Polyclonal antibody of porcine CENP-A protein as well as preparation method and application of polyclonal antibody

Abstract

The invention discloses a polyclonal antibody of porcine CENP-A protein as well as a preparation method and application of the polyclonal antibody, and relates to the technical field of bioengineering. The preparation method comprises the following steps: immunizing an animal by using a pig CENP-A protein, and collecting and separating to obtain serum; performing extraction and purification treatment on the serum to obtain the polyclonal antibody; the amino acid sequence of the pig CENP-A protein is as shown in SEQ ID NO. 1. The polyclonal antibody of the CENP-A protein is relatively good in specificity and relatively high in titer, and the polyclonal antibody of the CENP-A protein, provided by the invention, can be used for specifically recognizing endogenous CENP-A protein in pig tissues or pig small intestine epithelial cell lines in immunoblotting analysis, and can be applied to a centromere region sequence combined with CENP-A captured by ChIP-seq.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, in particular to a polyclonal antibody against porcine CENP-A protein and a preparation method and application thereof. Background Art

[0002] During cell division in eukaryotes, genetic material must be replicated with high fidelity and accurately distributed between the two daughter cells. This is fundamental to the survival of both cells and organisms. Cellular genetic material is primarily stored in chromosomes, and specific regions on chromosomes called centromeres serve as the structural foundation for this accurate chromosome segregation.

[0003] The function of the centromere is to recruit the kinetochore, which completes the pairing, connection, and separation of chromosomes and spindle microtubules. Centromeres and kinetochores are the core mechanisms that ensure chromosome segregation. Defects in centromeres or kinetochores can distort genetic information, leading to developmental disorders and related diseases. Recent bioinformatics, sequencing technologies, and chromosome mapping methods have revealed some of the molecular mechanisms underlying centromere function.

[0004] Centromeres are regions on chromosomes that bind specific histone proteins. During mitosis, these regions are responsible for connecting sister chromatids and recruiting kinetochore proteins, ensuring equal distribution of genetic information and maintaining genomic stability. Abnormalities in centromeres or kinetochores can lead to polyploidy, which in turn can cause various diseases, including cancer. Although centromeres are crucial for proper chromosome segregation, centromere DNA sequences are poorly conserved across species. Based on centromere DNA sequence and location, centromeres can be divided into three types: point centromeres, regional centromeres, and holocentromeres. Point centromeres typically contain a specific DNA sequence. For example, the centromeres of budding yeast and Saccharomyces cerevisiae contain a specific 125-base-pair DNA sequence. Regional centromeres contain numerous repetitive DNA sequences, primarily composed of satellite DNA. Human centromeres consist of a highly repetitive 171-base-pair α-satellite DNA sequence. Diffuse centromeres are mainly found in insects and plants and are distributed throughout the chromosome.

[0005] Centromere proteins form or regulate the function of centromeres, the central structures of chromosomes to which spindle fibers and tubulin are attached, enabling chromosome segregation during cell division. Known centromere proteins include constitutive proteins such as CENP-A, -B, -C, and -H, which are present at centromeres throughout the cell cycle, as well as transient proteins such as CENP-E, -F, INCENP, Mad1, Mad2, Bub1, Bub2, and RubR1, which appear after the onset of M phase. CENP-A (Centromere protein A, CENP-A) is another important centromere autoantigen besides CENP-B. It has a molecular weight of approximately 22 kDa and exhibits histone-like properties. It forms a polymeric structure with histones H3 and H4, participating in the formation of centromeric chromatin. The CENP-A gene encodes a histone H3 variant. The C-terminal two-thirds of CENP-A are highly homologous to histone H3, but the remaining amino-terminal third is unique to CENP-A. A domain consisting of histone homology sequences located in the C-terminal region is essential for targeting CENP-A to centromeres. The mouse CENP-A gene has been shown to be essential for gene targeting approaches. Cse4, a CENP-A homolog, has been isolated from Saccharomyces cerevisiae and has also been shown to be essential for chromosome segregation. Point mutation analysis has shown that the functional domains of Cse4p are distributed across a haplodomain on hist, which is required for interaction with histone H4 and the amino-terminal 33 peptide. CENP-A homologs have been identified in Caenorhabditis elegans, Drosophila melanogaster, and Streptococcus pombe and are also essential for chromosome segregation.

[0006] Centromere recruitment to the kinetochore is only possible when the H3 histone in the centromeric nucleosome is replaced by its variant, CENP-A (Centromeric Protein A). Therefore, CENP-A is also considered an epigenetic mark of the centromere. CENP-A has homologous proteins in various species, such as CSE4 in Saccharomyces cerevisiae, CNP1 in fission yeast, CID in Drosophila, and HCP-3 in Caenorhabditis elegans. The CENP-A nucleosome structure is similar to the classic H3 nucleosome. Histones H2A, H2B, and H4, along with CENP-A, form a histone octamer, around which DNA is wrapped in a left-handed orientation. CENP-A nucleosome assembly depends on the Holliday junction recognition protein (HJURP). The CBD (CENP-A binding domain) of HJURP specifically binds to the CATD (centromere-targeting domain) of CENP-A, facilitating the interaction of CENP-A with centromere DNA.

