A universal M13 phage probe for fully displaying scFv and its construction method and application

Abstract

The present invention discloses a monoclonal antibody, a nucleic acid molecule, an expression cassette, a recombinant vector, a recombinant cell, or a recombinant bacterium. The present invention also discloses a universal M13 phage probe that fully displays scFv, as well as its preparation method and application. The monoclonal antibody of the present invention has high titer, and the universal phage probe has low cost, high sensitivity, good stability, and a certain degree of versatility. It can be used as a novel biological detection probe to separate and detect trace biomarkers, pathogens, and the like in sample matrices.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a universal M13 phage probe for fully displaying scFv, and a construction method and application thereof. Background Art

[0002] Phage display was first described in 1985, when George P. Smith demonstrated that filamentous phages could display fusion proteins on the surface of virions after inserting foreign DNA fragments into the phage coat protein gene. Greg Winter and John McCafferty at the Laboratory of Molecular Biology, and Lerner and Barbas at the Scripps Research Institute, further developed and improved the technology. Among filamentous phages, the M13 phage is the most commonly used phage display technology, primarily due to its ability to integrate long fragments of foreign DNA into its genome. P3 phage libraries can display one to five copies of large foreign proteins on their surfaces. Despite this low copy number, they can be effectively used to display large peptides or proteins. However, conventional P3 display is often monovalent, which is inefficient. Approximately 2,700 copies of the P8 protein are densely packed on the phage coat, which means that by inserting a peptide coding sequence into the phage copy of the P8 gene, up to 2,700 copies can be presented. Peptides with a maximum length of 6 amino acids can be displayed along the phage capsid. However, P8 is well suited for displaying peptides and small proteins, but cannot display long peptides or large proteins.

[0003] Bacteriophages are unique nanomaterials. Genetic engineering can achieve site-specific universalization of specific coat proteins. Chemical modification, meanwhile, offers the advantage of further expanding the functionality of phages beyond that of native peptides and proteins. By leveraging the advantages of genetic engineering and chemical modification, different coat proteins can be manipulated to achieve desired functions, making the M13 phage a versatile sensing platform with diverse functional domains that can perform their functions without interfering with each other. Currently, in the biological field, the M13 phage has been widely used in vaccine preparation, targeted drug delivery, pathogen detection and treatment, disease diagnosis, and bio-scaffolds.

[0004] Alzheimer's disease (AD) and other cognitive impairment diseases are a group of neurodegenerative diseases that occur in the elderly and pre-elderly stages. They have an insidious onset and a progressive course. The core clinical manifestations are acquired cognitive impairment, involving cognitive domains such as memory, learning, orientation, comprehension, judgment, calculation, language, and visual-spatial functions. They affect daily living abilities and social and occupational functions to varying degrees, and eventually progress to global dementia.

[0005] Current research on Alzheimer's disease biomarkers focuses on body fluid markers (cerebrospinal fluid, blood, etc.) and imaging markers. Among them, cerebrospinal fluid Aβ1-42, cerebrospinal fluid phosphorylated Tau protein (P-Tau), cerebrospinal fluid total Tau protein (T-Tau), magnetic resonance imaging of hippocampal atrophy, and positron emission tomography (PET) imaging have confirmed their diagnostic value for Alzheimer's disease and are beginning to be used in clinical practice. However, the examination of imaging markers and cerebrospinal fluid markers has the problem of high cost and invasiveness, making them difficult for most people to accept.

[0006] Cerebrospinal fluid is obtained through lumbar puncture, which is very traumatic and difficult to perform multiple samplings in a short period of time, making it difficult to track the effectiveness of treatment. Blood is an easy-to-collect human sample. When extracting liquid samples to measure Alzheimer's disease biomarkers, blood sampling is more desirable than cerebrospinal fluid because it is easy to collect and less invasive. Changes in the level of Aβ1-42 in peripheral blood can be used as a potential biomarker to predict the progression of Alzheimer's disease in patients with preclinical and mild cognitive impairment. However, the level of Aβ1-42 in peripheral blood is relatively low, and new, more sensitive detection technologies are needed. Therefore, it is extremely important to develop a low-cost, highly sensitive, and stable phage probe. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a monoclonal antibody.

[0008] Another technical problem to be solved by the present invention is to provide a nucleic acid molecule encoding the monoclonal antibody.

[0009] The technical problem that the present invention also aims to solve is to provide an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium.

[0010] The technical problem that the present invention also aims to solve is to provide a universal M13 phage probe.

[0011] The technical problem that the present invention also aims to solve is to provide the use of the monoclonal antibody, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or recombinant bacteria, and the probe in preparing a detection kit for detecting Aβ1-42.

[0012] The final technical problem to be solved by the present invention is to provide a method for detecting Aβ1-42 in peripheral blood.

[0013] Technical solution: In order to solve the above technical problems, the present invention provides a monoclonal antibody, which includes monoclonal antibody 2E7 and / or monoclonal antibody 3B6.

[0014] The amino acid sequence of the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No. 1, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No. 2, the amino acid sequence of the light chain variable region of the monoclonal antibody 3B6 is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No. 4; the light chain variable region and heavy chain variable region of the 2E7 and 3B6 monoclonal antibodies are both composed of complementarity determining regions and framework regions, and the complementarity determining regions are both composed of CDR1, CDR2 and CDR3. The complementarity determining regions are shown in the following table:

[0015]

[0016] The present invention also includes a nucleic acid molecule encoding the monoclonal antibody. The nucleotide sequence of the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.5, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No.6, the nucleotide sequence of the light chain variable region of the monoclonal antibody 3B6 is shown in SEQ ID No.7, and the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3B6 is shown in SEQ ID No.8.

[0017] The present invention also includes an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium comprising the nucleic acid molecule.

[0018] The present invention also includes a universal M13 phage probe, wherein the universal M13 phage probe fully displays the monoclonal antibody at the N-terminus of the M13 P3 protein.

[0019] The universal M13 phage probe further comprises a recombinant phage obtained by performing site-directed mutation on the 31st amino acid of the P8 mature protein and performing biotinylation modification.

[0020] Wherein, the 31st amino acid of the P8 mature protein is mutated from valine to cysteine. Preferably, the phage probe further contains horseradish peroxidase-labeled streptavidin or other streptavidin labels.

