Duck CENPA antigen peptides, antibodies prepared from them, and their applications

Abstract

This invention relates to the fields of molecular biology and genomics, disclosing a duck CENPA antigen peptide, antibodies prepared from it, and their applications. The amino acid sequence of the duck CENPA antigen peptide is shown in SEQ ID NO.1. Adding a cysteine ​​residue to the N-terminus of the duck CENPA antigen peptide yields an immunopeptide, the amino acid sequence of which is shown in SEQ ID NO.2. The application of the immunopeptide in the preparation of anti-duck CENPA polyclonal antibodies is also disclosed. The anti-duck CENPA polyclonal antibody of this invention can specifically recognize the CENPA protein in duck chromatin and is suitable for epigenomic experiments such as CUT&Tag, thereby achieving efficient enrichment, localization, and resolution of the duck centromere region. It can be widely used in duck centromere sequence capture, centromere region delimitation, centromere structure analysis, and related genomics research.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genomics, and in particular to duck CENPA antigen peptides, antibodies prepared from them, and their applications. Background Technology

[0002] The centromere is a crucial functional region on eukaryotic chromosomes that ensures proper chromosome segregation. Its main function is to assemble kinetochores during cell division and mediate the connection between spindle microtubules and chromosomes, thereby ensuring the precise separation of sister chromatids or homologous chromosomes. Abnormal centromere function can lead to chromosomal instability, aneuploidy, and developmental abnormalities. Therefore, the composition, structure, and location of the centromere have always been core issues in genomics and chromosome biology research.

[0003] Unlike typical genomic regions, centromeres are typically rich in highly repetitive sequences, satellite DNA, and other complex repetitive elements, presenting significant challenges in assembly and annotation. Even under high-quality genome assembly conditions, relying solely on conventional sequence assembly and bioinformatics predictions often fails to accurately define the true boundaries, core functional regions, and sequence composition of centromeres. Therefore, centromere research depends not only on the genome sequence itself but also heavily on functional epigenetic markers for localization.

[0004] Among known centromere functional markers, CENPA (centromere protein A) is one of the most representative core molecules. CENPA is a variant of histone H3, specifically replacing the classic H3 in the nucleosome of the centromere region, and participates in establishing and maintaining centromere identity. Due to the high specific enrichment of CENPA on functional centromeres, immunological detection methods targeting CENPA, such as ChIP-seq, CUT&RUN, and CUT&Tag, have become important tools for elucidating centromere localization and structure. Compared to traditional ChIP-seq, CUT&Tag technology has advantages such as low background, high resolution, low starting material requirements, and greater sensitivity to repetitive region signals, making it particularly suitable for studying highly repetitive and difficult-to-resolve genomic regions such as centromeres.

[0005] However, most existing commercial antibodies against CENPA are derived from mammals or model organisms, with limited suitability for poultry, especially ducks. The main problems include: 1. Insufficient conservation across species: CENPA proteins, especially the N-terminal tail region, vary greatly among different species, and existing antibodies may not be able to effectively recognize duck CENPA; 2. Non-ideal specificity: Since CENPA has a certain degree of homology with conventional histone H3, if the antigen design is not reasonable, it may lead to cross-reaction of antibodies with H3 or other chromatin proteins; 3. Difficulty in supporting centromere functional localization: For species with high genomic repetition content and complex centromere regions, only anti-CENPA antibodies with high affinity and high specificity can form clear and reproducible centromere enrichment maps in experiments such as CUT&Tag. 4. Lack of dedicated antibodies for duck centromere research: Currently, there are few reports on dedicated antibodies and antigen sequences for the duck centromere region and their applications in functional omics localization, which limits systematic research on the structure, sequence composition and epigenetic characteristics of duck chromosome centromeres.

