Preparation method and application of quail CENP-A polyclonal antibody

Abstract

The invention discloses a preparation method and application of a quail CENP-A polyclonal antibody, and the preparation method of the quail CENP-A polyclonal antibody comprises the following steps: constructing a recombinant expression vector containing a CENP-A target gene segment as shown in SEQ ID NO.1; transforming the recombinant expression vector into a competent cell, inducing expression of a fusion protein, collecting and purifying to obtain a CENP-A recombinant protein; carrying out animal immune treatment by taking the CENP-A recombinant protein as an antigen, and collecting and separating an antibody in animal serum as a CENP-A polyclonal antibody. The invention also develops a method and a device for CUAMP; the quail CENP-A antibody sequenced by Tag fills up the blank of a quail commercial CENP-A antibody, is also an important basic work for further constructing a quail genome complete map, and provides a basic support for subsequently revealing how centromere related genes of vertebrates such as birds evolve among different species.

Description

Technical Field

[0001] The invention belongs to the field of immune biotechnology, and in particular relates to a method for preparing a quail CENP-A polyclonal antibody and application thereof. Background Art

[0002] As we all know, mitosis is the main way for eukaryotic cells to proliferate. Cells use mitosis to distribute the replicated genetic material equally to daughter cells. This process is mainly completed by the centromere to form the kinetochore. The centromere is the main constriction of the chromosome, that is, the thinner part of the chromosome, and the kinetochore is defined as a protein structure that enables the centromere DNA to attach to the spindle fibers. The kinetochore mediates the interaction between the chromosome and the spindle fibers, allowing the chromosomes to migrate during the division period. Under electron microscopy, the kinetochore is a three-layer disc-shaped structure. That is, the inner layer, the middle layer and the outer layer. The outer kinetochore is used to interact with microtubules, and the middle layer kinetochore mainly serves as a bridge connecting the inner and outer layers. The inner kinetochore is a specialized layer of chromatin, composed of centromere chromatin and basic proteins, and is always connected to the centromere DNA during the cell cycle.

[0003] CENP-A is a centromere-specific protein specifically expressed in animals, with a relative molecular mass of 17KDa. Compared with other histones, CENP-A is less conserved. Studies have shown that there are differences in the C-terminal folding of CENP-A histones even in similar species, so comparing the differences between CENP-A of various species has always been considered the basis for analyzing the epigenetic regulation mechanism of centromeres. Given the relative specificity of CENP-A, there are currently no commercial antibodies specifically for detecting quail CENP-A protein, and antibodies from other species cannot be used for deep sequencing due to homology issues, so further related and deeper functional mining is more difficult.

[0004] In addition to participating in normal cell division, CENP-A has also been shown to be closely related to the occurrence, development, invasion, metastasis and prognosis of cancer cells. Therefore, the development of quail CENP-A antibodies has important practical significance for the prediction of avian-related diseases and therapeutic targets. Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide a method for preparing a quail CENP-A polyclonal antibody, which solves the problem of lack of quail CENP-A sequencing-specific antibodies and provides an important tool for subsequent functional verification of the quail CENP-A gene.

[0006] The present invention also provides an application of a quail CENP-A polyclonal antibody in CUT&Tag sequencing. The quail CENP-A antibody used for CUT&Tag sequencing fills the gap in commercial quail CENP-A antibodies and is also an important basic work for further constructing a complete map of the quail genome.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the method for preparing a quail CENP-A polyclonal antibody of the present invention comprises the following steps:

[0008] (1) constructing a recombinant expression vector containing the CENP-A target gene fragment as shown in SEQ ID NO.1;

[0009] (2) transforming the recombinant expression vector into competent cells and inducing the expression of fusion protein, and collecting and purifying the CENP-A recombinant protein;

[0010] (3) Using the CENP-A recombinant protein as an antigen for animal immunization, and collecting serum antibodies as CENP-A polyclonal antibodies.

