An antigenic polypeptide, polyclonal antibody, and application of CENH3 protein from the East Asian scorpion.

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to an antigenic polypeptide, a polyclonal antibody, and their applications of the CENH3 protein from the East Asian scorpion. The antigenic polypeptide is derived from amino acid positions 60 to 148 of the CENH3 protein, and its amino acid sequence is shown in SEQ ID NO.3. This invention also discloses a polyclonal antibody against the CENH3 protein of the East Asian scorpion, prepared using the aforementioned antigenic polypeptide as an immunogen. The antigenic polypeptide provided by this invention exhibits good immunogenicity, and the prepared polyclonal antibody demonstrates high specificity and titer, capable of specifically recognizing the CENH3 protein of the East Asian scorpion. This provides an important detection tool for the functional verification of the CENH3 protein of the East Asian scorpion, filling a technological gap in this field.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an antigenic polypeptide, polyclonal antibody, and application of the CENH3 protein of the East Asian scorpion. Background Technology

[0002] Centromeres are key functional regions on eukaryotic chromosomes that mediate kinetochore assembly and proper chromosome segregation. Centromere protein CENH3, also known as CENP-A, is a specialized variant of histone H3 and a core epigenetic marker of centromere chromatin. CENH3 replaces classic histone H3 to construct centromere-specific chromatin structures, playing a crucial role in the stable inheritance of chromosomes during kinetochore assembly, mitosis, and meiosis.

[0003] Compared to classic histone H3, CENH3 protein exhibits lower sequence conservation across different species, particularly showing significant differences at the N-terminus and some variable regions. Therefore, antibodies prepared targeting CENH3 or CENP-A in other model organisms are often difficult to use directly for CENH3 detection in non-model species, or suffer from problems such as low recognition efficiency, high background, and unclear cross-reactivity.

[0004] The East Asian pincers scorpion is one of the common scorpion species in my country, widely distributed in Henan, Hebei, Shandong and other regions. It has important medicinal resource value and genomics research value. With the development of genomics, cytogenetics and chromosome biology research on the East Asian pincers scorpion, there is an urgent need for antibody tools that can identify the CENH3 protein of the East Asian pincers scorpion for protein expression detection, functional verification and centromere-related research.

[0005] However, there is currently a lack of specific antibodies targeting the CENH3 protein of the East Asian scorpion. Therefore, developing a polyclonal antibody capable of recognizing the CENH3 protein of the East Asian scorpion has clear technical requirements and application value. Summary of the Invention

[0006] The purpose of this invention is to provide an antigenic polypeptide of the CENH3 protein of the East Asian scorpion and a polyclonal antibody prepared therefrom, so as to solve the problem of the lack of antibody tools in the prior art that can detect the CENH3 protein of the East Asian scorpion.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an antigenic polypeptide of the CENH3 protein of the East Asian scorpion, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] Preferably, the antigenic polypeptide consists of amino acids from position 60 to position 148 of the CENH3 protein of the East Asian scorpion shown in SEQ ID NO.2.

[0009] The present invention also provides an immunogen comprising the antigenic polypeptide described above.

[0010] Preferably, the immunogen is a recombinant protein, which includes a protein tag and the antigenic polypeptide described above, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO.4.

[0011] The present invention also provides a polyclonal antibody against the CENH3 protein of the East Asian scorpion, wherein the polyclonal antibody is prepared from the antigenic polypeptide or the immunogen described above.

[0012] Preferably, the polyclonal antibody is a rabbit-derived polyclonal antibody.

[0013] Preferably, the polyclonal antibody is obtained through antigen-specific affinity purification.

