Anti-Populus tomentosa PtoWRKY40 antibody and application thereof

Abstract

The present invention provides an antibody against Populus tomentosa PtoWRKY40 and its application. Specifically, the present invention provides an antibody against Populus tomentosa PtoWRKY40 or an antigen-binding fragment thereof, wherein the antibody against Populus tomentosa PtoWRKY40 or an antigen-binding fragment thereof has very excellent specificity, can be used to effectively detect proteins that interact with PtoWRKY40 protein and the expression of PtoWRKY40 protein, and can be effectively applied to the study of the interaction between PtoWRKY40 protein and downstream genes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-Populus tomentosa PtoWRKY40 antibody and an application thereof. Background Art

[0002] Currently, most antibody libraries are prepared by genetic engineering and have the problem of low specificity, which will lead to the inability to effectively identify specific epitopes of target proteins, thus affecting the accuracy of research.

[0003] Some antibody libraries are small in scale and can only provide a limited number of antibodies, which makes it difficult to meet the needs of large-scale research. In this case, it is necessary to rely on the laboratory to carry out antibody preparation work on its own, but this method faces the problems of high cost and long cycle.

[0004] For some rare or trace proteins, existing antibody libraries are unlikely to provide sufficient antibody resources. In this case, it is necessary to find other alternatives, such as using mass spectrometry to identify protein interaction networks to make up for the limitations of antibody libraries.

[0005] Therefore, preparing antibodies with strong specificity is of great significance in this field. Summary of the invention

[0006] The purpose of the present invention is to: for the existing background technology, the existing antibody library may be difficult to provide sufficient antibody resources. In this case, if an antibody with good specificity for the target protein of this species is prepared, the interacting proteins of the protein are identified and mined by immunoprecipitation (IP) and mass spectrometry, the real protein interaction between the target protein and the interacting protein is confirmed by yeast two-hybrid and GST pulldown, and the interaction between the target protein and the downstream gene is confirmed by CHIP-PCR, thereby confirming the successful preparation of an antibody with good specificity for the target protein, making up for the limitation of the limited number of specific antibodies.

[0007] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof against Populus tomentosa PtoWRKY40 is provided, wherein the antibody or the antigen-binding fragment thereof against Populus tomentosa PtoWRKY40 has the following three complementarity determining regions CDR (HCDR) of the heavy chain variable region and three complementarity determining regions CDR (LCDR) of the light chain variable region:

[0008] HCDR1 shown in SEQ ID NO:4,

[0009] HCDR2 shown in SEQ ID NO:6,

[0010] HCDR3 shown in SEQ ID NO:8,

[0011] LCDR1 shown in SEQ ID NO:11,

[0012] LCDR2 shown in SEQ ID NO: 13, and

[0013] LCDR3 shown in SEQ ID NO:15.

[0014] In another preferred embodiment, the anti-populus tomentosa PtoWRKY40 antibody or antigen-binding fragment thereof has 6 complementary determining regions CDRs derived from the following amino acid sequences of the heavy chain variable region and the light chain variable region: SEQ ID NO: 1 and SEQ ID NO: 2;

[0015] The CDRs are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 determined by any one of the IMGT rules, Kabat rules, Chothia rules, AbM rules or Contact rules.

[0016] In another preferred example, the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment further includes four framework regions FR (H-FR) of the heavy chain variable region: H-FR1 shown in SEQ ID NO:3, H-FR2 shown in SEQ ID NO:5, H-FR3 shown in SEQ ID NO:7, and H-FR4 shown in SEQ ID NO:9; and / or four framework regions FR (L-FR) of the light chain variable region: L-FR 1 shown in SEQ ID NO:10, L-FR2 shown in SEQ ID NO:12, L-FR3 shown in SEQ ID NO:14; L-FR4 shown in SEQ ID NO:16.

[0017] In another preferred example, the amino acid sequence of the heavy chain variable region of the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:2.

[0018] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0019] (i) the anti-Populus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof according to the first aspect of the present invention; and

[0020] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0021] In another preferred embodiment, the tag includes an Fc tag, a FLAG tag, a 6His tag, or a combination thereof.

[0022] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0023] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0024] In the third aspect of the present invention, a nucleotide molecule is provided, wherein the nucleotide molecule encodes the anti-Populus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof according to the first aspect of the present invention.

[0025] In the fourth aspect of the present invention, a vector is provided, wherein the vector contains the nucleotide molecule described in the third aspect of the present invention.