[0006] Ducks are an important poultry species and a key subject of research in avian comparative genomics, chromosome evolution, and epigenetics. With the development of telomere-to-telomere (T2T) assembly technology and long-read sequencing, the continuity of the duck genome has significantly improved. However, the centromere region, due to its high repetition and complexity, still requires validation and analysis using functional markers. Therefore, developing a specific antigenic peptide and antibody against duck CENPA and applying it to CUT&Tag sequencing to accurately capture the duck centromere region is of great significance for establishing a standard method for locating duck centromeres, elucidating their sequence composition and structural characteristics, and advancing research in avian chromosome biology and breeding resources. Summary of the Invention

[0007] The purpose of this invention is to provide a duck CENPA antigenic peptide, antibodies prepared from it, and their applications. An immunopeptide is formed by adding a cysteine ​​residue to the N-terminus of the duck CENPA antigenic peptide. This immunopeptide can be used to prepare antibodies that specifically recognize the duck CENPA protein. Antibodies prepared based on this antigenic peptide can be applied to experiments such as CUT & Tag to enrich and locate centromere regions in the duck genome, and further, by combining methylation and other information, to analyze the sequence composition and structure of the duck centromere. This invention provides a key molecular tool for duck centromere research and has promising application prospects.

[0008] This invention screened a specific antigenic peptide sequence of 14 amino acids from the duck CENPA protein sequence, and successfully prepared an anti-CENPA polyclonal antibody for CUT&Tag experiments using this antigenic peptide. The enriched signal obtained based on this antibody can be used for precise identification of centromere regions in the duck genome, and can be further combined with multi-omics information such as methylation signals to systematically analyze the sequence composition and structure of centromeres on various duck chromosomes.

[0009] To achieve the above objectives, in a first aspect, the present invention provides a duck CENPA antigen polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] Secondly, the present invention provides an immune polypeptide by adding a cysteine ​​residue to the N-terminus of the duck CENPA antigen polypeptide to obtain an immune polypeptide, the amino acid sequence of which is shown in SEQ ID NO.2.

[0011] Thirdly, the present invention provides the application of the above-mentioned immune peptides in the preparation of anti-duck CENPA polyclonal antibodies.

[0012] Fourthly, the present invention provides a method for preparing an anti-duck CENPA polyclonal antibody, comprising the following steps: Step 1, Antigen Preparation: The immune polypeptide described in claim 2 was prepared by chemical synthesis and then coupled with a carrier protein to obtain an antigen. Step 2, animal immunization: The antigen is injected into a host animal to induce an immune response, thereby producing specific antibodies against the duck CENPA antigen polypeptide in the host animal; the host animal is a New Zealand rabbit. Step 3, collect serum: After the immunization cycle ends, host animal blood is collected and centrifuged to obtain antiserum; Step 4, antibody purification: Specific IgG in antiserum was purified to obtain anti-duck CENPA polyclonal antibody.

[0013] Fifthly, the present invention provides an anti-duck CENPA polyclonal antibody, which is prepared by the above-described preparation method.

[0014] In a sixth aspect, the present invention provides the use of the above-mentioned duck CENPA antigen polypeptide or the above-mentioned anti-duck CENPA polyclonal antibody in the preparation of reagents for detecting duck CENPA protein.

[0015] In a seventh aspect, the present invention provides the application of the above-mentioned duck CENPA antigen polypeptide or the above-mentioned anti-duck CENPA polyclonal antibody in the immune recognition and functional localization of the duck centromere region.

[0016] Eighthly, the present invention provides the application of the above-mentioned duck CENPA antigen polypeptide or the above-mentioned anti-duck CENPA polyclonal antibody in the capture of duck CENPA binding sites in the CUT&Tag experiment.

[0017] In a ninth aspect, the present invention provides the application of the above-mentioned duck CENPA antigen polypeptide or the above-mentioned anti-duck CENPA polyclonal antibody in the enrichment, boundary identification and structural analysis of centromere sequences in the duck genome.

[0018] In a tenth aspect, the present invention provides the application of the above-mentioned duck CENPA antigen polypeptide or the above-mentioned anti-duck CENPA polyclonal antibody in the comprehensive analysis of duck centromere regions by binding epigenetic signals.