[0007] The contribution of DNA sequences to centromere formation remains controversial (Marshall et al. 2008). Although the presence of numerous repetitive sequences (alpha sequences; alpha satellite DNA) within human centromere regions contributes to the efficient construction of human artificial chromosomes, analysis of human neocentromeres suggests that these repetitive sequences are not absolutely essential for centromere formation (Machsall et al. 2008). To understand the molecular basis of centromere formation, it is necessary to define the protein complexes associated with these regions and the underlying centromere DNA sequences.

[0008] Centromeres are essential for faithful chromosome segregation by providing a site for centromere assembly. While the role of centromeres is conserved throughout evolution, the DNA sequence associated with the centromere region is highly diverse between species, and how centromere DNA guides centromere formation remains to be determined. Despite prior research on chromosome segregation in pigs, the centromere sequence in pigs remains unknown.

[0009] Anticentromere antibodies (ACAs) are antibodies that recognize protein antigens present in the centromere region. CENP-B, CENP-A, and CENP-C are the three main autoantigens of ACA. ACAs are biomarkers for the diagnosis of several autoimmune diseases, particularly limited cutaneous systemic sclerosis (LSSc). Furthermore, ACAs are a valuable tool for cell biologists to explore and understand the highly organized structure of chromosomes and the regulatory mechanisms of cell division.

[0010] Porcine centromere DNA repeats are divided into two families, MC1 and AC2. MC1 consists of 340 bp and is distributed on the meso- and submeso-centromeres of porcine chromosomes (1-12) and the X chromosome. AC2 consists of a 12 bp monomeric repeat located on all acrocentric chromosomes (13-18). Currently, no porcine Y chromosome centromere DNA repeats have been reported. In 1990, Jantsch et al., while identifying porcine centromere clones, discovered that clone pAV1.5 hybridized specifically with porcine chromosome 1 and could be used as a specific probe for the porcine chromosome 1 centromere. In 1999, Jantsch et al. obtained a porcine chromosome 9 centromere DNA clone from a porcine genomic cosmid library. This clone consisted of a 3.3 kb repeat sequence composed of ten 340 bp monolayers concatenated together, with an average similarity of 79%. Pulse-field electrophoresis and southern hybridization analysis revealed that this 3.3 kb repeat sequence further concatenated to form a chromosomal region of approximately 2.2 Mb.

[0011] Since domestication, pigs have evolved into approximately 600 breeds through a combination of natural and artificial selection. This rich diversity is reflected not only in physical characteristics but also in important economic traits such as weight, growth rate, and intramuscular fat percentage. Pig breeds in different regions are adapted to local environments and farming needs. This diversity provides an excellent natural resource for studying the relationship between genetics and phenotype, contributing to a deeper understanding of the evolutionary paths of species under different selective pressures.

[0012] Chromosomal rearrangements are one of the major causes of subfertility in domestic pigs. To date, over 200 different chromosomal rearrangements have been identified in domestic pigs. The vast majority are balanced reciprocal translocations, accounting for over 90% of the described rearrangements. It is estimated that chromosomal rearrangements in domestic pigs occur spontaneously in 1 in 200 live births. The prevalence of chromosomal rearrangements in pig populations is thought to be between 0.5% and 1.5%, depending on the intensity of cytogenetic screening within these populations. Chromosomal inversions occur when a chromosome breaks at two locations within the chromosome. If the breakpoints are located on either side of the centromere, the inversion is termed a pericentric inversion. Inversions can sometimes result in dicentric chromosomes, which disrupt chromosome segregation during anaphase: the two centromeres of a dicentric chromosome migrate in opposite directions and form a bridge. If the bridge is not broken, chromosome segregation is altered, inducing the formation of dichromosomal or diploid cells. Although reciprocal translocations are quite common in pigs, their genetic and genomic causes remain unclear. Furthermore, the domestic pig karyotype (2n=38) consists of two sex chromosomes and 18 pairs of autosomes (5 metacentrics, 7 submetacentrics, and 6 pericentrics). This karyotype is relatively similar to that of humans. Therefore, the pig species is a better animal model than other mammalian species. Studying the DNA structure of pig centromere protein positions and the location of satellite DNA is crucial for studying karyotype changes and centromere repositioning events during species and family evolution.