[0026] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the fourth aspect of the present invention, or has the nucleotide molecule described in the third aspect of the present invention integrated into its genome, or expresses the anti-populus tomentosa PtoWRKY40 or its antigen-binding fragment described in the first aspect of the present invention.

[0027] In a sixth aspect of the present invention, a method for detecting Populus tomentosa PtoWRKY40 protein in a sample is provided, the method comprising the steps of:

[0028] (1) contacting a sample with the anti-Poplarus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention;

[0029] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of Populus tomentosa PtoWRKY40 protein in the sample.

[0030] In a seventh aspect of the present invention, a method for preparing the anti-Poplarus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof according to the first aspect of the present invention is provided, the method comprising the following steps:

[0031] (a) culturing the host cell according to the fifth aspect of the present invention under expression conditions, thereby expressing the anti-Populus tomentosa PtoWRKY40 antibody or antigen-binding fragment thereof;

[0032] (b) isolating and purifying the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment described in (a).

[0033] In an eighth aspect of the present invention, there is provided a use of the anti-Poplarus tomentosa PtoWRKY40 antibody or antigen-binding fragment thereof according to the first aspect of the present invention and the recombinant protein according to the second aspect of the present invention for preparing a reagent, a detection plate or a kit;

[0034] The reagent, detection plate or kit is used for detecting whether a sample contains the Populus tomentosa PtoWRKY40 protein, or for capturing the Populus tomentosa PtoWRKY40 protein.

[0035] In another preferred embodiment, the Populus tomentosa PtoWRKY40 protein is in a non-binding state with the interacting protein.

[0036] In another preferred embodiment, the Populus tomentosa PtoWRKY40 protein is in a binding state with an interacting protein.

[0037] In another preferred embodiment, the interacting protein is selected from the following group: CuZn-Superoxide dismutase (PtoSOD4), Elongation factor protein, Alpha-xylosidase family protein, Sucrosesynthase, Dynamin-related protein, box / FBD / LRR-repeat protein, and Zinc finger family protein.

[0038] In the ninth aspect of the present invention, a detection plate is provided, comprising a substrate (support plate) and a test strip, wherein the test strip contains the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention.

[0039] In another preferred embodiment, the test strip further comprises an antigen spotting area.

[0040] In another preferred embodiment, the test strip is composed of filter paper, chromatography material, nitrocellulose membrane and absorbent paper overlapped in sequence.

[0041] In a tenth aspect of the present invention, a kit is provided, comprising:

[0042] (1) a first container, wherein the first container contains the anti-Populus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0043] (2) a second container, wherein the second container contains a secondary antibody, wherein the secondary antibody is directed against the anti-Populus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and / or

[0044] (3) a third container, wherein the third container contains a cell lysis reagent;

[0045] or,

[0046] The kit contains the detection plate as described in the ninth aspect of the present invention.

[0047] In another preferred embodiment, the antibody in the first container is detectably labeled.

[0048] In another preferred embodiment, the antibody in the second container is detectably labeled.

[0049] Compared with the prior art, the present invention has the following beneficial effects: in the scheme of the present invention, by providing a method for preparing specific antibodies for specific proteins, the prepared antibodies with high specificity can be used to study the functions and interactions of the proteins. Recognizing proteins that interact with target proteins through antibodies with high specificity helps to reveal the complexity of protein interaction networks and lays a good foundation for identifying the functions of target proteins; in the study of protein and gene interactions, experiments such as immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) require antibodies, and the existence of antibody libraries makes these experiments more convenient and faster, improving the efficiency of research. Proteins are qualitatively or quantitatively detected through antibody libraries, such as Western blot and ELISA, to obtain information about protein expression, modification, and interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The specific identification of the PtoWRKY40 antibody and the screening of interacting proteins of the present invention are shown: (A) Specific screening of 12 PtoWRKY40 antibodies; (B) CoIP experiment was carried out on the cambium tissue of Populus tomentosa; (C) CoIP-MS screened 14 interacting proteins related to wood formation.

[0051] Figure 2 The schematic diagram of the interaction between PtoWRKY40 and PtoSOD4 of the present invention is shown: (A) Yeast two-hybrid assay shows that PtoWRKY40 can interact with PtoSOD4; (B) BiFC assay confirms that PtoWRKY40 can interact with PtoSOD4; (C) GST pulldown assay shows that PtoWRKY40 and PtoSOD4 interact with each other; (D) CoIP assay shows that PtoWRKY40 and PtoSOD4 interact with each other.