[0019] The advantages and positive effects of the duck CENPA antigen polypeptide, the antibody prepared from it, and its application described in this invention are as follows: This invention creates an immunopeptide by adding a cysteine ​​residue to the N-terminus of a duck CENPA antigenic peptide. This immunopeptide can be used to prepare antibodies that specifically recognize duck CENPA proteins. Antibodies prepared based on this antigenic peptide can be applied to experiments such as CUT & Tag to enrich and locate centromere regions in the duck genome, and further, by combining methylation and other information, to analyze the sequence composition and structure of duck centromeres. This invention provides a key molecular tool for duck centromere research and has promising application prospects.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 The above are the sequence alignment results of CENPA and Histone-H3 proteins in this embodiment of the invention. Figure 2 These are the repeatability analysis results from the CUT&Tag experiment in this embodiment of the invention; Figure 3 This is the result of CUT&Tag signal specific enrichment in an embodiment of the present invention (taking chrZ as an example); Figure 4 This is the result of chr22 CUT & Tag enrichment signal in an embodiment of the present invention; Figure 5 This refers to the chr1 CUT & Tag enriched signal in this embodiment of the invention; Figure 6 This refers to the chrZ CUT & Tag enriched signal in this embodiment of the invention; Figure 7 This is a genomic landscape map of the centromere and surrounding region of chr22 in an embodiment of the present invention. A represents the density distribution of methylation level, CUT & Tag enrichment signal, centromere monomers (Apl.133, Apl.202 and Apl.227), repeat elements and genes, and B represents a heatmap of centromere region sequence similarity. Figure 8This is a genomic landscape map of the chr1 centromere and surrounding region in an embodiment of the present invention. In this map, A represents the density distribution of methylation level, CUT & Tag enrichment signal, centromere monomers (Apl.1352_TE, Apl.1352), repeat elements, and genes, and B represents a heatmap of centromere region sequence similarity. Figure 9 This is a genomic landscape map of the centromere and surrounding region of chrZ in an embodiment of the present invention. In this map, A represents the density distribution of methylation level, CUT & Tag enrichment signal, centromere monomers (Apl.cenZ), repeat elements, and genes, and B represents a heatmap of centromere region sequence similarity. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0025] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0026] This invention discloses a specific targeting duck ( Anas platyrhynchos The antigenic polypeptide of CENP-A protein has the amino acid sequence: MPRPKDRSPRRRGP (SEQ ID NO.1). This antigenic polypeptide is derived from the duck CENPA protein sequence predicted by NCBI (SEQ ID NO.3).

[0027] The design and screening of this antigenic peptide sequence were based on strict bioinformatics and immunology co-design principles, exhibiting high specificity, excellent immunogenicity, and spatial accessibility. (1) Located in the N-terminal variable region of CENP-A, avoiding the core domain that is highly conserved with conventional histone H3: CENP-A, as a variant of histone H3, exhibits high homology between its C-terminal histone folding domain and that of conventional histone H3. Designing antigens in this conserved region can easily lead to cross-recognition of conventional histone H3 in the resulting antibodies. The antigenic peptide selected in this invention is located in the N-terminal non-core region of duck CENP-A. This region exhibits higher sequence diversity compared to the C-terminal conserved domain, making it a more suitable source of antigenic epitopes for distinguishing CENP-A from conventional histone H3.

[0028] (2) Significant sequence differences exist between the region corresponding to duck histone H3, which can form distinguishable epitopes ( Figure 1 ): Sequence alignment results showed that the antigenic peptide selected in this invention is MPRPKDRSPRRRGP (SEQ ID NO.1) in duck CENP-A, while the sequence of duck conventional histone H3 in the corresponding region is MARTKQTARKSTGG (SEQ ID NO.4). Although both contain some basic amino acid residues, they differ significantly at several key sites, including different arrangements of proline, aspartic acid, serine, and arginine, resulting in significant differences in the continuous sequence composition, local charge distribution, and spatial conformation of this region. Therefore, this peptide can serve as a distinguishing epitope between duck CENP-A and duck histone H3, and can be used to prepare specific antibodies with CENP-A recognition capabilities.

