Universal M13 bacteriophage probe for displaying scFv in complete value, and construction method and application of universal M13 bacteriophage probe

Abstract

The invention discloses a monoclonal antibody, a nucleic acid molecule, an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium. The invention further discloses a universal M13 bacteriophage probe for complete display of scFv and a preparation method and application of the universal M13 bacteriophage probe, the monoclonal antibody is high in titer, and the universal bacteriophage probe is low in cost, high in sensitivity and good in stability, has certain universality and can be used as a novel biological detection probe. Trace biomarkers, pathogens and the like in a sample matrix are separated and detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular 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 were able to display fusion proteins on the surface of virions after inserting foreign DNA fragments into the phage coat protein gene. Later, 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. The most commonly used phage display technology is the M13 phage among filamentous phages, mainly because it can integrate long fragments of foreign DNA into its genome. The P3 phage library can display 1 to 5 copies of large foreign proteins on its surface. Although the copy number is low, it can be effectively used to display large peptides or proteins. However, the usual P3 display is often a monovalent display, which is not very efficient. Approximately 2700 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 2700 copies can be presented, and peptides with a maximum length of 6 amino acids can be displayed along the phage capsid. However, P8 is very suitable for displaying peptides and small proteins, but cannot display long peptides or large proteins.

[0003] Bacteriophage is a unique nanomaterial that can achieve site-specific universality of a specific type of coat protein through genetic engineering, while chemical modification has the advantage of further expanding the function of phage, beyond natural peptides and proteins. By taking advantage of genetic engineering and chemical modification, different types of coat proteins can be manipulated to have the desired function, making the M13 phage a multifunctional sensing platform with various functional regions that can perform their functions without interfering with each other. Currently in the biological field, M13 phage has been widely used in vaccine preparation, targeted drug delivery, pathogen detection and treatment, disease diagnosis, biological scaffolds and other fields.

[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 clinical manifestations are characterized by acquired cognitive impairment, involving cognitive domains such as memory, learning, orientation, comprehension, judgment, calculation, language, and visual-spatial functions, which affect daily living abilities and social and occupational functions to varying degrees, and eventually progress to global dementia.

[0005] At present, the research on biomarkers of Alzheimer's disease mainly includes 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 hippocampal atrophy, and positron emission tomography (PET) imaging have confirmed their diagnostic value for Alzheimer's disease and have begun to be used in clinical practice. However, the examination of imaging markers and cerebrospinal fluid markers has the problems of high cost and invasiveness, so it is not easy 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, which is not conducive to tracking the treatment effect. Blood is a human sample that is easy to collect. 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 content of Aβ1-42 in peripheral blood can be used as a potential biomarker to predict the development of Alzheimer's disease in patients with preclinical and mild cognitive impairment. However, the content of Aβ1-42 in peripheral blood is relatively low, and a more sensitive new detection technology is 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 needs 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 needs 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 the recombinant bacteria, and the probe in the preparation of 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 complementary determining regions and framework regions, and the complementary determining regions are both composed of CDR1, CDR2 and CDR3. The complementary 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 as SEQ ID NO.5, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown as SEQ ID No.6, the nucleotide sequence of the light chain variable region of the monoclonal antibody 3B6 is shown as SEQ ID No.7, and the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3B6 is shown as 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 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.

[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 markers.

[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 in which the N1 and N2 genes in the M13P3 gene were deleted was cloned using primers p5E-F and p5E-R; fragment 2 of the single-chain antibody 2E7-scfv was cloned using primers 2E7-F and 2E7-R using the scFv gene of the monoclonal antibody 2E7 as a template, and fragment 1 and fragment 2 were homologously recombined to obtain plasmid p5E-P3CT scFv;

[0023] (2) Construction of plasmid M13KO7ΔP3-V31C: Primers ΔP3-F and ΔP3-R were used to delete aa 10-412 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, 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.

