A monoclonal antibody, a primer set, a kit, a recombinant vector and a recombinant engineering bacterium and their application

Abstract

The present invention relates to the technical field of monoclonal antibody preparation, and specifically discloses a monoclonal antibody, a primer set, a kit, a recombinant vector and a recombinant engineering bacterium and their applications. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the light chain is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10. The monoclonal antibody provided by the present invention has an affinity value of up to 1.01 pM for the phosphokinase region protein antigen, and the monoclonal antibody and related products can be used to prepare diagnostic reagents for diseases related to Klebsiella pneumoniae.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation, and in particular to a monoclonal antibody, a primer set, a kit, a recombinant vector and a recombinant engineering bacterium and applications thereof. Background Art

[0002] Klebsiella pneumoniae is a Gram-negative bacterium that is widely considered to be one of the important pathogens of hospital and community infections. It is usually found in the natural environment and in the human digestive tract, but can also cause a variety of infections, such as pneumonia, urinary tract infection, sepsis, etc. Among them, the capsule of Klebsiella pneumoniae is one of the important factors of its pathogenicity. The capsule is a polysaccharide-peptide complex that forms a protective layer on the surface of bacteria, which helps bacteria escape the attack of the host immune system and increases their resistance to antibiotics. The synthesis and assembly of the capsule is a complex process involving the interaction and regulation of multiple proteins. Among them, the Wzc phosphokinase domain protein plays a vital role in the capsule synthesis process.

[0003] Wzc phosphokinase domain protein is a transmembrane protein that is localized on the bacterial cell membrane and interacts with the capsule synthase complex through its extracellular domain, abbreviated as Wzc. The function of Wzc is mainly to regulate the sugar chain length and capsule yield during capsule synthesis. Studies have shown that Wzc may be a casein kinase that regulates the synthesis rate and quality of capsules by phosphorylating other capsule synthesis-related proteins during the synthesis process. The activity and function of Wzc are regulated by many factors. First, environmental factors affect the activity of Wzc. For example, temperature, pH value and nutritional conditions may affect the expression level and activity of Wzc. Secondly, intracellular signaling molecules can also regulate the function of Wzc. The activation or inhibition of intracellular signaling pathways will directly affect the phosphorylation activity of Wzc, thereby affecting the synthesis of capsules. In addition, other capsule synthesis-related proteins can also interact with Wzc to jointly regulate the capsule synthesis process.

[0004] In recent years, the study of Wzc phosphokinase domain proteins has become increasingly in-depth, revealing its important role in capsule synthesis. Through methods such as gene knockout, mutant analysis and protein interaction, scientists have gradually understood the functional mechanism and regulatory pathway of Wzc. For example, some studies have found that mutations in Wzc may lead to obstruction or abnormality of capsule synthesis, thereby affecting the pathogenicity and drug resistance of Klebsiella pneumoniae. Therefore, regulation of Wzc may become one of the focuses of future research. Research on Wzc is also of great significance in the development of antimicrobial drugs and the formulation of treatment strategies. On the one hand, by interfering with the activity or interaction of Wzc, the synthesis of the capsule can be inhibited, thereby weakening the pathogenicity of Klebsiella pneumoniae. Therefore, it is of great significance to develop an antibacterial drug with Wzc as a drug target or therapeutic target. Summary of the invention

[0005] In order to provide a product for efficiently detecting or treating Klebsiella pneumoniae infection, the present invention provides a monoclonal antibody, a primer set, a kit, a recombinant vector, a recombinant engineered bacterium and its application. The monoclonal antibody provided by the present invention has an affinity value of up to 1.01 pM for the protein antigen in the phosphokinase region, and the monoclonal antibody and related products can be used to prepare diagnostic reagents for Klebsiella pneumoniae-related diseases.

[0006] The present invention provides a monoclonal antibody, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region;

[0007] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the light chain is shown in SEQ ID NO.9, and its amino acid sequence is shown in SEQ ID NO.10.

[0008] The present invention also provides a pair of primer sets for amplifying the heavy chain of the monoclonal antibody. The sequences of the primer sets for amplifying the heavy chain are shown in SEQ ID NO.3 to SEQ ID NO.4.