[0013] As one of the world's primary farm animals, pigs provide a significant portion of human meat production and occupy a crucial position in the agricultural economy. Their traits, such as meat quality, growth rate, and reproductive capacity, are directly related to farming profitability and market supply. Therefore, research on pig genetic improvement is crucial for improving agricultural production efficiency, meeting consumer demand for high-quality meat, and promoting the sustainable development of the livestock industry. The breeding process has evolved through multiple stages, including phenotypic selection, best linear unbiased prediction (BLUP) selection, and genomic selection. Today, pig (Sus scrofa) breeding no longer relies solely on empirical observations, but rather on more scientific and efficient methods. Porcine genomic breeding is considered the most efficient breeding technology currently available, making in-depth genome analysis and information mining crucial. Genome assembly is a key step in deciphering pig genetic information. Advances in high-throughput sequencing technologies have enabled researchers to obtain high-resolution genomic data, enabling them to mine the rich genetic variation within the genome. The optimal strategy for constructing the most complete genome is to complete the ultimate genome assembly (T2T) of an individual using advanced sequencing technologies such as pedigree analysis, Hi-C, Nanopore ultra-long sequencing, and PacBio HiFi sequencing, as well as gap filling, telomere extension, and telomeric centromere identification. Currently, several high-quality porcine reference genomes exist, but they remain incomplete in genomic regions such as repetitive sequences, centromeres, and telomeres. A gapless genome is the ultimate goal of genome assembly and is crucial for improving the accuracy of read mapping and variant detection in individuals sequenced using both short- and long-read sequencing.

[0014] Currently, the most important technique for studying the regulatory landscape of histone modifications is the sequencing technology that combines chromatin immunoprecipitation with next-generation sequencing, or ChIP-seq. This technique, which combines ChIP and NGS, first uses antibodies specific for histone modifications to crosslink DNA and proteins within the cell (i.e., linking the target protein to chromatin). DNA regions bound to these histone modifications are then enriched. After purification and library construction, these regions are subjected to high-throughput sequencing to obtain the sequences of the DNA fragments. This technique is considered the best approach for genome-wide studies of histone modifications. It generally involves: cell / tissue fixation – chromatin fragmentation – chromatin immunoprecipitation – termination of the crosslinking reaction – DNA purification and identification. DNA-protein binding can be identified using qPCR or high-throughput sequencing.

[0015] However, ChIP research primarily relies on cell or tissue samples from animal models, and while the processing methods are relatively mature, its application in porcine muscle and adipose tissue is limited. Adipose tissue, in particular, requires extensive pre-processing, is difficult to fragment, and extracting intact nuclei is challenging, along with poor stability across samples. These factors hinder the application of this technique in porcine tissues. ChIP-seq is particularly expensive, and without effective quality control prior to analysis, relying solely on sequencing results after large-scale sample enrichment can be costly.

[0016] In view of the shortcomings of the existing technology, the present invention intends to mine the porcine CENP-A gene and its polyclonal antibody, so as to realize the application in porcine CENP-A sequence capture. Summary of the Invention

[0017] The present invention aims to provide a polyclonal antibody against porcine CENP-A protein, its preparation method, and its application, to address the aforementioned problems of the prior art. This polyclonal antibody against CENP-A protein has good specificity and high potency. The polyclonal antibody against CENP-A protein provided by the present invention can specifically recognize endogenous CENP-A protein in porcine tissues or porcine small intestinal epithelial cell lines in immunoblotting analysis and can be used in ChIP-seq to capture centromere region sequences bound by CENP-A.

[0018] To achieve the above object, the present invention provides the following solutions:

[0019] The present invention provides a gene encoding porcine CENP-A protein, the nucleotide sequence of the gene encoding is shown as SEQ ID NO.2.

[0020] The present invention also provides a recombinant expression vector containing the above-mentioned coding gene.

[0021] The present invention also provides a recombinant microbial strain comprising the above-mentioned recombinant expression vector.

[0022] The present invention also provides a method for preparing a polyclonal antibody against porcine CENP-A protein, comprising the following steps:

[0023] Animals were immunized with porcine CENP-A protein, and serum was collected and separated;

[0024] extracting and purifying the serum to obtain the polyclonal antibody;

[0025] The amino acid sequence of the porcine CENP-A protein is shown in SEQ ID NO.1.

[0026] Furthermore, the animal is a rabbit.

[0027] Furthermore, the number of immunizations of animals using the porcine CENP-A protein is 3-4 times, and the interval between immunizations is 1-2 weeks.

[0028] The present invention also provides a polyclonal antibody prepared according to the above preparation method.

[0029] The present invention also provides the use of the polyclonal antibody in preparing an ELISA kit for detecting porcine CENP-A protein.

[0030] The present invention also provides an ELISA kit for detecting porcine CENP-A protein, comprising the above-mentioned polyclonal antibody.

[0031] The present invention also provides the use of the polyclonal antibody in immunoblotting analysis, wherein the polyclonal antibody is used to specifically identify endogenous CENP-A protein in pig tissue or pig small intestinal epithelial cell line.

