Preparation and application of cotton fertility related protein Abrin polyclonal antibody

Abstract

The application discloses a polyclonal antibody for cotton Abrin protein and application thereof. The polyclonal antibody recognizes an antigen epitope in the cotton Abrin protein, and the amino acid sequence of the antigen epitope is as shown in SEQ ID NO:1, and the amino acid sequence of SEQ ID NO:1 is CDGFVEERWVFRSDGT. The polyclonal antibody can be used for Abrin protein immunoreaction signal detection in a cotton anther sample, and is used for comparing differences in the Abrin protein immunoreaction signal between a cotton cytoplasmic male sterile line and a maintainer line thereof and / or between different development periods of a cotton anther. The application further provides an immunogen for preparing the antibody, a preparation method, a Western blot detection kit and a detection method.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and molecular detection technology, specifically relating to the preparation and application of a polyclonal antibody against Abrin, a cotton fertility-related protein. Background Technology

[0002] Cytoplasmic male sterility in cotton is a crucial foundation for utilizing heterosis in cotton. Anther and pollen development is regulated by multiple factors, including gene expression, protein synthesis, energy metabolism, and organelle function. Comparing protein expression differences between cytoplasmic male-sterile lines and their maintainer lines, as well as at different anther developmental stages, helps in screening candidate proteins related to anther development and male sterility, and further analyzing the biological functions of these proteins.

[0003] Western blotting is the most commonly used method for the qualitative and quantitative detection of endogenous proteins in plant tissues, and this technique relies on antibodies with good specificity. Currently, there is still a lack of specific antibodies for detecting cotton abrin protein. Existing techniques, such as using full-length abrin protein to prepare antibodies, suffer from drawbacks including difficulties in prokaryotic expression and purification, abnormal folding, numerous contaminating bands after immunization, and insufficient specificity for detecting endogenous proteins in tissues.

[0004] Existing bioinformatics methods can predict potential linear epitopes of proteins, but most of the candidate epitope peptides predicted by software cannot induce animals to produce effective antibodies that can recognize endogenous natural proteins in plants. Furthermore, it is difficult to achieve stable Western blot detection in the complex total protein system of cotton anthers, thus failing to meet the need to compare the differences in Abrin protein expression between sterile lines and maintainer lines, as well as at different developmental stages of anthers.

[0005] Therefore, it is necessary to screen for Abrin protein epitopes suitable for antibody preparation and obtain antibodies that can be used to compare the differences in Abrin protein immune response signals between cotton cytoplasmic male sterile lines and their maintainer lines, as well as at different developmental stages of anthers. Summary of the Invention

[0006] The present invention aims to provide a polyclonal antibody targeting the cotton abrin protein. The polyclonal antibody specifically recognizes the antigenic epitope shown in SEQ ID NO: 1 of the cotton abrin protein, wherein the amino acid sequence shown in SEQ ID NO: 1 is CDGFVEERWVFRSDGT. The polyclonal antibody is used to detect the immunoreactivity signal of the abrin protein in cotton anther samples, and / or to compare the differences in the immunoreactivity signal of the abrin protein between cotton cytoplasmic male sterile lines and their maintainer lines, and / or between different developmental stages of cotton anthers.

[0007] Another object of the present invention is to provide an immunogen for preparing the polyclonal antibody, a method for preparing the polyclonal antibody, a Western blot assay kit containing the polyclonal antibody, and a method for detecting differences in the immunoreaction signal of Abrin protein in cotton anthers.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a polyclonal antibody targeting cotton abrin protein, wherein the polyclonal antibody specifically recognizes the antigenic epitope shown in SEQ ID NO: 1 of cotton abrin protein, and the amino acid sequence shown in SEQ ID NO: 1 is CDGFVEERWVFRSDGT; the polyclonal antibody is used to detect the abrin protein immunoreaction signal in cotton anther samples, and / or to compare the differences in abrin protein immunoreaction signal between cotton cytoplasmic male sterile lines and their maintainer lines and / or between different developmental stages of cotton anthers.

[0009] Furthermore, the polyclonal antibody is a rabbit-derived polyclonal antibody.

[0010] Furthermore, the polyclonal antibody was obtained by immunizing New Zealand rabbits with an immunogen formed by conjugating the antigenic epitope peptide shown in SEQ ID NO: 1 with a carrier protein.

[0011] Furthermore, the carrier protein is KLH.

[0012] Furthermore, the polyclonal antibody is obtained by affinity chromatography purification of immune serum.

[0013] In a second aspect, the present invention provides an immunogen for preparing the above-mentioned polyclonal antibody, the immunogen comprising a carrier protein and an antigenic epitope peptide coupled to the carrier protein, the antigenic epitope peptide being composed of the amino acid sequence shown in SEQ ID NO: 1.