[0052] Figure 3The schematic diagram of the interaction between PtoWRKY40 and PtoCCoAOMT1 / Pto4CL3 of the present invention is shown: (A) Yeast single hybridization experiment shows that PtoWRKY40 interacts with PtoCCoAOMT1 and Pto4CL3; (B) EMSA experiment shows that PtoWRKY40 can specifically bind to the promoter of PtoCCoAOMT1 containing the W-box core element; (C, D) ChIP-PCR (C) and ChIP-qPCR (D) show that PtoWRKY40 directly regulates the expression of downstream target genes PtoCCoAOMT1 and Pto4CL3; (E) EMSA experiment shows that PtoWRKY40 can specifically bind to the promoter of Pto4Cl3 containing the W-box core element.

[0053] Figure 4 A partial sequence diagram of the anti-Populus tomentosa PtoWRKY40 antibody of the present invention is shown.

[0054] Figure 5 The whole protein prediction analysis diagram of the present invention is shown, which gives the overall picture of the PtoWRKY40 protein structure and the location of the recommended epitope; the X-axis represents the number of amino acids in the protein, and Y is the algorithm value. The higher the score, the greater the possibility of the epitope; among them, the epitopes highlighted in blue are regions with higher immunogenicity and hydrophilicity scores (there is no signal peptide and transmembrane domain).

[0055] Figure 6 Six complementarity determining regions, CDRs, of preferred antibodies of the invention are shown. DETAILED DESCRIPTION

[0056] After extensive and in-depth research, the inventors obtained an anti-PtoWRKY40 antibody against Populus tomentosa through a large number of screenings. The experimental results show that the antibody of the present invention has excellent specificity. And the antibody of the present invention can effectively target and bind to the PtoWRKY40 protein in a bound state (and / or free state), and can effectively detect proteins that interact with the PtoWRKY40 protein. In addition, the antibody of the present invention can be effectively used in the study of the interaction between the PtoWRKY40 protein and downstream genes. Therefore, the expression of the PtoWRKY40 protein and the expression of proteins that interact with it can be detected by the antibody of the present invention, and can be used to study the expression of downstream genes that the PtoWRKY40 protein regulates and interacts with. The present invention was completed on this basis.

[0057] Specifically, the experiment of the present invention screened the PtoSOD4 protein that interacts with PtoWRKY40 through the PtoWRKY40 antibody; and carried out a CoIP experiment using the PtoWRKY40 antibody to confirm the interaction between the PtoWRKY40 protein and the PtoSOD4 protein; and carried out a ChIP-PCR experiment using the PtoWRKY40 antibody to confirm the interaction between the PtoWRKY40 protein and the two downstream genes PtoCCoAOMT1 and Pto4CL3.

[0058] the term

[0059] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0060] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0061] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0062] PtoWRKY40 and WRKY

[0063] PtoWRKY40 is a WRKY transcription factor in Populus tomentosa. WRKY transcription factors are a class of plant-specific transcription factors that regulate gene expression by binding to specific DNA sequences, thereby affecting plant growth and development and coping with environmental stress.

[0064] Antibody

[0065] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region, and the heavy chain constant region is composed of three domains CH1, CH2, and CH3. One end of each light chain has a variable region (VL) and the other end has a constant region, and the light chain constant region includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.

[0066] In the present invention, the terms "Fab" and "Fc" refer to the fact that papain can cleave antibodies into two identical Fab segments and one Fc segment. The Fab segment is composed of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of the antibody. The Fc segment is the crystallizable fragment (Fc), which is composed of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.

[0067] In the present invention, the term "scFv" refers to a single chain antibody fragment (scFv), which is composed of an antibody heavy chain variable region and a light chain variable region connected by a linker of usually 15 to 25 amino acids.

[0068] In the present invention, the term "variable" means that some parts of the variable region in the antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conservative part of the variable region is called the framework region (FR). The variable region of the natural heavy chain and light chain each contains four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)).