[0014] This invention also provides a method for preparing the above-mentioned polyclonal antibody against the CENH3 protein of the East Asian scorpion, characterized by comprising the following steps: (1) Obtain the antigenic polypeptide or the immunogen described above; and immunize experimental animals with the antigenic polypeptide or the immunogen. (2) Collect serum after immunization; (3) The serum was subjected to antigen-specific affinity purification to obtain polyclonal antibody against the CENH3 protein of the East Asian scorpion.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Based on the sequence characteristics of the CENH3 protein of the East Asian scorpion, this invention selects the amino acid region from position 60 to position 148 as an antigen fragment, and obtains an antigenic polypeptide suitable for preparing antibodies against the CENH3 protein of the East Asian scorpion; (2) The polyclonal antibody prepared in this invention showed a high titer when detected by ELISA, and a positive reaction could still be detected under the condition of 1:128000 dilution; (3) The polyclonal antibody prepared in this invention can recognize the endogenous CENH3 protein of East Asian scorpion in Western blot. The detection band is located at about 16 kDa, which is consistent with the theoretical molecular weight of East Asian scorpion CENH3 protein. It can be used for the detection of East Asian scorpion CENH3 protein expression, functional verification and related detection kit development. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The image shows the SDS-PAGE results of purified KSI-CENH3 recombinant protein containing the CENH3 antigen fragment of the East Asian scorpion; where MW is the protein molecular weight standard and 2 μg is the purified recombinant protein loaded with the sample.

[0018] Figure 2 This is a schematic diagram of the Western Blot results of polyclonal antibodies against CENH3 from the East Asian pincer scorpion and endogenous samples; where Marker: protein marker; MM1: male sample from population 1; MM2: female sample from population 1; MM3: juvenile sample from population 1; MM4: male sample from population 2; MM5: female sample from population 2. Detailed Implementation

[0019] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0021] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0022] Example 1: Design of the CENH3 antigen fragment from the East Asian scorpion and expression and purification of the KSI-CENH3 recombinant protein. 1.1 Sequence analysis of CENH3 protein from the East Asian pincers scorpion By aligning the human CENPA protein sequence (AAH02703.1) and the Drosophila melanogaster dCENP-A protein sequence (NP_523730.2) to the East Asian scorpion protein, several proteins that can be aligned to both target proteins were obtained and selected as candidate proteins.

[0023] The candidate protein sequence was compared with the two target protein sequences using MEGA software. Based on the specific site "QALL" of the CENH3 protein, the CENH3 protein sequence was identified from the East Asian pincer scorpion. The encoding gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the CENH3 protein it encodes is shown in SEQ ID NO.2.

[0024] SEQ ID NO.1: ATGGCTAAGAATAAACGAAAAACAGGATTGAGTCCACGTGATTCGAACGAATGATAGATCTTTTTCTTCTTCACCGAGATCTGTCAGAAGCTTTTCCAGTAGAAATGCAAGTCCTCTCACTAAACATAAAAGAATCCTTAATTCTCCTCCACGAAAGAAGCGTCGTTATCGTCCTGGTGTAAGAGCTTTAATGGAAATAAGAAAATATCAGAAAAGTTGTGATCTTCTT TTAAGAAAACTTCCATTTATGCGACTTGTAAAAGAAATCTGTGCTGAATTAACAGGCTGTTTTTATTGGAGAACCCAAGCACTTCTTGCTCTTCAAGAAGCTACTGAATCTTATCTTGTCAATTTATTTGAAGATTCTTATTTATGCTCTTTGCATGCTAAACGAGTTACTTTGATGCCATCAGATATTCGCTTGGCTCGAAGAATTCGTGGACGTAAAGATTTATAA SEQ ID NO.2: MAKNKRKTGLSPRDIRTNDRSFSSSPRSVRSFSSRNASPLTKHKRILNSPPRKKRRYRPGVRALMEIRKYQKSCDLLLRKLPFMRLVKEICAELTGCFYYWRTQALLALQEATESYLVNLFEDSYLCSLHAKRVTLMPSDIRLARRIRGRKDL The amino acid sequence of CENH3 protein varies greatly at the N-terminus among different species, while it is relatively conserved at the C-terminus. Based on this, the 60-148 region of the CENH3 protein from the East Asian scorpion was selected for the synthesis of the antigenic polypeptide. The amino acid sequence of this region is shown in SEQ ID NO.3.