[0014] Furthermore, the carrier protein is KLH, namely keyhole hemocyanin.

[0015] Thirdly, the present invention provides a method for preparing the above-mentioned polyclonal antibody, comprising the following steps: (1) The antigenic epitope peptide shown in SEQ ID NO: 1 is coupled with a carrier protein to prepare an immunogen; (2) Immunize the animals with the immunogen and collect the immune serum from the animals; (3) The immune serum was purified by affinity chromatography, the antibody eluent was collected, and the polyclonal antibody was obtained by neutralization and dialysis.

[0016] Furthermore, the carrier protein is KLH, and the animal is a New Zealand rabbit.

[0017] Furthermore, the immunization program includes primary immunization and booster immunizations at weeks 2, 4, and 5 after primary immunization; Freund's complete adjuvant is used for primary immunization and booster immunizations at week 2, while Freund's incomplete adjuvant is used for booster immunizations at weeks 4 and 5; for each immunization, the immunogen and the corresponding adjuvant are mixed and emulsified before subcutaneous injection.

[0018] Fourthly, the present invention provides a detection kit for detecting the immune response signal of cotton Abrin protein using Western blot, the detection kit comprising the aforementioned polyclonal antibody.

[0019] Furthermore, the polyclonal antibody is a rabbit-derived polyclonal antibody, and the detection kit also includes HRP-labeled anti-rabbit IgG secondary antibody and ECL chemiluminescent substrate.

[0020] Furthermore, the detection kit also includes one or more of the following: blocking solution, washing solution, primary antibody dilution solution, secondary antibody dilution solution, and protein transfer buffer.

[0021] Fifthly, the present invention provides a method for detecting differences in Abrin protein immunoreaction signals in cotton anthers, comprising the following steps: (1) Extract total protein from the cotton anther samples to be tested; (2) The total protein was subjected to SDS-polyacrylamide gel electrophoresis, and the separated protein was transferred to a solid membrane; (3) The above-mentioned polyclonal antibody was used as the primary antibody and incubated with the solid phase membrane; (4) Incubate with an enzyme-labeled secondary antibody that matches the polyclonal antibody and detect the signal; (5) Based on the intensity of the Abrin protein immune response signal, compare the differences in Abrin protein immune response signals between cotton cytoplasmic male sterile lines and their maintainer lines and / or between different developmental stages of cotton anthers.

[0022] Furthermore, the solid phase membrane is a PVDF membrane.

[0023] Furthermore, β-actin was used as an internal reference protein, and the relative intensity of the Abrin protein immune response signal was determined based on the gray value ratio of the Abrin protein immune response signal band to that of the β-actin band.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention screens and obtains the C-terminal antigenic epitope CDGFVEERWVFRSDGT suitable for antibody preparation by analyzing the antigenicity, hydrophilicity and surface accessibility of cotton Abrin protein, thus providing a clear target for the preparation of cotton Abrin protein antibodies.

[0025] (2) Rabbit polyclonal antibodies were prepared using the C-terminal epitope peptide of the present invention. ELISA detection showed that the antibody titer was not less than 1:128000 and had high immunoreactivity.

[0026] (3) The polyclonal antibody obtained by the present invention can obtain clear and stable immunoreaction bands in total protein samples of cotton anthers, and the detection effect is better than that of the antibody prepared by fusion expression of protein A and peptide B, which is suitable for detection of Abrin protein immunoreaction signal in cotton anthers.

[0027] (4) The polyclonal antibody obtained in this invention can be used to detect the differences in Abrin protein immunoreaction signals between cotton cytoplasmic male sterile lines and their maintainers and / or between different anther development stages of cotton, providing a detection tool for analyzing changes in the expression of cotton anther development and male sterility-related proteins. Attached Figure Description

[0028] Figure 1 The figure shows the predicted transmembrane structure of the cotton Abrin protein.

[0029] Figure 2 This is a schematic diagram of the structure of the recombinant expression vector pGEX-4T-1-Abrin.

[0030] Figure 3 The image shows the SDS-PAGE results of the purified fusion protein A, where M is the protein molecular weight standard and lane 1 represents the fusion protein A.

[0031] Figure 4 The graphs show the HPLC and mass spectrometry results of peptide B, where A represents the HPLC result of peptide B and B represents the mass spectrometry result of peptide B.

[0032] Figure 5 The graphs show the HPLC and mass spectrometry results of peptide C, where A represents the HPLC result of peptide C and B represents the mass spectrometry result of peptide C.

