Monoclonal antibody for detecting soybean phytochrome PhyB protein

Abstract

The invention discloses a monoclonal antibody for detecting soybean phytochrome PhyB protein, a heavy chain variable region of the monoclonal antibody comprises CDR-H1, CDR-H2 and CDR-H3, corresponding amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 are respectively shown as SEQ ID NO: 4-6, a light chain variable region of the monoclonal antibody comprises CDR-L1, CDR-L2 and CDR-L3, corresponding amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 are respectively shown as SEQ ID NO: 7-9, and the light chain variable region of the monoclonal antibody comprises CDR-L1, CDR-L2 and CDR-L3. The method has the advantages that the affinity and the specificity to the PhyB protein of the soybean phytochrome are high, the PhyB protein can be accurately recognized, cross reaction with other proteins is avoided, a reliable tool is provided for expression dynamic tracking, subcellular localization and protein interaction research of the PhyB protein, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and particularly relates to a monoclonal antibody for detecting soybean phytochrome PhyB protein. BACKGROUND

[0002] Soybean phytochrome PhyB protein is a key photoreceptor protein for sensing red and far-red light signals in soybean, and belongs to the core member of the phytochrome family. It widely exists in different tissues and growth stages of soybean, receives external light signals, and changes in conformation (transforms into an active state under red light excitation and reverses under far-red light irradiation), thereby starting downstream signal pathways and regulating core physiological processes such as flowering time, plant type formation, photosynthetic efficiency and stress resistance of soybean. It is an important molecular hub connecting external light environment and internal growth and development regulation network of soybean, and its expression level and activity change directly affect the adaptability of soybean to the environment and yield and quality-related traits.

[0003] The existing methods for detecting soybean phytochrome PhyB protein have obvious limitations, which restricts the related research and application promotion. In the traditional method, the detection technology based on fluorescence labeling is easily interfered by the autofluorescence of soybean tissue, has insufficient specificity and low sensitivity, and is difficult to accurately quantify low-abundance PhyB protein. Conventional immunodetection such as Western blotting relies on polyclonal antibodies, which is prone to non-specific binding to cause false positive results, and the stability of the antibodies between batches is poor, which cannot guarantee the repeatability of the detection results. In addition, methods such as mass spectrometry are complex to operate and costly, and require professional equipment and technical personnel, which are not suitable for large-scale sample rapid screening and are difficult to meet the needs of breeding practice and field application.

[0004] It has important practical significance and application value to research a monoclonal antibody with high specificity and high sensitivity for detecting soybean phytochrome PhyB protein. The monoclonal antibody can accurately recognize PhyB protein and avoid cross-reaction with other proteins, effectively solving the problems of poor specificity and large signal interference of traditional detection methods, and providing a reliable tool for dynamic tracking of PhyB protein expression, subcellular localization and protein interaction research. At the same time, based on the antibody, ELISA kits, detection test papers and other efficient detection products can be developed to realize high-throughput, low-cost rapid screening of large-scale soybean samples, help rapid screening and breeding process acceleration of light-responsive excellent soybean genotypes, provide molecular detection support for optimizing soybean cultivation strategies and improving stress resistance and yield, and promote soybean molecular breeding and agricultural production quality and efficiency. SUMMARY

[0005] Therefore, the present application aims to provide a monoclonal antibody for detecting soybean phytochrome PhyB protein, wherein the heavy chain variable region of the monoclonal antibody comprises CDR-H1, CDR-H2 and CDR-H3, the corresponding amino acid sequences of which are shown in SEQ ID NO: 4-6; and the light chain variable region of the monoclonal antibody comprises CDR-L1, CDR-L2 and CDR-L3, the corresponding amino acid sequences of which are shown in SEQ ID NO: 7-9.

[0006] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions.

[0007] The first aspect of the present application provides a monoclonal antibody for detecting soybean phytochrome PhyB protein.

[0008] Further, the amino acid sequences of CDR1-3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NO: 4-6, respectively; and the amino acid sequences of CDR1-3 of the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 7-9, respectively.

[0009] Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2 or has at least 80% homology with SEQ ID NO: 2.

[0010] Further, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3 or has at least 80% homology with SEQ ID NO: 3.

[0011] In the present application, the homology refers to the similarity between the target amino acid sequence and the amino acid sequence shown in SEQ ID NO: 2 (heavy chain variable region) or SEQ ID NO: 3 (light chain variable region), which is quantified by the proportion of identical amino acid residues in the total sequence length after global alignment by a sequence alignment tool (such as BLAST, ClustalW), and >80% homology means that the proportion of identical amino acid residues between the target sequence and the corresponding SEQ ID NO sequence is not less than 80%. Such homology allows conservative amino acid substitution (such as exchange of amino acids with similar properties) in the non-CDR region, but ensures that the sequence after variation can still encode a monoclonal antibody with the ability to specifically bind soybean PhyB protein, which not only retains the core function but also provides a reasonable range for sequence optimization. The functional homologous variants obtained in this way also fall within the protection scope of the present application.