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

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

[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) adding HRP-conjugated secondary antibody of corresponding affinity, incubating, and washing;

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

[0047] Invention mechanism: The present invention prepares phage probes through a dual-plasmid system, and the P3 gene is knocked out of the plasmidated M13KO7 genome, so that the P3CT scFv fusion protein expressed by p5E-P3CT scFv becomes the only P3 protein source for phage assembly, thereby realizing full-valence display of single-chain antibodies; at the same time, the 31st amino acid of the P8 mature protein of the plasmidated M13KO7 genome is site-directedly mutated and biotinylated, and the quantitative advantage of the M13 phage P8 protein (about 2700 copies) and the strong binding force of biotin-streptavidin are utilized to realize further universal modification of the probe to cope with different purification, enrichment and detection requirements.

[0048] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention provides a pair of monoclonal antibodies 2E7 and 3B6, and the present invention also provides a universal M13@scFv@Biotin probe, which realizes the full-valent display of scFv on M13 P3 protein and the full-valent modification of biotin on P8 protein. By simply replacing the scFv gene on the plasmid p5E-P3CTscFv, specific binding to different antigens can be achieved; at the same time, the approximately 2700 biotin modified on the probe can bind to any streptavidin-labeled molecules and materials (such as HRP-SA, SA-AuNPs, SA-MNPs, etc.) with high affinity to meet the needs of different detection methods, and the application value is very wide. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a Western Blot verification diagram of the M13 probe displaying scFv described in the embodiment 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 This is a P8 protein spectrum analysis diagram of the 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 part of the embodiments of the present invention and should not be considered as limiting the scope of the present invention. In the examples, the specific conditions not particularly specified are carried out according to the conditions of normal conditions or manufacturer recommendations. The reagents or instruments used are not indicated by the manufacturer and 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 Novozyme Biotechnology Co., Ltd., with the catalog number P525-01; FastPure Viral DNA / RNAMini Kit was purchased from Nanjing Novozyme 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-biotinylamino-4-[4`-(methylmaleimide)cyclohexanecarboxamide]butane) was purchased from Beijing Biolab Technology Co., Ltd., Catalog No.: GS4327; horseradish peroxidase-labeled Streptavidin (HRP-labeled Streptavidin) was purchased from Shanghai Biotime Biotechnology Co., Ltd.; horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (HRP-labeled goat anti-mouse IgG (H+L)) was purchased from Shanghai Biotime Biotechnology Co., Ltd., Catalog No.: A0216; anti-M13 / fd / F1 Filamentous Phages mouse monoclonal, B62-FE2, HRP Conjugate was purchased from PROGEN, USA, with the catalog number 61097-HRPS. -. -Aβ1-42 monoclonal antibodies 2E7 and 3B6 were prepared in this 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 NEB, USA, with the catalog number E8033S; primer synthesis and sequencing services were provided by Beijing Qingke Biotechnology Co., Ltd.

[0054] BALB / C mice were purchased from the Comparative Medicine Center of Yangzhou University; SP2 / 0 was the cell line preserved in this experiment; Freund's incomplete adjuvant, catalog number: P2031-10ml; Freund's complete adjuvant, catalog number: P2036-10ml; DMEM, purchased from Gibco, USA; catalog number: 11965126; polyethylene glycol 1450 (PEG1450), catalog number: P7181-5X5ML, Hybri-Max TM HAT medium supplement (50×), catalog number H0262-10VL, HT additive (100X), catalog number: 11067030, were all purchased from Sigma, USA; Australian fetal bovine serum, purchased from Suzhou Shuanglu Biotechnology Co., Ltd., catalog number: A511-001; 96-well cell culture plate, catalog number: 701002, 48-well cell culture plate, catalog number: 748002, ELISA plate, catalog number: 514201, were all purchased from Wuxi Nice Life Science Co., Ltd.; Penicillin-streptomycin solution (100X), catalog number: C0222, Protein A+G Agarose (Fast Flow, 1ml) pre-packed column, catalog number: P2028, were all purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; Total RNA extraction kit, catalog number: RC411-01, cDNA first-strand synthesis special kit, catalog number: R212-01, Phanta Max Super-Fidelity DNA Polymerase, catalog number: P505-d1, were purchased from Nanjing Novozymes Biotech 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]]> 19μl 2×Phanta Max Master Mix(Dye Plus) 25μl Upstream primer (10 μM) 2μl Downstream primer (10 μM) 2μl Template DNA 2μl

[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 embodiment, beta amyloid 1-42 (Aβ1-42) polypeptide is selected as the immunogen. When synthesizing the Aβ1-42 polypeptide, cysteine ​​C is added to the N-terminus for labeling the carrier protein hemocyanin KLH. When synthesizing the Aβ1-42 polypeptide, the carrier protein bovine serum albumin BSA is connected to the N-terminus through cysteine ​​for titer detection 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 be synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.

