Antigen polypeptide and polyclonal antibody for preparing specific binding HERVH capsid protein and application of antigen polypeptide and polyclonal antibody

Abstract

The invention provides an antigen polypeptide for preparing a specific binding HERVH capsid protein, a polyclonal antibody and application thereof. The amino acid sequence of the antigen polypeptide is as shown in SEQ ID NO: 2. The polypeptide is prepared into an immunogen immune animal and purified to obtain a high-titer specific antibody. The antibody can specifically recognize HERVH capsid protein in tissues and body fluid samples (such as serum and exosome). The antibody and the kit provided by the invention are high in sensitivity and strong in specificity, can be used for early diagnosis, auxiliary diagnosis, prognosis evaluation and risk stratification of colorectal cancer, and have remarkable clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to antigenic polypeptides and polyclonal antibodies for the preparation of HERVH capsid proteins and their applications. Background Technology

[0002] Colorectal cancer (CRC) is a prevalent malignant tumor worldwide, ranking among the top in both incidence and mortality. Epidemiological data shows that early diagnosis of CRC is crucial for improving patient survival. Currently, the clinical diagnosis of CRC mainly relies on colonoscopy, pathological biopsy, and serum tumor marker (such as CEA and CA19-9) testing. However, colonoscopy has drawbacks such as being invasive, expensive, and having poor patient compliance, making it difficult to use as the first-line method for large-scale screening; while existing serum markers often lack sufficient sensitivity and specificity in the early diagnosis of CRC, easily leading to missed or misdiagnosed cases. Therefore, the search for novel, highly sensitive, and specific biomarkers, especially those that can be used for non-invasive detection, is of significant clinical importance.

[0003] Human endogenous retroviruses (HERVs) comprise approximately 8% of the human genome and are genetic remnants left behind after ancient retroviruses infected human germ cells. Among them, HERVH (H family endogenous retroviruses) is one of the more active subfamilies. Current research indicates that HERVH transcripts (mRNA) play a role in maintaining the pluripotency of embryonic stem cells and exhibit abnormally high transcription levels in various tumor tissues, including colorectal cancer.

[0004] Although existing literature reports the upregulation of HERVH mRNA in colorectal cancer, current detection methods mainly focus on the nucleic acid level (such as qPCR). However, mRNA levels do not always perfectly correlate with protein levels, and nucleic acid detection has limitations in sample processing and stability. More importantly, systematic research is lacking regarding the expression pattern of the capsid protein encoded by the HERVH env gene in colorectal cancer, whether it assembles into virus-like particles and is released extracellularly, and whether it can serve as a diagnostic biomarker at the protein level. This is largely due to the lack of highly specific and high-affinity antibody reagents targeting the HERVH capsid protein. Given the large number of members in the HERV family and their high sequence similarity, preparing antibodies that specifically recognize the HERVH capsid protein without cross-reacting with other family members presents significant technical challenges. The lack of effective immunological detection tools limits the development and application of HERVH protein as a clinical diagnostic biomarker. Summary of the Invention

[0005] The purpose of this invention is to provide antigenic peptides and polyclonal antibodies for the preparation of specific binding HERVH capsid proteins and their applications. The antigenic peptides, antibodies and detection kits provided by this invention offer a novel, sensitive and specific method for the screening, diagnosis and prognostic assessment of colorectal cancer, with broad clinical application prospects.

[0006] The first aspect of the present invention provides an antigenic polypeptide for preparing a polyclonal antibody that specifically binds to the HERVH capsid protein, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] A second aspect of the present invention provides an immunogen for preparing a polyclonal antibody that specifically binds to the HERVH capsid protein, which is prepared using the above-mentioned antigenic polypeptide as a hapten.

[0008] In one embodiment of the present invention, the immunogen is prepared by mixing the above-mentioned antigenic peptide with a pre-maleylated carrier protein in a buffer solution at pH 6.5 to 7.5, wherein the molar ratio of the antigenic peptide to the carrier protein is 20:1 to 50:1; reacting at room temperature for 1 to 3 hours; and after the reaction, removing the uncoupled antigenic peptide by dialysis or gel filtration to obtain the immunogen.

[0009] A third aspect of the present invention provides a polyclonal antibody for the specific detection of HERVH capsid protein, which is prepared by immunizing non-human mammals with the above-mentioned immunogen.