[0021] The present invention also includes a method for preparing the universal M13 phage probe, comprising the following steps:

[0022] 1) Construction of plasmid p5E-P3CT scFv: Vector fragment 1 containing the N1 and N2 genes deleted from the M13P3 gene was cloned using primers p5E-F and p5E-R. Using the scFv gene of the monoclonal antibody 2E7 as a template, fragment 2 of the single-chain antibody 2E7-scfv was cloned using primers 2E7-F and 2E7-R. Fragments 1 and 2 were homologously recombined and ligated to generate the plasmid p5E-P3CT scFv.

[0023] (2) Construction of plasmid M13KO7ΔP3-V31C: Primers ΔP3-F and ΔP3-R were used to delete aa10-412 of the P3 gene, leaving only the first 9 amino acids of the signal peptide and the last 12 amino acids of the CT region, resulting in plasmid M13KO7ΔP3. Then, using M13KO7ΔP3 as a template, primers V31C-F and V31C-R were used to clone the plasmid M13KO7ΔP3-V31C in which the valine 31 of the M13 P8 mature protein was mutated to cysteine.

[0024] (3) Preparation of the universal M13@scFv@Biotin probe: The plasmids obtained in steps (1) and (2) were co-transformed into Escherichia coli, and a bacterial solution containing the double plasmids was cultured to assemble into the phage M13@scFv. M13@scFv was incubated with BMCC-Biotin at room temperature, and then separated through a desalting column to obtain the recombinant phage M13@scFv@Biotin.

[0025] Wherein, the sequences of primers p5E-F, p5E-R, 2E7-F and 2E7-R in step 1) are as follows:

[0026] p5E-F:

[0027] CCACCACCACCACCACGCACCGGCACCGGCGCCGgcacctgccccggattttgattatgaaaagatggcaaac

[0028] p5E-R:

[0029] GCAAGCCAGCGGCTGCCGTAGGCAATAGGtatttcatggttaatttctcctctttaatgaattctgtgtgaaattgttatccgctcacaat

[0030] 2E7-F:

[0031] CCTATTGCCTACGGCAGCCGCTGGCTTGCtgctgctggcagctcagccggccatggcgaagcttgtgaagctgcagcagtcaggacctgagct

[0032] 2E7-R:

[0033] CGGCGCCGGTGCCGGTGCGTGGTGGTGGTGGTGGtgggatccaggtggtccaccatcagcccg;

[0034] Preferably, the sequences of primers ΔP3-F, ΔP3-R, V31C-F and V31C-R in step 2) are as follows:

[0035] ΔP3-F: AATTATTATTCGCAATTCCTTCTACGTTTGCTAACATACT

[0036] ΔP3-R: AGTATGTTAGCAAACGTAGAAGGAATTGCGAATAATAATT

[0037] V31C-F:GGTTGTTTGCATTGTCGGCGCAACTATCGGTA

[0038] V31C-R: GACAATGCAAACAACCATCGCCCACGCATAAC.

[0039] The present invention also includes the use of the monoclonal antibody, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or recombinant bacteria, and the probe in preparing a detection kit for detecting Aβ1-42.

[0040] The present invention also includes a method for detecting Aβ1-42 in peripheral blood, which preferably comprises the following steps:

[0041] (1) Add monoclonal antibody 3B6 to the ELISA plate, incubate, and rinse;

[0042] (2) Add blocking solution to the ELISA plate, incubate, and rinse;

[0043] (3) Add the sample / standard to be tested to the ELISA plate, incubate, and rinse;

[0044] (4) Adding probes or other control samples to the ELISA plate, incubating, and washing;

[0045] (5) Add HRP-conjugated secondary antibody with corresponding affinity, incubate, and wash;

[0046] (6) Add TMB colorimetric solution to develop color, stop, read OD450, and determine the content of the sample to be tested according to the standard curve

[0047] Invention Mechanism: The present invention uses a dual-plasmid system to prepare phage probes. The P3 gene is knocked out of the plasmidated M13KO7 genome, making the P3CT scFv fusion protein expressed by the p5E-P3CT scFv the sole source of P3 protein for phage assembly, thereby achieving full-valence display of the single-chain antibody. Simultaneously, the 31st amino acid of the P8 mature protein of the plasmidated M13KO7 genome is site-directedly mutagenized and biotinylated. Utilizing the quantitative advantage of the M13 phage P8 protein (approximately 2,700 copies) and the strong binding affinity of biotin-streptavidin, further universal modification of the probe can be achieved to meet different purification, enrichment, and detection requirements.

[0048] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: It provides a pair of monoclonal antibodies 2E7 and 3B6, and a universal M13@scFv@Biotin probe that enables full-valent display of scFv on the M13 P3 protein and full-valent modification of biotin on the P8 protein. By simply replacing the scFv gene on the plasmid p5E-P3CTscFv, specific binding to different antigens can be achieved. Furthermore, the approximately 2,700 biotin modified on the probe can bind with high affinity to any streptavidin-labeled molecules and materials (such as HRP-SA, SA-AuNPs, SA-MNPs, etc.), adapting to the needs of different detection methods, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Western blot analysis of the M13 probe displaying scFv described in the examples of the present invention. M: protein molecular weight marker; 1: M13KO7@scFv; 3: Hyperphage@scFv; 5: M13@scFv@Biotin; 2, 4: wild-type M13 P3 protein.

[0050] Figure 2 Graphs showing protein spectrum analysis of P8 for wild-type M13KO7 (M13 P8 WT), M13@scFv (M13P8 V31C), and M13@scFv@Biotin (M13 P8 BMCC) described in the examples of the present invention.

[0051] Figure 3 This is the ELISA quantitative standard curve of M13@scFv@Biotin and 2E7 monoclonal antibody described in the examples of the present invention. DETAILED DESCRIPTION

[0052] The following detailed and comprehensive description of the embodiments of the present invention is provided in conjunction with specific examples. Obviously, the described examples are only intended to illustrate a portion of the present invention and should not be construed as limiting the scope of the invention. In the examples, the specific conditions not otherwise specified are all carried out according to conventional conditions or manufacturer's recommendations. Reagents or instruments used that do not indicate the manufacturer are all commercially available conventional products.