[0032] The present invention discloses the following technical effects:

[0033] The present invention produces a polyclonal antibody to porcine CENP-A protein by immunizing animals with a recombinant CENP-A protein. The CENP-A recombinant protein exhibits stable immune responses and high antibody levels, and the resulting polyclonal antibody exhibits high specificity and titer. The polyclonal antibody to CENP-A protein provided by the present invention can specifically recognize endogenous CENP-A protein in porcine tissues or porcine small intestinal epithelial cell lines in immunoblotting analysis and can be used in ChIP-seq to capture centromere region sequences bound by CENP-A. This invention is of great significance for detecting centromere region sequences in the porcine genome and for improving the porcine genome map. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is a schematic diagram of the analysis results of the porcine CENP-A protein signal peptide;

[0036] Figure 2 This is a schematic diagram of the prediction results of the hydrophilicity of porcine CENP-A protein;

[0037] Figure 3 This is a schematic diagram of the prediction results of the transmembrane region of the porcine CENP-A protein;

[0038] Figure 4 Schematic diagram of the prediction results of the intrinsic disordered region of porcine CENP-A protein;

[0039] Figure 5 This is the predicted result diagram of the nuclear localization sequence of pig CENP-A protein;

[0040] Figure 6 The results are from sequencing using universal primers after codon optimization and ligation into the vector;

[0041] Figure 7 This is the map of the pET-30a vector;

[0042] Figure 8 Schematic diagram of the verification results of porcine CENP-A truncated recombinant protein; M1 represents protein marker Bio-rad, Cat. No. 1610374S; BSA represents bovine serum albumin; R represents porcine CENP-A truncated recombinant protein;

[0043] Figure 9 This is a graph showing the specificity of the polyclonal antibody against porcine CENP-A;

[0044] Figure 10 This is a graph showing the test results of the high efficiency of the polyclonal antibody against porcine CENP-A;

[0045] Figure 11 This is the centromere peak enrichment result of ChIP-seq using porcine CENP-A polyclonal antibody. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1 Expression and purification of recombinant protein

[0052] 1. Sequence Analysis of Porcine CENP-A Protein

[0053] The porcine CENP-A gene sequence information NM_001006196.3 was obtained from the NCBI database, and the porcine CENP-A protein signal peptide was analyzed using the SignalP tool (http: / / www.cbs.dtu.dk / services / SignalP-4.1 / ). Figure 1 ), and the hydrophilicity analysis was performed using the Novopro online tool (https: / / www.novopro.cn / tools / protein-hydrophilicity-plot.html) Figure 2 ), TMHMM 2.0 Server (http: / / www.cbs.dtu.dk / services / TMH-MM / ) was used to predict the transmembrane domain of the receptor ( Figure 3 ), and use the NOVOPRO online tool (https: / / www.novopro.cn / tools / disordered.html) to predict the intrinsic disordered regions of proteins ( Figure 4 ), and the protein nuclear localization sequence was predicted using the NOVOPRO online tool (https: / / www.novopro.cn / tools / disordered.html) Figure 5 ).

[0054] Results showed that the porcine CENP-A protein consists of 137 amino acids (see SEQ ID NO. 1), lacking a signal peptide or transmembrane region and possessing a disordered region. The protein is relatively hydrophobic, and amino acids 3 to 44 are located within a nuclear localization sequence. Given the short protein sequence and the absence of highly complex GC regions, the full-length protein was selected for recombinant protein construction. The NCBI accession number for the gene encoding the porcine CENP-A protein is XM_013996268.2.

[0055] SEQ ID NO.1:

[0056] MGPRRRRRKPETPRRRPSPAPAAPRRGPPLGPPSSRQLGPRRHRVLREIRILQKSTHLLLR KNPFCRLAREICVQFTRGVDFNWQAQALLALQEAAEAFLVHLFEDAYLLSLHAGRVTLFPK DIQLARRIRGIQEGLG.

[0057] 2. Construction and identification of recombinant vectors

[0058] Based on the obtained pig CENP-A gene coding sequence, codon optimization was performed on the gene sequence encoding the pig CENP-A recombinant protein. The optimized sequence is shown in SEQ ID NO.2.

[0059] SEQ ID NO.2:

[0060] CATATGCATCACCACCATCACCACGGACCCAGGCGTCGCCCGCGCAAGCCGGAAACCCCGAGACGTCGCCCAGCTAGCCCGGCACCGGCGGCTCCGCGTCGTGGCCCTCCGCTGGGTCCGCGTCGTCATCGTGTTCTGCGTGAAATTCGTATTCTGCAAAAATCCACGCATTTGCTCTTGCGCAAGAACCCGTTCTGCCGGCTGGCCCGTGAG ATCTGTGTTCAGTTTACCCGTGGCGTGGACTTCAATTGGCAGGCGCAGGCCCTGTTAGCGTTGCAAGAGGCGGCAGAAGCGTTTTTGGTGCACCTGTTCGAGGACGCATACCTGCTGAGCCTGCACGCGGGTCGTGTCACCCTGTTTCCGAAAGATATTCAGCTGGCGCGTCGCATCCGCGGCATCCAAGAGGGTCTGGGTTAATGAAAGCTT.