[0010] The fourth aspect of this invention provides a method for preparing the above-mentioned polyclonal antibody, comprising the following steps: S1. Immunize non-human mammals with the above-mentioned immunogens and screen for high-titer immunized animals; S2. Collect whole blood from the selected immunized animals, separate serum, and use a solid-phase medium conjugated with the polypeptide shown in SEQ ID NO:2 for antigen affinity purification to obtain polyclonal antibodies.

[0011] In one embodiment of the present invention, the immunized animal is an immunized rabbit, the number of immunizations is 3 to 5, and the immunized animal selected for detection and screening refers to an immunized rabbit whose antigen polypeptide detection titer reaches 1:10000 or higher using the ELISA method.

[0012] The fifth aspect of the present invention provides the use of the above-described polyclonal antibody in the preparation of products for the diagnosis, auxiliary diagnosis, prognosis assessment or monitoring of colorectal cancer.

[0013] In one embodiment of the present invention, the product is used to detect the expression level of HERVH capsid protein in a biological sample by means of an immunological reaction; the immunological reaction includes ELISA, Western blot, or immunohistochemistry.

[0014] In one embodiment of the present invention, the biological sample is selected from tumor tissue, adjacent normal tissue, blood, serum, plasma, exosomes, or cell culture supernatant.

[0015] A sixth aspect of the present invention provides a colorectal cancer diagnostic kit, the kit comprising the aforementioned polyclonal antibody.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The antigenic peptide provided in this invention does not use the full-length sequence of the HERVH capsid protein for immunization. Instead, through bioinformatics analysis and structural prediction, a short peptide (SEQ ID NO:2, LDRSSKTSPDISHQC) located on the protein surface and with high conservation was screened as the antigenic epitope. An immunogen was prepared using a specific conjugation method, and combined with antigen affinity purification technology, a high-purity polyclonal antibody specifically binding to the HERVH capsid protein was successfully prepared. Experimental results show that the antibody has an ELISA detection titer of over 1:10000 and can specifically recognize the target protein in tumor tissue, effectively avoiding non-specific interference between endogenous retrovirus families, providing a key tool for the detection of HERVH protein levels.

[0017] Immunohistochemical (IHC) analysis of clinical tissue samples using the polyclonal antibody prepared in this invention showed that the expression level (Hscore) of HERVH capsid protein in colorectal cancer tissue was significantly higher than that in adjacent normal tissue (p < 0.05). This indicates that the polyclonal antibody provided by this invention can clearly distinguish between cancerous and normal tissues, has excellent clinical and pathological diagnostic value, and can serve as a powerful supplement to existing pathological diagnostic indicators.

[0018] This invention not only validated the expression of the biomarker at the tissue level, but also, for the first time, demonstrated through various methods such as Western blotting, immunoelectron microscopy, and ddPCR that the HERVH capsid protein can assemble into virus-like particles and be secreted extracellularly (in cell culture supernatant / exosomes) in colorectal cancer cells with high HERVH expression (especially ARID1A deletion). This discovery indicates that the HERVH capsid protein can exist as a secretory biomarker in blood, serum, plasma, or exosomes, thereby supporting the development of body fluid detection kits based on methods such as ELISA using the antibodies of this invention, enabling non-invasive early screening and disease monitoring of colorectal cancer.

[0019] The antibody detection method of this invention, which detects the level of HERVH capsid protein, not only helps in the diagnosis of colorectal cancer, but may also be used to stratify the risk of molecular subtypes with poor prognosis, such as ARID1A mutant or deletion types, thereby guiding individualized clinical treatment decisions. Attached Figure Description

[0020] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram showing the location of the antigenic polypeptide epitope in the three-dimensional protein structure model of the present invention; the red part is the location of the antigenic polypeptide shown in SEQ ID NO: 2; Figure 2 This describes the results of immunohistochemical staining of colorectal cancer tissue microarrays using the polyclonal antibodies of this invention, along with H-score statistical charts. Figure 2 In the image, 'a' represents an example of a stained section of adjacent normal tissue. Figure 2 In the image, b is an example of a stained section of tumor tissue. Figure 2 In the middle, 'c' represents the corresponding statistical analysis bar chart; Figure 3 The figure shows the results of a Western Blot experiment using the polyclonal antibody of this invention to detect the expression level of HERVH capsid protein in the lysate (Cell) and cell culture supernatant (CM) of ARID1A knockdown (KD) and wild-type (WT) HCT116 cells; in the figure, CD81 is a marker of extracellular vesicles and Calnexin is an intracellular marker. Figure 4 To verify the detection results of HERVH virus-like particle release, the following diagram is used: Figure 4 Image a shows the results of digital droplet PCR (ddPCR) for detecting HERVH viral RNA copy number in cell culture supernatant. Figure 4 In the middle b, the results of immunoelectron microscopy (Immuno-TEM) observation of virus-like particles labeled with HERVH capsid protein are shown. Detailed Implementation

[0021] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0022] Unless otherwise specified in the following examples, the biochemical reagents are all conventional reagents in the art, which can be prepared by conventional methods in the art or obtained commercially, and are of laboratory purity grade.