[0025] SEQ ID NO.3: GVRALMEIRKYQKSCDLLLRKLPFMRLVKEICAELTGCFYYWRTQALLALQEATESYLVNLFEDSYLCSLHAKRVTLMPSDIRLARRIR 1.2 Construction of KSI-CENH3 recombinant protein expression vector Based on the antigenic polypeptide sequence shown in SEQ ID NO.3, Pujian Biotechnology (Wuhan) Co., Ltd. was commissioned to synthesize its encoding DNA template de novo. The target fragment was obtained by PCR amplification, and the target fragment and the enzyme digestion vector were ligated by homologous recombination: the expression system was the E. coli expression system, the protein tag was the KSI large tag, the enzyme digestion site was BamHI / EcoRI, and the expression vector was the pET28b-KSI-CENH3 vector.

[0026] The recombinant protein sequence is shown in SEQ ID NO.4, with positions 1-145 being the protein tag. The predicted size of the recombinant protein is 26.29 kDa.

[0027] SEQ ID NO.4 MGSSHHHHHHSSGLVPRGSHMHTPEHITAVVQRFVAALNAGDLDGIVALFADDATVEDPVGSEPRSGTAAIREFYANSLKLPLAVELTQEVRAVANEAAFAFTVSFEYQGRKTVVAPI DHFRFNGAGKVVSIRALFGEKNIHACQGSGVRALMEIRKYQKSCDLLLRKLPFMRLVKEICAELTGCFYYWRTQALLALQEATESYLVNLFEDSYLCSLHAKRVTLMPSDIRLARRIR Add an appropriate amount (1-2 μL) of the expression vector pET28b-KSI-CENH3 to pre-prepared competent cells, place on ice for 30 min, and then heat shock at 42°C for 90 s. Add an appropriate amount of antibiotic-free LB liquid medium and incubate at 37°C and 150 rpm for 40 min. Spread the mixture onto LB solid culture dishes with the appropriate antibiotics and incubate overnight at 37°C. Select a single white colony for sequencing, and compare the sequencing results with the sequence of SEQ ID NO.4. Positive single clones are Escherichia coli containing pET28b-KSI-CENH3, i.e., recombinant bacteria.

[0028] 1.3 Low-level induction and preliminary identification of KSI-CENH3 recombinant protein Pick single clones from the culture dish and transfer them to 3 ml of the corresponding resistant LB medium. Incubate at 37°C and 200 r / min for 3-4 h. Transfer 1.5 ml of the bacterial culture to a new 15 ml centrifuge tube and add 1.5 μl of IPTG (final concentration 1 mM). Incubate at 37°C and 200 r / min for 4 h. Add 1.5 μl of IPTG to the remaining 1.5 ml of bacterial culture and incubate at 16°C and 200 r / min overnight. Transfer the induced bacterial culture to a 1.5 ml centrifuge tube, centrifuge at 10000 g for 1 min, and discard the supernatant. Add 1 ml of 1×PBS (containing 8 M urea) to the pellet and sonicate to lyse the cells. Add 25 μl of 5× reducing loading buffer to the sample, boil for 10 min, and then perform SDS-PAGE analysis.

[0029] Mix the above sample with reduced 5×SDS test sample buffer, heat to 100℃ for denaturation, and cool to room temperature for later use. Use a 12% gradient PAGE gel. After the stacking gel has fully polymerized (approximately 30 minutes), fix the gel onto the electrophoresis apparatus, carefully remove the sample comb, fill the electrophoresis tank with 1× electrophoresis buffer, and add the prepared test sample solution and protein standard molecular weight to the sample wells using a pipette. Perform constant voltage electrophoresis, with an initial voltage of 80 volts, adjusting to 180 volts when entering the separating gel. When the bromophenol blue reaches the bottom of the gel, turn off the power and stop electrophoresis. After electrophoresis, remove the gel (strip), place it in the fixative for 30 minutes, then remove the gel (strip) and place it in a container containing staining solution (the staining solution should cover the gel surface), and stain on a shaker for at least 60 minutes. Discard the staining solution, place the gel in destaining solution, and destain on a shaker until the background blue has mostly faded. Place the completely destaining gel into a gel imaging system for imaging. The results show ( Figure 1 A positive band for recombinant CENH3 protein was observed at 26 kDa, indicating high specificity and high expression levels.