[0061] The optimized sequence was delivered to GenScript Biotech to synthesize the DNA template from scratch. The target fragment was amplified by PCR and then ligated with the enzyme-digested vector using homologous recombination to obtain the eukaryotic expression vector pET-30a-His-CENP-A.

[0062] pET-30a (GenScript Biotech, C7282UZRG0-2) was selected as the ligation vector (see vector map for details). Figure 7 ), selecting NdeI (5'-CAT↓ATG-3') and HindIII (5'A↓AGCTT 3') as restriction endonucleases for the insert fragment. 1 μg of pET-30a plasmid was mixed with 1 μL each of the restriction endonucleases NdeI and HindIII. 5 μL of FlyCut buffer was added, and the volume was made up to 50 μL with nuclease-free water. The digestion product was incubated at 37°C for 15 minutes, and the digestion product was recovered by gel electrophoresis. At a molar ratio of 3:1 between the insert fragment and the vector, 1 μL of 5× T4 DNA ligase and 2 μL of buffer were added. The amplified CENP-A fragment was ligated into the pET-30a vector at 16°C overnight to generate the eukaryotic expression vector pET-30a-His-CENP-A.

[0063] Take 2 μL of the eukaryotic expression vector pET-30a-His-CENP-A to transform BL21 (DE3) competent cells, spread on LB resistance plates, and culture inverted at 37°C overnight. Select a single white colony for sequencing, and compare the sequencing results with the full-length sequence of the CENP-A gene ( Figure 6 The positive single clone is the E. coli containing pET-30a-His-CENP-A, i.e. the recombinant bacteria.

[0064] 3. Extraction and Preliminary Identification of Porcine CENP-A Recombinant Protein

[0065] Pick up the recombinant bacteria and place them in a test tube containing 4 mL of LB medium containing kanamycin, and culture them at 37°C and 220 rpm until the bacteria grow.

[0066] OD 600 The cell count was approximately 1.2. The treatment group was treated with the inducer IPTG to a final concentration of 0.5 mM. The control group was not treated with IPTG. The cells were cultured at 15°C for more than 16 hours. The cells were centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, the pellet was collected, and resuspension buffer was added and pipetted until completely resuspended. The pellet was then disrupted in an ice bath using an ultrasonic disruptor and centrifuged at 10000 rpm for 5 minutes. The supernatant was then analyzed by SDS-PAGE and western blot. The specific steps were as follows:

[0067] Take 100 μL of the above sample, add 25 μL of reduced SDS-Loading buffer (300 mM Tris-HCl, 10% SDS, 30% glycerol, 0.5% bromophenol blue, 250 mM DTT, pH 6.8), heat denaturation at 100 ° C for 10 min, centrifuge at 12000 rpm for 2 min, use 4% to 20% gradient PAGE gel, take the denatured protein samples of the treatment group and control group (BSA) for electrophoresis, constant voltage 140 V, after 60 min, transfer the protein on the SDS-PAGE gel to a 0.22 μm PVDF membrane by wet transfer; add rapid blocking solution and block the membrane at room temperature for 2 h; use His-tag polyclonal antibody (Bioworld, AP0032) as the primary antibody and incubate at 4 ° C for 12 h, and then use Mouse-anti-His mAb (GenScript, Cat. No. A00186) was used as the secondary antibody and incubated at room temperature for 2 h; the protein was developed using an ECL luminescence kit (Thermo Fisher, A34580).

[0068] The results show that ( Figure 8 ): A positive band of CENP-A recombinant protein appeared at 17kDa, with strong specificity and high expression level.

[0069] 4. Scale-up of CENP-A recombinant protein expression and Ni 2+ Affinity column purification

[0070] After induction, the cells were resuspended in lysis buffer and then sonicated. 2+ The target protein is purified by affinity column and stored in buffer. After sterilization through a 0.22μm filter, it is aliquoted and stored in phosphate buffer (pH 8.0). The specific steps are as follows:

[0071] (1)Ni 2+ Preparation of affinity columns

[0072] First, Ni 2+ The affinity filler is suspended to make a 50% suspension. The suspension is aspirated and loaded into the column with a column bed volume of 1 mL. The column bed is washed with 8 to 10 column volumes of distilled water, and then rinsed with 5 to 6 column volumes of 100 mmol / L nickel sulfate until the column liquid turns blue-green. Finally, the column bed is washed with 8 to 10 column volumes of distilled water to remove all unbound Ni. 2+ .