[0069] As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., refers to those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. The light chain and heavy chain of immunoglobulins each have four FRs, respectively referred to as L-FR1, L-FR2, L-FR3, L-FR4 and H-FR1, H-FR2, H-FR3, H-FR4. Accordingly, the light chain variable domain can therefore be referred to as:

[0070] (L-FR1)-(L-CDR1)-(L-FR2)-(L-CDR2)-(L-FR3)-(L-CDR3)-(L-FR4) and the heavy chain variable domain can therefore be expressed as (H-FR1)-(H-CDR1)-(H-FR2)-(H-CDR2)-(H-FR3)-(H-CDR3)-(H-FR4). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0071] Knowing the amino acid sequences of the CDRs, one skilled in the art can easily determine the framework regions L-FR1, L-FR2, L-FR3, L-FR4 and / or H-FR1, H-FR2, H-FR3, H-FR4.

[0072] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0073] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light chain and the heavy chain to fold into an exchange dual variable region immunoglobulin. In the present invention, preferred linkers refer to linkers Linker1 and Linker2, wherein Linker1 connects the VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect scFv to the heavy chain of another antibody.

[0074] Examples of suitable linkers include single glycine (Gly), or serine (Ser) residues. The identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element that is desired to be achieved in the linker.

[0075] In the present invention, the antibody of the present invention also includes conservative variants thereof, which refers to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the bispecific antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0076] Table A

[0077]

[0078] In the present invention, the terms "anti", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed. Typically, an antibody binds to the antigen with an equilibrium dissociation constant (KD) less than about 10-7M, for example, less than about 10-8M, 10-9M, 10-10M, 10-11M or less. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, the binding affinity of an antibody to an antigen is measured in a BIACORE instrument using surface plasmon resonance (SPR) or the relative affinity of an antibody to an antigen is measured using ELISA.

[0079] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.

[0080] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention 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 DNA can be a coding strand or a non-coding strand.

[0081] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0082] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0083] Test Uses and Kits

[0084] The antibodies of the present invention can be used in detection applications, such as for detecting samples, thereby providing detection information.

[0085] In the present invention, the samples used include cells and tissue samples.

[0086] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0087] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0088] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0089] General Methods

[0090] Preparation and transformation of competent yeast

[0091] (1) First, streak AH109 yeast on YPDA medium and grow at 30℃ for 2-3 days until a single colony grows out; (2) Pick several fresh yeast clones with a diameter of 2-3 mm and inoculate them into 1mL YPDA liquid medium. Vigorously shake for 5 minutes to evenly disperse the cell clumps; (3) Transfer to 50mL YPDA medium and culture overnight at 30℃ and 250rpm for 16-18 hours until the cell growth state reaches the stable phase (OD600>1.5); (4) Dilute the overnight culture solution with YPDA medium at a ratio of 1:20 to an OD600 of 0.2-0.3; (5) Continue to culture at 30℃ and 230rpm for about 3 hours. Right, until OD600 is 0.4-0.6; (6) Transfer the cells to a 50 mL centrifuge tube and centrifuge at room temperature at 1,000 g for 5 min; (7) Discard the supernatant, resuspend the cell pellet with sterile ultrapure water, and combine them in a centrifuge tube with a total volume of approximately 25 mL; (8) Centrifuge at room temperature at 1,000 g for 5 min and discard the supernatant; (9) Resuspend with 1.5 mL of freshly prepared 1×TE / 1×LiAc to obtain yeast competent cells; (10) Add 0.1 μg of plasmid DNA and 0.1 μg of salmon sperm DNA to a 1.5 mL centrifuge tube and mix thoroughly; (11) Add 0.1 mL of yeast competent cells and shake to mix; (12) Add 0.6 mL PEG / LiAc solution (each 1mL PEG / LiAc contains: 800μL 50% PEG, 100μL LiAc, 100μL 10×TE), pipette to mix; (13) incubate at 30℃, 200rpm for 30min; (14) add 70μL DMSO and gently invert to mix; (15) heat shock in a 42℃ water bath for 15min, invert to mix every 5min; (16) place on ice for 1-2min; (17) centrifuge at 14,000rpm for 5s at room temperature and discard the supernatant; (18) resuspend the cells in 0.5mL sterile 1×TE; (19) take 100μL of the resuspended cells and spread them on a plate containing an appropriate SD-deficient culture medium, and spread them evenly with a spreader; (20) incubate the plate inverted in a 30℃ incubator for about 2-4 days.

[0092] Yeast One Hybrid

[0093] (1) Yeast competent cells transformed with bait-pHis2.1 and PtoWRKY40-pGADT7 were evenly spread on the SD / -Leu-Trp medium, with 400 μL of bacterial solution spread on each medium;

[0094] (2) The culture medium was placed in a 30°C incubator for two days until colonies were clearly visible. Single yeast colonies of uniform size were picked with a sterilized toothpick and resuspended in 200 μL of 0.9% NaCl solution;

[0095] (3) Spot 5 μL of the resuspended bacterial solution on the double-deficient medium (SD / -Leu-Trp) and triple-deficient medium (SD / -Leu-Trp-His, 20 mM 3-AT), incubate at 30°C, observe and take pictures.