[0011] Furthermore, in step (1), the CENP-A target gene fragment shown in SEQ ID NO.1 is recombined between the double restriction sites of the pet22b vector.

[0012] Furthermore, the method of inducing expression of the fusion protein in step (2) is: picking the constructed recombinant expression strain and culturing it in LB liquid culture medium until the bacterial liquid concentration OD 600 When the pH value is 0.6-0.8, IPTG is added to induce the expression of fusion protein for 16-18h.

[0013] Furthermore, the animal subjected to the immunization treatment in step (3) is a mouse.

[0014] Furthermore, the animal immunization treatment method in step (3) is: taking the purified CENP-A recombinant protein and emulsifying it with Freund's adjuvant, injecting it into the test animal, and after 6 immunizations, collecting the test animal serum 70 days after the last final immunization, and collecting the serum to obtain the antiserum.

[0015] Furthermore, the purified CENP-A recombinant protein and Freund's adjuvant have the same volume, Freund's complete adjuvant is used for the primary immunization, and Freund's incomplete adjuvant is used for the additional immunization.

[0016] The quail CENP-A polyclonal antibody of the present invention is prepared by the quail CENP-A polyclonal antibody preparation method.

[0017] The invention discloses an application of the quail CENP-A polyclonal antibody in quail CUT&Tag sequencing and detecting the expression level of CENP-A protein in quail tissues.

[0018] Furthermore, the application comprises the following steps:

[0019] (1) taking quail tissue samples, lysing the tissues, obtaining a cell nucleus suspension, and combining the cell nuclei with a magnetic bead system;

[0020] (2) The bound cell nuclei are sequentially incubated with primary antibodies, secondary antibodies, and pAG-Tn5 transposase, followed by DNA fragmentation;

[0021] (3) performing PCR amplification on the recovered transposition products, sequencing, and constructing a library;

[0022] (4) Perform quality control, comparison, and analysis on the constructed library.

[0023] Preferably, the application comprises the following steps:

[0024] (1) Take quail tissue samples, lyse the tissues with a cell nuclear lysis buffer to obtain a cell nuclear suspension, and take 100,000 cell nuclei to combine with the magnetic bead system;

[0025] (2) The cell nuclei were incubated with primary antibodies, secondary antibodies, and pAG-Tn5 transposase in sequence, followed by DNA fragmentation;

[0026] (3) The recovered transposition products were PCR amplified using a system containing NEBNext High-Fidelity 2X PCR Master Mix and primers for sequencing adapters, and sequenced using Illumina novaseq 6000 to construct a library;

[0027] (4) Screening of sequencing library fragments and detection of library concentration and fragment distribution.

[0028] Furthermore, the tissue in step (1) is quail ovary tissue.

[0029] The invention discloses an application of the polyclonal antibody in detecting the expression of CENP-A protein in quail tissues.

[0030] The invention develops quail CENP-A antibodies for CUT&Tag sequencing, which fills the gap of commercial quail CENP-A antibodies and also helps to further construct a complete map of the quail genome. In addition, in addition to participating in normal cell division, CENP-A has also been proven to be closely related to the occurrence, development, invasion, metastasis and prognosis of cancer cells. Therefore, the development of quail CENP-A antibodies has important practical significance for the prediction of poultry-related diseases and therapeutic targets.

[0031] The present invention provides a method for preparing a quail CENP-A antibody that can be used for CUT&Tag sequencing and its application, which can quickly and accurately detect the expression level of CENP-A protein in quail tissues and can be used for CUT&Tag sequencing. In addition, the present invention first proposes the use of the gene sequence shown in SEQ ID NO.1 in preparing a quail CENP-A antibody for CUT&Tag sequencing and detecting the expression level of CENP-A protein in quail tissues.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0033] The present invention provides a method for preparing a quail CENP-A polyclonal antibody and an application thereof, which can be used for WB detection and CUT&Tag sequencing. The polyclonal antibody production process is simple to operate and the results are reliable, filling the gap in quail CENP-A polyclonal antibodies, and can provide convenience for deeper research on epigenetic regulation of quail centromeres and centromere-related disease mechanisms, and also provide basic support for subsequent analysis of the centromere structure and evolutionary process of vertebrates such as birds, and revealing how centromere-related genes evolve among different species.