[0033] Figure 6 The image shows the results of indirect ELISA detection of pre-immunization serum and purified antibodies. RB431 and RB432 are purified antibodies obtained by immunizing animals with peptide B, RB433 and RB434 are purified antibodies obtained by immunizing animals with peptide C, and RB639 and RB640 are purified antibodies obtained by immunizing animals with fusion-expressed protein A.

[0034] Figure 7 The figure shows the Western blot screening results of total anther proteins LD6A and LD6B by antibodies-A, antibody-B, and antibody-C.

[0035] Figure 8 The figure shows the results of Western blot detection and fold change analysis of Abrin protein immunoreaction signals at different developmental stages of cotton anthers by antibody-C. In the figure, A represents the Western blot detection results, and B represents the fold change analysis results of Abrin protein immunoreaction signals. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. The following embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Unless otherwise stated, the reagents and instruments used in the embodiments are commercially available; experimental procedures not explicitly described can be performed according to the instructions for the corresponding reagents or instruments.

[0037] Example 1: Sequence analysis of cotton abrin protein and screening of candidate antigenic peptides Differential gene expression analysis was performed based on cotton anther de novo RNA-seq data. The transcriptome data was accessed via the NCBI Sequence Read Archive (PRJNA577562). The analysis identified an Abrin gene highly expressed in sterile lines at the anther abortion stage. Sequence alignment results showed that this gene sequence was identical to the cotton Abrin gene sequence recorded in GenBank accession number XM_016849659.

[0038] ORFfinder analysis of the open reading frame of the Abrin gene revealed that the Abrin protein consists of 577 amino acid residues, with the amino acid sequence shown in SEQ ID NO: 2. ExPASy ProtParam analysis of the Abrin protein predicted its molecular weight to be 64.71 kDa and its theoretical isoelectric point to be 5.21.

[0039] The transmembrane structure of the Abrin protein was analyzed using TMHMM Server v.2.0. The results are shown below. Figure 1 The prediction results show that the Abrin protein contains a single transmembrane structure.

[0040] DNASTAR 11.0 was used to comprehensively analyze the antigenicity, hydrophilicity, and surface accessibility of the Abrin protein amino acid sequence. The analysis results showed that there were differences in antigenicity, hydrophilicity, and surface accessibility in different regions of the Abrin protein. Based on the above prediction results, one theoretical candidate antigenic epitope peptide was identified in each of the N-terminal, C-terminal, and mid-segment regions of the Abrin protein, for a total of three theoretical candidate antigenic epitope peptides.

[0041] (1) The natural sequence at N-terminus 40-57 is RFTTERATRNSYLMFMKD, SEQ ID NO: 6. To facilitate coupling with the carrier protein, a cysteine ​​residue was added to the C-terminus to synthesize the polypeptide sequence RFTTERATRNSYLMFMKDC, named polypeptide B, corresponding to SEQ ID NO: 4; this peptide was subsequently synthesized and used for animal immunization.

[0042] (2) The sequence at C-terminus 521-536 is CDGFVEERWVFRSDGT. This sequence contains cysteine ​​and can be directly used for conjugation. It is named polypeptide C, corresponding to SEQ ID NO: 1. This peptide was subsequently synthesized and used for animal immunization.

[0043] (3) The natural sequence of the middle segment is SAQPTDPRQYVLVELSNG, SEQ ID NO: 7. To adapt to vector conjugation, a cysteine ​​residue was added to the N-terminus to obtain the sequence CSAQPTDPRQYVLVELSNG, denoted as SEQ ID NO: 5. This peptide segment is only used as a theoretical candidate for bioinformatics, and no peptide synthesis, vector conjugation, or animal immunization experiments have been carried out.

[0044] To set up a full-length protein control immunogen, the complete coding sequence of the Abrin protein shown in SEQ ID NO: 3 was ligated into the prokaryotic expression vector pGEX-4T-1 with a GST tag to construct the recombinant expression vector pGEX-4T-1-Abrin (vector map shown). Figure 2 The GST fusion protein obtained by vector-induced expression was named fusion expression protein A.

[0045] Based on the three types of immunogens mentioned above, three groups of polyclonal antibodies were prepared and named: (1) Antibody obtained by immunization with fusion-expressed protein A: antibody-A; (2) Antibody-B obtained by immunization with polypeptide B-KLH conjugate; (3) Antibody-C obtained by immunization with polypeptide C-KLH conjugate.

[0046] Subsequent animal immunization, antibody titer detection, and Western blot screening experiments in this invention only use three types of immunogens: (1) Fusion expression of protein A; (2) Peptide B-KLH conjugate; (3) Peptide C-KLH conjugate.

[0047] Example 2: Synthesis and detection of peptides B and C Based on the screening results of Example 1, peptides B and C were selected for solid-phase synthesis.