[0009] The present invention also provides a pair of primer sets for amplifying the light chain of the monoclonal antibody. The sequences of the primer sets for amplifying the light chain are shown in SEQ ID NO.5 to SEQ ID NO.6.

[0010] The present invention also provides a kit comprising the monoclonal antibody.

[0011] Furthermore, the kit also includes any one of a solid phase carrier, a coating buffer, a blocking solution, a sample processing solution or a color developing solution, or a combination of at least two thereof.

[0012] The present invention also provides a Wzc phosphokinase region protein antigen that specifically binds to the monoclonal antibody, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding Wzc is shown in SEQ ID NO.2.

[0013] The present invention also provides a recombinant vector, which is obtained by cloning the coding gene Wzc into a pET28b vector, and the sequence of the coding gene Wzc is shown in SEQ ID NO.2.

[0014] The present invention also provides a recombinant engineering bacterium, wherein the recombinant engineering bacterium comprises the recombinant vector.

[0015] The present invention also provides a use of the monoclonal antibody, the primer set or the kit in preparing a disease diagnosis preparation;

[0016] The pathogen of the disease is Klebsiella pneumoniae.

[0017] Furthermore, the disease is pneumonia, urinary tract infection or sepsis.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The monoclonal antibody provided by the present invention has an affinity value of up to 1.01 pM with Wzc, which is extremely high and can be applied to the detection and research of the capsule synthesis mechanism and inhibitors of Klebsiella pneumoniae. The monoclonal antibody and related products can be used to prepare diagnostic reagents for Klebsiella pneumoniae-related diseases (pneumonia, urinary tract infection or sepsis). It provides a drug basis for the prevention and treatment of Klebsiella pneumoniae-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is an SDS-PAGE diagram of the purified Wzc phosphokinase region protein antigen in Example 1 of the present invention. From left to right, the lanes are protein Marker (M), supernatant of host bacteria lysate expressing Wzc (lane 1), flow-through (lane 2), washing solution (lane 3), E1-E9 eluate (lanes 4-12) and the final protein purified Wzc (lane 13).

[0022] Figure 2This is a graph showing the WB specificity identification results of the monoclonal antibody in Example 2 of the present invention, wherein M represents a protein marker, and its bands are 250 kDa, 150 kDa, 100 kDa, 75 kDa, 60 kDa, 45 kDa, 35 kDa and 25 kDa from top to bottom, M1 and M2 are the same markers, lane 1 is the total protein of Klebsiella pneumoniae (containing the natural, full-length Wzc phosphokinase domain protein), and lane 2 is the purified phosphokinase domain protein.

[0023] Figure 3 MW is the amplification result of the antibody gene in Example 3 of the present invention, wherein VH and VL refer to the PCR amplification results of the heavy chain and light chain, respectively.

[0024] Figure 4 This is the SPR detection diagram of antibody affinity in Example 4 of the present invention, the black lines are fitting values, and the colored lines are measured values. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] Example 1: Expression and purification of Wzc phosphokinase region antigen protein.

[0027] 1. Design of Wzc phosphokinase region antigen protein expression plan:

[0028] According to the amino acid sequence of the Wzc phosphokinase region (shown in SEQ ID NO.1), the Wzc phosphokinase region gene, referred to as the Wzc gene, was directly synthesized. Its gene sequence is shown in SEQ ID NO.2. The Wzc phosphokinase region was expressed using an E. coli system.

[0029] SEQ ID NO.1:

[0030] MIMTVAIVTTRFFNNAIQNTAFHNHATLFEAFHAHVDFFNGIFFEACNQQTGTGIAANNSSIGHCQNRRGVDNDQIIILTCPIQQLFEARMHQQFCRIRRNLTTTNIIELAAFNFINNHFCGSLTTQELAQALIVFMAEHFMCIAFTQICVNEQNFFAGNCNGGCQIAANEGFTCARACTADHQHIIFGFHHCEVQAGAQTTNGFHCQIGRITHCQQGIALIFTNFTALTCIFCQPFANRDTGIHINANFFQLFRTFNTSTHM