[0023] The bioinformatics sequences involved in this invention include the full-length amino acid sequence of the HERVH capsid protein, which is referenced in UniProtKB / Swiss-Prot database entry Q9N2J8; and the antigenic polypeptide sequence used for immunization, as shown in SEQ ID NO: 2.

[0024] The present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1 This embodiment provides the design, synthesis, and immunogen preparation of anti-HERVH capsid protein antigen peptides. 1. Design of antigenic peptides: Based on the three-dimensional structure prediction and hydrophilicity analysis of the HERVH capsid protein (UniProtKB: Q9N2J8), a region on the protein surface with high exposure and conservation was selected as the antigenic epitope. Specifically, 14 consecutive amino acid residues starting from amino acid 115 were selected, and a cysteine ​​residue was introduced at its C-terminus for coupling, resulting in the antigenic polypeptide with the amino acid sequence LDRSSKTSPDISHQC (i.e., SEQ ID NO: 2 in the sequence listing). Figure 1 The model diagram is arranged by structural units (e.g., transmembrane subunits and surface subunits are represented by different colors), and the position of the antigenic peptide in the spatial structure is marked with a prominent color (red in the figure).

[0026] The amino acid sequence of the HERVH capsid protein in this embodiment is shown in SEQ ID NO: 1.

[0027] SEQ ID NO: 1: MILAGRAPSNTSTLMKFYSLLLYSLLFSFPFLYHPLPLPSYLHHTINLTHSLPAASNPSLANNCWLCISLSSSAYIAVPTLQTDRATSPVSLHLRTSFNSPHLYPPEELIYFLDRSSKTSPDISHQPAAALLHIYLKN LSPYINSTPPIFGPLTTQTTIPVAAPLCISRQRPTGIPLGNISPSRCSFTLHLQSPTTHVTETIGVFQLHIIDKPSINTDKLKNVSSNYCLGRHLPYISLHPWLPSPCSSDSPPRPSSCLLTPSPQNNSERLLVDTQRF LIHHENRTSSSMQLAHQSPLQPLTAAALAGSLGVWVQDTPFSTPSHPFSLHLQFCLTQGLFFLCGSSTYMCLPANWTGTCTLVFLTPKIQFANGTKELPVPLMTLTPQKRVIPLIPLMVGLLGSASTIALSTGIAGIST SVTTFRSPSNDFSASITDISQTLSVLQAQVDSLAAVVLQNRRGLGLSILLNEECCFYLNQSGLVYENIKKLKDRAQKLANQASNYAESPWALSNWMSWVLPILSPLIPIFLLLLFGPCIFHLVSQFIQNRIQAITNHSI The amino acid sequence of the antigenic polypeptide in this embodiment is shown in SEQ ID NO: 2.

[0028] SEQ ID NO: 2:LDRSSKTSPDISHQC 2. Synthesis of antigenic polypeptides: The polypeptide shown in SEQ ID NO: 2 was synthesized using the Fmoc solid-phase polypeptide synthesis method. After synthesis, it was purified by HPLC (high performance liquid chromatography) to a purity of ≥95%, and then freeze-dried for later use.

[0029] 3. Preparation of immunogen (conjugate): Since SEQ ID NO: 2 is a small short peptide, direct immunization is unlikely to induce a strong immune response. Therefore, it is conjugated with a carrier protein to prepare an immunogen.

[0030] Carrier selection: Hemocyanin (KLH) was selected as the carrier protein.

[0031] Coupling process: Dissolve the pre-maleylated carrier protein in PBS buffer (pH 7.2); dissolve the synthesized antigen peptide (SEQ ID NO: 2) in the same buffer and quickly add it to the KLH solution, so that the molar ratio of antigen peptide to carrier protein is controlled between 30:1 and 50:1.

[0032] Reaction conditions: The mixture was stirred magnetically at room temperature in the dark for 2 hours. After the reaction, the reaction solution was placed in a dialysis bag (molecular weight cutoff 10 kD) and dialyzed in PBS for 24 hours (with the medium changed 3 times during the process) to remove uncoupled free peptides and small molecule byproducts, finally obtaining the antigen peptide-KLH conjugate, which is the immunogen. After aliquoting, it was stored at -80℃.