[0034] The quail CENP-A polyclonal antibody prepared by the method has high titer and high sensitivity and can specifically identify quail ovarian tissue CENP-A. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The difference analysis diagram of CENP-A gene sequence among different species, (A) is the alignment diagram of CENP-A gene sequence among quail, chicken, duck and turkey, (B) the evolutionary tree analysis of CENP-A gene among different species;

[0036] Figure 2 This is the SDS-PAGE gel image of the purified protein product;

[0037] Figure 3 This is a Western Blot image. The target band of the immunoblot in the image is between 10 and 25 kDa, which is consistent with the theoretical molecular weight (17 kDa) of quail CENP-A protein.

[0038] Figure 4 (A) is the quality inspection peak diagram of CUT&Tag sequencing library, (B) is the quality inspection peak diagram of IgG detection sample library;

[0039] Figure 5These are the quality control analysis diagrams of sequencing data. (A) is the repetitive sequence level diagram of CENP-A detection samples, (B) is the base quality distribution diagram of CENP-A detection samples, (C) is the base content distribution diagram of CENP-A detection samples, (D) is the inserted fragment distribution diagram of CENP-A detection samples, and (E) is the TSS enrichment analysis diagram of CENP-A detection samples. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0042] Example 1

[0043] 1. Materials and Methods

[0044] 1.1 Sequence analysis of quail CENP-A

[0045] In the NCBI database, it was found that the CENP-A gene has a known transcript (XM_015878589.2) with a length of 479bp. Using Snapgene to compare the CENP-A gene sequences of quail, chicken, duck and turkey, it was found that the CENP-A genes of quail and chicken have a high degree of similarity. Subsequently, Mega7.0 was used to perform an evolutionary tree analysis on the CENP-A genes of multiple species. The results showed that the quail is most closely related to the chicken, followed by the turkey and the mallard. The results are as follows Figure 1 As shown, the CENP-A gene of quail and chicken is highly similar, and the evolutionary tree analysis also shows that for the CENP-A gene, quail and chicken are most closely related, followed by turkey and duck.

[0046] 1.2 Synthesis and purification of quail CENP-A antigen

[0047] According to the quail gene sequence information (Gene ID: 107321566), a specific sequence was selected to add 6 histidines at the end of the sequence, and codon optimization was performed. After optimization, the gene nucleotide sequence for quail CENP-A detection was obtained as shown in SEQ ID NO.1:

[0048] ATGCCGCGTCCGAAACCGCGTAGTCCGCGTCGTCGTGGTCGTCCGCCGCCTGCAGCTCCTCCTCCTCCTCCAGCCCGTCCGCGTGCACGTCGTTATCGTCCGGGTCAGCGTGCCCTGCGTGAAATTCGCCGTTATCAGAGTAGTACCGCACTGCTGCTGCGTCGCCAGCCGTTTGCCCGTGTTGTTCGCGAAATTTGCCTGCTGTTTACC CGTGGCGTTGATTATCGCTGGCAGGCCATGGCCCTGCTGGCCTTACAGGAAGCCGCAGAAGCCTTTCTGGTGCATCTGCTGGAAGATGCCTATCTGTGTAGCCTGCATGCACGTCGCGTGACCCTGTATCCGAAAGATCTGCAGCTGGCACGCCGTCTGCGTGGCCTGCAGGGTGAAGGCTTTCTCGAGCACCACCACCACCACCACTGA

[0049] The corresponding encoded amino acid sequence is shown in SEQ ID NO.2:

[0050] MPRPKPRSPRRRGRPPPAAPPPPPARPRARRYRPGQRALREIRRYQSSTALLLRRQ PFARVVREICLLFTRGVDYRWQAMALLALQEAAEAFLVHLLEDAYLCSLHARRVTLY PKDLQLARRLRGLQGEGFLEHHHHHH.