[0031] SEQ ID NO.2:

[0032] ATGCGTAGAGGTATTGAAAGTCCGGAACAGCTGGAAGAAATTGGCATTAATGTGTATGCCAGTATCCCGATTAGCGAATGGCTGGCAAAAAATGCACGTCAGAGCGGTAAAGTTCGTAAAAATCAGAGCGATACCCTGCTGGCAGTGGGTAATCCGGCCGATCTGGCAGTGGAAGCCATTCGTGGTCTGCGCACCAGCCTGCACTTCGCAATGATGGAAGCCAAAAATAATGTGCTGATGATCAGCGGTGCAAGCCCGAGCGCAGGTAAAACCTTCATTAGCAGCAATCTGGCAGCCACCATTGCAATTACCGGCAAAAAAGTTCTGTTCATTGACGCAGATCTGCGTAAAGGCTATGCACATAAAATGTTCGGCCATAAAAACGATAAGGGCCTGAGCGAGTTCCTGAGTGGTCAGGCTGCCGCAGAAATGGTTATTGATAAAGTTGAAGGCGGCGAGTTCGATTATATTGGTCGTGGTCAGATTCCGCCGAATCCGGCAGAACTGCTGATGCATCCGCGCTTCGAACAGCTGCTGAATTGGGCAAGTCAGAATTATGATCTGATCATTATCGACACCCCGCCGATTCTGGCAGTGACCGATGCTGCTATTATTGGCCGCTATGCCGGTACCTGTCTGCTGGTTGCACGCTTCGAAAAAAATACCGTTAAAGAAATCGACGTGAGCATGAAACGCTTCGAACAAAGTGGCGTGGTTGTGAAAGGCTGTATTCTGAATGGCGTTGTTAAAAAAGCGAGTAGTTACTATGGCTACGGTCATAATCATTACGGTTATAGCTACAGTGACAAGAAAATCGATTAAGAATTC

[0033] 2. Preparation of pWzc plasmid:

[0034] A GSlinker with the sequence ATGGGCAGCAGC (amino acid sequence MGSS) and a 6*His tag (amino acid sequence: HHHHHHH, nucleotide sequence: CATCATCATCATCATCATCAT) were added to the N-terminus of the Wzc gene sequence, and the Wzc gene was cloned into the pET28b vector through the NdeI / EcoRI restriction site to obtain the pET28b-Wzc recombinant vector, referred to as pWzc plasmid.

[0035] 3. Expression and purification of Wzc phosphokinase domain protein

[0036] (1) Construction of genetically engineered Escherichia coli carrying the pWzc plasmid.

[0037] The pWzc plasmid was mixed with competent Escherichia coli DH5α, placed in an ice-water mixture for 30 minutes, and the pWzc plasmid was added for heat shock at 42°C for 45 seconds, and 900 μL of SOC medium preheated at 37°C was added, and the mixture was shaken at 37°C and 150 rpm for 1 hour, and evenly coated on a plate containing 50 μg / L kanamycin, cultured overnight, and positive clones were selected. The Escherichia coli DH5α carrying the Wzc gene was determined by PCR. The Escherichia coli DH5α carrying the pWzc plasmid was cultured at 37°C and 220 rpm for 16 hours, centrifuged at 12000g for 5 minutes, and the bacterial precipitate was used to extract the pWzc plasmid using the SteadyPure plasmid extraction kit (Hunan Eric Biotechnology). The specific operation was carried out according to the instructions.

[0038] The pWzc plasmid was mixed with competent Escherichia coli BL21 (DE3), placed in an ice-water mixture for 30 min, heat-shocked at 42°C for 45 s, added with 900 μL of SOC medium preheated at 37°C, shaken at 37°C at 150 rpm for 1 h, evenly spread on a plate containing 50 μg / L kanamycin, cultured overnight, selected positive clones, and confirmed by PCR that the Escherichia coli BL21 (DE3) carried the Wzc gene.