[0073] (2) Ni of target protein 2+ Column affinity chromatography purification

[0074] Wash the column bed with lysis buffer using 5 column volumes (CV). Add 2.5 mL of lysate supernatant to the column bed. Control the flow rate to ensure the sample flows through the column bed as slowly as possible. Repeat the flow-through twice. Collect the flow-through (FT), aspirate 60 μL, add 15 μL of 5× SDS-loading buffer, and mix thoroughly. Wash the column bed with 5 CV of elution buffer to elute the target protein and collect fractions. Collect approximately 600 μL per EP tube and take eluted samples for gel analysis. Take 60 μL of each fraction and add 15 μL of 5× SDS-sample buffer to mix thoroughly.

[0075] Example 2 Preparation and purification of polyclonal antibodies

[0076] In this example, the porcine CENP-A polyclonal antibody was prepared by immunizing an animal with the porcine CENP-A recombinant protein prepared in Example 1 as an antigen, as detailed below:

[0077] Immunization: Three healthy female New Zealand white rabbits (randomly divided into two groups, negative control was injected with serum) were immunized with porcine CENP-A recombinant protein at a dose of 200 μg / rabbit (maximum 300 μg / rabbit) injected subcutaneously on the back. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for subsequent immunizations.

[0078] Booster immunization: Immunization intervals are 1 week (maximum 2 weeks), 4 immunizations (minimum 3), with subcutaneous injections at 5 points on the rabbit's back and venous blood drawn to obtain polyclonal antibody serum. Each booster immunization uses an emulsion of antigen mixed with Freund's incomplete adjuvant.

[0079] Blood collection: About 1-2 weeks after the last booster immunization, collect blood from the rabbit through the vein to obtain a sufficient amount of blood.

[0080] Serum separation: The collected blood is allowed to stand and coagulate to obtain blood clots and serum. The serum is separated by centrifugation.

[0081] Antibody purification: Use antigen affinity purification to extract and purify polyclonal antibodies from serum, and remove polyclonal antibodies against his tag by decross-adsorption. Evenly suspend the prepared antigen affinity resin (Protein A) in a test tube and transfer the resin to a clean empty column. Let the resin settle, drain the buffer from the column, and balance the resin with 10 resin bed volumes of PBS. Connect the resin column to the nucleic acid protein detector, slowly pass the serum sample through the column, and monitor the A 280 The absorbance of the effluent at A was measured and the resin was washed with PBS until the absorbance at A 280After the absorbance stabilizes at 4°C, drain the PBS and add elution buffer to the column. Collect the antibody-containing eluate in a test tube based on the reading of the nucleic acid-protein detector. Immediately neutralize the antibody eluate with neutralization buffer. Add 20 resin bed volumes of PBS to the column to equilibrate the resin. Dialyze the antibody eluate against phosphate-buffered saline (PBS) overnight at 4°C.

[0082] Storage: Aliquot the purified antibody solution into ProClin 300 and store at -80°C to avoid repeated freezing and thawing.

[0083] Example 3 Specificity and effectiveness analysis of polyclonal antibodies

[0084] This example analyzes the specificity and effectiveness of the porcine CENP-A polyclonal antibody prepared in Example 2, as detailed below:

[0085] 1. Effectiveness testing of porcine CENP-A polyclonal antibodies

[0086] ELISA detection of porcine CENP-A polyclonal antibody titer:

[0087] Preparation of related solutions: Coating Buffer (8.5 g NaCl, 1.4 g Na2HPO4, 0.2 g NaH2PO4, add ddH2O to make 1000 mL of pH 7.4 solution); Washing Buffer (0.5 mL Tween-20, 1000 mL PBS Buffer);

[0088] Blocking Buffer (1000 mL Washing Buffer, 10 g BSA) was prepared; Stop Solution (83 mL 12 mol / L HCl, 917 mL ddH2O) was prepared; TMB Reagent (GenScript Cat. No. M00078) was used to detect horseradish peroxidase (HRP) activity, producing a blue color (Amax = 370 nm and 652 nm), which turned yellow (Amax = 450 nm) after adding sulfuric acid or phosphoric acid stop solution.

[0089] Coating: The porcine CENP-A protein purified in Example 1 was assayed for protein concentration using the BCA assay. The protein was diluted to 3 μg / mL using the antigen coating method with Coating Buffer and added to a 96-well plate at 100 μL per well, with triplicate wells. The plate was sealed with sealing film and coated at 37°C for 1 hour, then incubated at 4°C overnight. After removing the coating solution, the plate was washed twice with 250 μL of Washing Buffer per well.

[0090] Blocking: Add 150 μL Blocking Buffer to each well, seal the plate with sealing film and incubate at 37°C for 1 hour. Remove the blocking solution and wash twice with the prepared Washing Buffer, 250 μL per well.