[0048] 2.1 Synthesis and Detection of Peptide B The amino acid sequence of polypeptide B is: RFTTERATRNSYLMFMKDC. The sample number of polypeptide B is 993233, and the batch number is P220520-SY993233.

[0049] Purity was determined by HPLC using a reversed-phase C18 column. The column size was 4.6 mm × 250 mm. Mobile phase A was 0.1% trifluoroacetic acid / acetonitrile, mobile phase B was 0.1% trifluoroacetic acid / water, the flow rate was 1.0 mL / min, the detection wavelength was 220 nm, and the injection volume was 10 μL.

[0050] HPLC detection results are shown in Figure 4 The results showed that the retention time of the main peak of peptide B was 10.279 min, and the peak area accounted for 90.13%. The remaining peaks were low-content impurity peaks, indicating that the purity of peptide B was 90.13%.

[0051] The molecular weight of peptide B was determined by ESI mass spectrometry, and the results are shown in [Figure number missing]. Figure 4 The mass spectrometry results showed that peptide B had a measured molecular weight of 2370.72 Da and [M+2H] was detected. 2+ [M+3H] 3+ and [M+4H] 4+ Characteristic ion peaks were observed. The detection results were consistent with the designed sequence of peptide B, indicating that the synthesized peptide B product meets the requirements for subsequent carrier protein conjugation and animal immunization.

[0052] 2.2 Synthesis and Detection of Peptide C The amino acid sequence of polypeptide C is: CDGFVEERWVFRSDGT. The sample number of polypeptide C is 993234, and the batch number is P220601-SY993234.

[0053] The purity of peptide C was determined by HPLC using a reversed-phase C18 column. The column size was 4.6 mm × 250 mm. Mobile phase A was 0.1% trifluoroacetic acid / acetonitrile, mobile phase B was 0.1% trifluoroacetic acid / water, the flow rate was 1.0 mL / min, the detection wavelength was 220 nm, and the injection volume was 10 μL.

[0054] HPLC detection results are shown in Figure 5 The results showed that the retention time of the main peak of peptide C was 6.673 min, and the peak area accounted for 90.96%. The remaining peaks were low-content impurity peaks, indicating that the purity of peptide C was 90.96%.

[0055] The molecular weight of peptide C was determined by ESI mass spectrometry, and the results are shown in [Figure number missing]. Figure 5 Mass spectrometry analysis of peptide C showed that its molecular weight was 1903.03 Da, and [M+2H] was detected. 2+ and [M+3H] 3+ Characteristic ion peaks were observed. The detection results were consistent with the designed sequence of peptide C, indicating that the synthesized peptide C product meets the requirements for subsequent carrier protein conjugation and animal immunization.

[0056] The above results indicate that peptides B and C meet the design requirements and can be used for subsequent carrier protein conjugation and animal immunization.

[0057] Example 3: Prokaryotic expression and purification of fusion protein A 3.1 Construction of Recombinant Expression Vectors The Abrin protein coding sequence shown in SEQ ID NO: 3 was ligated into the pGEX-4T-1 vector to obtain the recombinant expression vector pGEX-4T-1-Abrin with a GST tag. The vector structure is shown in [link to vector structure]. Figure 2 .

[0058] 3.2 Small-scale induction of expression pGEX-4T-1-Abrin was transformed into Escherichia coli BL21 competent cells.

[0059] Single colonies were picked and inoculated into LB liquid medium and cultured with shaking at 37°C and 200 rpm. When the bacterial culture OD... 600 When the concentration reaches 0.5–0.6, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.8 mmol / L, and continue induction culture at 37℃ and 200 rpm for 4 h.

[0060] After induction, bacterial cells were collected, and the expression of fusion protein A was detected by SDS-PAGE.

[0061] 3.3 Expanded culture and induced expression Select single clones that meet the expression requirements, inoculate them into 3 mL of LB liquid medium, and culture overnight at 37°C and 200 rpm with shaking.

[0062] Inoculate the overnight culture solution into 300 mL of LB liquid medium at a volume ratio of 1:100 and incubate at 37°C with shaking at 200 rpm. When the OD of the culture solution... 600 When the concentration reaches 0.5–0.6, add IPTG to a final concentration of 0.8 mmol / L, and continue induction culture at 37℃ and 200 rpm for 4 h.

[0063] After induction, the bacterial culture was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. The bacterial cells were washed twice with PBS, and then resuspended in PBS. After adding an appropriate amount of lysozyme for 30 min, the cells were sonicated to disrupt the bacterial structure.

[0064] The lysate was centrifuged at 10,000 rpm for 10 min, and the supernatant and precipitate were collected separately. SDS-PAGE was used to detect the distribution of fusion protein A in the supernatant and precipitate. Fusion protein A was mainly found in the supernatant.