[0039] (2) Expression of Wzc phosphokinase domain protein

[0040] Reagents used:

[0041] Buffer A formula: 50mmol / L tris(hydroxyaminomethane), 300mmol / L sodium chloride, pH 7.4;

[0042] Washing buffer: 50mmol / L tris(hydroxyaminomethane), 300mmol / L sodium chloride, 50mmol / L imidazole, pH 7.4;

[0043] Elution buffer: 50mmol / L tris(hydroxyaminomethane), 300mmol / L sodium chloride, 500mmol / L imidazole, pH 7.4.

[0044] Pick the positive clones on the plate and inoculate them into LB culture medium containing 50 μg / L kanamycin; shake at 37°C 220 rpm until the bacterial OD 600 The concentration of IPTG was 0.7, and then IPTG was added to a final concentration of 1 mmol / L to induce the expression of the fusion protein. The culture was continued for 6 h, and the mixture was centrifuged at 4°C and 6,000 g for 30 min. The bacterial precipitate was collected and resuspended in buffer A to obtain a bacterial suspension.

[0045] The bacterial suspension was placed in an ice-water bath and ultrasonically disrupted for 10 minutes at a power of 750W, working for 1 second, and resting for 2 seconds. Then, the supernatant (supernatant of the host bacterial lysate expressing Wzc) was collected after centrifugation at 4°C and 6000g for 30 minutes, and the supernatant was passed through a Ni column. After the Ni-column was washed three times with a washing buffer (flow-through solution), the protein was eluted with an elution buffer (washing solution) to obtain the Wzc phosphokinase region protein antigen. 2 μg of Wzc phosphokinase region protein was taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., SDS-PAGE.

[0046] The results are as follows Figure 1 As shown, after SDS-PAGE, the obtained Wzc phosphokinase domain protein has a high purity.

[0047] Example 2: Preparation and detection of mouse monoclonal antibodies.

[0048] 1. Animal immunization and establishment of indirect ELISA

[0049] The Wzc phosphokinase domain protein in Example 1 was used as an immunogen to immunize 4 6-week-old BALBc mice, with each immunization dose of 100 μg / mouse. The immunization method was subcutaneous multi-point injection on the back, immunization once every 2 weeks, the first immunization was an immunogen plus an equal volume of Freund's complete adjuvant, and the second and third immunizations were immunogens plus an equal volume of Freund's incomplete adjuvant. Seven days after the third immunization, blood was collected from the mouse eyeballs and serum was collected. Three days before fusion, pure antigen was injected intraperitoneally for booster immunization, i.e., four immunizations. The specific process of the mouse immunization process is described in Table 1. The determination of the optimal antigen, antibody working concentration and yin and yang critical values ​​of indirect ELISA was determined by matrix titration.

[0050] Table 1 Immunization process of mice

[0051] Immunization time dose Day 1 100μg / each Second Free (Day 14) 100μg / each Three Free (Day28) 100μg / each Antiserum titer detection (Day 35) ELISA test serum titer greater than 1:32000 Four Free (Day42) 100μg / each Antiserum titer detection (Day 49) ELISA test serum titer greater than 1:32000

[0052] 2. Cell fusion and subclone screening

[0053] The immune sera from the four mice immunized four times were diluted 1:4000, 1:8000, and 1:16000, respectively, and the serum titers were determined according to the established indirect ELISA.4 The above can be used for fusion. Three days after booster immunization, mouse spleen cells were taken and fused with SP20 myeloma cells. After fusion, the supernatant was tested with the established ELISA. Then, the positive hybridoma cells were subcloned by the limiting dilution method and the square array titration method. The established ELISA was used to detect whether the subcloning was successful. The positive well cells were cloned three times in succession according to the above method, and the positive hybridoma wells with high OD value, good cell viability and stable antibody secretion were selected for expansion culture and timely freezing to obtain hybridoma cells.