[0033] Example 2 This embodiment provides the preparation and titer determination of specific polyclonal antibodies. 1. Animal immunization: Two healthy New Zealand white rabbits (numbered A and B), female, weighing 2.0-2.5 kg, were selected.

[0034] Primary immunization: The immunogen (containing approximately 200 μg of antigenic polypeptide) prepared in Example 1 was fully emulsified with an equal volume of Freund's complete adjuvant to form a water-in-oil emulsion, which was then injected subcutaneously at multiple points on the back.

[0035] Booster immunization: Booster immunizations are administered every 2 weeks after the initial immunization. The immunogen (containing approximately 100 μg of antigenic peptide) is emulsified with an equal volume of Freund's incomplete adjuvant (FIA) and injected, for a total of 4 booster immunizations.

[0036] 2. Antibody purification: Ten days after the last immunization, blood was drawn from the heart, allowed to stand at room temperature to coagulate, and then centrifuged to separate the serum.

[0037] Step S1 (crude purity): Total IgG in serum is adsorbed using a Protein A / G affinity chromatography column.

[0038] Step S2 (purification): Prepare an agarose gel affinity chromatography column conjugated with the polypeptide shown in SEQ ID NO: 2. Pass the total IgG obtained in step S1 through the column. The specific antibody binds to the polypeptide on the column, while other proteins flow through. Elute the specific antibody with glycine-HCl buffer (pH 2.5) and immediately neutralize with Tris-HCl (pH 8.0). Dialyze to replace with PBS buffer to obtain a high-purity anti-HERVH capsid protein polyclonal antibody.

[0039] 3. Potency testing (indirect ELISA): Coating: Using the free polypeptide shown in SEQ ID NO: 2 as the antigen, dilute with carbonate buffer (pH 9.6) to 1 μg / mL, coat a 96-well microplate at 100 μL / well, and incubate overnight at 4°C.

[0040] Blocking and incubation: Block with 5% skim milk powder the next day. Add the purified antibody to be tested, which has been serially diluted (starting concentration 1 μg / mL, dilution range 1:1,000 to 1:512,000), and incubate at 37°C for 1 hour.

[0041] Color development: Add HRP-labeled goat anti-rabbit IgG (secondary antibody), incubate, add TMB substrate for color development, terminate the reaction with stop solution, and measure the OD450 value.

[0042] The results are shown in Table 1. Table 1 presents the ELISA detection results (OD450 values) of rabbit polyclonal antibodies prepared against the immunogenic antigen (SEQ ID NO:2, peptide antigen) at a series of dilution gradients, and gives the endpoint titer calculated according to the signal / blank (S / B) ≥ 2.1 standard. The first row of the table shows the theoretical antibody concentration (ng / ml) corresponding to the antigen coating; the second row shows the corresponding dilution factor (from 1:1,000 to 1:512,000); the third row shows the measured OD450 value (antibody sample); the bottom shows the OD450 value of the negative control (NC) at the corresponding dilution; Blank is the average OD value of the blank wells (without primary antibody). Note: The starting concentration is 1 μg / ml; titer is determined according to "highest dilution and S / B (Signal / Blank) ≥ 2.1" (S is sample OD450, B is blank OD450), and the blank OD450 is calculated using the average value of repeated tests.

[0043] Table 1 The results showed that at a dilution of 1:512,000, the ratio of the OD450 value (signal S) to the OD450 value (background B) of the blank control (S / B) was still greater than 2.1. The antibody maintained good binding activity even at extremely high dilutions, indicating that the prepared polyclonal antibody possesses extremely high titer (Titer > 1:500,000) and affinity.

[0044] The negative control (NC) had an endpoint titer of approximately 1:8,000 at the highest decision dilution, indicating that the antibody signal was significantly better than background / nonspecific binding after immunization.

[0045] The signal gradually decreased with dilution, and the curve shape was consistent with the expected antibody binding kinetics, supporting that the polyclonal antibody has high titer and good specificity.

[0046] The above method demonstrates that a polyclonal antibody with high affinity and good specificity for HERVH capsid protein can be obtained using only a 15-amino acid antigenic peptide starting from N115, providing a foundation for its subsequent application in clinical sample testing and functional studies.