[0065] 3.4 GST affinity purification 250 mL of E. coli expressing fusion protein A was collected by centrifugation, and the bacterial cells were resuspended in 10–15 mL of lysis buffer and then disrupted by freeze-thaw and / or sonication.

[0066] The lysis buffer contains 20–50 mmol / L Tris-HCl and 0.25 mol / L NaCl, with a pH of 7.5–8.5. EDTA, PMSF, or other protease inhibitors may be added as appropriate.

[0067] After centrifugation of the lysate, the supernatant was collected and added to a pre-equilibrated GST-resin affinity purification column. After loading, the purification column was washed with 10–20 column volumes of lysis buffer.

[0068] The fusion-expressed protein A was eluted using 2–4 mL of elution buffer. The elution buffer consisted of lysis buffer and freshly prepared reduced glutathione, with a final concentration of 6 mmol / L for the reduced glutathione.

[0069] The eluent was collected, and the purification results were analyzed by SDS-PAGE. The results are shown in the figure below. Figure 3 The results showed that the purified fusion-expressed protein A formed a clear band near the target molecular weight, which can be used for subsequent animal immunization.

[0070] After purification, the column was washed with 20 column volumes of 1% acetic acid, then the pH was equilibrated with PBS / TBS, the column was washed with plenty of pure water, and finally 5 mL of 20% ethanol was added and stored at 4°C.

[0071] Example 4: Coupling of peptides B and C with KLH The cross-linking agent MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester, 3-maleimide benzoic acid-N-hydroxysuccinimide ester) was used to couple peptide B and peptide C with KLH to prepare peptide B-KLH conjugates and peptide C-KLH conjugates, respectively.

[0072] KLH was prepared into a 10 mg / mL solution using PBS buffer (pH 7.2–7.4). The KLH solution was dialyzed at room temperature for 2 h or at 4°C overnight.

[0073] MBS was dissolved in DMF (Dimethyl Formamide) to prepare an MBS solution with a concentration of 10 mg / mL.

[0074] Mix KLH solution and MBS solution at a mass ratio of 10:1 and incubate at room temperature for 30 min. Shake the bottle during incubation to ensure KLH activation.

[0075] The activated KLH solution was treated with Sephadex G-25 gel filter media to remove excess MBS and reaction byproducts.

[0076] Peptide B and peptide C were prepared into 10 mg / mL peptide solutions using PBS buffer (pH 7.2). Activated KLH was then mixed with either peptide B or peptide C at a 1:1 mass ratio and incubated at room temperature for 3 h to obtain peptide B-KLH conjugates and peptide C-KLH conjugates.

[0077] The B-KLH conjugate and C-KLH conjugate were placed in PBS and dialyzed overnight at 4°C. After dialyzing, they were aliquoted and stored at -20°C for later use.

[0078] Example 5 Animal Immunization The animal immunization experiments were commissioned to Saixin (Hainan) Biotechnology Co., Ltd.

[0079] Age-appropriate male New Zealand rabbits were selected as immunization animals. Two male New Zealand rabbits were immunized with each immunogen, for a total of six male New Zealand rabbits.

[0080] Before immunization, 2-3 mL of blood was collected from the ear vein of each male New Zealand rabbit, and the pre-immunization serum was separated as a negative control for subsequent indirect ELISA detection.

[0081] Based on the different immunogens, the experimental animals were divided into the following three groups: The first group was immunized with fusion-expressed protein A, and the resulting antibody was named antibody-A. The second group was immunized with a peptide B-KLH conjugate, and the resulting antibody was named antibody-B. The third group was immunized with a peptide C-KLH conjugate, and the resulting antibody was named antibody-C.

[0082] For the initial immunization, 0.5 mg of fusion expressed protein A, peptide B-KLH conjugate, or peptide C-KLH conjugate were added to 0.5 mL of PBS, then mixed thoroughly with an equal volume of Freund's complete adjuvant and emulsified. The mixture was then administered subcutaneously to immunize male New Zealand rabbits.

[0083] The first booster immunization was administered two weeks after the primary immunization. The first booster immunization used the same immunogen dose and injection method as the primary immunization, with Freund's complete adjuvant as the adjuvant.

[0084] Booster immunizations were administered at weeks 4 and 5 following the initial immunization. Both booster immunizations used Freund's incomplete adjuvant, and the immunogen dosage and injection method were the same as for the initial immunization.

[0085] At weeks 6, 7, 8, and 9 following the initial immunization, the cells were treated with injections of 0.9% NaCl solution. A small amount of blood was collected at week 7, serum was separated, and the titer of the immune serum was determined using indirect ELISA.

[0086] Blood was collected from rabbits via carotid artery pleophoresis 10 weeks after the initial immunization to prepare serum.