[0030] 1.4 Scale-up expression and purification of KSI-CENH3 recombinant protein Cells were lysed using an appropriate amount of lysis buffer (PBS pH 7.5, 10% Glyerol, 1 mM PMSF). After thorough mixing, cells were sonicated for 20 min. The cells were then centrifuged at 12000 g for 10 min, the supernatant was separated, and the precipitate was retained. The precipitate was resuspended in 1×PBS (pH 7.5) containing 8 M urea and sonicated for 20 min. After lysis, the cells were centrifuged at 12000 g for 10 min, the supernatant was separated, and the precipitate was discarded. A certain amount of Ni resin was added to the supernatant, and the cells were incubated in a shaker for 30 min. The resin was collected, and the permeation buffer (FT) was temporarily retained. Purification was then performed according to the steps in Table 1. Table 1 Purification Steps

[0031] Add 1% Triton-114 to the purified protein and mix by rotation at 4°C for 30 minutes; after mixing by rotation, incubate in a water bath at 37°C for 10 minutes; after the water bath, centrifuge at 25°C and 9000 g for 10 minutes; carefully aspirate the clear supernatant after centrifugation, which is the endotoxin-free target protein; filter aseptically and perform SDS-PAGE analysis, the purity of the target protein is 80%.

[0032] Example 2: Preparation and purification of polyclonal antibody against CENH3 protein of East Asian scorpion. In this embodiment, the East Asian scorpion CENH3 polyclonal antibody was prepared by immunizing animals with the recombinant East Asian scorpion CENH3 protein obtained in Example 1 as an antigen, as detailed below: Immunization: Two healthy New Zealand white rabbits were immunized with the CENH3 protein antigen from the East Asian scorpion. The immunization schedule is shown in Table 2. Table 2 Protein antigen immunization procedure

[0033] Blood was collected after three immunizations according to the immunization protocol (DAY 22). Rabbit blood was left to stand at room temperature for several hours, then incubated overnight at 4°C. The blood clots were gently loosened with a wooden pry bar and detached from the side wall of the centrifuge tube. The blood was transferred to a suitable centrifuge tube and centrifuged at 5000 g for 10 min to remove residual red blood cells and other debris.

[0034] Antibody purification: The purification method is antigen-specific affinity purification. Polyclonal antibodies are extracted and purified from serum, and cross-adsorption and removal of polyclonal antibodies targeting the KSI tag are performed.

[0035] Example 3: ELISA titer detection of polyclonal antibody against CENH3 protein of East Asian scorpion. 3.1 ELISA Antigen Coating Experimental Procedure (1) Coating: Calculate the required antigen volume, coating liquid volume, protein coating concentration of 1-2 ug / ml, and peptide coating concentration of 1-5 ug / ml.

[0036] (2) Blocking: Take the coated microplate out of the 37°C incubator or the 4°C refrigerator and shake the coating solution into the water tank. Then add 5% Milk in 300ul per well. Cover the plate and incubate at 37°C for 1 hour. If the blocking is done after 4 pm, incubate at 4°C overnight.

[0037] (3) Washing: Remove the coated and blocked ELISA plate from the 37℃ incubator or the 4℃ refrigerator, shake the blocking solution into a water bath, and wash 3 times with PBST. Arrange the plates in the order of project number and sequence number, and bag them. Store at -20℃ for use. Shelf life is 2 months. Discard the plates after 2 months. If primary antibody needs to be added immediately, add the primary antibody directly for incubation. There is no need to store the plates at -20℃.