[0029] (3) Rich in hydrophilic and charged residues, possessing good antigenicity and epitope accessibility: The antigenic polypeptide is rich in charged amino acids such as arginine, lysine, and aspartic acid, and contains multiple proline residues. This type of sequence typically exhibits high hydrophilicity and flexibility, tending to be located in exposed regions of the protein surface, facilitating antibody recognition. Furthermore, this polypeptide is located in the N-terminal tail region of CENP-A, theoretically making it more readily exposed to the native chromatin environment than the core structure within the nucleosome, thus promoting stable binding in experimental systems such as CUT&Tag that rely on native conformation recognition.

[0030] (4) Avoid extremely low-complexity repetitive regions to improve the quality of antigen preparation: The N-terminus of duck CENP-A contains a low-complexity region rich in glycine and proline. While such repetitive sequences are species-specific, they are not conducive to obtaining a stable and specific immune response. The antigenic peptide selected in this invention is located after this low-complexity region, which preserves the differential characteristics of the duck CENP-A N-terminal region while avoiding the non-specificity and unstable immune effects that may result from directly using highly repetitive low-complexity sequences as antigens.

[0031] (5) Possesses experimentally verified technical effects: Antibodies prepared based on the aforementioned antigenic peptides can effectively recognize duck CENP-A and obtain significant centromere enrichment signals in CUT&Tag sequencing. Combined with methylation sequencing results, it is possible to locate, analyze the sequence composition, and characterize the centromere regions of the entire duck chromosome.

[0032] The above results demonstrate that the antigenic polypeptides selected in this invention have clear functionality and practical application value.

[0033] This invention provides a method for preparing anti-duck CENPA antibody, comprising the following steps: The above-mentioned antigenic polypeptide was synthesized by adding a cysteine ​​residue to its N-terminus and then conjugated with a carrier protein to prepare an antigen for animal immunization. The antigen was injected into a host animal to induce an immune response; the preferred host animal was the New Zealand rabbit. Immunization could be performed using a combination of primary immunization and multiple booster immunizations to improve antibody titer and affinity. After immunization, blood was collected from the host animal, and serum was separated to obtain antiserum. Antibodies in the antiserum were screened, enriched, and purified using methods such as affinity chromatography to obtain high-purity anti-duck CENPA polyclonal antibodies. The antibody titer, specificity, and quality were detected and evaluated using methods such as ELISA. Preferably, the obtained antibody can be used to recognize the CENPA protein in the duck cell nucleus and produces a significant enrichment signal in the centromere region in the CUT&Tag experiment.

[0034] The present invention also provides the use of the antigenic polypeptide or the antibody in the following applications: Reagents for preparing the detection of duck CENPA protein; (1) Used for immune recognition and functional localization of the centromere region in ducks; (2) Used for capturing duck CENPA binding sites in CUT&Tag experiments; (3) Used for enrichment, boundary identification and structural analysis of centromere sequences in the duck genome; (4) Used to perform comprehensive analysis of the centromere region of ducks by combining epigenetic signals such as DNA methylation; (5) Used for research related to avian chromosome biology, comparative genomics, epigenetics and genetic breeding.

[0035] Example 1: Design of duck CENPA antigen peptides Based on the duck CENPA protein sequence (NCBI Accession Number: XP_038043914.2), its amino acid sequence was analyzed, and fragments located in the N-terminal variant region with high antigenicity and suitable for peptide immunization were screened as candidate antigens. The following 14 aa peptide was ultimately determined as the core sequence for the immunogenicity: MPRPKDRSPRRRGP (SEQ ID NO.1). To facilitate coupling with the carrier protein, a cysteine ​​residue was added to the N-terminus of the above sequence, yielding the immunogenic peptide: CMPRPKDRSPRRRGP (SEQ ID NO.2). This sequence has a clear origin, is located in the N-terminal region of the duck CENPA protein, and exhibits a strong enrichment of basic amino acids, which is conducive to the formation of a good immunogenic epitope. The characteristics of the immunogenic peptide are shown in Table 1. Table 1. Characteristics of immune peptides

[0036] Example 2: Preparation of anti-duck CENPA polyclonal antibody (1) Antigen preparation: The immunogenic peptide CMPRPKDRSPRRRGP (SEQ ID NO.2) was prepared by chemical synthesis and conjugated with a carrier protein to obtain an immunogen.