[0091] Incubation: Dilute the primary antibody or antiserum (1 μg / mL) in Blocking Buffer, add serial dilutions (1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000) of unimmunized negative control serum, and 100 μL / well. Incubate at 37°C for 1 hour. Wash twice with Washing Buffer, 250 μL per well. Add 100 μL of a 1:1000 dilution of secondary antibody (Goat anti-Rabbit IgG (H+L)) to each well. Seal the plate with film and incubate at 37°C for 1 hour. Wash three times with Washing Buffer. Finally, add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 15 minutes. Stop the reaction with 50 μL of Stop Solution and measure absorbance at 450 nm on a microplate reader for 30 minutes. The results (Table 1) show that the antibody maintains a high titer at a 1:512,000 dilution compared to the control.

[0092] Table 1 Results of ELISA detection of porcine CENP-A polyclonal antibody titer

[0093]

[0094] 2. Specificity Detection of Porcine CENP-A Polyclonal Antibodies

[0095] HindIII and BamHI and protective bases were added to the upstream and downstream primers of the porcine CENP-A gene respectively for vector subcloning. The vector was handed over to Jima Biotechnology Company to construct the pcDNA3.1-Flag-CENP-A eukaryotic expression plasmid and transfected into the porcine small intestinal epithelial cell line. The cells were placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. After adding 100 μL of RIPA (Beyotime Company, P0013B), the cells were lysed for 10 minutes and then centrifuged at 4°C and 12000 rpm for 10 minutes, and the supernatant was aspirated. Anti-FLAG antibody (1:10000) (Proteintech, 20543-1-AP) was used to incubate overnight at 4°C, and β-actin was used as an internal control at a ratio of (1:5000) (Abmart, T40104). The strips were washed 3 times with TBST buffer and incubated with HRP-labeled goat anti-rabbit IgG (1:5000) (Abmart, ab205718) at room temperature for 2 hours. The strips were detected using an ultra-sensitive ECL detection kit. The results showed ( Figure 9 ) The protein sample of cells transfected with pcDNA3.1-Flag-CENP-A showed a strong positive band at 17 kDa, indicating that the prepared polyclonal antibody was specific.

[0096] 20 mg each of pig heart, liver, spleen, lung, and kidney tissues were collected and ground with liquid nitrogen. 200 μL of RIPA (Beyotime, P0013B) was added and lysed on ice for 10 minutes. The protein solution was ultrasonically disrupted (50% output power, 5 times for 10 seconds each time), and centrifuged at 4°C, 12000 rpm, for 10 minutes to aspirate the supernatant. BCA quantification was performed, and 1 / 5 times the volume of 5×SDA protein sample loading buffer was added to the above-extracted tissue protein solution, and the solution was denatured by boiling at 100°C for 10 minutes; the protein was separated by 15% SDS-PAGE electrophoresis, and then the protein was transferred to a 0.22μm PVDF membrane by wet transfer; 5% skim milk powder was blocked at room temperature for 2 hours; the appropriate band was sheared according to the predicted protein molecular weight, and the porcine CENP-A polyclonal antibody (1:100) prepared in Example 2 was used for incubation at 4°C overnight, and β-actin was used as an internal reference according to the ratio (1:5000) (Abmart Company, T40104) for incubation; the strips were washed 3 times with TBST buffer and incubated at room temperature for 2 hours with HRP-labeled goat anti-rabbit IgG (1:5000) (Abmart Company, ab205718); and the ultra-sensitive ECL detection kit was used for detection. Results ( Figure 10 ) showed that each tissue protein sample had a strong positive band at 17 kDa, indicating that the prepared CENP-A polyclonal antibody was specific and highly effective.

[0097] Example 4 ChIP-seq validation of porcine CENP-A polyclonal antibodies

[0098] Cross-linking: Prepare PBS solution containing protease inhibitors (Solution A): Add 20 μL of prepared PIS solution and 40 μL of PMSF solution to 10 mL of PBS, mix thoroughly, and place on ice; add 560 μL of 37% formaldehyde solution (final formaldehyde concentration is 1%) to each 15 cm culture dish containing 20 mL of culture medium, rotate the culture dish slightly to mix, and place at room temperature for 10 minutes to cross-link; after the cross-linking is completed, add 1.37 mL of 2 M glycine solution to each 15 cm culture dish and rotate it slightly to mix, incubate at room temperature for 5 minutes to terminate the cross-linking; discard the culture medium and rinse twice with ice-cold PBS solution (pre-cooled in advance), and discard the PBS solution; add 3 mL of ice-cold prepared Solution A to each 15 mm culture dish of cells, scrape and count the cells, and count them according to 10 cells per tube. 7 The cells were aliquoted and centrifuged at 1000 g for 5 min at 4°C to precipitate the cells.

[0099] Nuclei treatment: After cross-linking the sample, grind it in liquid nitrogen and add 1 mL of cell lysis buffer (10 mM Tris, 10 mM NaCl, 0.2% NP-40 [pH 8.0], 1× protease inhibitor). Centrifuge at 4°C to extract the nuclei. Add 200 μL of 1% SDS solution containing protease inhibitors. Resuspend the nuclei by pipetting and incubate on ice for 10 min.