[0054] 3. Ascites preparation and monoclonal antibody titer

[0055] 7-week-old BALBc mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was intraperitoneally injected with 1 × 10 6 1 mL of hybridoma cells with a concentration of 100 / mL was added. After 14 days, ascites was collected and the supernatant was centrifuged to obtain the hybridoma cell culture supernatant, which was purified using the ProteinLResin kit and the concentration of the purified ascites was determined using the BCAProteinAssayKit kit. The monoclonal antibody was titered by indirect ELISA and the titer was 1:2000. The purified Wzc phosphokinase domain protein was used as a positive control, and the SP20 myeloma cell supernatant and the serum of mice inoculated with any antigen were used as negative controls. Each sample was repeated 3 times.

[0056] 4. Western blot (WB) of native protein detected by monoclonal antibodies:

[0057] Preparation of TBST buffer: Add 8 g of sodium chloride, 0.2 g of potassium chloride, and 0.5 mL of Tween 80 to 50 mL of 1 mol / L, pH 7.5 tris(hydroxyaminomethane)-hydrochloric acid, and add distilled water to make up to 1 L.

[0058] Take 5 μg of Klebsiella pneumoniae total protein (containing natural Wzc phosphokinase domain protein) and the purified Wzc phosphokinase domain protein of the present invention, perform SDS-PAGE, and after transfer, block with TBST buffer containing 5% bovine serum albumin at room temperature for 1 hour, use hybridoma cell culture supernatant (1:2000) as primary antibody, and incubate overnight at 4°C on a low-temperature shaker. The next day, after washing the membrane, add horseradish peroxidase-labeled Goat Anti-Mouse IgG (1:5000, Lianke Bio, catalog number: 70-GAM0072) as secondary antibody, and use eECL high-sensitivity luminescent reagent (Kangwei Century, catalog number: CW0049S) for detection.

[0059] Test results such as Figure 2As shown, M is a protein marker, and the molecular weights are 250, 150, 100, 70, 50, 40, 35, and 20 kDa from top to bottom; lanes 1 and 2 are the total protein of Klebsiella pneumoniae (containing the natural Wzc phosphokinase domain protein), artificially expressed, and purified Wzc phosphokinase domain proteins, respectively. Figure 2 It can be seen that obvious luminescent bands are visible at the positions of 70-100 kDa of Klebsiella pneumoniae total protein, artificially expressed and purified Wzc phosphokinase domain proteins, indicating that the obtained monoclonal antibody can bind to natural Wzc phosphokinase domain protein and artificially expressed Wzc phosphokinase domain protein.

[0060] Example 3: Determination of monoclonal antibody gene sequence.

[0061] 1. Sequencing method

[0062] Total RNA was extracted from the hybridoma cells prepared in Example 2 and reverse transcribed into cDNA. The cDNA was used as a template and amplified using the following primers.

[0063] The heavy chain primers include the upstream primer H5 shown in SEQ ID NO.3 and the downstream primer H3 shown in SEQ ID NO.4; the light chain primers include the upstream primer L5 shown in SEQ ID NO.5 and the downstream primer L3 shown in SEQ ID NO.6.

[0064] Upstream primer H5: 5'-GAAGTGMAGCTCGAGGAGTCTGGGGGA-3' (SEQ ID NO. 3);

[0065] Downstream primer H3: 5'-AGGCTTACTAGTACAAATCCCTGGGCACAAT-3' (SEQ ID NO. 4);

[0066] Light chain primer:

[0067] Upstream primer L5: 5'-CCTCTAGACAGGCTGTTGTGACTCAGGAATC-3' (SEQ ID NO. 5);

[0068] Downstream primer L3: 5'-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3'

[0069] (SEQ ID NO.6).

[0070] The amplified PCR products were connected to T vectors, and colony PCR was performed after transformation, and positive clones were selected for sequencing analysis. The sequences obtained by sequencing were analyzed using online software Vbase2 and IMGT / V Quest.

[0071] 2. Monoclonal antibody gene and amino acid sequence

[0072] The gene sequence and amino acid sequence of the monoclonal antibody were analyzed and the results were as follows: the cDNA sequence of the heavy chain variable region was shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region was shown in SEQ ID NO.8.