[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 includes the following steps:

[0069] 1. Animal immunization

[0070] For the first immunization, Freund's complete adjuvant was mixed with the antigen in equal amounts in a 1:1 ratio. After full emulsification, 50 μg Aβ1-42-KLH antigen was administered to each BALB / C mouse. Subsequently, the protein was emulsified with Freund's incomplete adjuvant in the same way, 50 μg per mouse, and subcutaneous multi-point immunization was performed. Each immunization was separated by 2 to 3 weeks. On the 7th day after the third immunization, the mouse tail tip blood was collected to test the titer. After the mouse tail blood was collected, it was placed at 4°C for 30 minutes. After the serum was precipitated, the serum was collected, and blank mouse serum was set up as a control. The treatment method was the same as that of mouse serum. When the OD450nm ratio of the experimental group to the control group was greater than 2.1, the maximum serum dilution multiple was the mouse serum titer. After the serum titer reached 1:12800, mice with high titer were selected and immunized by intraperitoneal injection of an equal volume emulsion (200 μl) of 50 μg antibody and Freund's incomplete adjuvant. The mice were killed 3 days later for cell fusion.

[0071] 2. Cell fusion

[0072] HAT medium configuration: 1mL Hybri-Max TMHAT culture medium supplement (50×), 5mL Australian fetal bovine serum, 500ul penicillin-streptomycin solution, 43.5mL DMEM culture medium, mix well. Collect blood from the eyeballs of mice that have completed booster immunization, and separate the serum as a positive control. After the mice were killed by cervical dislocation, they were soaked 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 upward, 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 from 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 use a filter to filter it, which is the spleen cell. SP2 / 0 and spleen cells were counted, spleen cells and SP2 / 0 were mixed at a cell number ratio of 8:1, inverted to mix, and centrifuged at 1000rpm for 4min; in a 37°C water bath, the precipitated cells were tapped to make them evenly distributed at the bottom of the tube, and allowed to stand for 1min. Within 1min, 1ml PEG1450 was added to the centrifuge tube and allowed to stand for 1min. Within 30s, 1ml 37°C preheated DMEM culture medium was added along the tube wall to terminate the cell fusion reaction; the tip of the gun was inserted under the liquid surface, 1ml DMEM culture medium was added within 1min, and this step was repeated until 20ml DMEM culture medium was added; 30ml DMEM culture medium was slowly added, and centrifuged at 800rpm for 4min, the supernatant was discarded, and 50ml DMEM culture medium was added, and centrifuged at 800rpm for 4min, the supernatant was discarded, and HAT culture medium was added. The cells were gently blown up and added to a 96-well cell culture plate (200μl / well), and marked; indirect ELISA identification was performed 7 days later.

[0073] 3. Screening of positive hybridoma cells

[0074] Configuration of HT culture medium: 500μL HT additive (100X), 5mL Australian fetal bovine serum, 500ul penicillin-streptomycin solution, 44mL DMEM culture medium, mix well. Take the hybridoma cell supernatant and screen the positive hybridoma cells by indirect ELISA. Select the wells with high OD450nm values ​​and only single cell clumps, discard the culture medium, add 200μl HT culture medium, blow the cells and count them, take about 200 cells to spread half a 96-well plate, and pass the remaining cells to a 48-well plate for continued expansion and culture, and freeze. After 7 days, the monoclonal cells were identified by ELISA, and the above method was used for subcloning again. After 3 subclones, single cell clumps with higher OD450nm values ​​were selected and cloned again according to the above method. If the OD450nm values ​​of the wells with cells are all high, and the OD450nm values ​​of the wells without cell clumps are not higher than the negative control, it is considered to be a hybridoma cell line that can 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 The hybridoma cells were injected into the peritoneal cavity of mice, and the ascites was collected 7 days later.