[0012] It should be noted that the scope of protection of the present application covers all CDR sequences obtained after numbering the monoclonal antibody heavy chain variable region and light chain variable region of the present application according to any CDR numbering scheme (including but not limited to commonly used numbering systems such as Kabat, Chothia, IMGT, AbM, Contact, etc.), regardless of the slight differences in the starting / ending sites or amino acid residue ranges of the CDR sequences caused by the differences in the definition rules, as long as these CDR sequences are derived from the heavy chain variable region (such as SEQ ID NO: 2 or its homologous sequence) and the light chain variable region (such as SEQ ID NO: 3 or its homologous sequence) as described above, and can guarantee the core function of the antibody specifically binding to the core function of the soybean phytochrome PhyB protein, which belongs to the protection scope of the present application and is not limited by the selection of the specific numbering scheme.

[0013] The second aspect of the present application provides a polynucleotide encoding the monoclonal antibody of the first aspect of the present application.

[0014] Optionally, the polynucleotide is a DNA molecule.

[0015] In the present application, the polynucleotide can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The present application also includes degenerate variants of the nucleotide sequence encoding the monoclonal antibody, i.e. nucleotide sequences that encode the same amino acid sequence but have different nucleotide sequences.

[0016] According to the amino acid sequence and codon, those skilled in the art can easily obtain the coding sequence of the monoclonal antibody. The field also changes the expression of polypeptides in different species through codon optimization, and the codon preference of different species belongs to the conventional technology in the field.

[0017] The third aspect of the present application provides a recombinant vector comprising the polynucleotide of the second aspect of the present application.

[0018] Optionally, the recombinant vector is a eukaryotic expression vector.

[0019] Optionally, the eukaryotic expression vector is a pcDNA vector, a pCMV vector, a pEGFP-N1 / pEGFP-C1 vector, a pIRES vector, a pCDNA6 vector, a yeast eukaryotic expression vector, a Saccharomyces cerevisiae expression vector or an insect cell eukaryotic expression vector.

[0020] In some embodiments, a mammalian cell eukaryotic expression vector can be selected, which can meet the requirements of post-translational modification of antibody expression and ensure the activity of the antibody, and specifically includes pcDNA series vectors (such as pcDNA3.1, pcDNA3.4, pcDNA4, pcDNA5, etc.), pCMV series vectors (such as pCMV-HA, pCMV-Myc, pCMV-Tag series), pEGFP-N1 / pEGFP-C1 series vectors, pIRES series vectors (such as pIRES2-EGFP), pCDNA6 series vectors, etc.; a yeast eukaryotic expression vector can also be selected, such as a Pichia pastoris expression vector (pPIC3.5K, pPIC9K, pPICZ alpha-A), a Saccharomyces cerevisiae expression vector (pYES2, pYES3, pESC series); an insect cell eukaryotic expression vector can also be selected, such as a pFastBac series vector, a pVL1393 vector, etc. The above-mentioned vectors all have core elements required for eukaryotic expression (such as a promoter, a terminator, a multiple cloning site, a screening marker gene, etc.), can effectively carry the above-mentioned polynucleotide, and can realize stable or transient expression of the antibody in a corresponding eukaryotic host cell. A person skilled in the art can select a specific vector according to the type of target host cell (such as a HEK293 cell, a CHO cell, a yeast cell, an insect cell, etc.) and the expression requirement.

[0021] The fourth aspect of the present application provides a recombinant host cell comprising the recombinant vector of the third aspect of the present application.

[0022] Optionally, the host cell is a eukaryotic cell.

[0023] Optionally, the host cell is a mammalian cell, a yeast cell or an insect cell.

[0024] Optionally, the mammalian cell is a CHO cell, a HEK293 cell (such as a HEK293T, a HEK293F), an NS / 0 myeloma cell, a Sp2 / 0 myeloma cell, a Cap cell or a COS cell.

[0025] Optionally, the yeast cell is a Pichia pastoris (such as a GS115, a KM71 strain) or a Saccharomyces cerevisiae.

[0026] Optionally, the insect cell is an Sf9 / Sf21 cell or a High Five cell.

[0027] In some embodiments, the mammalian cell is a preferred host for expression of the recombinant antibody of the present application, and the post-translational modification system thereof is highly consistent with the natural expression environment of the antibody, which can maximize the structural integrity and binding activity of the antibody.

[0028] In some embodiments, the yeast cell, as a single-cell eukaryote, has the advantages of easy cultivation, fast growth and low cost of prokaryote and the basic post-translational modification ability of eukaryote, and is suitable for laboratory small-scale preparation or industrial production of antibodies.

[0029] In some embodiments, the insect cell is suitable for baculovirus expression system, which can realize efficient expression of antibodies and protein folding and modification close to eukaryotes, and is suitable for large-scale preparation of recombinant proteins.