[0096] Yeast two-hybrid

[0097] The PtoWRKY40-pGBKT7, PtoSOD4-pGADT7, pGADT7, and pGBKT7 expression vectors were transformed into yeast AH109 competent cells using the LiAc-mediated method, and then grown on double-deficient (SD / -His / -Trp) plates for 2-3 days. Positive single clones were picked and grown on SD / -Ade / -His / -Trp / -Leu quadruple-deficient plates. After culturing for 3-4 days, β-galactosidase activity was detected.

[0098] Bimolecular fluorescence complementation (BiFC)

[0099] The N-terminus of YFP was fused with PtoWRKY40 into the vector pSPYNE173, and the C-terminus of PtoSOD4 was fused with YFP into the vector pSPYCE(M), and the tobacco leaf cells were co-transformed. After 48 hours of culture, the tobacco leaves were observed and images were collected using a laser confocal fluorescence microscope, with an excitation wavelength of 488nm.

[0100] Protein expression, purification and identification

[0101] Transform the expression bacteria, mix the expression plasmid with 50 μL competent cells of the expression bacteria, and incubate on ice for 30 minutes; ② Heat shock at 42°C for 45 seconds; ③ Instantly transfer to ice and incubate for 5 minutes; ④ Mix the competent cells with 1 mL of LB medium and shake culture at 37°C for 1 hour; ⑤ Plate.

[0102] ② Preservation of expression bacteria. Pick a single clone from the plate and inoculate it into 3-4 mL of LB medium containing the corresponding antibiotics, and culture it at 220 rpm and 37°C overnight; ② Add 1 / 5 volume of 60% glycerol to the bacteria, mix thoroughly, and store it in separate tubes at -80°C for later use;

[0103] Expand the culture, inoculate into 50-100 mL LB medium at a ratio of 1:50-1:200, and add the corresponding antibiotics; ② Culture at 220 rpm, 37 ° C for about 2 hours, observe at any time, measure the OD600 value of the bacterial solution, and induce expression when it reaches the range of 0.6-0.8;

[0104] IPTG induction, take 1-2ml bacterial solution, centrifuge at 4000g for 2min, remove the supernatant medium and only leave 100μL; ② add 1 / 5 volume of protein loading buffer, mix thoroughly and boil in boiling water for 10min; ③ centrifuge at 12000g for 10min at room temperature, the supernatant sample is the induced expression control sample; ④ add 1 / 1000 volume of 1M IPTG inducer to the remaining bacterial solution to make the final concentration at 1mM; ⑤ induce expression and culture at 220rpm, 37℃ for 1~3h;

[0105] For expression identification, take 1-2 ml of induced expression bacterial solution, centrifuge at 4000g for 2 min, remove the supernatant culture medium and only leave 100 μL; ② add 1 / 5 volume of protein loading buffer, mix thoroughly and boil in boiling water for 10 min; ③ centrifuge at room temperature at 12000g for 10 min, the supernatant sample is the induced expression sample;

[0106] Expression form identification: the remaining samples of expression identification were centrifuged at 4000g for 2min, and the supernatant culture medium was removed; ② After adding the culture volume of 1×PBS and mixing, centrifuged at 4000g for 2min, and the supernatant culture medium was removed; ③ Add 1 / 3 to 1 / 2 of the culture volume of 1×PBS and PMSF, and mix by blowing; ④ Set the ultrasonic conditions (time: 30min, ultrasonic time: 5s, interval time: 10s, power: 40%, total time is 30 to 1h) and ultrasonically mix the sample on ice until the solution is transparent; ⑤ Ultrasonic disruption of the sample was performed at 4°C and centrifuged at 12000g for 15min; ⑥ Take 100μL of the supernatant and add 1 / 5 volume of protein loading buffer, mix well and boil in boiling water for 10min to obtain the supernatant sample, and the remaining supernatant samples were stored at 4°C for future use; ⑦ Add 100μL of 1×PBS to the precipitate and mix by blowing, add 1 / 5 volume of protein loading buffer, mix well and boil in boiling water for 10min to obtain the precipitate sample;

[0107] Prepare PAGE gel of corresponding concentration according to the size of target protein, the default is 12%; ② Run the gel in the order of Marker (not added in the pre-experimental stage), induced control sample, induced sample, supernatant sample, and precipitate sample (the sample can be boiled and centrifuged again before loading); ③ Electrophoresis at 100V for 15min, then at 150V for 45min; ④ Stain with Coomassie Brilliant Blue solution, shake and stain at room temperature for 10-1h until the gel turns blue; ⑤ Recover the Coomassie Brilliant Blue solution, wash the gel twice with tap water, add 15mL of water and microwave on medium heat for 5min to determine the expression and expression form.