[0053] Helper phage M13KO7 was purchased from NEB, USA, with the catalog number N0315S; pCANTAB 5E was purchased from Wuhan Miaoling Biotechnology Co., Ltd., with the catalog number P1099; Hyperphage M13 KO7ΔpIII was purchased from PROGEN, USA, with the catalog number PRHYPE; 2×Phanta Max Master Mix (Dye Plus) was purchased from Nanjing Novozymes Biotechnology Co., Ltd., with the catalog number P525-01; FastPure Viral DNA / RNA Mini Kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd., with the catalog number RC311; -Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No. CU201-02); TG1 competent cells were purchased from Shanghai Tolo Harbor Biotechnology Co., Ltd., Catalog No. CC96170; DH5α competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., Catalog No. DL1001S; BMCC-Biotin (1-biotinamido-4-[4'-(methylmaleimide)cyclohexanecarboxamide]butane) was purchased from Beijing Biolab Technology Co., Ltd., Catalog No. GS4327; horseradish peroxidase-conjugated Streptavidin (HRP-conjugated Streptavidin) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (HRP-conjugated goat anti-mouse IgG (H+L)) was purchased from Shanghai Beyotime Biotechnology Co., Ltd., Catalog No. A0216; anti-M13 / fd / F1 Filamentous Phages mouse monoclonal, B62-FE2, HRP Conjugate was purchased from PROGEN, USA, Catalog No. 61097-HRPS. Aβ1-42 monoclonal antibodies 2E7 and 3B6 were prepared in our laboratory. Aβ1-42, Aβ1-42-KLH, and Aβ1-42-BSA peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.; anti-M13-pIII monoclonal antibody was purchased from New England Biolabs, USA, Catalog No. E8033S. Primer synthesis and sequencing services were provided by Beijing Qingke Biotechnology Co., Ltd.

[0054] BALB / C mice were purchased from the Center for Comparative Medicine of Yangzhou University. SP2 / 0 was the cell line used in this experiment. Freund's incomplete adjuvant, catalog number: P2031-10 ml; Freund's complete adjuvant, catalog number: P2036-10 ml; DMEM, purchased from Gibco, USA, catalog number: 11965126; polyethylene glycol 1450 (PEG1450), catalog number: P7181-5X5 ml, Hybri-Max TM HAT medium supplement (50×), Catalog No. H0262-10VL, HT supplement (100×), Catalog No. 11067030, were purchased from Sigma, USA; Australian fetal bovine serum, Catalog No. A511-001, was purchased from Suzhou Shuanglu Biotechnology Co., Ltd.; 96-well cell culture plates, Catalog No. 701002, 48-well cell culture plates, Catalog No. 748002, and ELISA plates, Catalog No. 514201, were purchased from Wuxi Nice Life Science Co., Ltd.; Penicillin-Streptomycin Solution (100×), Catalog No. C0222, Protein A+G Agarose (Fast Flow, 1 ml) prepacked column, Catalog No. P2028, were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Total RNA extraction kit, Catalog No. RC411-01, cDNA single-strand synthesis kit, Catalog No. R212-01, Phanta Max Super-Fidelity DNA Polymerase, product number: P505-d1, were purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0055] The following are the primer sequences, PCR amplification system, and PCR amplification conditions used in the PCR amplification process of the embodiments of the present invention.

[0056] 1. Primers

[0057] Table 1 Primers used in the present invention

[0058]

[0059]

[0060] 2. 2×Phanta Max Master Mix (Dye Plus) PCR amplification system and amplification procedure

[0061] Table 2 Preparation of high-fidelity enzyme PCR system with a total volume of 50 μL

[0062] Components volume <![CDATA[ddH2O]]> 19 μl 2×Phanta Max Master Mix(Dye Plus) 25 μl Upstream primer (10 μM) 2 μl Downstream primer (10 μM) 2 μl Template DNA 2 μl

[0063] PCR reaction program: 95°C for 3 min; 95°C for 15 s, 56°C for 15 s, 72°C for 30-60 sec / kb, for a total of 35 cycles; 72°C for 5 min.

[0064] Example 1

[0065] In this example, the amyloid-β protein 1-42 (Aβ1-42) peptide was selected as the immunogen. When synthesizing the Aβ1-42 peptide, a cysteine ​​C was added to the N-terminus for labeling with the carrier protein hemocyanin KLH. When synthesizing the Aβ1-42 peptide, the carrier protein bovine serum albumin (BSA) was linked to the N-terminus via a cysteine ​​for titer testing of animal immune serum and subsequent detection of rabbit monoclonal antibody expression supernatant. Aβ1-42, Aβ1-42-KLH, and Aβ1-42-BSA were all commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis.

[0066] Aβ1-42 synthetic peptide, the sequence is:

[0067] CDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

[0068] This example provides a method for preparing a monoclonal antibody against Aβ1-42. The preparation method is based on hybridoma technology and specifically comprises the following steps:

[0069] 1. Animal immunization

[0070] For the first immunization, Freund's complete adjuvant was mixed with the antigen in equal parts at a 1:1 ratio. After thorough emulsification, 50 μg of Aβ1-42-KLH antigen was administered to each BALB / C mouse. Subsequent immunizations were performed using the same method, emulsified with Freund's incomplete adjuvant, at a rate of 50 μg per mouse. Multiple immunizations were performed subcutaneously, with an interval of 2-3 weeks between each immunization. Seven days after the third immunization, tail tip blood was collected to test the titer. The tail blood was then placed at 4°C for 30 minutes. After serum precipitation, it was collected and a blank mouse serum control was established, processed in the same manner as the mouse serum. The maximum serum dilution factor at which the OD450nm ratio between the experimental and control groups was greater than 2.1 was used to determine the mouse serum titer. Once the serum titer reached 1:12,800, mice with the highest titer were selected and boosted intraperitoneally with an equal volume (200 μl) of an emulsion of 50 μg antibody and Freund's incomplete adjuvant. Three days later, the mice were sacrificed and cell fusion was performed.