[0038] 3.2 ELISA titer assay (1) Primary antibody incubation: Add primary antibody according to experimental requirements, 100 μL per well, cover and incubate at 37°C for 60 minutes. Remove the primary antibody, wash 3 times with PBST, and pat dry on absorbent paper towels.

[0039] (2) Secondary antibody incubation: Add the corresponding species secondary antibody according to the experimental requirements, 100 μL per well, cover and incubate at 37°C for 30 minutes. Remove the secondary antibody, wash 3 times with PBST, and pat dry on absorbent paper towels.

[0040] (3) TMB color development: Pour the prepared TMB color development solution into a clean sample container covered with PE gloves. Use a multi-channel pipette to add 100 μL to each well of the microplate sequentially. Cover the plate and place it in an incubator for 5-10 minutes to observe the color development.

[0041] (4) Termination: Turn on the microplate reader, preheat for 1 minute, take out the microplate from the incubator, and add 2 M HCl to each well in 50 μL.

[0042] (5) Reading: Open the Thermo microplate reader software, select the wavelength of 450-620 nm, wipe the bottom of the microplate with an absorbent towel and place it in the microplate reader slot, then click start to read the value.

[0043] 3.3 ELISA test results The results, as shown in Table 3, indicate that the antibody titer is greater than 128K.

[0044] Table 3 Results of antibody indirect ELISA titer assay

[0045] As shown in Table 3, when the antibody was diluted to 1:128000, the detection result using KSI-CENH3[Gly60-Arg148] as the coating antigen was still positive, with a P / N value of 7.00. Therefore, within the detection range of this experiment, the highest positive dilution of the polyclonal antibody against CENH3 protein of the East Asian scorpion prepared in this invention is 1:128000, and the antibody titer is not lower than 1:128000.

[0046] Meanwhile, when KSI-tagged protein was used as the coating antigen, the overall antibody response was lower than that when KSI-CENH3 recombinant protein was used as the coating antigen, suggesting that after cross-adsorption treatment, the resulting antibody mainly recognizes the CENH3 antigen fragment of the East Asian scorpion.

[0047] Example 4: Validation of the polyclonal antibody against the CENH3 protein of the East Asian scorpion using Western blot. Five samples from different sexes and developmental stages of two populations were selected as endogenous samples for Western blot analysis of the CENH3 protein of the East Asian scorpion.

[0048] Population 1 consisted of three samples: adult male pedipalps (MM1), adult female pedipalps (MM2), and whole juveniles (MM3, sex unknown); Population 2 consisted of two samples: adult male pedipalps (MM4) and adult female pedipalps (MM5). All samples were used to extract total protein and perform Western blot analysis.

[0049] The specific steps are as follows: 4.1 Protein Extraction (1) Take 50-100 mg of tissue and grind it into powder quickly in liquid nitrogen; (2) Add 0.5-1 ml of pre-cooled RIPA lysis buffer (containing protein phosphatase inhibitors). (3) Then place it in an ice bath for 10 minutes and vortex it for 30 seconds every 5 minutes; (4) Centrifuge at 12000g at 4℃ for 10 minutes, and transfer the supernatant to a new centrifuge tube to obtain the total protein product of the tissue; 4.2 Protein concentration determination For standard preparation, BSA standards were used with concentrations of 0.5 ug / ul, 0.4 ug / ul, 0.3 ug / ul, 0.2 ug / ul, 0.1 ug / ul, 0.05 ug / ul, and 0.025 ug / ul, for a total of 8 gradients as standard controls.

[0050] For BCA protein quantification, mix 200 μL of BCA solution with 4 μL of copper sulfate solution per well to prepare the working solution for BCA protein quantification.

[0051] Load 20 μL of standard and sample into each well, then add BCA working solution and incubate at 37°C for 30 min. Read the OD value at 562 nm using a microplate reader.

[0052] Plot a standard curve with the standard concentration (ug / ul) on the horizontal axis and the OD value on the vertical axis. Draw a linear equation with a linear correlation greater than 0.995. Substitute the measured OD value of the sample into the standard curve equation to calculate the sample concentration (ug / ul).