[0030] In some embodiments, the type of host cell is not particularly limited in the present application. HEK293 or COS cell can be selected for rapid verification in laboratory stage; NS / 0 or Sp2 / 0 cell can be selected for small-scale high-purity preparation; CHO or Cap cell can be selected for industrial production; Pichia pastoris or Sf9 cell can be selected for cost-sensitive scenarios. As long as the cell contains the recombinant vector of the third aspect of the present application and can realize efficient expression of the monoclonal antibody against soybean PhyB (retaining specific binding activity), the selection of the above-mentioned cells all belongs to the protection scope of the present application.

[0031] The fifth aspect of the present application provides a detection reagent for detecting soybean phytochrome PhyB protein.

[0032] Further, the detection reagent contains the monoclonal antibody of the first aspect of the present application.

[0033] In some embodiments, the monoclonal antibody of the first aspect of the present application can be in naked antibody form according to actual detection needs, or be labeled and modified with fluorescein (FITC, Cy3, etc.), enzyme (HRP, AP), nanoparticles (gold nanoparticles, quantum dots), etc. in advance, and directly undertake the function of specific recognition of soybean PhyB protein.

[0034] In some embodiments, the detection reagent further comprises a sample processing component, an immune response auxiliary component, a signal detection component, a quality control and stabilization component. Among them, the sample processing component includes protein extraction buffer (such as RIPA, Tris-HCl), protease / phosphatase inhibitor (to prevent PhyB protein degradation or modification changes), sample diluent (BSA-containing PBS, to maintain protein stability), for efficient extraction and protection of PhyB protein in the sample. The immune response auxiliary component includes blocking solution (skimmed milk powder, BSA, etc., to reduce non-specific binding), washing solution (PBST / TBST, to remove unbound impurities), antibody diluent (containing stabilizer and preservative, to accurately control the antibody concentration), and some reagents also match the corresponding secondary antibody (such as enzyme-labeled goat anti-mouse secondary antibody) to realize signal amplification. The signal detection component includes corresponding substrate according to the type of label, such as HRP-labeled antibody matched with TMB / chemiluminescence substrate, fluorescent-labeled antibody matched with fluorescent quenching inhibitor, enzyme-labeled antibody matched with color developing termination solution, to ensure that the detection signal can be stably read and quantified. The quality control and stabilization component includes PhyB protein standard (for quantitative calibration), positive / negative control samples (to verify the effectiveness of the detection), preservative (sodium azide, ProClin), and stabilizer (glycerol, sucrose), to ensure the shelf life of the reagent and the repeatability of the detection.

[0035] In some embodiments, the detection reagent includes but is not limited to: ELISA special detection reagent, Western Blot special reagent, immunofluorescence detection reagent, flow cytometry special reagent, immunochromatography rapid detection reagent, immunosensor chip matching reagent.

[0036] In some embodiments, the ELISA special detection reagent includes labeled monoclonal antibody (such as HRP labeling), coating buffer, blocking solution, washing solution, TMB developing solution, termination solution, PhyB standard. It can be used for quantitative detection of PhyB protein in soybean leaf, seed, root and other tissue samples, suitable for laboratory batch sample analysis (such as comparison of PhyB expression of different varieties of soybeans, dynamic monitoring of PhyB expression under different light environments), can quickly obtain quantitative data, and support soybean light response mechanism research and breeding material screening.

[0037] In some embodiments, the Western Blot special reagent includes naked monoclonal antibody, enzyme-labeled secondary antibody, protein extraction buffer, protease inhibitor, electrophoresis loading buffer, membrane transfer buffer, chemiluminescent substrate. It can be used for qualitative verification and relative quantification of PhyB protein in soybean cells or tissues, for example, to verify the correctness of the expression of recombinant PhyB protein, to detect the expression difference of PhyB protein in soybean mutants, and is a core tool for PhyB protein identification in molecular biology experiments.

[0038] In some embodiments, the immunofluorescence detection reagent comprises a fluorescein-labeled monoclonal antibody, a sample fixing solution (paraformaldehyde), a permeation solution (Triton X-100), a fluorescence quencher, a blocking solution, a washing solution. It can be used for subcellular localization observation of PhyB protein in soybean root tip, cotyledon and other tissue sections or protoplast, and is suitable for fluorescence microscopy or confocal microscopy, which can directly show the distribution characteristics of PhyB protein in the nucleus and cytoplasm, and help the research on protein localization mechanism in the light signal pathway.

[0039] In some embodiments, the flow cytometry special reagent comprises a fluorescence-labeled monoclonal antibody, a cell fixing solution, a permeation solution, a washing buffer, a negative control antibody. It can be used for rapid quantification and positive cell sorting of PhyB protein in soybean suspension cells or protoplast, and is suitable for high-throughput screening of large-scale cell samples, such as isolation of cell populations with different PhyB expression levels, and research on the correlation between cell heterogeneity and light response difference.