[0037] (2) Animal immunization: New Zealand rabbits were selected as the host animal, and the above-mentioned immunogens were administered subcutaneously or by multiple injections according to the immunization program. After the initial immunization, multiple booster immunizations were performed to induce the body to produce specific antibodies against duck CENPA antigen peptides.

[0038] (3) Collect serum: Rabbit blood was collected after the immunization cycle ended, and antiserum was obtained by centrifugation.

[0039] (4) Antibody purification: Specific IgG in antiserum was purified by affinity chromatography to obtain anti-duck CENPA polyclonal antibody.

[0040] (5) Antibody verification: The antibody titer and its specific recognition ability against antigenic peptides were detected by ELISA to evaluate antibody quality.

[0041] In some implementations, Western blot, immunofluorescence, or CUT&Tag pre-experiments can be used to further verify its application performance.

[0042] Example 3: Application of anti-duck CENPA antibody in CUT&Tag The anti-duck CENPA antibody prepared in this invention was used to perform CUT&Tag experiments on cell nuclei isolated from the heart tissue of Beijing ducks to locate the CENPA-enriched region in the duck genome.

[0043] (1) Sample preparation: Heart tissue from Peking ducks was collected and homogenized in 1 mL of 1× Homogenization Buffer (HB). The homogenate was filtered and centrifuged at 4°C and 500×g for 5 min. The resulting precipitate was further centrifuged using a density gradient for 10 min to separate high-purity cell nuclei. The nucleus suspension was quantified using a LUNA-FL automated cell counter (Logos Biosystems).

[0044] (2) Construction of CUT&Tag library: Library preparation was performed according to the standard CUT & Tag workflow. The brief steps are as follows: The isolated cell nuclei were bound to Concanavalin A-coated magnetic beads and permeabilized using didigitonin. Subsequently, the anti-duck CENPA primary antibody prepared according to this invention was added for incubation, allowing the antibody to bind to CENPA in the chromatin. Then, pA-Tn5 transposase was added to insert adapters near the antibody binding sites. The resulting DNA fragments were amplified by PCR to construct sequencing libraries, which were then purified using AMPure XP beads (Beckman Coulter). Library quality was assessed using an Agilent 2100 Bioanalyzer. Cluster generation was performed using Illumina cBot, and 150 bp paired-end sequencing was performed on the Illumina NovaSeq platform. IgG was used as a negative control, and two technical replicates were set up for both the CENPA and IgG groups.

[0045] (3) Bioinformatics analysis: After quality control and adapter removal of the raw CUT & Tag sequencing data, clean reads were aligned to the HAUpekinT2T genome. Alignment was performed using Bowtie2 (v2.5.4) with the following parameters: --very-sensitive --end-to-end --no-discordant --no-mixed -k 10.

[0046] SAMTools (v1.22) was used to preserve correctly paired reads; Picard (v3.4.0) was used to mark duplicates; based on the resulting BAM file, the callpeak and bdgcmp modules of MACS3 (v3.0.4) were used to identify CENPA enrichment peaks and calculate enriched signals.

[0047] (4) Usage description: The CUT&Tag experiments described above revealed significant CENPA-enriched regions in the duck genome, corresponding to functional centromere regions. Combined with reference genome assembly results and DNA methylation signals, the sequence composition, boundary features, and structural patterns of centromeres on various duck chromosomes can be further analyzed.

[0048] Example 4: Validation of antibody specificity and CUT & Tag enrichment effect To verify the titer, specificity, and application performance of the anti-duck CENPA antibody prepared in this invention in the CUT&Tag experiment, the purified antibody was tested by ELISA, and the repeatability and enrichment characteristics of the CUT&Tag sequencing results were analyzed.

[0049] (1) ELISA detection of antibody titer and specificity: The recognition ability of pre-immune serum and purified antibodies for antigenic peptides was detected using an indirect ELISA method. Free peptides were used as the coating antigen at a concentration of 4 μg / mL, with 100 μL per well. The coating buffer was pH 7.4 phosphate-buffered saline. HRP-conjugated anti-rabbit IgG secondary antibody was used as the secondary antibody.