[0053] The results are shown in Table 4.

[0054] Table 4 Results

[0055] 4.3 SDS-PAGE Adhesive Preparation Based on the molecular weight of the target protein, select an appropriate gel concentration. For example, to prepare a 10% gel, one gel requires 5 ml of separating gel. The preparation method is as follows: H2O 1.95 ml, 30% Acrylamide 1.7 ml, 1.5 M Tris-HCl pH=8.8 1.3 ml, 10% SDS 52 μl, 10% APS 50 μl, TEMED 2 μl. Thoroughly mix the gel, avoiding air bubbles. After pouring the gel, seal with 2 ml of deionized water.

[0056] Prepare the stacking gel. One gel requires 2ml of stacking gel. The preparation method is as follows: 1.4ml H2O, 0.33ml 30% Acrylamide, 250ul 1.0M Tris-HCl pH=6.8, 20ul 10% SDS, 20ul 10% APS, and 2ul TEMED. After pouring in the stacking gel, immediately insert a gel comb to avoid air bubbles in the pore areas. If air bubbles are present, gently tilt the comb and tap to remove them. Allow the gel to solidify before use.

[0057] 4.4 Sample Preparation Equilibrate 5x Loading Buffer to room temperature, ensuring the SDS is fully dissolved. The fused Loading should be a clear, bright blue-purple color. Add the Loading to the adjusted sample concentration at a 1:5 ratio, gently pipetting repeatedly to ensure thorough mixing of the Loading and protein solution in the pipette tip. Insert the sample tube into a float plate, incubate in a boiling water bath for 10 minutes, then cool the sample on ice. After mixing, aliquot the sample and store at -80°C.

[0058] 4.5 Sample loading and electrophoresis Through preliminary experiments, a suitable range of sample loading volume is determined. Before loading, it is necessary to check whether the gel wells are blocked. Electrophoresis conditions: constant voltage of 80V for stacking gel. After bromophenol blue runs out of the stacking gel, adjust the voltage to 120V. Stop electrophoresis when bromophenol blue reaches the bottom of the gel plate. Add ice cubes or ice packs outside the electrophoresis tank during electrophoresis to avoid excessively high electrode and solution temperatures caused by prolonged electrophoresis.

[0059] 4.6 Transfer of film After electrophoresis, use a gel separator to gently remove the gel, discarding the stacking gel and retaining only the separating gel. Open the transfer core, place a layer of sponge pad, then three sheets of medium-speed filter paper soaked in transfer buffer. Carefully place the gel on the filter paper, cut an NC membrane to the appropriate size, completely covering the gel, and add three more sheets of filter paper. Use a gel roller to gently remove any air bubbles repeatedly. Throughout the process, add transfer buffer intermittently to keep the entire sandwich structure moist. Then place the entire transfer core into the transfer tank, fill it with transfer buffer pre-chilled at 4 degrees Celsius, and place a frozen ice pack in the outer tank for cooling.

[0060] Wet transfer conditions: 300mA constant current, transfer time depends on the size of the target protein molecule, 1 minute for every 1kDa molecule, for example, if the target protein molecule is 100kDa, then 100 mins of transfer is required.

[0061] 4.7 Closed Wash the membrane after transfer with TBST for 5 minutes, discard the washing solution, and add blocking solution. Commonly used blocking solutions include 5% BSA and 5% skim milk powder / TBST. Phosphorylated proteins need to be blocked with BSA, while non-phosphorylated proteins can be blocked with skim milk powder.

[0062] 4.8 Primary Antibody Incubation Primary antibody incubation: Dilute the primary antibody with antibody diluent (the antibody diluent is usually the same as the blocking buffer) and incubate overnight at 4°C on a shaker. Refer to the instructions for the dilution ratio. For example, a 1:500 dilution means adding 8 μL of antibody stock solution to 4000 μL of antibody diluent. 4000 μL of diluted antibody working solution is enough to submerge one membrane for incubation.