[0040] In some embodiments, the immunochromatographic rapid detection reagent comprises a gold nanoparticle-labeled monoclonal antibody, a test strip (containing a detection line coated with a monoclonal antibody and a quality control line), and a sample lysate. It can be used for rapid qualitative screening of the presence or absence of PhyB protein in soybean samples in the field or laboratory, and the operation does not require professional equipment, and the results can be obtained within a few minutes, which is suitable for preliminary screening of breeding materials (such as rapid identification of high PhyB expression varieties) or on-site detection of sample collection.

[0041] In some embodiments, the immunosensor chip kit reagent comprises a naked monoclonal antibody, a chip activation buffer, a sample diluent, a signal detection buffer, and a standard. It can be used for high-sensitivity rapid quantification of PhyB protein in a small amount of soybean sample (such as tender tissue extract), and is suitable for an immunosensor chip detection platform, without complex labeling process, and can realize accurate detection of trace PhyB protein, and is suitable for analysis of valuable samples or low-abundance PhyB expression samples.

[0042] The sixth aspect of the present application provides a detection product for detecting soybean phytochrome PhyB protein.

[0043] Further, the detection product comprises the monoclonal antibody of the first aspect of the present application or the detection reagent of the fifth aspect of the present application;

[0044] Optionally, the detection product is a detection kit, a detection chip, a detection test paper or a detection probe;

[0045] Optionally, the detection kit is an ELISA detection kit, an immunoblotting detection kit, an immunofluorescence detection kit or an immunohistochemical detection kit;

[0046] Optionally, the detection chip is a protein detection chip or an immunosensor chip.

[0047] Optionally, the detection test paper is an immunochromatographic detection test paper.

[0048] Optionally, the detection probe is a monoclonal antibody probe modified by a penetrating peptide.

[0049] In some embodiments, the ELISA detection kit integrates monoclonal antibodies, enzyme-labeled secondary antibodies, blocking solution, washing solution, TMB developing solution, termination solution, and PhyB standard, etc., and is suitable for quantitative detection of PhyB protein in soybean samples, standardization of operation, and batch sample analysis in a laboratory.

[0050] In some embodiments, the Western Blot special kit contains monoclonal antibodies, enzyme-labeled secondary antibodies, protein extraction buffer, protease inhibitors, and chemiluminescent substrate, etc., and is used for qualitative and relative quantitative verification of PhyB protein after gel electrophoresis.

[0051] In some embodiments, the immunofluorescence detection kit is equipped with fluorescently labeled secondary antibodies, fluorescence quenching inhibitors, sample fixing solution, and permeation solution, etc., and is suitable for subcellular localization observation of PhyB protein in soybean cells or tissue sections, and fluorescence microscope detection.

[0052] In some embodiments, the immunohistochemical detection kit contains monoclonal antibodies, enzyme-labeled secondary antibodies, DAB developing solution, hematoxylin counterstaining solution, and dewaxing and hydration reagents, etc., and is used for localization and semi-quantitative analysis of PhyB protein in soybean tissue sections, and pathological section observation scenarios.

[0053] In some embodiments, the protein detection chip is to fix monoclonal antibodies on a glass chip or a nitrocellulose membrane carrier, and can simultaneously detect the expression level of PhyB protein in multiple soybean samples, realizing high-throughput rapid screening.

[0054] In some embodiments, the immunosensor chip is to fix monoclonal antibodies on a sensor interface (such as a gold electrode, an optical sensor chip) by combining biosensor technology, to realize high-sensitivity and rapid quantification of PhyB protein through specific binding-induced signal changes, without complex labeling processes.

[0055] In some embodiments, the immunochromatographic detection test paper is to use a nitrocellulose membrane as a carrier, to preset a detection line (T line) and a quality control line (C line) coated with monoclonal antibodies, and to be equipped with gold nanoparticle-labeled secondary antibodies, which is suitable for on-site rapid qualitative screening of PhyB protein in soybean samples, and is simple to operate without professional equipment.

[0056] In some embodiments, the penetrated peptide modified monoclonal antibody probe (combined with fluorescent or bioluminescent markers) can penetrate the soybean tissue barrier, specifically bind to the target protein, and be suitable for monitoring the dynamic distribution of PhyB protein in soybean living tissues.

[0057] Further, the penetrated peptide is a kind of short peptide (usually 10-30 amino acids) that can carry macromolecular substances (such as antibodies and probes) to penetrate cell membranes and even tissue barriers. The core function is to help the antibody probe break through the physical barrier of soybean tissue and realize the detection of PhyB protein in living tissues.

[0058] Further, the penetrated peptide is connected to the monoclonal antibody of the present application by chemical coupling, and is combined with fluorescent or bioluminescent markers. This helps the antibody to break through the barrier of soybean tissue and cell membrane, and can track its distribution in living bodies through marker signals, ultimately realizing real-time monitoring of the dynamic changes of PhyB protein, and solving the problem that traditional antibodies cannot penetrate plant tissues and are difficult to detect in living bodies.