[0071] 2. Cell fusion

[0072] HAT medium configuration: 1 mL Hybri-Max TMHAT culture medium supplement (50×), 5mL Australian fetal bovine serum, 500ul penicillin-streptomycin solution, 43.5mL DMEM culture medium, mix well. Blood was collected from the eyeballs of mice that had completed the booster immunization, and the serum was separated as a positive control. After the mice were killed by cervical dislocation, they were immersed in 75% ethanol for 10 minutes, and the mice were fixed on the dissection table in the biosafety cabinet. Use sterilized tweezers to lift the abdominal skin, use sterile scissors to cut a small piece of skin from the lower abdomen upwards, separate the skin and peritoneum, carefully open other internal organs, separate the spleen, gently remove it, and place it in a culture dish containing 20ml DMEM culture medium. Use a syringe filled with 20ml DMEM culture medium to pierce the top of the spleen, penetrate the spleen, gently push the syringe, blow out the spleen cells in the culture dish, repeat several times until the spleen no longer changes color, and filter it through a filter to obtain spleen cells. SP2 / 0 and splenocytes were counted and mixed at a cell ratio of 8:1. The mixture was inverted and centrifuged at 1000 rpm for 4 min. In a 37°C water bath, the precipitated cells were tapped to evenly distribute them at the bottom of the tube and allowed to stand for 1 min. Within 1 min, 1 ml of PEG1450 was added to the centrifuge tube and allowed to stand for 1 min. Within 30 s, 1 ml of 37°C preheated DMEM was added along the tube wall to terminate the cell fusion reaction. The pipette tip was inserted below the liquid surface and 1 ml of DMEM was added over 1 min. This step was repeated until 20 ml of DMEM was added. 30 ml of DMEM was slowly added and the tube was centrifuged at 800 rpm for 4 min. The supernatant was discarded. 50 ml of DMEM was added and the tube was centrifuged at 800 rpm for 4 min. The supernatant was discarded, and HAT medium was added. The cells were gently aspirated and plated into a 96-well cell culture plate (200 μl / well) and labeled. After 7 days, indirect ELISA was performed.

[0073] 3. Screening of positive hybridoma cells

[0074] HT medium was prepared by mixing 500 μL of HT supplement (100X), 5 mL of Australian fetal bovine serum, 500 μL of penicillin-streptomycin solution, and 44 mL of DMEM. Hybridoma cell supernatant was collected and screened for positive hybridoma cells using indirect ELISA. Wells with high OD450 nm values ​​and only single cell clumps were selected, the medium discarded, and 200 μL of HT medium was added. The cells were pipetted and counted. Approximately 200 cells were plated onto half a 96-well plate. The remaining cells were passaged to a 48-well plate for further expansion and cryopreserved. After 7 days, single clones were identified by ELISA and subcloned again using the above method. After three subcloning cycles, single cell clumps with high OD450 nm values ​​were selected and cloned again using the above method. If all wells with cells had high OD450 nm values ​​and the OD450 nm values ​​of wells without cell clumps were no higher than those of the negative control, the hybridoma cell line was considered to secrete monoclonal antibodies.

[0075] 4. Preparation of Monoclonal Antibodies

[0076] The cell line obtained in step 3 was injected into the peritoneal cavity of mice, the mice were cultured, and ascites was extracted from the peritoneal cavity of the mice for purification. The specific operation steps are as follows: 500 μl of Freund's incomplete adjuvant was injected into the peritoneal cavity of the mice. After 24 hours, approximately 1×10 7 Hybridoma cells were injected into the peritoneal cavity of mice, and ascites was collected 7 days later.

[0077] Prepare the following purification buffers: Binding buffer: phosphate buffered saline (PBS, 0.01M, pH 7.4). Prepare by dissolving 8g NaCl, 1.44g Na2HPO4, 0.24g KH2PO4, and 0.2g KCl in 800ml of ultrapure water. Adjust the pH to 7.4 with 0.1M NaOH, and then dilute to 1L. Neutralization buffer: 1M Tris-HCl, pH 8.8. Prepare by weighing 121.1g Tris and dissolving it in 800ml of ultrapure water. Adjust the pH to 8.8 with concentrated hydrochloric acid, and then dilute to 1L. Elution buffer: 100mM glycine, pH 2-3. Prepare by dissolving 7.507g glycine in 800ml of ultrapure water, adjusting the pH to 2.5 with concentrated hydrochloric acid, and then dilute to 1L.

[0078] Antibodies were purified using a Biyuntian Protein A+G Agarose (Fast Flow, 1 ml) pre-packed column. The specific steps were as follows: centrifuge the ascites at 10,000 rpm for 10 min, collect the supernatant, and take 20 μl to prepare a sample; filter the Binding buffer, Neutralization buffer, and Elution buffer with a 0.45 μm filter for later use; dilute the ascites twice with Binding buffer; fill a syringe with 10 ml of Binding buffer and connect it to the purification column. After removing bubbles, slowly push the piston to remove the storage solution; draw 10 ml of Binding buffer at a flow rate of 1 ml / min to equilibrate the column; draw the diluted ascites with a syringe at a flow rate of 1 ml / min to allow the antibody to bind to the column; draw up 10 ml of Binding buffer to wash away unbound antibodies until the outflowing liquid is colorless; draw 5 ml of Elution buffer to elute the antibodies bound to the column, and adjust the collected eluate to pH 7.5 with Neutralization buffer. 20 μl of sample was taken from each tube to prepare a sample for use in identifying the purified monoclonal antibody.

[0079] 5. Determination of monoclonal antibody cell titer

[0080] (1) Dissolve the Aβ1-42-BSA conjugate complex in coating solution (0.15 g sodium carbonate, 0.29 g sodium bicarbonate, 0.02 g sodium azide, add double-distilled water to 100 ml, adjust to pH 9.6) to 5 μg / ml. Add 100 μl of coating solution to each well and incubate at 4°C overnight. Wash three times with immersion for 3 minutes each time.

[0081] (2) Add 200 μl of blocking solution (5% skim milk powder) to each well and incubate at 37°C for 2 hours; then soak and wash three times, each for 3 minutes;

[0082] (3) Add serially diluted 2E7 and 3B6 cell supernatants and negative mouse serum, 100 μl / well, incubate at 37°C for 1 hour, and then soak and wash three times for 3 minutes each time.