[0108] Immunoblotting

[0109] 1. Protein sample preparation

[0110] ① Centrifuge the bacterial solution at 10,000 rpm to obtain the precipitate, wash the bacteria with 1×PBS at pH 7.0, remove the supernatant by centrifugation, add 100 ul of 1×PBS, pipet and mix well, then add an appropriate amount of 5×loading buffer and boil in water for 10 min.

[0111] 2. Protein quality detection

[0112] ① Add 5× protein loading buffer containing 5% β-mercaptoethanol (sample: buffer = 4:1) to the protein sample and boil it in boiling water for 10 minutes for denaturation; ② Take 10ul of protein sample for gel electrophoresis and store the prepared sample at -20℃; ③ Stain with Coomassie Brilliant Blue R250 and shake on a shaker for 30min-60min; ④ Add water and decolorize on high heat in a microwave oven for 5min, 2-3 times.

[0113] 3. Polyacrylamide gel electrophoresis

[0114] ① Prepare separation gel and concentration gel. The concentration of separation gel is determined according to the molecular weight of the protein (12-60KD with 12%, 20-80KD with 10%). ② The amount of protein loaded per well is about 50ug or 15-20ul, and 3ul of protein marker is loaded. ③ First, the voltage is 80V for 15min, then the voltage is changed to 120V and the electrophoresis is continued for 40min.

[0115] 4. Transfer

[0116] ① Cut the PVDF membrane according to the size of the gel (8 samples can be cut into 1.5cm×8cm), cut the corners to distinguish the front and back, and then soak the membrane in methanol for activation. ② Place the splint on the transfer plate with the black side facing down, and place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in order; ③ The gel should be cut according to the molecular weight of the target protein. ④ Clamp the splint, put 2 ice bags in the transfer tank, fill it with transfer buffer, and then transfer the membrane in a 4℃ refrigerator.

[0117] 5. Closure

[0118] ①After the transfer, take out the membrane, turn the front side up, and wash it with TBST 2-3 times, 5 minutes each time. ②Add blocking solution and block at 4℃ overnight or shake on a shaker at room temperature for 2-3 hours.

[0119] 6. Antibody incubation

[0120] ①After blocking, wash the membrane with TBST 3 times, 5 minutes each time. ②Refer to the antibody manual to determine the dilution ratio and dilute the primary antibody to about 2 ml, seal the membrane with a film sealer and PE gloves, and shake at room temperature for 2 hours. ③Recover the primary antibody and wash the membrane with TBST 3 times, 10 minutes each time. ④1:5000 dilution of the secondary antibody, about 2 ml, seal the membrane with a film sealer and PE gloves, and shake at room temperature for 1 hour. ⑤Recover the secondary antibody and wash the membrane with TBST 3 times, 10 minutes each time.

[0121] 7. Development

[0122] ① Mix equal amounts of super-sensitive luminescent liquid, about 200ul is needed for one membrane. ② In the darkroom, open the transparent thin bag on the splint, put the membrane, add the luminescent liquid, cover the thin bag, then put the X-ray film, and then cover the splint. ③ The pressing time is determined by the brightness of the luminescence. If the fluorescence is very strong, it can be pressed for 10-60S, and if it is weak, it needs to be pressed for 5-10min. ④ Put the X-ray film in the developer to develop until clear strips can be seen by the naked eye; ⑤ Move the X-ray film to the fixer and fix it for 5-10min.

[0123] Example 1 Antibody Screening

[0124] According to the PtoWRKY40 sequence, 12 monoclonal antibodies were prepared. The specificity of each antibody was tested by IP and Western blot on the total protein of Populus tomentosa cambium. The results showed that antibody c1 (22472-1-9-3B10-1) had the best specificity.