[0059] Further, the penetrated peptide suitable for soybean tissue detection includes TAT peptide (derived from HIV-1 Tat protein), RGD peptide (with both cell integrin targeting and penetrating functions), Penetratin peptide (derived from fruit fly antennal foot protein), CPP9 peptide, etc. Among them, TAT peptide is the most commonly used type in plant tissue detection due to its high penetration efficiency and wide application range.

[0060] Further, the penetrated peptide modification process includes probe pretreatment (antibody activation, membrane penetrating peptide and marker molecule pretreatment), covalent coupling reaction (using crosslinking agent mediated coupling, directional coupling optimization), purification and activity verification.

[0061] The seventh aspect of the present application provides any one of the following methods:

[0062] (1) A method for preparing the monoclonal antibody of the first aspect of the present application, the method comprising the following steps: culturing the recombinant host cell of the fourth aspect of the present application, and isolating and obtaining the monoclonal antibody.

[0063] Further, the isolation and obtaining of the monoclonal antibody comprises: after the end of the culture, removing cell debris and impurities by centrifugation or filtration, and collecting the supernatant containing the antibody; using affinity chromatography to enrich the antibody, such as using Ni 2+Affinity chromatography column, Protein A / G chromatography column for conventional antibody, capture antibody by specific binding, elute target antibody with elution buffer (such as glycine buffer at pH 3.0-3.5); further purify the primary purified antibody by gel filtration chromatography (such as Superdex 200 column) or ion exchange chromatography to remove impurities, aggregates and residual impurities, and obtain high-purity antibody (purity ≥ 95%).

[0064] (2) A method for detecting soybean phytochrome PhyB protein for non-diagnostic purposes, comprising the following steps: contacting the sample to be tested with the monoclonal antibody of the first aspect of the application, and detecting the formation of antigen-antibody immune complexes.

[0065] Further, according to the detection requirements, the corresponding detection methods are selected, and the core is to convert the formation of immune complexes into a readable signal, and the specific methods include but are not limited to:

[0066] 1) Enzyme-linked immunosorbent assay (ELISA): After adding enzyme-labeled secondary antibody (such as HRP-labeled goat anti-mouse secondary antibody) and incubating, TMB color developing solution is added, the enzyme on the immune complex catalyzes the substrate to develop color, and the absorbance value at 450 nm is detected by an enzyme-labeled instrument, and the quantitative concentration of PhyB protein in the sample is calculated through a standard curve;

[0067] 2) Western Blot: After electrophoresis and membrane transfer, the sample is incubated with the antibody, and a chemiluminescent substrate is added, and the immune complex emits a fluorescent signal, which is captured by an imaging system to determine whether PhyB protein exists according to the presence or absence of the band, and the relative quantitative analysis is carried out according to the band gray value;

[0068] 3) Immunofluorescence method: The fluorescence-labeled monoclonal antibody is incubated, and then the fluorescence signal is observed directly by fluorescence microscope or confocal microscope, and the localization and distribution of PhyB protein in cells or tissues are directly presented;

[0069] 4) Immunochromatography: Add the sample to the test strip, and the sample migrates and binds to the gold nanoparticle-labeled antibody, and if it contains PhyB protein, an immune complex is formed, which is enriched and colored at the detection line, and the qualitative detection is realized by observing whether the detection line is colored.

[0070] Further, the antigen-antibody contact reaction specifically includes: mixing the sample to be tested, the positive control, and the negative control with the diluted monoclonal antibody respectively, and incubating under suitable conditions. The liquid phase reaction (such as ELISA) can be incubated at 37°C for 1-2 hours, and the solid phase reaction (such as immunoblotting, chip detection) needs to be fixed on the carrier (enzyme-labeled plate, blotting membrane, chip) first, then blocked, and then the antibody is added and incubated to ensure that the antibody is fully combined with the PhyB protein in the sample to form an immune complex. After incubation, the unbound free antibody is removed by repeatedly washing with a washing solution (such as PBST) for 3-5 times, and the background signal interference is reduced.

[0071] The eighth aspect of the present application provides the application of any one of the following aspects:

[0072] (1) The application of the monoclonal antibody of the first aspect of the present application in the preparation of a detection reagent for detecting soybean phytochrome PhyB protein;

[0073] (2) The application of the monoclonal antibody of the first aspect of the present application or the detection reagent of the fifth aspect of the present application in the preparation of a detection product for detecting soybean phytochrome PhyB protein;

[0074] Optionally, the detection product is a detection kit, a detection chip, a detection test paper, or a detection probe;

[0075] Optionally, the detection kit is an ELISA detection kit, an immunoblotting detection kit, an immunofluorescence detection kit, or an immunohistochemical detection kit;

[0076] Optionally, the detection chip is a protein detection chip or an immunosensor chip;

[0077] Optionally, the detection test paper is an immunochromatography test paper;

[0078] Optionally, the detection probe is a monoclonal antibody probe modified by a penetrating peptide.