[0051] The sequence was artificially synthesized by Nanjing Baode Biotechnology Co., Ltd. After the artificially synthesized CENP-A gene and pet22b vector were digested with restriction endonucleases NdeI and XhoI, the target fragment was recovered by 1% agarose gel electrophoresis, and the target fragment was connected with T4 ligase and transformed into Escherichia coli DH5α competent cells, plated and cultured overnight, and a single colony was picked the next day. The correctly identified recombinant plasmid was named pet22b-CENP-A after sequencing and stored at -20°C for use. The expression plasmid was added to the BL21 (DE3) competent cells for transformation, and after heat shock and cold shock, the bacterial solution was evenly spread on the LB plate containing ampicillin resistance and cultured in a constant temperature incubator at 37°C for 12-14h; the next day, the seed solution was prepared, and the single clone was picked in a shake flask containing LB culture medium containing ampicillin, and cultured at 37°C and 220rpm for 16h; the seed solution was transferred to a shake flask containing culture medium containing ampicillin at a ratio of 1:100 and cultured until the bacterial OD 600is 0.6-0.8, add IPTG to induce expression at 20°C and 220rpm for 16-18h. Collect the bacterial sludge by centrifugation, add buffer to resuspend the bacterial sludge, and repeat the homogenization and crushing cycle 3 times; take the sample after crushing and run the gel, after centrifugation, take the supernatant for Ni column affinity chromatography (manufacturer: Changzhou Tiandi Renhe Biotechnology Co., Ltd., item number: SA00501L). Use 250mM imidazole buffer to elute the protein, and the eluted protein is CENP-A recombinant protein. Take the above-mentioned ultrasonic and purified samples, add reduced loading, heat and boil in a water bath, and perform SDS-PAGE analysis. See the gel image of the finished protein. Figure 2 As shown, the size of the purified homemade quail CENP-A antigen protein band is between 10-25 kDa, which is consistent with the theoretical molecular weight (17 kDa) of quail CENP-A protein. The target protein has a good expression effect, clear bands, and high purity. After sequencing, it corresponds to SEQ ID NO.1 and 2.

[0052] Example 2

[0053] Preparation of CENP-A polyclonal antibody

[0054] 1.1 Immunization of experimental animals

[0055] 12 healthy BALB / c mice aged 6-8 weeks were selected as immunized animals, weighing 20±2g, purchased from Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing. Antigens were emulsified according to the steps, and the specific process was as follows: the antigen prepared in Example 1 was diluted to 1mg / mL using PBS, 50uL or 25uL of antigen was taken, and the same volume of Freund's adjuvant (Freund's complete adjuvant was used for primary immunization, and Freund's incomplete adjuvant was used for additional immunization) was added, and the antigen and adjuvant were quickly mixed to emulsify them through the antigen emulsification joint connection. According to the immunization cycle and the corresponding immunization dose (see Table 1).

[0056] Table 1 Antigen immunization schedule

[0057]

[0058]

[0059] Positive serum was collected 7 days after the last final immunization, centrifuged at 3500 rpm for 15 min, and the supernatant was collected and preserved after two centrifugations. The serum antibodies were collected as CENP-A polyclonal antibodies.

[0060] 1.2 ELISA method to determine the titer of antibodies

[0061] Coating: Dilute the antigen to 2 μg / mL with coating diluent (PBS, pH 7.4), mix well and add to the ELISA plate, 100 μL per well. Cover with a cover film and incubate in a 4°C refrigerator overnight. Take out the ELISA plate the next day, wash the plate 3 times with PBST, and pat dry for later use.

[0062] Blocking: Add 200 μL / well blocking solution (5% skim milk powder) to the ELISA plate and incubate at 37°C for 2 h. Take out the ELISA plate, wash it once with PBST, and pat it dry for later use.