[0083] (4) Add horseradish peroxidase-labeled goat anti-mouse IgG (H+L), 100 μl / well, incubate at 37°C for 1 hour; soak and wash three times, 3 minutes each time;

[0084] (5) Add 100 μl of TMB colorimetric solution to each well and incubate at 37°C in the dark for 20 min.

[0085] (6) Add 50 μl of stop solution (2 M / L H2SO4) to each well and read the OD450 absorbance using a microplate reader.

[0086] potency 2E7 3B6 Negative serum 1:800 1.1104 1.8394 0.1163 1:1600 0.6562 1.0012 0.0933 1:3200 0.3762 0.5289 0.0867 1:6400 0.2456 0.3014 0.0829 1:12800 0.1661 0.191 0.0805 1:25600 0.1216 0.1318 0.0812

[0087] From the above results, it can be seen that the titer of the 2E7 monoclonal antibody and the 3B6 monoclonal antibody of the present application reached 12,800.

[0088] Example 2 Obtaining Monoclonal Antibody Sequences

[0089] 1. Hybridoma cell culture and total RNA extraction

[0090] Hybridoma cells were cultured in HT medium at 37°C and 5% carbon dioxide until the cell count reached 1×10 7 Total RNA was extracted from cells using a total RNA extraction kit.

[0091] 2. Synthesis of the first strand of cDNA

[0092] cDNA was synthesized using a cDNA single-strand synthesis kit using the extracted total RNA as an amplification template.

[0093] 3. Design upstream and downstream primers for light chain variable region and heavy chain variable region for gene amplification.

[0094] Heavy chain variable region upstream primer: TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC;

[0095] Heavy chain variable region downstream primer: AGGTSMARCTGCAGSAGTCWGG;

[0096] Light chain variable region upstream primer 1: CCGTTTGATTTCCAGCTTGGTGCC;

[0097] Light chain variable region upstream primer 2: CCGTTTTATTTCCAGCTTGGTCCC;

[0098] Light chain variable region upstream primer 3: CCGTTTTATTTCCAACTTTGTCCC;

[0099] Light chain variable region upstream primer 4: CCGTTTCAGCTCCAGCTTGGTCCC;

[0100] Light chain variable region downstream primer: GACATTGAGCTCACCCAGTCTCCA.

[0101] The cDNA reverse transcribed by a special cDNA single-strand synthesis kit was used as a template, and the heavy chain variable region and light chain variable region primers were used to amplify the heavy chain variable region and light chain variable region for gene sequencing.

[0102] 4. Cloning and screening of PCR amplification products

[0103] The sequencing results of the heavy and light chain variable regions are as follows:

[0104] 2E7 light chain variable region amino acid sequence (SEQ ID No. 1)

[0105] DILMTQTPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIKRADGGPP

[0106] 2E7 light chain variable region nucleotide sequence (SEQ ID NO.5)

[0107] GACATTCTGATGACACAGACTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGG AATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGGTGGACCACCTTGA

[0108] 2E7 heavy chain variable region amino acid sequence (SEQ ID No. 2)

[0109] VKLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYGGTSYNQKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARGTKSDYWGQGTTVTVSS

[0110] 2E7 heavy chain variable region nucleotide sequence (SEQ ID No. 6)

[0111] GTGAAGCTGCAGCAGTCAGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACAACATGAACTGGGTGAAGCAGAGCAATGGAAAGAGCCTTGAGTGGATTGGAAATATTGATCCTTACTATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGTACTAAGTCGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0112] Amino acid sequence of the variable region of the 3B6 light chain (SEQ ID No. 3)

[0113] DILVTQTPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPPTFGGGTKLEIKRADGGPTLIQG

[0114] Nucleotide sequence of the variable region of the 3B6 light chain (SEQ ID No. 7)

[0115] GACATTCTGGTGACACAGACTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTAAATAGTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGTCAGCAACATTATAGCACTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGGTGGACCAACCTTGATCCAAGGG

[0116] 3B6 heavy chain variable region amino acid sequence (SEQ ID No. 4)

[0117] VKLQESGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATPYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSALYYCARTGGLLRPQVMDYWGQGTSVTVSS

[0118] 3B6 heavy chain variable region nucleotide sequence (SEQ ID No. 8)

[0119] GTGAAGCTGCAGGAATCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTGAAGCAAAGCCATGTAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTTACAATGGTGCTACTCCCTACAAC CAGAATTTCAAGGACAAGGCCAGCTTGACTGTAGATAAGTCCTCCAGTACAGCCTACATGGAACTCCACAGCCTGACATCTGAGGACTCTGCACTCTATTACTGTGCAAGAACGGGGGGCCTCTTACGGCCCCAGGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCTTCA

[0120] Among them, the light chain variable region and heavy chain variable region of the 2E7 and 3B6 monoclonal antibodies are both composed of complementary determining regions and framework regions. The complementary determining regions are both composed of CDR1, CDR2 and CDR3. The complementary determining regions are shown in the table below:

[0121]

[0122] 5. Obtain scFv fragments by PCR

[0123] RNA was extracted from spleen cells of immunized mice, and the concentration of RNA was measured using a Nanodrop ultramicro spectrophotometer. The purity of the extracted RNA was determined based on the A260 / A280 ratio of pure RNA = 2.0. The RNA was reverse transcribed into cDNA, and 50 μL was taken out for subsequent PCR. The rest was aliquoted and stored at -20°C.

[0124] Using 2E7-VH cDNA (SEQ ID NO. 6) as a template, primers 2E7-F and 2E7-LR were used to amplify the homology arm containing linker (G4S)4, resulting in fragment 2E7-VHL. Using 2E7-VL cDNA (SEQ ID NO. 5) as a template, primers 2E7-LF and 2E7-R were used to amplify the homology arm containing linker (G4S)4, resulting in fragment 2E7-VLL. The reaction conditions used the 2× Phanta Max Master Mix (Dye Plus) high-fidelity PCR amplification system and protocol. After PCR amplification, PCR products were subjected to agarose gel electrophoresis to confirm the amplified DNA fragment, and the target fragment was recovered using a gel recovery kit.

[0125] The antibody light and heavy chain variable region fragments were amplified with linker (G4S) 4 and scFv was obtained by overlap extension PCR using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Novozymes).