[0079] In some embodiments, the present application also provides the application of the monoclonal antibody of the first aspect of the present application in the fields of soybean light signal regulation mechanism research, genetic breeding assistance, cultivation optimization, and related technical development. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 : Electrophoresis result diagram corresponding to the antibody 2F3 targeting the soybean phytochrome PhyB protein;

[0081] Figure 2 : HPLC result diagram corresponding to the antibody 2F3 targeting the soybean phytochrome PhyB protein;

[0082] Figure 3Figure 1 is a result chart of ELISA detection of the binding activity of the antibody 2F3 targeting soybean phytochrome PhyB protein. DETAILED DESCRIPTION

[0083] The application will be further described below in conjunction with specific examples. The following specific examples are only used to explain the application and should not be understood as limiting the application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to the examples without departing from the principles and purposes of the application, and the scope of the application is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the application are easily obtained by those skilled in the art, and can be obtained from commercial channels if not otherwise specified, and the experimental methods of the application not specified in the application are usually implemented according to conventional conditions or according to the conditions suggested by the manufacturers. In particular, the following examples are only used to illustrate the application and should not limit the scope of the application in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the application and should not and will not limit the application described in detail in the claims.

[0084] Example 1: Screening of antibodies targeting soybean phytochrome PhyB protein

[0085] 1. Immunogen recombinant expression

[0086] (1) Synthesize the sequence of the soybean phytochrome gene PhyB expression protein, construct into the pCDNA3.1 vector, extract the plasmid for transfection, transfect into HEK293 cells, culture the cells for 7 days, harvest the supernatant, purify by Ni column, replace the buffer after concentration, and obtain the soybean phytochrome gene PhyB expression protein.

[0087] The sequence information of the recombinant soybean phytochrome gene PhyB expression protein is as follows:

[0088]

[0089] 2. Immunize mice, SP2 / 0 fusion, screening, subclone

[0090] (1) Immunize mice: the first time with Freund's complete adjuvant, 100 μg per mouse, intraperitoneal injection, total dose 0.5 mL per mouse, the second time immunization interval 3 weeks; the second time use Freund's incomplete adjuvant, the dose is 50 μg / 0.5 mL per mouse, the third time immunization interval 2 weeks; 10 days after the third injection, prepare cell fusion.

[0091] (2) SP2 / 0 fusion: take feeder cells, which can be used according to 10 5 cells / well, 10 5 cells / 100 μL / well plated one day before fusion; take mouse immune spleen cells and prepared myeloma cells for fusion with fusion agent PEG, and plate into 96 cell culture plates with added feeder cells, 100 μL / well.

[0092] (3) Positive screening: positive well screening is carried out by ELISA detection method, plate recombinant soybean phytochrome PhyB protein overnight; wash the plate, add skim milk powder to block, 37°C, 1 h; wash the plate, add 100 μL 96 well culture supernatant, 37°C, incubate for 1 h; wash the plate, add HRP labeled goat anti-mouse secondary antibody, 37°C, incubate for 30 min; wash the plate, add color developing solution, color develop for 10 min, add stop solution, read OD450 value; screen high expression cell strains for subclone culture.

[0093] 3. Sequence fishing

[0094] Collect cells, extract total RNA with Trizol, use oligo (dT) 20 as primer to generate cDNA by reverse transcription. Then use specific primers to amplify the heavy and light chain variable region genes respectively. The PCR products are purified by electrophoresis, then inserted into the vector by TA cloning, transformed, and positive clones are selected for sequencing.

[0095] 4. Experimental results

[0096] The target soybean phytochrome PhyB protein antibody 2F3 is screened, and the corresponding sequence information is shown in Table 1 and Table 2.

[0097] Table 1 Heavy chain variable region and light chain variable region sequences of the target soybean phytochrome PhyB protein antibody 2F3

[0098]

[0099] Table 2 CDR sequences of the target soybean phytochrome PhyB protein antibody 2F3

[0100]

[0101] Example 2 Detection of affinity and specificity of antibody targeting soybean phytochrome PhyB protein

[0102] 1. Antibody expression

[0103] The amino acid sequence of 2F3 as described in Example 1 was optimized according to human codons, and was synthesized by GenScript and constructed into a pcDNA3.4 vector. The plasmid was prepared for large-scale extraction.

[0104] One day before transfection, well-conditioned 293 cells (adherent cell confluence of 70-80%, and the density of suspension cells was 3x10 5 cells / mL) were inoculated in culture vessels. On the day of transfection, endotoxin-free plasmids were prepared in a ratio of HC:LC=1:1-1.2, and the amount of PEI was calculated according to N / P=3:1. DNA and PEI were diluted with serum-free medium and mixed, and the complex was incubated at room temperature for 15-20 min. The complex was added to the cells, which were cultured at 37°C, 5% CO2. The medium was changed after 6-12 h, and the supernatant was collected after 48-96 h of continuous culture. The concentration and activity of the antibody were detected by ultraviolet UV 280 nm, ELISA, and SDS-PAGE methods.