[0077] Prepare the buffer used for purification: Binding buffer: phosphate buffer (PBS, 0.01M, pH 7.4), preparation method: NaCl 8g, Na2HPO4 1.44g, KH2PO4 0.24g, KCl 0.2g dissolved in 800ml ultrapure water, adjust the pH to 7.4 with 0.1M NaOH, and then dilute to 1L. Neutralization buffer: 1M Tris-HCl, pH8.8, preparation method: weigh 121.1g Tris and dissolve in 800ml ultrapure water, adjust to 8.8 with concentrated hydrochloric acid, and dilute to 1L. Elution buffer: 100mM glycine, pH2-3, preparation method: 7.507g glycine dissolved in 800ml ultrapure water, adjust to 2.5 with concentrated hydrochloric acid, and dilute to 1L.

[0078] The antibody was purified using a pre-packed column of Protein A+G Agarose (Fast Flow, 1 ml) from Bio-Tech. The specific steps were as follows: centrifuge the ascites at 10,000 rpm for 10 min, collect the supernatant, and take 20 μl to make 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, remove the bubbles and slowly push the piston to remove the storage solution; draw 10 ml of Binding buffer at a flow rate of 1 ml / min to balance 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 10 ml of Binding buffer to wash away the unbound antibody until the outflowing liquid is colorless; draw 5 ml of Elution buffer to elute the antibody bound to the column, and adjust the pH of the collected eluate to 7.5 with Neutralization buffer. 20 μl of sample was taken from each tube to prepare samples for later use and to identify the purified monoclonal antibodies.

[0079] 5. Determination of monoclonal antibody cell titer

[0080] (1) Dissolve the Aβ1-42-BSA coupling complex to 5 μg / ml using coating solution (sodium carbonate 0.15 g, sodium bicarbonate 0.29 g, sodium azide 0.02 g, add double distilled water to 100 ml, adjust to pH 9.6), add 100 μl of coating solution to each well, and let stand at 4°C overnight; soak and wash 3 times, 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, 3 minutes each time;

[0082] (3) Add gradient dilutions of 2E7 and 3B6 cell supernatants and negative mouse serum, 100 μl / well, incubate at 37°C for 1 hour; soak and wash 3 times, 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 3 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 (2M / 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] It can be seen from the above results 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 number 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] A cDNA single-strand synthesis kit was used to synthesize cDNA 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 one-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 the 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 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 complementary determining regions are shown in the following table:

[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 ultra-micro spectrophotometer. The purity of the extracted RNA was determined based on the A260 / A280 of pure RNA = 2.0. The RNA was reverse transcribed into cDNA, 50 μL was taken out for subsequent PCR, and the rest was 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 with linker (G4S) 4, and the fragment 2E7-VHL was obtained; using 2E7-VL cDNA (SEQ ID NO.5) as a template, primers 2E7-LF and 2E7-R were used to amplify the homology arm with linker (G4S) 4, and the fragment 2E7-VLL was obtained; the reaction conditions were 2×Phanta Max Master Mix (Dye Plus) high-fidelity enzyme PCR amplification system and amplification program. After the PCR amplification was completed, the PCR product was taken for agarose gel electrophoresis to confirm the amplified DNA fragment, and the target fragment was recovered using a gel recovery kit.

[0125] The light and heavy chain variable region fragments of the antibody were added with linker (G4S) 4, and scFv was obtained by overlap extension PCR amplification. The high-fidelity enzyme used in overlap extension PCR was Phanta Max Super-Fidelity DNA Polymerase (Novozyme).

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

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

[0128] The reaction conditions are: pre-denaturation at 95°C for 3 min, followed by 12 cycles of 95°C for 15 s, 56°C for 15 s, and 72°C for 1 min. After the reaction is complete, the second step of overlap extension PCR is performed using the following reaction system:

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

[0130] The template is the PCR product of the first step of overlap extension PCR. The reaction conditions are: pre-denaturation at 95°C for 3 min; 12 cycles of 95°C for 15 s, 56°C for 15 s, and 72°C for 1 min. Extension at 72°C for 10 min. 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 recovery 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, so that the fusion protein P3CT scFv was similar in size to the wild-type P3, reducing the resistance to post-translational protein translocation and phage assembly and increasing the display efficiency of scFv.