[0126] The first step of overlap extension PCR was performed using the following reaction system:

[0127] 2×buffer 25 μL dNTP 1 μL High-fidelity enzyme 1 μL 2E7-VLL 5μL 2E7-VHL 5μL water 13μL

[0128] The reaction conditions were: initial denaturation at 95°C for 3 minutes, followed by 12 cycles of 95°C for 15 seconds, 56°C for 15 seconds, and 72°C for 1 minute. After the reaction was complete, the second step of overlap extension PCR was performed using the following reaction system:

[0129] 2×buffer 25 μL dNTP 1 μL High-fidelity enzyme 1 μL template 10 μL R 2μL F 2μL water 9μL

[0130] The template was the PCR product from the first step of overlap extension PCR. The reaction conditions were: initial denaturation at 95°C for 3 minutes; 12 cycles of 95°C for 15 seconds, 56°C for 15 seconds, and 72°C for 1 minute. Extension was performed at 72°C for 10 minutes. After PCR amplification, the PCR product was subjected to agarose gel electrophoresis to confirm the amplified DNA fragment, and the target fragment was recovered using a gel extraction kit.

[0131] Example 3 Construction of plasmids p5E-P3CT scFv and M13KO7ΔP3-V31C

[0132] 1. Construction of plasmid p5E-P3CT scFv

[0133] The pel B signal peptide and bacterial ribosome binding site RBS were introduced into the plasmid pCANTAB 5E, and the N1 and N2 regions of the M13P3 gene were deleted. This made the fusion protein P3CT scFv similar in size to the wild-type P3, reduced the resistance to post-translational protein translocation and phage assembly, and increased the display efficiency of the scFv.

[0134] Specifically, taking the synthesis of the Aβ1-42-specific monoclonal antibody 2E7 in Example 2 as an example, the principle of homologous recombination was used to insert pel B, RBS, and scFv and delete the N1 and N2 genes in the M13 P3 gene. The pCANTAB 5E vector was cloned by PCR amplification using primers p5E-F / R, and the cloned 2E7 scFv was amplified by PCR using primers 2E7-F / R. Then, the pCANTAB 5E vector and the fragment 2E7 scFv were connected using a homologous recombination kit. The connection product was transformed into DH5α competent cells by chemical transformation. The plasmid was extracted from the bacterial solution that was positive for sequencing verification to obtain the recombinant plasmid p5E-P3CT scFv. The specific primers were designed as follows (the uppercase bold part is the homology arm):

[0135] Upstream primer p5E-F:

[0136] CCACCACCACCACCACGCACCGGCACCGGCGCCGgcacctgccccggattttgattatgaaaagatggcaaac

[0137] Downstream primer p5E-R:

[0138] GCAAGCCAGCGGCTGCCGTAGGCAATAGGtatttcatggttaatttctcctctttaatgaattctgtgtgaaattgttatccgctcacaat

[0139] Upstream primer 2E7-F:

[0140] CCTATTGCCTACGGCAGCCGCTGGCTTGCtgctgctggcagctcagccggccatggcgaagcttgtgaagctgcagcagtcaggacctgagct

[0141] Downstream primer 2E7-R:

[0142] CGGCGCCGGTGCCGGTGCGTGGTGGTGGTGGTGGtgggatccaggtggtccaccatcagcccgt

[0143] 2. Construction of plasmid M13KO7ΔP3-V31C

[0144] Homologous recombination was used to delete the P3 gene and point mutate the P8 gene in the M13KO7 genome. Using the M13KO7 genome as a template, PCR amplification with primers ΔP3-F / R deleted most of the P3 gene (10-412 aa), retaining only the first 9 amino acids of the signal peptide and the last 12 amino acids of the CT region to generate the M13KO7ΔP3 fragment. Using the M13KO7ΔP3 fragment as a template, PCR amplification with primers V31C-F / R mutated the valine (Val) at position 31 of the mature P8 protein (removing the 23 aa signal peptide) to cysteine ​​(Cys), generating the M13KO7ΔP3-V31C fragment for subsequent biotinylation. The M13KO7ΔP3-V31C fragments were then ligated using a homologous recombination kit. The ligation product was then chemically transformed into DH5α competent cells. Plasmids were extracted from the bacterial suspension that was positive for sequencing to obtain the recombinant plasmid M13KO7ΔP3-V31C. The specific primers were designed as follows (the bolded portion indicates the location of the point mutation):

[0145] Upstream primer ΔP3-F: AATTATTATTCCGCAATTCCTTCTACGTTTGCTAACATACT Downstream primer ΔP3-R: AGTATGTTAGCAAACGTAGAAGGAATTGCGAATAATAATT Upstream primer V31C-F: GGTTGTTTGCATTGTCGGCGCAACTATCGGTA

[0146] Downstream primer V31C-R: GACAATGCAAACAACCATCGCCCACGCATAAC

[0147] Example 4 Preparation of M13@scFv@Biotin Universal Probe

[0148] Plasmids p5E-P3CT scFv and M13KO7ΔP3-V31C were co-transformed into TG1 competent cells. After sequencing confirmation, the cells were cultured overnight at 37°C in 2×YT (16 g of peptone, 10 g of yeast extract, and 5 g of NaCl were weighed and added to ddH2O to a volume of 1 L) containing 60 μg / mL ampicillin and 50 μg / mL kanamycin. The cells were centrifuged at 8000 rpm for 10 min at 4°C to remove the bacteria. The phages were then precipitated by centrifugation at 48000 rpm for 4 h at 4°C. The pellet was resuspended in PBS and briefly centrifuged to remove cell debris to obtain the phage M13@scFv.

[0149] An appropriate volume of 8 mM BMCC-Biotin was added to M13@scFv, mixed well, and incubated at room temperature for 2 h. Unreacted BMCC-Biotin was separated using a desalting column to obtain the recombinant phage M13@scFv@Biotin.

[0150] Example 5: Verification of full-valence display of M13@scFv@Biotin universal probes P3 and P8

[0151] The pel B signal peptide and bacterial ribosome binding site (RBS) were introduced into the plasmid pCANTAB 5E. The pCANTAB 5E vector portion was cloned by PCR amplification using primers p5E-F1 / p5E-R, and the 2E7 scFv portion was cloned by PCR amplification using primers 2E7-F / 2E7-R1. The pCANTAB 5E vector and the 2E7 scFv fragment were then ligated using a homologous recombination kit. The ligation product was chemically transformed into DH5α competent cells, and the plasmid was extracted from the bacterial solution verified to be positive by sequencing to obtain the recombinant plasmid p5E-scFv.