[0087] Example 6 Affinity chromatography purification of immune serum Rabbit immune serum obtained by immunization with fusion-expressed protein A, peptide B-KLH conjugate, and peptide C-KLH conjugate was purified by affinity chromatography. Based on the source of the immunogen, the purified products were named antibody-A, antibody-B, and antibody-C, respectively.

[0088] Rabbit immune serum obtained from immunization with the peptide C-KLH conjugate was purified using CNBr-activated Sepharose 4B affinity resin conjugated to peptide C. Rabbit immune serum obtained from immunization with the fusion expression protein A and peptide B-KLH conjugate was purified using affinity resin conjugated to the corresponding antigens.

[0089] Mix the affinity resin thoroughly, pack the resin into the chromatography column, allow the resin to settle naturally and drain the preservation solution.

[0090] Add 10 column volumes of PBS buffer to equilibrate the chromatography column.

[0091] Immune serum was added to the equilibrated chromatography column, the eluent was collected, and the absorbance of the eluent at 280 nm was monitored using a nucleic acid protein detector.

[0092] After sample loading, the chromatography column was washed with PBS buffer until the absorbance of the eluent at 280 nm remained stable.

[0093] Drain the PBS from the chromatography column, add 0.1 mol / L glycine-hydrochloric acid elution buffer (pH 2.7), and elute the antibody bound to the affinity resin.

[0094] Collect the antibody eluent in separate tubes and immediately add 1 mol / L Tris-HCl neutralization buffer (pH 8.5) to adjust the pH of the eluent to neutral.

[0095] The elution fractions containing antibodies were combined, placed in a dialysis bag, and dialyzed overnight in PBS at 4°C to obtain purified antibodies.

[0096] Add sodium azide to the purified antibody to achieve a final sodium azide concentration of 0.02%, and store at 4°C for later use.

[0097] Example 7 Indirect ELISA titer detection of purified antibodies The antibody titers of antibody-A, antibody-B, and antibody-C were detected using indirect ELISA.

[0098] The enzyme-labeled plates were coated with detection antigens corresponding to each immunogen. The detection antigens were diluted to 2 μg / mL with carbonate buffer, and 100 μL was added to each well. The plates were incubated overnight at 4°C.

[0099] Remove the antigen coating solution, wash away the unbound antigen from the plate, and wash three times with washing buffer.

[0100] Add 200 μL of blocking buffer to each well and incubate at room temperature for 2 h. Discard the blocking buffer and wash the plate three times with washing buffer.

[0101] The purified antibody was diluted to 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000 using blocking buffer, and 100 μL was added to each well.

[0102] Pre-immunization serum was diluted 1:1000 and used as a negative control.

[0103] After adding the sample, incubate at room temperature for 60 min. Discard the antibody solution and wash the plate three times with washing buffer.

[0104] HRP-labeled goat anti-rabbit IgG secondary antibody was diluted 1:5000 with blocking buffer containing 1%–3% BSA in PBS (pH 7.4), and 100 μL was added to each well and incubated at 37°C for 45 min.

[0105] Discard the secondary antibody solution and wash the plate three times with washing buffer.

[0106] Add 100 μL of TMB substrate to each well and incubate at room temperature in the dark for 10 min. Stop the colorimetric reaction by adding 50 μL of 2 mol / L sulfuric acid stop solution. Read the absorbance at 450 nm on an ELISA plate reader.

[0107] The positive criterion is P / N ≥ 2.1, where P is the absorbance of the antibody well and N is the absorbance of the pre-immunization serum control well. The maximum antibody dilution that meets the positive criterion is used as the antibody titer.

[0108] Test results are shown Figure 6 RB431 and RB432 are purified antibodies obtained by immunizing animals with peptide B; RB433 and RB434 are purified antibodies obtained by immunizing animals with peptide C; RB639 and RB640 are purified antibodies obtained by immunizing animals with fusion expressed protein A.

[0109] RB431, RB432, RB433, RB434, RB639, and RB640 all met the positive criteria at a dilution of 1:128000, indicating that the titers of the six purified antibodies were not lower than 1:128000.

[0110] Example 8: Western blot screening of antibodies prepared from different immunogens 8.1 Extraction of total protein from cotton anthers Anther samples were collected from cotton cytoplasmic male sterile line LD6A and its isonuclear maintainer line LD6B, and total protein was extracted from the anthers using the TCA / acetone method.

[0111] The concentration of total protein in each anther sample was determined using the Bradford Protein Quantitative Reagent Kit, and the protein concentrations of each sample were adjusted to be consistent.

[0112] 8.2 SDS-PAGE Prepare an SDS-PAGE gel with a mass concentration of 12.5% ​​according to standard methods.