[0105] The results showed that the concentration of the 2F3 clone was 436 mg / L.

[0106] 2. Physicochemical properties

[0107] (1) Detection of antibody purity by SDS-PAGE method

[0108] Main instruments: chemiluminescence imager, purchased from Tanon, model Tanon-5200; electrophoresis instrument, purchased from BIO-RAD, model poweerpac basic; electrophoresis tank, purchased from BIO-RAD, model DYC-Mini4.

[0109] Main reagents: 1 M Tris-HCl buffer, purchased from Beijing Solabio Technology Co., Ltd.; 1.5 M Tris-HCl buffer, purchased from Beijing Solabio Technology Co., Ltd.; 10% SDS, purchased from Beijing Solabio Technology Co., Ltd.; FastStain, purchased from Gene Universal; 30% gel preparation solution (29:1), purchased from Beijing Solabio Technology Co., Ltd.; Rainbow 180 broad-spectrum protein marker, purchased from Beijing Solabio Technology Co., Ltd.

[0110] Sample preparation: 20 μL of sample was mixed with 5 μL of 5x reducing buffer, heated at 95℃ for 5 min, and cooled; 20 μL of sample was mixed with 5 μL of 5x non-reducing buffer.

[0111] Electrophoresis: The gel was prepared, an appropriate amount of electrophoresis buffer was added, the sample was added, and electrophoresis was performed.

[0112] Staining and destaining: After electrophoresis, the gel was taken out and placed in an appropriate amount of coomassie brilliant blue staining solution, and stained at room temperature for 1 h or more; the staining solution was poured out, an appropriate amount of coomassie brilliant blue staining and destaining solution was added, and destaining was performed at room temperature for 4-24 h. After destaining was completed, the gel was soaked with ddH2O, compared with the marker protein, and the gel of the desired protein component was cut off, collected, and the protein to be purified was separated from the gel.

[0113] (2) HPLC was used to detect the purity of the antibody

[0114] Main instruments: liquid chromatograph, purchased from Agilent Technologies Co., Ltd., model Agilent 1100 / 1200 / 1260; liquid chromatograph column, purchased from Tosoh Corporation, model WieTSKgel G3000SWxl, 7.8 mm I.D. x 30 cm; pH meter, purchased from Sartorius Scientific Instrument Co., Ltd.; electronic balance, purchased from Sartorius Scientific Instrument Co., Ltd.

[0115] Main reagents: dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride, all purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0116] Preparation of mobile phase: dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride were added to about 900 mL of purified water, stirred and dissolved, made up to 1 L, measured with a pH meter, and the pH was determined to be between 6.2±0.1. 0.22 μm filter membrane was used for filtration, and the solution was stored at room temperature.

[0117] Sample preparation: system suitability sample: the standard was diluted with the mobile phase to 2 mg / mL; test sample: the sample to be tested was diluted with the mobile phase to 2 mg / mL.

[0118] Chromatographic conditions: injection volume was 25 μL, injection temperature was 6℃, running time was 30 min, flow rate was 0.5 mL / min, column temperature was 30℃, detection wavelength was 280 nm (VWD) or detection wavelength was 280 nm, bandwidth was 4 nm, reference wavelength was 360 nm, bandwidth was 80 nm (DAD), and peak width was 0.025 min (0.5 s).

[0119] The electrophoresis result chart and liquid chromatography result chart of the antibody 2F3 targeting soybean phytochrome PhyB protein are shown in Figures 1 and 2, respectively. Figure 1 (the bands from left to right are marker, reduced band, respectively), Figure 2 As shown in Figure 2, the results show that the detection purity of the 2F3 is greater than 95%.

[0120] 3. ELISA detection of the binding activity of the antibody

[0121] Coating: dilute the antigen (soybean phytochrome PhyB protein) to 2 μg / mL with the coating solution, mix well, add to the 96-well coating plate, 100 μL / well, seal and block at 4°C overnight. Wash 3 times with the plate washer, and the last time should not have liquid left on the plate. Dry the liquid on the surface of the plate with a water-absorbing paper.

[0122] Blocking: add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL / well, incubate at 37°C for 1 h, and wash the plate 3 times according to the above procedure. Dilute the antibody in gradient, 100 μL / well, react at 37°C for 1 h, and wash the plate 3 times according to the above procedure.

[0123] Add secondary antibody: dilute with DPBS at 1:2000, add to the 96-well plate, 100 μL / well, react at 37°C for 1 h, and wash the plate 3 times according to the above procedure.

[0124] Color development: add TMB, 100 μL / well, develop at room temperature in the dark for 10 min.