[0152] Phage display procedures: Transform TG1 competent cells with the p5E-P3CT scFv plasmid. After sequencing confirmation, expand the TG1 cells to 200 mL of 2×YT medium. When the OD600 of the culture reaches 0.6, add M13KO7 phage. Mix well, incubate at 37°C for one hour, and then incubate overnight at 37°C, 220 rpm. The next day, remove the cells by centrifugation at 8000 rpm for 10 minutes at 4°C. Sediment the phage by centrifugation at 48000 rpm for 4 hours at 4°C, resuspend the pellet in PBS, and briefly centrifuge to remove cell debris. This yields the phage M13KO7@scFv. The display procedures for Hyperphage@scFv are the same as for M13KO7@scFv, except that M13KO7 is replaced with Hyperphage when adding phage.

[0153] Western Blot was used to verify the full display of scFv on P3CT. Western Blot experiments were performed using M13KO7@scFv (based on phagemid p5E-scFv and helper phage M13KO7 display), Hyperphage@scFv (based on phagemid p5E-scFv and commercial improved helper phage Hyperphage M13 KO7ΔpIII display), and M13@scFv@Biotin (based on dual plasmid system p5E-P3CT scFv and M13KO7ΔP3-V31C display). At the same time, M13 P3 protein (obtained in M13KO7 phage) was set as a control, anti-M13-pIII monoclonal antibody was used as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. The results are shown in Figure 2. Figure 1As shown, most of M13KO7@scFv is wild-type P3, and the proportion of P3-scFv fusion protein is about 5%; the proportion of P3-scFv fusion protein in Hyperphage@scFv is about 32%; M13@scFv@Biotin has only one band of P3CT-scFv fusion protein, with a proportion of 100%, proving that the probe achieves full-valence display of exogenous scFv on M13 P3 protein.

[0154] The MALDI Biotyper was used to perform mass spectrometry analysis on the P8 proteins of wild-type M13KO7 (M13 P8WT, NEB, USA), M13@scFv (M13 P8 V31C, containing the V31C mutation of the P8 protein), and M13@scFv@Biotin (M13 P8 BMCC, containing the biotinylated modification of the P8 protein). The results showed that in the range of 1000-10000 m / z, the P8 protein of wild-type M13KO7 had a single peak at 5242.581, M13@scFv had a single peak at 5245.952, and M13@scFv@Biotin had a single peak at 5780.122, indicating that all P8 proteins were successfully point-mutated and all were successfully modified with biotin (e.g. Figure 2 shown).

[0155] Example 6 Establishment of a sandwich ELISA method for detecting Aβ1-42 based on the M13@scFv@Biotin universal probe

[0156] The specific application of the M13@scFv@Biotin universal probe described in the present invention in ELISA detection is to establish a sandwich ELISA detection method using the Aβ1-42-specific monoclonal antibody pair 2E7 and 3B6 synthesized in Example 2 as an example (recognizing Aβ1-14 and Aβ22-42, respectively).

[0157] To prepare phosphate buffered saline (PBS, 0.01M, pH 7.4): Dissolve 8g NaCl, 1.44g Na2HPO4, 0.24g KH2PO4, and 0.2g KCl in 1L ultrapure water. Adjust the pH to 7.4 with 0.1M NaOH. To prepare PBST: Dissolve 8g NaCl, 1.44g Na2HPO4, 0.24g KH2PO4, 0.2g KCl, and 0.5mL Tween-20 in 1L ultrapure water. Adjust the pH to 7.4 with 0.1M NaOH.

[0158] (1) Coat the ELISA plate with humanized monoclonal antibody 3B6 (1 μg / ml), add 100 μl per well, and incubate overnight at 2-8°C.

[0159] (2) Wash with PBST three times, 5 min each time, add 200 μl of blocking solution to each well, and block at 37°C for 2 h;

[0160] (3) Wash with PBST five times for 5 min each time, and add 100 μl of 1 ng / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.25 pg / ml, 15.625 pg / ml, and 7.8125 pg / ml of Aβ1-42 peptide and PBS (blank control) to each well, with one row for each dilution, and incubate at 37°C for 1 h;

[0161] (4) Wash with PBST 5 times, 5 min each time, and add 100 μl of M13@scFv@Biotin (containing 2E7scFv, about 10 9 phages), mouse 2E7 monoclonal antibody (positive control, 3 ng / ml, about 10 9 monoclonal antibody molecules), wild-type M13KO7 (negative control, about 10 9 phages), 3 columns each, incubated at 37°C for 1 h;

[0162] (5) Wash with PBST five times for 5 min each time, add 100 μl of commercial horseradish peroxidase-labeled Streptavidin to the M13@scFv@Biotin wells; add 100 μl of commercial horseradish peroxidase-labeled goat anti-mouse IgG (H+L) to the mouse 2E7 monoclonal antibody wells; add 100 μl of commercial anti-M13 / fd / F1 Filamentous Phages mouse monoclonal HRP Conjugate to the wild-type M13KO7 wells, and incubate at 37°C for 1 h;

[0163] (6) Wash with PBST five times, 5 min each time, add 100 μl of TMB color development solution to each well, and develop at 37°C in the dark for 20 min;

[0164] (7) Add 50 μl of stop solution (2M / L H2SO4) to each well and read the OD450 absorbance using a microplate reader. Quantitative standard curves for M13@scFv@Biotin and 2E7 monoclonal antibody were established based on the OD values ​​(e.g. Figure 3 shown).

