10 escherichia coli competence, and the plasmid is amplified.

Adding the obtained heavy and light chain expression plasmids of the monoclonal antibody into an Opti-Mem transfection medium according to a proportion of 1:1, fully mixing, adding a transfection reagent PEI with the mass 4 times that of DNA, mixing, standing at room temperature in a dark place for 30min, and then adding into 293T cells; after incubation for 6h, removing the transfection system, adding FreeStyleTM293 expression culture medium, purifying the expressed antibody supernatant by using AKTA Protein purification system and adopting an affinity purification (Protein A) method to obtain the monoclonal antibody of the anti-NDM enzyme, and the specific steps are as follows: (1) Centrifuging the expressed antibody supernatant at 2500 Xg for 10min at room temperature, and removing the precipitate; (2) The affinity purification column containing Protein A was sufficiently eluted with 10-fold volume of Binding Buffer; (3) Passing the expression supernatant through a purification column at a flow rate of 5 mL/min; (4) Washing the purification column thoroughly with 20 times the volume of the purification column in Binding Buffer; (5) Eluting the purification column with 0.1M citric acid buffer solution with pH =3.0-3.5 until the elution peak is reduced to an equilibrium state, and adjusting pH to 7.0 with 1M Tris-HCl buffer solution with pH = 9.0; (6) And (3) concentrating the purified monoclonal antibody by using a concentration centrifugal column, using PBS as a buffer for storing the antibody, and finally determining the concentration of the concentrated antibody by using a BSA protein concentration detection method.

Monoclonal antibody 1 (antibody 1 for short) and monoclonal antibody 2 (antibody 2 for short) were finally obtained by the above-described method. Wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody 1 are as follows:

signal peptide-VH:

MDWTWRFLFVVAAATGVQSEVQLVESSGGLVKPGGSLKLSCAASGFTFSSYSMNSVRQAPEKGLEWVASISASSSYIFYADSLKGRFTISFDNAKNTLFLQDTSLRSEDTAMYYCARERTVYTGRYSRLYYYGLDVWGQGTTVTVSS(SEQ ID NO.21)。

signal peptide-VL:

MDMRVPAQLLGLLLLWLSGARCDIVMTHATASLSFSLGTTATLSCRAGQSVGSNLAWTQQKAEQVPRGLIHGASTKATGVPVRFRYSGSGTDFTLTISGLEHEDAALYYCQQYDNWPPTFGQGTKVEIK(SEQ ID NO.22)。

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

signal peptide-VH:

MDWTWRFLFVVAAATGVQSQVQVQESGPGLVKPSGSLFLVCSITGFPITSGDYYWTWIRQLPGKPLEWMGYISHSGGPFYNPSLKSPISTTREAAKNQFFLQLNSVTTQDAAMYYCAGDLDYDVGHNYYYGMDVWGQGTTVTVSS(SEQ ID NO.23)。

signal peptide-VL:

MDMRVPAQLLGLLLLWLSGARCDISLTQAPASLSFSLGETATLPCRASQSVTNGLLAWVQQKAEQVPRLLIHGPSNRATGFPVRFSGTGSGTDFTLTISSLEPEDAAVYFCQQYGSSPRTFGQGTKVEIK(SEQ ID NO.24)。

example 6: determination of molecular weight

The molecular weight of the monoclonal antibody was identified by SDS-PAGE electrophoresis using an amount of 5. Mu.g per lane, and using a standard series of known molecular weights as a reference, first electrophoresis at 90V for 20min, then at 140V until the indicator was completely removed, the gel was removed, stained with Coomassie Brilliant blue, and the stained gel was analyzed for molecular weight of the biological material, as shown in FIG. 1 (first column of marker, second column of antibody 1, and third column of antibody 2).

Example 7: potency assay

The ELISA method is adopted to detect the affinity activity (titer) of the monoclonal antibody to NDM enzyme, and the main steps are as follows: (1) NDM enzyme antigen is diluted to 1 ng/mu L by PBS, added into a 96-hole enzyme label plate by 100 mu L per hole, and coated for 2h at 37 ℃; (2) Discard the supernatant, wash the plate 3 times with 0.01M PBST, prepare a blocking solution containing 3% BSA using PBST, add 100. Mu.L per well, block for 2h at 37 ℃; (3) Discard the supernatant, wash 5 times with PBST, perform gradient dilution of the purified and concentrated antibody from 1; (4) Discarding the antibody diluent, washing with PBST for 6 times, diluting goat anti-rabbit IgG-HRP with 1; (5) Discarding the secondary antibody diluent, washing with PBST for 6 times, adding TMB at a concentration of 100 μ L/hole, and standing at 37 deg.C in a dark place for 15min; (6) The reaction was stopped by adding 50. Mu.L of 1M dilute sulfuric acid to each well, and the absorbance was measured at 450 nm. As shown in fig. 2 (in the figure, 1# is

antibody

1,2# is antibody 2), the selected monoclonal antibody has strong binding ability to NDM enzyme, and the titer to NDM enzyme antigen reaches 1 1280000 (OD > 0.5).