[0050] ELISA results showed that the purified anti-duck CENPA antibody prepared in this invention exhibited high reactivity against the antigenic peptide. Under serial dilution conditions from 1:1000 to 1:512000, the OD of the purified antibody... 450 The values ​​were all significantly higher than those of the pre-immunization negative control serum. Specifically, the OD values ​​of the purified antibody at dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000 were significantly higher. 450 The values ​​were 2.631, 2.653, 2.757, 2.693, 2.574, 2.586, 2.494, 2.220, 1.815, and 1.341, respectively; corresponding to the OD values ​​of the pre-immunization negative control serum. 450 The values ​​are 0.087, 0.058, 0.058, 0.052, 0.061, 0.053, 0.059, 0.056, 0.053, and 0.058, respectively. Blank hole OD 450 The value is 0.051.

[0051] Based on the criterion of a Signal / Blank ratio ≥ 2.1, the ELISA titer of the purified antibody of this invention is greater than 1:512000, while the titer of the pre-immunization negative control serum is less than 1:1000. These results indicate that the anti-duck CENPA antibody prepared by this invention has a high titer and good specificity, and can effectively recognize the antigenic polypeptide.

[0052] (2) Repeatability analysis in CUT & Tag experiments: The anti-duck CENPA antibody prepared according to this invention was further used to conduct CUT&Tag experiments, with IgG as a negative control. Two technical replicates were set up for each group. After standardizing the sequencing data, cluster analysis and repeatability analysis were performed on the CENPA group and the IgG group samples.

[0053] The results showed that ( Figure 2 The two CENPA-based replicates showed good consistency and were clearly separated from the IgG negative control group, indicating that the antibody of this invention can stably generate reproducible specific enrichment signals in the CUT&Tag experiment, rather than random background noise. This result demonstrates that the prepared antibody has good experimental stability and reproducibility, making it suitable for subsequent high-resolution localization analysis of the duck centromere region.

[0054] (3) Validation of CUT & Tag enrichment patterns: Using the Z chromosome as an example, the sequencing depth distribution of CENPA_1, CENPA_2, IgG_1, and IgG_2 is displayed in a genome browser. The results show ( Figure 3 The CENPA antibody group showed a clear single main peak signal in the target area, while the IgG negative control group mainly showed scattered multi-peak low background signals, and no strong enriched main peak corresponding to the CENPA group was observed.

[0055] Further visualization analysis of the enrichment signals of chr1, chr22, and chrZ showed that ( Figures 4-6 The aforementioned chromosomes all exhibited significant CENPA main enrichment peaks, with concentrated peak positions and prominent signals, clearly distinguishing them from the background regions. Combined with the genome-wide distribution results, it is evident that the CENPA enrichment signal is primarily located in the centromere candidate regions of each duck chromosome, indicating that the anti-duck CENPA antibody prepared in this invention can effectively identify functional centromere regions of duck chromosomes and can be used for specific capture of duck centromere sequences.

[0056] Example 5: Genome-wide identification of the duck centromere region To systematically identify the centromere regions of each chromosome in the duck genome, based on the CUT&Tag enrichment signals obtained from the anti-duck CENPA antibody of this invention, combined with DNA methylation patterns, repetitive sequence composition, transposon element distribution, gene annotation information, and sequence similarity analysis, the centromere regions of the entire duck genome were comprehensively defined.

[0057] (1) Identification of centromere enrichment peaks across the entire genome: Based on the CENPA CUT & Tag signal, distinct CENPA enrichment peaks were identified on all duck chromosomes. These enrichment peaks constitute the main criterion for determining the centromeric core region. Furthermore, the centromeric core region defined by CENPA was combined with the pericentromeric domains on both sides rich in satellite repeat sequences, and the overall boundary of the centromeric region was defined by incorporating the changes in DNA methylation (5mC) signal.

[0058] In this embodiment, the centromere region of the duck genome is defined as: the centromere core region containing CENPA enrichment, and the adjacent near-centromere region that has satellite repeat sequence enrichment characteristics and is supported by methylation patterns.