[0125] 22472-1-9-3B10-1 PtoWRKY40 antibody strain sequencing results:

[0126] Heavy chain number: 22472-1-9 / 3B10-1----HA-1;

[0127] Light chain number: 22472-1-9 / 3B10-1----LG-1;

[0128] VH template clone number: 22472-1-9 / 3B10-1----HA-1;

[0129] VH - amino acid sequence:

[0130] EVQLVESGGGLVKPGGSLKLSCEVSGFTFSTYAMSWVRQSPDKRLEWVA EISRGGTYTYYPDTVTGRFTISRDNAKNTLYLEMNSLRSEDTATYYCTRNRDY DGDSYGMDYWGQGTSVTVSS(SEQ ID NO:1);

[0131] VL template clone number:

[0132] 22472-1-9 / 3B10-1----LG-1;

[0133] VL - amino acid sequence:

[0134] QIVLTQSPAIMSASPGEKVTMICSASSSVSWIHWYQQKSGTSPKRWIYDT SKLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNNPLTFGAGTKL ELK (SEQ ID NO: 2).

[0135] The antibody information of Populus tomentosa PtoWRKY40 of the present invention is as follows Figure 4 shown.

[0136] Heavy chain sample number: 22472-1-9 / 3B10-1----HA-1 (heavy chain)

[0137] The sequence information of the heavy chain variable region is shown in Table 1 below.

[0138] Table 1

[0139]

[0140] Light chain sample number: 22472-1-9 / 3B10-1----LG-1 (light chain)

[0141] The sequence information of the light chain variable region is shown in Table 2 below.

[0142] Table 2

[0143]

[0144] Example 2 Antibody effectiveness test experiment and its results

[0145] Based on the c1 antibody with the best specificity, CoIP experiments were carried out on the immature xylem and cambium tissues of Populus tomentosa. Figure 1 B), combined with mass spectrometry sequencing, 494 PtoWRKY40 interacting proteins were screened, including 14 proteins related to wood formation ( Figure 1 C).

[0146] The results showed that PtoWRKY40 protein may interact with 18 secondary wall development-related proteins from the cambium tissue of Populus tomentosa, including CuZn-Superoxidedismutase (PtoSOD4), Elongation factorprotein, Alpha-xylosidase familyprotein, Sucrosesynthase, Dynamin-related protein, box / FBD / LRR-repeat protein and Zincfinger familyprotein.

[0147] PtoWRKY40 and PtoSOD4 have protein interactions. Based on the previous use of the most specific PtoWRKY40 antibody, the PtoWRKY40 protein obtained by CoIP technology screening library can interact with 18 secondary wall development-related proteins. This paper focuses on the interaction between PtoWRKY40 and the interested PtoSOD4.

[0148] Further studies using yeast two-hybrid, BiFC, GSTpμLl down and CoIP showed that PtoWRKY40 interacted with PtoSOD4. Figure 2 AD), since the interacting protein PtoSOD4 was identified by screening the library with the PtoWRKY40 antibody, this result also confirmed the effectiveness of the PtoWRKY40 antibody.

[0149] Yeast one-hybrid, EMSA (Electrophoretic Mobility Shift Assay) and ChIP-qPCR experiments showed that PtoWRKY40 bound to the promoters of PtoCCoAOMT1 and Pto4CL3.

[0150] Using the self-made c1 antibody with the best specificity, ChIP-PCR experiments were carried out on the xylem tissue of Populus tomentosa, and it was found that the expression levels of the two genes PtoCCoAOMT1 (primer 1 and primer 2 are two pairs of primers for amplifying the promoter region) and Pto4CL3 were relatively high, indicating that PtoCCoAOMT1 and Pto4CL3 may be two downstream genes directly regulated by PtoWRKY40 ( Figure 3 CD).

[0151] ChIP-PCR experiments also verified the yeast one-hybrid ( Figure 3 A) and EMSA ( Figure 3 B, E) results show that the homemade PtoWRKY40 antibody with the best specificity can be used for ChIP-PCR ( Figure 3 C) and ChIP-qPCR experiments, and can confirm the reliability of yeast one-hybrid and EMSA results.