[0134] Specifically, taking the Aβ1-42-specific monoclonal antibody 2E7 synthesized in Example 2 as an example, the homologous recombination principle was used to insert pel B, RBS, 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, and the connection product was transformed into DH5α competent cells by chemical transformation. The plasmid was extracted from the bacterial solution that was positive after 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] The P3 gene on the M13KO7 genome was deleted and the P8 gene was mutated using the principle of homologous recombination. Using the M13KO7 genome as a template, PCR amplification was performed with primers ΔP3-F / R to delete most of the P3 gene (10-412aa), leaving only the first 9 amino acids of the signal peptide and the last 12 amino acids of the CT region to obtain the M13KO7ΔP3 fragment. Using the M13KO7ΔP3 fragment as a template, PCR amplification was performed with primers V31C-F / R to mutate the 31st valine (Val) of the P8 mature protein (removing the 23aa signal peptide) to cysteine ​​(Cys) to obtain the M13KO7ΔP3-V31C fragment for subsequent biotinylation modification. Then, the M13KO7ΔP3-V31C fragments were connected using a homologous recombination kit, and the connection products were 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 M13KO7ΔP3-V31C. The specific primers were designed as follows (the bold part is the point mutation position):

[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] The plasmid p5E-P3CT scFv and M13KO7ΔP3-V31C were co-transformed into TG1 competent cells. After sequencing and identification, they 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 make up to 1 L) containing 60 μg / mL ampicillin and 50 μg / mL kanamycin. The bacteria were removed by centrifugation at 8000 rpm for 10 min at 4°C, and the phages were precipitated by centrifugation at 48000 rpm for 4 h at 4°C. The precipitate 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, 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-price 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 part was cloned by PCR amplification using primers p5E-F1 / p5E-R, and the 2E7 scFv part was cloned by PCR amplification using primers 2E7-F / 2E7-R1. 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 verified to be positive by sequencing to obtain the recombinant plasmid p5E-scFv.

[0152] Phage display steps: Transform the plasmid p5E-P3CT scFv into TG1 competent cells. After sequencing and identification, expand TG1 to 200mL 2×YT medium. When the bacterial solution OD600 is 0.6, add M13KO7 phage, mix well, let stand at 37℃ for one hour, and culture overnight at 37℃ 220rpm. The next day, centrifuge at 8000rpm for 10min at 4℃ to remove the bacteria, centrifuge at 48000rpm for 4h at 4℃ to precipitate the phage, resuspend the precipitate in PBS, and centrifuge briefly to remove cell debris to obtain the phage M13KO7@scFv. The display steps of Hyperphage@scFv are the same as M13KO7@scFv, except that M13KO7 is replaced by Hyperphage when adding phage.

[0153] Western Blot was used to verify the full-price display of scFv on P3CT. Western Blot experiments were performed using M13KO7@scFv (displayed based on phagemid p5E-scFv and helper phage M13KO7), Hyperphage@scFv (displayed based on phagemid p5E-scFv and commercialized improved helper phage Hyperphage M13 KO7ΔpIII), and M13@scFv@Biotin (displayed based on the dual-plasmid system p5E-P3CT scFv and M13KO7ΔP3-V31C). M13 P3 protein (obtained from 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 mass spectrometer for rapid identification of microorganisms and proteins (MALDI Biotyper) was used to perform mass spectrometry analysis on the P8 protein 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 (such as Figure 2 shown).

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

[0156] The specific application of the M13@scFv@Biotin universal probe of the present invention in ELISA detection is to establish a sandwich ELISA detection method by taking 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] Preparation method of phosphate buffer (PBS, 0.01M, pH 7.4): 8g NaCl, 1.44g Na2HPO4, 0.24g KH2PO4, 0.2g KCl were dissolved in 1L ultrapure water, and the pH value was adjusted to 7.4 with 0.1M NaOH. Preparation method of PBST: 8g NaCl, 1.44g Na2HPO4, 0.24g KH2PO4, 0.2g KCl, 0.5mL Tween-20 were dissolved in 1L ultrapure water, and the pH value was adjusted 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, 5 min each time, 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, 7.8125 pg / ml Aβ1-42 peptide and PBS (blank control) to each well, one row for each dilution, and incubate at 37°C for 1 h;

[0161] (4) Wash with PBST five 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, 3ng / 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, 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 colorimetric solution to each well, and color 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 of M13@scFv@Biotin and 2E7 monoclonal antibody were established based on the OD values ​​(e.g. Figure 3 shown).