[0059] (2) Centromere core region length and sequence composition characteristics: Analysis revealed that the length of the centromere core region of each chromosome in the duck genome typically ranges from 28.50 to 222.06 kb. These core regions are mainly composed of three types of repeating monomers with lengths of 133, 202, and 227 bp, respectively, and are defined as Apl.133, Apl.202, and Apl.227.

[0060] Furthermore, three types of additional complex repeating monosomy were identified in the centromere regions of chromosomes 1 and Z. These results indicate that the duck centromere core region possesses a relatively conserved yet chromosome-specific pattern of repeating sequences.

[0061] (3) Landscape analysis of representative chromosome centromere regions: Using chr1, chr22, and chrZ as representatives, a genomic landscape of their centromere regions is presented. The presentation includes: distribution of DNA methylation signals; distribution of transposon elements (TEs); gene annotation distribution; and sequence similarity heatmaps.

[0062] The results show that ( Figure 7-9The centromere regions of representative chromosomes all exhibited significant CENPA enrichment cores, accompanied by characteristic methylation patterns, enrichment of repetitive sequences / transposon elements, and localized low gene density. Sequence similarity analysis further revealed the organization patterns and homology relationships of repetitive units within the centromere region, providing a basis for the sequence structure analysis of duck centromeres.

[0063] In summary, the anti-duck CENPA antibody prepared based on this invention and its CUT&Tag enrichment signal can stably identify centromere regions of duck chromosomes at the whole genome scale, and can further analyze the core region length, repetitive sequence composition and regional structural characteristics, indicating that the antibody is suitable for the systematic identification and structural study of duck centromere sequences.

[0064] Therefore, this invention creates an immunopeptide by adding a cysteine ​​residue to the N-terminus of the duck CENPA antigenic peptide. This immunopeptide can be used to prepare antibodies that specifically recognize the duck CENPA protein. Antibodies prepared based on this antigenic peptide can be applied to experiments such as CUT&Tag to enrich and locate centromere regions in the duck genome, and further, by combining methylation and other information, to analyze the sequence composition and structure of the duck centromere. This invention provides a key molecular tool for duck centromere research and has promising application prospects.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A duck CENPA antigen polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. An immune polypeptide, characterized in that: Adding a cysteine ​​residue to the N-terminus of the duck CENPA antigen polypeptide according to claim 1 yields an immune polypeptide, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. The use of the immunopeptide according to claim 2 in the preparation of anti-duck CENPA polyclonal antibody.

4. A method for preparing an anti-duck CENPA polyclonal antibody, characterized in that, Includes the following steps: Step 1, Antigen Preparation: The immune polypeptide described in claim 2 was prepared by chemical synthesis and then coupled with a carrier protein to obtain an antigen. Step 2, animal immunization: The antigen is injected into a host animal to induce an immune response, thereby producing specific antibodies against the duck CENPA antigen polypeptide in the host animal; the host animal is a New Zealand rabbit. Step 3, collect serum: After the immunization cycle ends, host animal blood is collected and centrifuged to obtain antiserum; Step 4, antibody purification: Specific IgG in antiserum was purified to obtain anti-duck CENPA polyclonal antibody.

5. A polyclonal antibody against duck CENPA, characterized in that: It is prepared by the preparation method described in claim 4.

6. The use of the duck CENPA antigen polypeptide of claim 1 or the anti-duck CENPA polyclonal antibody of claim 5 in the preparation of reagents for detecting duck CENPA protein.

7. The application of the duck CENPA antigen polypeptide of claim 1 or the anti-duck CENPA polyclonal antibody of claim 5 in the immune recognition and functional localization of the duck centromere region.

8. The use of the duck CENPA antigen polypeptide of claim 1 or the anti-duck CENPA polyclonal antibody of claim 5 in the capture of duck CENPA binding sites in the CUT&Tag experiment.

9. The application of the duck CENPA antigen polypeptide of claim 1 or the anti-duck CENPA polyclonal antibody of claim 5 in the enrichment, boundary identification and structural analysis of the centromere sequence of the duck genome.

10. The application of the duck CENPA antigen polypeptide of claim 1 or the anti-duck CENPA polyclonal antibody of claim 5 in the comprehensive analysis of duck centromere regions by binding epigenetic signals.

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