[0113] Take 20 μg of cotton protein sample from each material and add it to the vertical electrophoresis tank.

[0114] Electrophoresis was started at a constant voltage of 60 V. After the sample entered the separating gel, the voltage was adjusted to 80 V, and electrophoresis continued until the bromophenol blue indicator band was close to the bottom of the gel.

[0115] 8.3 Transfer of film Proteins in a gel were transferred to a PVDF membrane using a semi-dry transfer method.

[0116] Before use, PVDF membranes should be soaked in methanol for 10–15 seconds and then equilibrated in transfer buffer.

[0117] During transfer, the flow rate was 1.5 mA / cm² according to the gel area. 2 Set the total current and the film transfer time to 40 min.

[0118] 8.4 Blocking and Primary Antibody Incubation After the transfer was completed, the PVDF membrane was placed in TBST containing 5% skim milk powder and sealed at 37°C for 2 hours.

[0119] The membrane was washed four times with PBS containing 0.3% Tween 20 for 5 minutes each time.

[0120] Antibody-A, antibody-B, and antibody-C were diluted at a ratio of 1:500 using TBST containing 5% skim milk powder.

[0121] Different PVDF films were incubated overnight at 4°C with antibody-A, antibody-B, or antibody-C, respectively.

[0122] 8.5 Secondary antibody incubation and signal detection After the primary antibody incubation was completed, the membrane was washed four times with PBS containing 0.1% Tween 20, each time for 5 minutes.

[0123] HRP-labeled anti-rabbit IgG secondary antibody was diluted 1:1000 with TBST containing 5% skim milk powder and incubated at 37°C for 1 h.

[0124] After the secondary antibody incubation was completed, the membrane was washed four times with PBS containing 0.1% Tween 20, each time for 5 minutes.

[0125] Add ECL chemiluminescent substrate and react at room temperature for 2–5 min. Expose and image using a Syngene gel imaging system, or expose film for 5 min to obtain Western blot results.

[0126] 8.6 Screening Results The Western blot (WB) screening experiment used the following uniform experimental conditions: 20 μg of total anther protein was loaded per lane, the primary antibody was diluted 1:500, the HRP-labeled goat anti-rabbit secondary antibody was diluted 1:1000, and ECL chemiluminescence was developed at room temperature for 2–5 min.

[0127] Test results are shown Figure 7 The β-actin internal control bands showed that the sample loading amount and transfer efficiency were basically consistent in each lane.

[0128] The Western blot results of antibody-A, antibody-B, and antibody-C for total protein samples from LD6A and LD6B anthers showed differences. Antibody-C was able to obtain relatively clear immunoreaction bands in both LD6A and LD6B anther samples, with the main immunoreaction band located around 53 kDa near the protein marker, and an additional accompanying immunoreaction band observed around 34 kDa.

[0129] Compared with antibody-A and antibody-B, antibody-C showed higher band clarity and lower background interference. Therefore, antibody-C was selected for subsequent detection of Abrin protein immune response signals at different developmental stages of cotton anthers.

[0130] Example 9: Detection of differences in Abrin protein immunoreaction signals at different developmental stages of cotton anthers Referring to the cotton anther development stage classification method published by Zheng et al., the anther development stage was determined based on the anther diameter and combined with the anther development morphological characteristics; the reference is: Zheng J et al., Comparative Transcriptome Analysis between a Novel Allohexaploid Cotton Progeny CMS Line LD6A and Its Maintainer Line LD6B, International Journal of Molecular Sciences, 2019, 20(24):6127, DOI:10.3390 / ijms20246127. Anther samples of LD6A and LD6B were collected at the pollen mother cell (PMC), tetrad (Td), uninucleate (Uni), and mature pollen (MP) stages, respectively.

[0131] Total protein was extracted from each anther sample using the TCA / acetone method, and protein concentration was determined using the Bradford method.

[0132] Western blot analysis was performed according to the method described in Example 8. 20 μg of total anther protein was added to each lane. Antibody-C was used as the primary antibody for Abrin protein detection, with a primary antibody dilution ratio of 1:500. HRP-labeled anti-rabbit IgG secondary antibody was used as the secondary antibody, with a secondary antibody dilution ratio of 1:1000.

[0133] β-actin was used as an internal reference protein.

[0134] The results of the Western blot are shown below. Figure 8 In the study, antibody-C was able to obtain clear immunoreaction bands in samples from different anther development stages of LD6A and LD6B. The band signal intensity varied between different anther development stages.

[0135] ImageJ software was used to... Figure 8 Gray-scale analysis was performed on the approximately 53 kDa immunoreaction band and the β-actin band in A.