[0073] SEQ ID NO.7:

[0074] GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTTCTACATTCACTGGGTGAAGCAGAGCCATGGAAAGGGCCTTGAGTGGATTGGACGTGTTAATCCTAACAATGGTGGTACT GACTACAACCAGAAGTTCAAGGGCAAGGCCATATTAACTGTAGACAAGTCATCCACCACAGCCTACATGGAACTCCGCAGCCTGACATCTGACGACTCTGCGGTCTATTACTGTGCAAGATCAGACTGGGCCCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0075] SEQ ID NO.8:

[0076] EVQLQQSGPDLVKPGASVKISCKAS GYSFTGF IHWVKQSHGKGLEWIGR VNPNNGGT DYNQKFKGKAILTVDKSSTTAYMELRRSLTSDDSAVYYC ARSDWAPDY WGQGTTLTVSS.

[0077] CDR regions are underlined.

[0078] The cDNA sequence of the light chain variable region is shown in SEQ ID NO.9, and the amino acid sequence encoded by it is shown in SEQ ID NO.10.

[0079] SEQ ID NO.9:

[0080] GACATTTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTAACTCGGCATCCTATCG GAACAGCGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0081] SEQ ID NO.10:

[0082] DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALINSASYRNSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGAGTKLELK

[0083] Example 4: Affinity identification of monoclonal antibodies.

[0084] 1. Experimental Preparation

[0085] (1) According to OpenSPR TM Instrument standard operating procedure to install COOH chip.

[0086] (2) Start running at maximum flow rate (150 μL / min) with PBST (pH 7.4) as the detection buffer.

[0087] (3) After reaching the signal baseline, load 200 μL of IPA (isopropanol) and run for 10 seconds to remove bubbles. After reaching the baseline, rinse the sample loop with buffer and evacuate it with air.

[0088] (4) After the signal reaches the baseline, adjust the buffer flow rate to 20 μL / min.

[0089] (5) Load EDC / NHS (1:1) solution to activate the chip.

[0090] (6) Load 200 μL of ligand diluted in immobilization buffer and run for 4 min. Rinse the sample loop with buffer and evacuate it with air.

[0091] (7) Load 200 μL of Blocking solution, rinse the sample loop with buffer, and evacuate it with air.

[0092] (8) Observe the baseline for 5 minutes to ensure stability.

[0093] (9) The analyte was diluted with phosphate buffer solution (PBST) at pH 7.4 to the following concentrations: 0.5 nmol / L, 1 nmol / L, 2 nmol / L, 4 nmol / L, 8 nmol / L, and 16 nmol / L, respectively, and loaded at 40 μL / min. The binding time between the analyte and the ligand was 120 s, and the natural dissociation time was 240 s.

[0094] (10) The analysis software used for this experimental result is: TraceDrawer (Ridgeview Instrumentsab, Sweden), and the analysis method is the One To One analysis model.

[0095] (11) Fit the curve and calculate the affinity to obtain the antibody affinity value K d It is 1.01 pM, indicating that the monoclonal antibody of the present invention has a very high affinity to Wzc and can be applied to the detection and research of the synthesis mechanism and inhibitors of Klebsiella pneumoniae capsule.

[0096] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0097] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the light chain is shown in SEQ ID NO.9, and its amino acid sequence is shown in SEQ ID NO.

10.

2. The monoclonal antibody according to claim 1, characterized in that The sequences of the primer sets for amplifying the heavy chain of the monoclonal antibody are shown in SEQ ID NO.3 to SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1, characterized in that The sequences of the primer sets for amplifying the light chain of the monoclonal antibody are shown in SEQ ID NO.5 to SEQ ID NO.

6. A kit comprising the monoclonal antibody according to claim 1.

5. The kit according to claim 4, characterized in that The kit further comprises any one or a combination of at least two of a solid phase carrier coating buffer, a blocking solution, a sample processing solution and a color developing solution.

6. A Wzc phosphokinase region protein antigen that specifically binds to the monoclonal antibody according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence encoding gene Wzc is shown in SEQ ID NO.

2.

7. A recombinant vector, characterized in that: The recombinant vector is obtained by cloning the coding gene Wzc according to claim 6 into a pET28b vector, and the sequence of the coding gene Wzc is shown in SEQ ID NO.

2.

8. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the recombinant vector according to claim 7.

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