[0100] Chromatin digestion: Sonicate the DNA (8 pulses, 60 seconds on, 120 seconds off) to fragments of 200-1500 bp. Centrifuge at 13,000 rpm for 10 min at 4°C. Transfer the supernatant to a fresh 2 mL centrifuge tube and discard the pellet. Dilute the sonicated supernatant with 10x ChIP diluent (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.01% SDS, 1% Triton X-100, 1x protease inhibitors). Add 200 μL of supernatant to 1.8 mL of ChIP diluent to a final volume of 2 mL. To remove nonspecific proteins, add 75 μL of Salmon Sperm DNA / Protein AAgarose-50% Slurry and incubate at 4°C for 60 min. Centrifuge at 1000 rpm for 3 min to pellet the Salmon Sperm DNA / Protein AAgarose-50% Slurry and collect the supernatant.

[0101] Chromatin immunoprecipitation: Add 10 μg of antibody to the supernatant and mix overnight at 4°C. Add 60 μL of Salmon Sperm DNA / Protein AAgarose-50% Slurry to precipitate the antibody / antigen complex and rotate at 4°C for 60 minutes. Centrifuge at 1000 rpm for 3 minutes at 4°C to collect the pellet, remove the supernatant, and begin the elution process.

[0102] Elution: Low-salt immune complex elution buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100 [pH 8.1]), rotate for 5 minutes, centrifuge at 1000 rpm for 3 minutes to collect the precipitate. High-salt immune complex elution buffer (20 mM Tris, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100 [pH 8.1]), rotate for 5 minutes, centrifuge at 1000 rpm for 3 minutes to collect the precipitate. LiCl immune complex elution buffer (10 mM Tris, 0.25 M LiCl, 1 mM EDTA, 1% NP-40 [pH 8.1]), rotate for 5 minutes, centrifuge at 1000 rpm for 3 minutes to collect the precipitate. TE buffer, rotate for 5 minutes, centrifuge at 1000 rpm for 3 minutes to collect the precipitate, twice. You now have the protein A / antibody / histone / DNA complex. Prepare fresh elution buffer (1% SDS, 0.1M NaHCO₃). Add 250 μL of elution buffer to the pellet, mix thoroughly, and rotate at room temperature for 15 minutes. Centrifuge at 1000 rpm for 3 minutes. Transfer the supernatant to a new centrifuge tube and repeat the above process, resulting in a final supernatant volume of approximately 500 μL.

[0103] Decrosslinking: Add 20 μL of 5 M sodium chloride to decrosslink and incubate at 65°C overnight. Then add 10 μL of 0.5 M EDTA, 20 μL of 1 M Tris-HCl (pH 6.5), and 2 μL of 10 mg / mL Proteinase K and rotate at 55°C for 4 hours. Add an equal volume of phenol / chloroform to extract the DNA and centrifuge at 14,000 g for 10 minutes. Collect the supernatant and avoid aspirating the filamentous protein. Add 2.5 times the volume of pure ethanol and 1 / 10 volume of sodium acetate to precipitate the DNA and centrifuge at 14,000 g for 10 minutes. Collect the precipitate, wash with 80% alcohol, and air dry with the lid open. Dissolve in 50 μL of TE buffer.

[0104] The quality control and data analysis of the offline data were performed to obtain the enrichment peak of the DNA fragments bound to the genomic position of the target protein pulled down by the pig CENP-A protein polyclonal antibody ( Figure 11 ).

[0105] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A gene encoding porcine CENP-A protein, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

2. A recombinant expression vector, characterized in that: Comprising the encoding gene according to claim 1.

3. A recombinant microbial strain, characterized in that Comprising the recombinant expression vector according to claim 2.

4. A method for preparing a polyclonal antibody against porcine CENP-A protein, characterized in that: The following steps are involved: Animals were immunized with porcine CENP-A protein, and serum was collected and separated; extracting and purifying the serum to obtain the polyclonal antibody; The amino acid sequence of the porcine CENP-A protein is shown in SEQ ID NO.

1.

5. The preparation method according to claim 4, characterized in that The animal is a rabbit.

6. The preparation method according to claim 4, characterized in that The number of immunization times for animals immunized with the porcine CENP-A protein is 3-4 times, and the immunization interval is 1-2 weeks.

7. A polyclonal antibody prepared according to the preparation method according to any one of claims 4 to 6.

8. Use of the polyclonal antibody according to claim 7 in preparing an ELISA kit for detecting porcine CENP-A protein.

9. An ELISA kit for detecting porcine CENP-A protein, characterized in that: Comprising the polyclonal antibody according to claim 7.

10. Use of the polyclonal antibody according to claim 7 in immunoblotting analysis, characterized in that: The polyclonal antibody is used for specifically recognizing endogenous CENP-A protein in pig tissues or pig small intestinal epithelial cell lines.

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