[0136] For each anther developmental stage, the ratio of the gray values ​​of the approximately 53 kDa immunoreaction band to the β-actin band in LD6A and LD6B samples was calculated as the relative immunoreaction signal intensity of the corresponding samples; then the ratio of the relative immunoreaction signal intensity of LD6A to that of LD6B was calculated as the fold change for that developmental stage.

[0137] The calculation formula is as follows: The relative immunoreaction signal intensity of LD6A = the gray value of the approximately 53 kDa immunoreaction band in LD6A / the gray value of the β-actin band in LD6A; The relative immune response signal intensity of LD6B = the gray value of the approximately 53 kDa immune response band in LD6B / the gray value of the β-actin band in LD6B; Difference factor = LD6A relative immune response signal intensity / LD6B relative immune response signal intensity.

[0138] The grayscale analysis results are shown below. Figure 8 The results showed that the fold difference in Abrin protein immunoreaction signals between LD6A and LD6B varied at different anther development stages, indicating that antibody-C can be used to compare the differences in Abrin protein immunoreaction signals between cotton cytoplasmic male sterile lines and their maintainer lines at different anther development stages. This difference can be used to characterize changes in Abrin protein-related expression.

Claims

1. A polyclonal antibody against cotton abrin protein, characterized in that, The polyclonal antibody is a rabbit-derived polyclonal antibody, obtained by immunizing New Zealand rabbits with an immunogen formed by covalently conjugating the antigen epitope peptide shown in SEQ ID NO: 1 with KLH. The primary immunization and the booster immunization at week 2 after the primary immunization were administered subcutaneously after emulsifying 0.5 mg of the immunogen with Freund's complete adjuvant. The booster immunizations at weeks 4 and 5 after the primary immunization were administered subcutaneously after emulsifying 0.5 mg of the immunogen with Freund's incomplete adjuvant. The amino acid sequence of the epitope peptide shown in SEQ ID NO: 1 is CDGFVEERWVFRSDGT. The polyclonal antibody is used to detect the Abrin protein immunoreaction signal in cotton anthers and / or to compare the differences in Abrin protein immunoreaction signals between cotton cytoplasmic male sterile lines and their maintainer lines and / or between different developmental stages of cotton anthers.

2. An immunogen for preparing the polyclonal antibody of claim 1, characterized in that, The immunogen includes a carrier protein and an antigenic epitope peptide covalently coupled to the carrier protein, the antigenic epitope peptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

3. The immunogen according to claim 2, characterized in that, The carrier protein is KLH.

4. A method for preparing the polyclonal antibody of claim 1, characterized in that, Includes the following steps: (1) The antigenic epitope peptide shown in SEQ ID NO: 1 was covalently coupled with KLH to prepare an immunogen; (2) New Zealand rabbits were immunized with the immunogen described above. For the first immunization and the booster immunization in the second week after the first immunization, 0.5 mg of the immunogen was mixed with Freund's complete adjuvant and emulsified before being injected subcutaneously. For the booster immunizations in the fourth and fifth weeks after the first immunization, 0.5 mg of the immunogen was mixed with Freund's incomplete adjuvant and emulsified before being injected subcutaneously. (3) Collect immune serum from New Zealand rabbits; (4) The immune serum is purified by affinity chromatography, the antibody eluent is collected, and after neutralization and dialysis, the polyclonal antibody of claim 1 is obtained.

5. The method according to claim 4, characterized in that, In step (4), the immune serum was purified by affinity chromatography using CNBr-activated Sepharose 4B affinity resin conjugated with the antigen epitope peptide shown in SEQ ID NO:

1.

6. A detection kit for detecting the immunoreactivity signal of cotton abrin protein using Western blot, characterized in that, Includes the polyclonal antibody as described in claim 1.

7. The detection kit according to claim 6, characterized in that, The polyclonal antibody is a rabbit-derived polyclonal antibody, and the detection kit also includes HRP-labeled anti-rabbit IgG secondary antibody and ECL chemiluminescent substrate.

8. A method for detecting differences in Abrin protein immunoreaction signals in cotton anthers, characterized in that, Includes the following steps: (1) Extract total protein from the cotton anther samples to be tested; (2) The total protein was subjected to SDS-polyacrylamide gel electrophoresis, and the separated protein was transferred to a solid membrane; (3) The polyclonal antibody described in claim 1 is used as the primary antibody and incubated with the solid phase membrane; (4) Incubate with an enzyme-labeled secondary antibody that matches the polyclonal antibody and detect the signal; (5) Based on the intensity of the Abrin protein immune response signal, compare the differences in Abrin protein immune response signals between cotton cytoplasmic male sterile lines and their maintainer lines and / or the differences in Abrin protein immune response signals between different developmental stages of cotton anthers.

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