[0165] The results show that the universal M13@scFv@Biotin probe of the present invention can modify thousands of HRP molecules due to the quantitative advantage of M13 bacteriophage P8 protein (about 2700 copies) and the strong binding force of biotin-streptavidin, significantly amplifying the detection signal of ELISA, and can replace the HRP-labeled secondary antibody in traditional ELISA. In a specific embodiment, the detection limit of the positive control (traditional antibody) is 62.5pg / ml. In contrast, the detection limit of the universal M13@scFv@Biotin probe constructed by the present invention is 7.8125pg / ml, which is more sensitive than the traditional method.

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.4; 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, and the complementary determining regions are shown in the following table:

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 as SEQ ID NO.5, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2E7 is shown as SEQ ID No.6, the nucleotide sequence of the light chain variable region of the monoclonal antibody 3B6 is shown as SEQ ID No.7, and the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3B6 is shown as 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 is a monoclonal antibody according to claim 1 that is fully displayed 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 and biotinylation modification on the 31st amino acid of the P8 mature protein.

6. The universal M13 phage probe according to claim 5, characterized in that: The amino acid at position 31 of the P8 mature protein is mutated from valine to cysteine. Preferably, the phage probe further contains horseradish peroxidase-labeled streptavidin or other streptavidin markers.

7. The method for preparing the universal M13 phage probe according to any one of claims 4 to 6, characterized in that: The following steps are involved: (1) Construction of plasmid p5E-P3CT scFv: The vector fragment 1 in which the N1 and N2 genes in the M13 P3 gene were deleted was cloned using primers p5E-F and p5E-R; the fragment 2 of the single-chain antibody 2E7-scfv was cloned using primers 2E7-F and 2E7-R using the scFv gene of the monoclonal antibody 2E7 as a template, and the fragment 1 and fragment 2 were homologously recombined to obtain the plasmid p5E-P3CT scFv; (2) Construction of plasmid M13KO7ΔP3-V31C: Primers ΔP3-F and ΔP3-R were used to delete aa 10-412 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, 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 M13@scFv@Biotin universal probe: The plasmids obtained in steps (1) and (2) were co-transformed into Escherichia coli, and a bacterial solution containing two plasmids was cultured to assemble the bacterial solution into bacteriophage M13@scFv. M13@scFv was incubated with BMCC-Biotin at room temperature, and then separated through a desalting column to obtain the recombinant bacteriophage M13@scFv@Biotin.

8. The method for preparing a universal M13 phage probe according to claim 5, characterized in that: 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: CGGCGCCGGTGCCGGTGCGTGGTGGTGGTGGTGGtgggatccaggtggtccaccatcagc ccg; Preferably, the sequences of primers ΔP3-F, ΔP3-R, V31C-F and V31C-R in step (2) are as follows: ΔP3-F: AATTATTATTCCGCAATTCCTTCTACGTTTGCTAACATACT ΔP3-R: AGTATGTTAGCAAACGTAGAAGGAATTGCGAATAATAATT V31C-F:GGTTGTTTGCATTGTCGGCGCAACTATCGGTA V31C-R: GACAATGCAAACAACCATCGCCCACGCATAAC.

9. 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 bacteria according to claim 3, and the probe according to any one of claims 4 to 6 in the preparation of a detection kit for detecting Aβ1-42.

10. A method for detecting Aβ1-42 in peripheral blood, characterized in that: The detection using the probe according to any one of claims 4 to 6 preferably comprises the following steps: (1) Add monoclonal antibody 3B6 to the ELISA plate, incubate, and wash; (2) Add blocking solution to the ELISA plate, incubate, and rinse; (3) Add the sample / standard to be tested to the ELISA plate, incubate, and rinse; (4) adding probes or other control samples to the ELISA plate, incubating, and washing; (5) adding HRP-conjugated secondary antibody of corresponding affinity, incubating, and washing; (6) Add TMB colorimetric solution for color development, stop the reaction, read OD450, and determine the content of the sample to be tested based on the standard curve.

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