Example 8: comparison with existing antibodies

The affinity activity (titer) to NDM enzyme was measured by ELISA using the antibody obtained by the present invention, a natural antibody and a published murine monoclonal antibody (purchased from Zhuhai Bomei Biotech Co., ltd.), as described in example 7. The results are shown in Table 3, which shows that the monoclonal antibody of the present invention has increased affinity and enhanced biological activity compared with the prior art.

TABLE 3

Note:

antibody

1,2# for antibody 1 is antibody 2.

Example 9: cross reaction

Respectively coating the enzyme label plate with KPC, NDM, VIM, IMP and OXA-48 enzyme, wherein the coating amount of each hole is 50ng; diluting the monoclonal antibody to 10ng/mL, adding the diluted monoclonal antibody into each enzyme label plate, adding 100 mu L of monoclonal antibody into each hole, and incubating for 1h at 37 ℃; adding HRP (horse radish peroxidase) -labeled goat anti-rabbit secondary antibody after washing, adding 100 mu L of HRP-labeled goat anti-rabbit secondary antibody into each hole, and incubating for 0.5h at 37 ℃; after washing, TMB was added and incubation was carried out at 37 ℃ for 15min, and the reading was terminated. The results are shown in FIGS. 3 and 4, which indicate that the monoclonal antibody does not cross-react with other carbapenemases and has strong specificity.

Example 10: antibody pairing validation

One of the two antibodies is used as a capture antibody, the other is used as a labeled antibody (HRP enzyme label), the capture antibody is also labeled by the HRP enzyme at the same time, the capture antibody is used as a control group, the capture antibody is coated on an antigen plate, the antigen diluted by multiple times is added firstly, the unbound antigen is washed off after incubation, the unbound labeled antibody is washed off after the addition of the labeled antibody for incubation, and finally, a developing solution is added for developing. If the color can be developed, the specific binding of the labeled antibody and the antigen is indicated, and the capture antibody and the labeled antibody are a pair of paired antibodies. If the color is not developed, it indicates that the labeled antibody is not bound to the antigen and is eluted, and the capture antibody and the labeled antibody are not the partner antibody, and the results are shown in Table 4, which indicates that the two antibodies have the best ability to bind to the antigen.

TABLE 4

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A binding protein for neodrime beta-lactamase, wherein said binding protein comprises a variable region a or a variable region B;

2. The binding protein according to claim 1, wherein said variable region a comprises a heavy chain variable region VH having an amino acid sequence shown as SEQ ID No.13 and a light chain variable region VL having an amino acid sequence shown as SEQ ID No. 14.

3. The binding protein according to claim 1, wherein said variable region B comprises a heavy chain variable region VH having an amino acid sequence shown as SEQ ID No.15 and a light chain variable region VL having an amino acid sequence shown as SEQ ID No. 16.

4. Use of a binding protein according to any one of claims 1 to 3 for the preparation of a neo-darrietal beta-lactamase assay product.

5. A colloidal gold immunochromatographic test strip for detecting neodrime beta-lactamase, comprising the binding protein of any one of claims 1 to 3.

6. Use of a binding protein according to any one of claims 1 to 3 for the preparation of a purified product of neodrime beta-lactamase.

7. A kit for purifying neodrimetallo beta-lactamase, wherein the kit comprises the binding protein of any one of claims 1-3.

8. A nucleic acid encoding the binding protein of any one of claims 1 to 3, wherein the nucleic acid has the nucleic acid sequence shown as SEQ ID No.17 and SEQ ID No. 18;

or the nucleic acid has a nucleic acid sequence shown as SEQ ID NO.19 and SEQ ID NO. 20.

9. A biomaterial characterized by being selected from one or more of the following (n 1) to (n 3):

(n 1) a vector comprising the nucleic acid of claim 8;

(n 2) a recombinant microorganism comprising the nucleic acid of claim 8, or a recombinant microorganism comprising the vector of (n 1);

(n 3) a cell line comprising the nucleic acid of claim 8, or a cell line comprising the vector of (n 1).

10. A method of producing a binding protein according to any one of claims 1 to 3, comprising: culturing the recombinant microorganism or cell line of claim 9 in a culture medium and isolating the binding protein.