[0152] discuss

[0153] The present invention provides an anti-Poplar tomentosa PtoWRKY40 antibody and its application. Specifically, the present invention provides an anti-Poplar tomentosa PtoWRKY40 antibody (variable region information such as Figure 6As shown in FIG. 1 ), it can effectively target and bind to the PtoWRKY40 protein in a bound state (and / or free state), thereby effectively detecting the protein interacting therewith and the expression of the PtoWRKY40 protein (epitope analysis of the PtoWRKY40 protein is shown in FIG. Figure 5 As shown). In addition, the antibody of the present invention can be effectively used in the study of the interaction between PtoWRKY40 protein and downstream genes, and can effectively detect the expression of downstream genes that PtoWRKY40 protein regulates and interacts with. The heavy chain variable region and light chain variable region information of the preferred antibody of the present invention are as follows Figure 4 shown.

[0154] The anti-Poplarus tomentosa PtoWRKY40 antibody of the present invention has a wide range of applications. It plays an important role in studying protein functions, interactions and modifications. By screening specific antibodies against a specific protein, the protein network that interacts with the target protein is identified, and its complexity is revealed. By providing specific antibodies against a specific protein, it can be used to study the function, interaction and modification of the protein. It has a wide range of applications.

[0155] The anti-Populus tomentosa PtoWRKY40 antibodies of the present invention can be used to develop detection reagents. Specifically, these antibodies can be used to detect the levels of WRKY proteins in related plants.

[0156] The antibodies of the present invention can be used to develop plant growth and development detection reagents to detect changes in expression associated with WRKY proteins during plant growth and development. For example, they can detect whether the expression of endogenous WRKY proteins changes when plants are at a certain growth stage or in response to certain environmental stresses.

[0157] The antibodies of the present invention can be used for the study of plant hormones or signal molecules. For example, if the epitope is related to plant hormones or certain signal molecules, these antibodies can be used to study the role of these molecules in plant growth and development.

[0158] The antibodies of the present invention can be used for transgenic plant detection. For example, if the epitope is present in a transgenic plant, these antibodies can be used to quickly detect the presence of the transgene.

[0159] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail in the various embodiments referred to in this specification, the present invention is not limited to specific implementation methods. Therefore, any modification or replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against Populus tomentosa PtoWRKY40, characterized in that: The anti-Populus tomentosa PtoWRKY40 antibody or antigen-binding fragment thereof has the following three complementarity determining regions CDR (HCDR) of the heavy chain variable region and three complementarity determining regions CDR (LCDR) of the light chain variable region: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 8, LCDR1 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 15; The amino acid sequence of LCDR2 is DTS.

2. A recombinant protein, characterized in that The recombinant protein has: (i) the anti-Populus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof according to claim 1; and (ii) Tag sequences that facilitate expression and / or purification.

3. A nucleotide molecule, characterized in that The nucleotide molecule encodes the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment according to claim 1.

4. A carrier, characterized in that The vector contains the nucleotide molecule according to claim 3.

5. A host cell, characterized in that The host cell contains the vector of claim 4, or has the nucleotide molecule of claim 3 integrated into its genome, or expresses the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment as claimed in claim 1.

6. A method for detecting Populus tomentosa PtoWRKY40 protein in a sample, the method comprising the steps of: (1) contacting a sample with the anti-Poplarus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof as claimed in claim 1; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of Populus tomentosa PtoWRKY40 protein in the sample.

7. A method for preparing the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment according to claim 1, the method comprising the following steps: (a) culturing the host cell according to claim 5 under expression conditions, thereby expressing the anti-Populus tomentosa PtoWRKY40 antibody or antigen-binding fragment thereof; (b) isolating and purifying the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment described in (a).

8. Use of the anti-Populus tomentosa PtoWRKY40 antibody or antigen-binding fragment thereof according to claim 1 and the recombinant protein according to claim 2 for preparing a reagent, a detection plate or a kit; The reagent, detection plate or kit is used for detecting whether a sample contains the Populus tomentosa PtoWRKY40 protein, or for capturing the Populus tomentosa PtoWRKY40 protein.

9. A detection board, characterized in that: The detection plate comprises a substrate and a test strip, wherein the test strip contains the anti-Populus tomentosa PtoWRKY40 antibody or its antigen-binding fragment as claimed in claim 1, or the recombinant protein as claimed in claim 2.

10. A kit, characterized in that: The kit includes: (1) a first container, wherein the first container contains the anti-Populus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof according to claim 1; and / or (2) a second container, wherein the second container contains a secondary antibody directed against the anti-Populus tomentosa PtoWRKY40 antibody or an antigen-binding fragment thereof according to claim 1; and / or (3) a third container, wherein the third container contains a cell lysis reagent; Alternatively, the kit contains the detection plate as described in claim 9.

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