[0063] Primary antibody: Add the corresponding primary antibody (serum antibody obtained in step 1.1) to the blocked ELISA plate, dilute the serum with PBS (pH 7.4) in multiples, with the initial dilution ratio of 1:1000 and the final dilution ratio of 1:320000. Set PBS (pH 7.4) as a negative control, add to the 96-well plate, and incubate at 37°C for 1 hour. Take out the ELISA plate, wash it 3 times, and pat it dry.

[0064] Secondary antibody: Add enzyme-labeled secondary antibody (goat anti-mouse IgG HRP (Nanjing Biotech Co., Ltd., BS12478), 1:20000 dilution) to the ELISA plate, 100 μL per well, incubate at 37°C for 1 h, take out the ELISA plate, wash the plate and pat dry.

[0065] Color development and termination: Add TMB color development solution to the ELISA plate, 100 μL / well, incubate at room temperature for 15 minutes, add 50 μL / well 1M HCl to the ELISA plate to terminate the reaction. Read immediately at 450nm on the ELISA reader. As shown in Table 2, the detection control is normal, and the titers of 3#, 6#, and 10# are above 1:160000.

[0066] Table 2 ELISA method to determine the titer of quail CENP-A polyclonal antiserum

[0067]

[0068] 1.3 Western Blot Analysis of the Specificity of Quail CENP-A Polyclonal Antibody

[0069] Quail individuals raised normally in the market were randomly selected, and ovarian tissues were collected after bleeding to death. Protein lysis buffer (Biyuntian, item number: P0013B) was added to ultrasonically extract the protein in the tissue. The protein concentration was detected using a BCA kit (Biyuntian, item number: P0012), and the tissue lysis buffer (Biyuntian, item number: P0013B) was used to dilute it to 10 mg / mL. SDS-PAGE gel was prepared according to the standard method, 10 μL of tissue lysis buffer with a protein concentration of 10 mg / mL was added to the loading well of the vertical electrophoresis tank, and the constant voltage was 80V for electrophoresis. After the sample ran through the concentrated gel, it was changed to a constant voltage of 120V, and the electrophoresis was terminated when the bromophenol blue indicator ran to the bottom of the separation gel. The wet transfer method was used for constant voltage 90V for 90 minutes to transfer the protein to the NC membrane. The quail CENP-A polyclonal antibody obtained in this example was used as the primary antibody, diluted 1:1000, shaken at 4°C overnight, and then HRP-labeled goat anti-mouse antibody (Nanjing Biotech Co., Ltd., BS12478, diluted 1:10000) was shaken at room temperature for 2 hours, and the ultra-sensitive ECL chemiluminescence kit (Biyuntian, catalog number: P0018S) was used for color development, and the immunoblot results were obtained after exposure for 30 seconds. The results are shown in Figure 2. Figure 3 The size of the immunoblot band of the antibody obtained from the serum on the quail ovary protein is between 10-25 kDa, which is consistent with the theoretical molecular weight (17 kDa) of the quail CENP-A protein, and there is basically no mixed band, indicating that the homemade quail CENP-A antibody has high accuracy and specificity in WB detection. It shows that the quail CENP-A polyclonal antibody in the present invention has high sensitivity, and can detect a band with a molecular weight between 10-25 kDa in the quail ovary tissue lysate, which is consistent with the expected relative molecular weight of the quail CENP-A protein.