[0063] The next day, the membrane was taken out at 4 degrees Celsius and equilibrated on a shaker at room temperature for 15 minutes. After the solution returned to room temperature, the antibody incubation solution was discarded, and 5 ml of TBST was added to wash the membrane 3 times, 5 minutes each time. After the last wash, the TBST was poured off, the incubation box was inverted on absorbent paper, and the residual liquid was absorbed in preparation for adding the secondary antibody. 4.9 Secondary Antibiotic Incubation Dilute the secondary antibody with antibody diluent, add the HRP-labeled secondary antibody that recognizes the corresponding primary antibody. If the host of the primary antibody is Mouse, then use Goat-Anit-Mouse IgG-HRP, diluted 1:5000. Shake gently at room temperature for 1 hour and 10 minutes, remove the secondary antibody, add 5 ml of TBST to wash the membrane 3 times, 5 minutes each time. After the last wash, do not discard the TBST, immerse the washed membrane in it, equilibrate at 4 degrees Celsius for 10 minutes, and then expose to sunlight.

[0064] 4.10 ECL color development and result analysis This experiment used automatic exposure. Before use, the chemiluminescence analyzer was pre-cooled. The membrane was placed in the darkroom photographic platform of the instrument, the luminescent liquid was added, the chamber door was closed, and automatic exposure was performed to take a picture.

[0065] The results are as follows Figure 2 As shown, each adult protein sample exhibits a strong positive band at a size of 16 kDa, indicating that the prepared CENH3 polyclonal antibody demonstrates specificity and high efficiency.

[0066] In summary, based on the sequence characteristics of the CENH3 protein from the East Asian scorpion, this invention designed and obtained an antigenic polypeptide derived from amino acid positions 60 to 148 of this protein. A polyclonal antibody against the CENH3 protein of the East Asian scorpion was then prepared using a KSI-CENH3 recombinant protein containing this antigenic polypeptide. ELISA results showed that the antibody remained positive at a dilution of 1:128000. Western blot results showed that the antibody could recognize an endogenous CENH3 protein band of approximately 16 kDa in total protein samples from East Asian scorpion tissue, and this molecular weight is consistent with the theoretical molecular weight of the CENH3 protein. These results indicate that the polyclonal antibody prepared by this invention has good immunoreactivity and detection application value, and can be used for the expression detection, functional verification, and preparation of related detection reagents or kits for the CENH3 protein of the East Asian scorpion.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antigenic polypeptide of the CENH3 protein of the East Asian scorpion, characterized in that, The amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO.

3.

2. The antigenic polypeptide according to claim 1, characterized in that, The antigenic polypeptide consists of amino acids from position 60 to position 148 of the CENH3 protein of the East Asian scorpion, as shown in SEQ ID NO.

2.

3. An immunogen, characterized in that, The immunogen comprises the antigenic polypeptide of claim 1 or 2.

4. The immunogen according to claim 3, characterized in that, The immunogen is a recombinant protein, which includes a protein tag and the antigenic polypeptide as described in claim 1 or 2, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO.

4.

5. A polyclonal antibody against the CENH3 protein of the East Asian scorpion, characterized in that, The polyclonal antibody is prepared from the antigenic polypeptide of claim 1 or 2 or the immunogen of claim 3 or 4.

6. The polyclonal antibody according to claim 5, characterized in that, The polyclonal antibody is a rabbit-derived polyclonal antibody.

7. The polyclonal antibody according to claim 5 or 6, characterized in that, The polyclonal antibody was obtained through antigen-specific affinity purification.

8. A method for preparing a polyclonal antibody against the CENH3 protein of the East Asian scorpion according to any one of claims 5-7, characterized in that, Includes the following steps: (1) Obtain the antigenic polypeptide of claim 1 or 2, or the immunogen of claim 3 or 4; and immunize experimental animals with the antigenic polypeptide or immunogen; (2) Collect serum after immunization; (3) The serum was subjected to antigen-specific affinity purification to obtain polyclonal antibody against the CENH3 protein of the East Asian scorpion.

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