[0125] Termination: add 2N H2SO4, 100 μL / well. Measure OD 450 within 10 min.

[0126] The result chart of the ELISA detection of the binding activity of the antibody 2F3 targeting soybean phytochrome PhyB protein is shown in Figure 3. Figure 3 The results show that the antibody 2F3 can specifically bind to soybean phytochrome PhyB protein and show concentration dependence, and the corresponding EC 50 is 0.1357 μg / mL, with high affinity.

[0127] 4. Specific detection of the antibody

[0128] Coat the same concentration of Arabidopsis PhyB, rice PhyB, corn PhyB, sorghum PhyB, pea PhyB, potato PhyB, and other common crop phytochrome gene B expression proteins, add the antibody, add the secondary antibody, develop color, read, and analyze data.

[0129] The specificity detection results of the antibody 2F3 targeting the soybean phytochrome gene PhyB expression protein are shown in Table 3. The results show that only the soybean PhyB protein develops color normally, and the rest of the antigens have no positive reaction, that is, the antibody 2F3 targeting the soybean phytochrome gene PhyB expression protein can specifically bind to the soybean phytochrome gene PhyB expression protein, and has high specificity.

[0130] Table 3 Specificity detection results of the antibody 2F3 targeting the soybean phytochrome gene PhyB expression protein

[0131]

Claims

1. A monoclonal antibody for detecting soybean phytochrome PhyB protein, characterized by, The amino acid sequences of CDR1-3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NO: 4-6, respectively; the amino acid sequences of CDR1-3 of the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 7-9, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2 or has at least 80% homology with SEQ ID NO:

2.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3 or has at least 80% homology with SEQ ID NO:

3.

4. A polynucleotide encoding the monoclonal antibody of any one of claims 1-3; Optionally, the polynucleotide is a DNA molecule.

5. A recombinant vector comprising the polynucleotide of claim 4; Optionally, the recombinant vector is a eukaryotic expression vector; Optionally, the eukaryotic expression vector is a pcDNA vector, a pCMV vector, a pEGFP-N1 / pEGFP-C1 vector, a pIRES vector, a pCDNA6 vector, a yeast eukaryotic expression vector, a Saccharomyces cerevisiae expression vector, or an insect cell eukaryotic expression vector.

6. A recombinant host cell comprising the recombinant vector of claim 5; Optionally, the host cell is a eukaryotic cell; Optionally, the host cell is a mammalian cell, a yeast cell, or an insect cell; Optionally, the mammalian cell is a CHO cell, a HEK293 cell, an NS / 0 myeloma cell, a Sp2 / 0 myeloma cell, a Cap cell, or a COS cell; Optionally, the yeast cell is Pichia pastoris or Saccharomyces cerevisiae; Optionally, the insect cell is an Sf9 / Sf21 cell or a High Five cell.

7. A detection reagent for detecting a soybean phytochrome PhyB protein, characterized by, The detection reagent comprises the monoclonal antibody of any one of claims 1-3.

8. A detection product for detecting a soybean phytochrome PhyB protein, characterized by, The detection product comprises the monoclonal antibody of any one of claims 1-3 or the detection reagent of claim 7; Optionally, the detection product is a detection kit, a detection chip, a detection test paper, or a detection probe; Optionally, the detection kit is an ELISA detection kit, an immunoblotting detection kit, an immunofluorescence detection kit, or an immunohistochemical detection kit; Optionally, the detection chip is a protein detection chip or an immunosensor chip; Optionally, the detection test paper is an immunochromatographic detection test paper; Optionally, the detection probe is a monoclonal antibody probe modified by a penetrating peptide.

9. Any one of the following methods: (1) A method for preparing the monoclonal antibody of any one of claims 1-3, the method comprising the following steps: culturing the recombinant host cell of claim 6, and isolating to obtain the monoclonal antibody; (2) A method for detecting soybean phytochrome PhyB protein for non-diagnostic purposes, the method comprising the following steps: contacting a sample to be tested with the monoclonal antibody of any one of claims 1-3, and detecting the formation of an antigen-antibody immune complex.

10. Any one of the following aspects for use: (1) Use of the monoclonal antibody according to any one of claims 1-3 in the preparation of a detection reagent for detecting soybean phytochrome PhyB protein; (2) Use of the monoclonal antibody according to any one of claims 1-3 or the detection reagent according to claim 7 in the preparation of a detection product for detecting soybean phytochrome PhyB protein; Optionally, the detection product is a detection kit, a detection chip, a detection test paper or a detection probe; Optionally, the detection kit is an ELISA detection kit, an immunoblot detection kit, an immunofluorescence detection kit or an immunohistochemical detection kit; Optionally, the detection chip is a protein detection chip or an immunosensor chip; Optionally, the detection test paper is an immunochromatographic detection test paper; Optionally, the detection probe is a monoclonal antibody probe modified by a penetrating peptide.

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