[0070] Example 3

[0071] CUT&Tag Detection Analysis of Quail CENP-A Antibody

[0072] The self-made quail CENP-A antibody was used for CUT&Tag sequencing, and the detection object was quail ovarian tissue. The detection steps are as follows:

[0073] The tissue sample was ground with liquid nitrogen for about 30 minutes to ensure that it was fully ground to a uniform powder. About 0.02 g of the tissue sample was weighed, 980 μL of cell nucleus lysis buffer (containing 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Triton X-100) was added, and 20 μL of 50×PI (protease inhibitor) was added and mixed by blowing to separate 100,000 cell nuclei. The collected 100,000 cell nuclei were combined with the ConcanavalinA magnetic bead system at room temperature for 20 minutes. The cell magnetic bead binding product was incubated with the quail CENP-A polyclonal antibody prepared in Example 2 as a primary antibody and IgG at room temperature for 3 hours. After incubation with the primary antibody system, the primary antibody system was discarded on the magnetic stand, and the secondary antibody system (Goat pAb to Rb IgG, abcam, catalog number: ab6702) was incubated at room temperature for 1 hour. After incubation with the secondary antibody system, wash three times with Dig-Wash Buffer (Hyperactive Universal CUT & Tag Assay Kit for Illumina, Vazyme, Catalog No.: TD903-01), and then switch to pG-Tn5 (YZseq CUT & Tag library prep kit, Shadow Gene) and incubate at room temperature for 1 hour. After incubation with the pG-Tn5 system, wash three times with 1× Dig-300 Buffer, and then switch to the magnesium ion activation system (Invitrogen TM , Catalog No.: AM9530G) and incubated at 37°C for 1 h. TM , Catalog No.: 15553-027) was used to denature the activation system, and the DNA product was extracted using Tagment DNA Extract Beads (novoprotein, Catalog No.: N245-01L). The recovered DNA product was PCR amplified using a system containing NEBNext High-Fidelity 2X PCR Master Mix (NEB, Catalog No.: M0541L) and adapter sequencing adapter primers (N5-adapter (forward): CTAAGCCT, N5-adapter (reverse): AGGCTTAG). The amplified PCR product was purified and recovered and fragments were sorted using KAPA Pure Beads (KAPABIOSYSTEMS, Catalog No.: KS8002). As shown in Table 3, the size and concentration of the library fragments were detected using an Agilent 2100 bioanalyzer. After passing the quality inspection, the library was sequenced using an Illumina novaseq 6000 to construct the library, and the sequencing strategy was PE150.

[0074] Table 3 Library quality inspection results

[0075]

[0076] The quality inspection peaks of the sequencing libraries of quail CENP-A antibody and IgG were constructed to ensure the availability of samples before loading. The results are shown in Figure 4 Compared with IgG, the quality inspection peak graph of CENP-A has a peak between 15-100bp, indicating that CENP-A has a specific binding signal here.

[0077] To ensure the availability of antibodies, the sequencing data was analyzed. The original off-machine sequencing data generally contains unqualified data such as sequencing adapters, low-quality bases, and sequencing reads with a high N ratio. Therefore, we performed quality control on the original sequencing data and used the cutadapt tool to perform quality control on the original sequencing data, including removing adapter sequences, low-quality sequencing bases, and sequences with a high proportion of unknown sequencing bases (N) in the original sequencing data, as well as reads shorter than 5bp after the above quality control. Download the genome fasta file and gtf annotation file of the species from the genome website. Use Bowtie2 to build an index for the reference genome and align the valid data of the double ends to the reference genome. As shown in Table 4, picard was used to mark duplicate reads, and the script program was used to count the alignment indicators (including overall alignment rate, unique alignment rate, mitochondrial read ratio, etc.). Based on the bam file after marking duplicates, samtools was used to count the length of the inserted fragment. According to the sequencing data, the reads with the best alignment position with the genome were retained, and other multiple alignment reads were discarded. Deeptools was used to analyze their enrichment near the gene TSS.