[0165] Results showed that the universal M13@scFv@Biotin probe described in this invention, due to the quantitative advantage of the M13 phage P8 protein (approximately 2,700 copies) and the strong binding affinity of biotin-streptavidin, can modify thousands of HRP molecules, significantly amplifying the ELISA detection signal and replacing the HRP-labeled secondary antibody in traditional ELISA. In a specific embodiment, the detection limit of the positive control (traditional antibody) was 62.5 pg / ml. In comparison, the detection limit of the universal M13@scFv@Biotin probe constructed in this invention was 7.8125 pg / ml, which is more sensitive than traditional methods.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibodies include monoclonal antibody 2E7 and / or monoclonal antibody 3B6, the amino acid sequence of the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No. 1, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No. 2, the amino acid sequence of the light chain variable region of the monoclonal antibody 3B6 is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No. As shown in No. 4; the light chain variable region and heavy chain variable region of the 2E7 and 3B6 monoclonal antibodies are composed of complementary determining regions and framework regions, and the complementary determining regions are composed of CDR1, CDR2 and CDR3. The amino acid sequence of the CDR1 light chain variable region of the monoclonal antibody 2E7 is QSLLYSSNQKNY, the amino acid sequence of the CDR1 heavy chain variable region of the monoclonal antibody 2E7 is GYSFTGYN, the amino acid sequence of the CDR2 light chain variable region of the monoclonal antibody 2E7 is WAS, the amino acid sequence of the CDR2 heavy chain variable region of the monoclonal antibody 2E7 is IDPYYGGT, the amino acid sequence of the CDR3 light chain variable region of the monoclonal antibody 2E7 is QQYYSYPYT, and the amino acid sequence of the CDR2 heavy chain variable region of the monoclonal antibody 2E7 is The amino acid sequence of the CDR3 heavy chain variable region of the monoclonal antibody 2E7 is ARGTKSDY, the amino acid sequence of the CDR1 light chain variable region of the monoclonal antibody 3B6 is QSLLNSSNQKNY, the amino acid sequence of the CDR1 heavy chain variable region of the monoclonal antibody 3B6 is GYSFTGYY, the amino acid sequence of the CDR2 light chain variable region of the monoclonal antibody 3B6 is FAS, the amino acid sequence of the CDR2 heavy chain variable region of the monoclonal antibody 3B6 is INPYNGAT, the amino acid sequence of the CDR3 light chain variable region of the monoclonal antibody 3B6 is QQHYSTPPT, and the amino acid sequence of the CDR3 heavy chain variable region of the monoclonal antibody 3B6 is ARTGGLLRPQVMDY.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody according to claim 1, the nucleotide sequence of the light chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID NO.5, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown in SEQ ID No.6, the nucleotide sequence of the light chain variable region of the monoclonal antibody 3B6 is shown in SEQ ID No.7, and the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3B6 is shown in SEQ ID No.

8.

3. An expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium, characterized in that: Comprising the nucleic acid molecule of claim 2.

4. A universal M13 phage probe, characterized in that: The universal M13 phage probe fully displays the monoclonal antibody according to claim 1 at the N-terminus of the M13 P3 protein.

5. The universal M13 phage probe according to claim 4, characterized in that: The universal M13 phage probe also includes a recombinant phage obtained by performing site-directed mutation on the 31st amino acid of the P8 mature protein and performing biotinylation modification.

6. The universal M13 phage probe according to claim 5, characterized in that: The 31st amino acid of the P8 mature protein is mutated from valine to cysteine.

7. The universal M13 phage probe according to claim 6, characterized in that: The phage probe further contains horseradish peroxidase-labeled streptavidin or other streptavidin labels.

8. The method for preparing the universal M13 phage probe according to any one of claims 4 to 7, characterized in that: The following steps are involved: (1) Construction of plasmid p5E-P3CT scFv: Using primers p5E-F and p5E-R, vector fragment 1 containing the N1 and N2 genes deleted from the M13 P3 gene was cloned. Using the scFv gene of the monoclonal antibody 2E7 as a template, primers 2E7-F and 2E7-R were used to clone fragment 2 of the single-chain antibody 2E7-scfv. Fragments 1 and 2 were homologously recombined and ligated to obtain the plasmid p5E-P3CT scFv. (2) Construction of plasmid M13KO7ΔP3-V31C: Primers ΔP3-F and ΔP3-R were used to delete the 10th to 412th amino acids of the P3 gene, leaving only the first 9 amino acids of the signal peptide and the last 12 amino acids of the CT region, to obtain plasmid M13KO7ΔP3. Then, using M13KO7ΔP3 as a template, primers V31C-F and V31C-R were used to clone the plasmid M13KO7ΔP3-V31C in which the valine 31 of the M13 P8 mature protein was mutated to cysteine. (3) Preparation of the universal probe M13@scFv@Biotin: The plasmids obtained in steps (1) and (2) were co-transformed into Escherichia coli, and a bacterial solution containing the double plasmids was cultured to assemble into the phage M13@scFv. M13@scFv was incubated with BMCC-Biotin at room temperature, and then separated through a desalting column to obtain the recombinant phage M13@scFv@Biotin.

9. The method for preparing the universal M13 phage probe according to claim 8, wherein: The sequences of primers p5E-F, p5E-R, 2E7-F, and 2E7-R in step (1) are as follows: p5E-F: CCACCACCACCACCACGCACCGGCACCGGCGCCGgcacctgccccggattttgattatgaaaagatggcaaac p5E-R: GCAAGCCAGCGGCTGCCGTAGGCAATAGGtatttcatggttaatttctcctctttaatgaattctgtgtgaaattgttatccgctcacaat 2E7-F: CCTATTGCCTACGGCAGCCGCTGGCTTGCtgctgctggcagctcagccggccatggcgaagcttgtgaagctgcagcagtcaggacctgagct 2E7-R: CGGCGCCGGTGCCGGTGCGTGGTGGTGGTGGTGGtgggatccaggtggtccaccatcagcccg.

10. The method for preparing a universal M13 phage probe according to claim 8, wherein: The sequences of primers ΔP3-F, ΔP3-R, V31C-F, and V31C-R in step (2) are as follows: ΔP3-F: AATTATTATTCGCAATTCCTTCTACGTTTGCTAACATACT ΔP3-R: AGTATGTTAGCAAACGTAGAAGGAATTGCGAATAATAATT V31C-F:GGTTGTTTGCATTGTCGGCGCAACTATCGGTA V31C-R: GACAATGCAAACAACCATCGCCCACGCATAAC.

11. Use of the monoclonal antibody according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette, recombinant vector, recombinant cell or recombinant bacterium according to claim 3, or the probe according to any one of claims 4 to 7 in the preparation of a detection kit for detecting Aβ1-42.

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