[0078] Table 4 Sequencing data quality control statistics

[0079]

[0080] like Figure 5 (A) is the repetitive sequence of CENP-A sample. The results show that the repetitive sequence of CENP-A sample has low richness and high representativeness. Figure 5 (B) is the base quality distribution. The quality value reflects the accuracy of high-throughput sequencing. The results show that the sequencing data is normal. Figure 5 (C) is the base content distribution. The content distribution of ATCG can reflect whether the sequencing is normal to a certain extent. According to the principle of base complementarity, the ratio of A to T should be close, and the ratio of C to G should also be close. Due to the inherent characteristics of the second-generation sequencing, the base content of the first dozen bp will fluctuate. It can be seen that the base content distribution of the present invention is basically the same after a dozen bp, and the content of A and T, G and C is basically the same, indicating that the base content of the sequencing data is normal. Figure 5(D) The insert fragment distribution and the logarithmic transformation of the normalized read density, respectively. These two figures together provide detailed information about the distribution of DNA fragment lengths in the homemade antibody CUT&Tag experiment. The left figure provides an intuitive read density distribution, and the right figure enhances the observation of low read density areas through logarithmic transformation. According to the CUT&Tag library construction principle, a qualified experiment should at least detect open areas without nucleosomes and areas occupied by one nucleosome. Here, we also observed two peaks of 0-200bp and 200-400bp. Figure 5 (E) Insertion TSS enrichment analysis. The results showed that the homemade quail CENP-A antibody had a higher peak in the target area, indicating that the antibody has strong sensitivity and high specificity, and can be used for quail CUT&Tag sequencing and detection of CENP-A protein expression levels in quail tissues.

Claims

1. A method for preparing a quail CENP-A polyclonal antibody, characterized in that: The steps include: (1) constructing a recombinant expression vector containing the CENP-A target gene fragment as shown in SEQ ID NO.1; (2) transforming the recombinant expression vector into competent cells and inducing the expression of fusion protein, and collecting and purifying the CENP-A recombinant protein; (3) Using the CENP-A recombinant protein as an antigen for animal immunization, and collecting serum antibodies as CENP-A polyclonal antibodies.

2. The method for preparing quail CENP-A polyclonal antibody according to claim 1, characterized in that: In step (1), the CENP-A target gene fragment shown in SEQ ID NO.1 is recombined between the double restriction sites of the pet22b vector.

3. The method for preparing quail CENP-A polyclonal antibody according to claim 1, characterized in that: The method for inducing expression of the fusion protein in step (2) is: picking up the constructed recombinant expression strain and culturing it in LB liquid culture medium until the bacterial liquid concentration OD 600 When the pH value is 0.6-0.8, IPTG is added to induce the expression of fusion protein for 16-18h.

4. The method for preparing quail CENP-A polyclonal antibody according to claim 1, characterized in that: The animal immunization treatment method in step (3) is as follows: the purified CENP-A recombinant protein is emulsified with Freund's adjuvant and injected into the test animal. After 6 immunizations, the serum of the test animal is collected 70 days after the last final immunization, and the collected serum is the antiserum.

5. The method for preparing quail CENP-A polyclonal antibody according to claim 4, characterized in that: The purified CENP-A recombinant protein and Freund's adjuvant have the same volume, Freund's complete adjuvant is used for the primary immunization, and Freund's incomplete adjuvant is used for the additional immunization.

6. A quail CENP-A polyclonal antibody, characterized in that: The polyclonal antibody is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the quail CENP-A polyclonal antibody according to claim 6 in quail CUT&Tag sequencing and detecting the expression level of CENP-A protein in quail tissues.

8. The use according to claim 7, characterized in that: The application comprises the following steps: (1) taking quail tissue samples, lysing the tissues, obtaining a cell nucleus suspension, and combining the cell nuclei with a magnetic bead system; (2) The bound cell nuclei are sequentially incubated with primary antibodies, secondary antibodies, and pAG-Tn5 transposase, followed by DNA fragmentation; (3) performing PCR amplification on the recovered transposition products, sequencing, and constructing a library; (4) Perform quality control, comparison, and analysis on the constructed library.

9. The use according to claim 8, characterized in that: The tissue in step (1) is quail ovarian tissue.

10. Use of a gene sequence shown in SEQ ID NO.1 in the preparation of quail CENP-A antibodies for CUT&Tag sequencing and detecting the expression level of CENP-A protein in quail tissues.

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