[0047] Example 3 This embodiment provides the application of immunohistochemical detection of colorectal cancer tissue samples, and verifies the application value of the prepared antibody in clinical pathological diagnosis.

[0048] 1. Sample Source Surgically removed tumor tissue and corresponding adjacent normal tissue were collected from 96 patients diagnosed with colorectal cancer (CRC) and fabricated into a tissue microarray.

[0049] 2. Detection Method After dewaxing and hydration, the sections were subjected to high-temperature antigen retrieval using citrate buffer (pH 6.0).

[0050] Use 3% H2O2 to block endogenous peroxidase.

[0051] Add the polyclonal antibody prepared in Example 2 (dilution 1:500) dropwise and incubate overnight at 4°C.

[0052] The next day, after rewarming, HRP-labeled secondary antibody polymer was added, DAB staining was performed, and hematoxylin counterstaining was used to stain the cell nuclei.

[0053] 3. Result Determination The H-score scoring system was used (H-score = ∑(intensity × positive rate), range 0-300).

[0054] The results are as follows Figure 2 As shown: In adjacent normal tissue, HERVH capsid protein shows only extremely weak staining or is negative ( Figure 2 a).

[0055] In colorectal cancer tumor tissue, significant brownish-yellow granular deposits are visible, mainly located in the cytoplasm, with some cell membranes also showing staining. Figure 2 b).

[0056] Statistical analysis showed that the H-score of tumor tissue was significantly higher than that of adjacent normal tissue (P = 0.0183 < 0.05). Figure 2 c).

[0057] In summary, the antibody prepared by this invention can specifically recognize HERVH capsid protein in tissues and can clearly distinguish between cancerous tissues and normal tissues, making it suitable for the auxiliary diagnosis of colorectal cancer.

[0058] Example 4 This embodiment verifies the feasibility of HERVH capsid protein as a secretory marker. The purpose of this embodiment is to investigate whether the HERVH capsid protein can form virus-like particles and be secreted extracellularly, thereby verifying its potential as a biomarker for liquid biopsy.

[0059] 1. Cell model construction and Western blot detection HCT116 colorectal cancer cell line (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected, and ARID1A gene knockdown (KD) stable transgenic lines (KO1#, KO2#) and control line (WT) were constructed using CRISPRi technology.

[0060] Collect cell lysate and cell culture supernatant (and enrich exosome / particle components by ultracentrifugation).

[0061] The antibody prepared in Example 2 was used for Western blotting detection.

[0062] The results are as follows Figure 3 As shown, compared with the WT group, the HERVH capsid protein (HERVH-Env) in the ARID1A knockdown group was significantly upregulated both intracellularly and in the culture supernatant. Furthermore, CD81 (an exosome marker) was detected in the supernatant fraction, but Calnexin (an endoplasmic reticulum marker) was not detected, demonstrating that the detected HERVH protein was released via the secretory pathway, rather than through cell rupture contamination.

[0063] 2. Morphological and molecular verification of virus-like particles (VLPs) Digital droplet PCR: RNA was extracted from cell culture supernatant, reverse transcribed, and then quantified absolutely using ddPCR targeting a specific HERVH sequence. Results are as follows: Figure 4 a: The HERVH viral RNA copy number was significantly increased in the supernatant of ARID1A-deficient / HERVH-overexpressing cells.

[0064] Immunoelectron microscopy: Cells and concentrated culture supernatant samples were stained with gold immunochromatographic markers (primary antibody was the polyclonal antibody of this invention, and secondary antibody was a gold particle-labeled antibody). Results are as follows: Figure 4 b: Under an electron microscope, virus-like particles (VLPs) with a diameter of about 100 nm were observed to bud from the cell membrane surface or be free in the extracellular space, and gold particles were specifically attached to the surface of these particles.

[0065] The results are as follows Figure 3As shown, experiments demonstrate that HERVH capsid protein is not only highly expressed intracellularly in colorectal cancer (especially the ARID1A-deficient type), but can also assemble into virus-like particles and be secreted extracellularly. This indicates that the antibody of the present invention can not only be used in tissue biopsy (IHC), but also has the potential to develop non-invasive detection kits (such as ELISA kits) based on blood / serum / exosomes for detecting free HERVH protein or viral particles in liquid samples.

[0066] Example 5 This embodiment provides the assembly of a colorectal cancer diagnostic kit. Based on the above embodiments, this embodiment also provides a colorectal cancer diagnostic kit.

[0067] This kit contains: Capture / Detection Antibody: Purified polyclonal antibody prepared in Example 2 (concentration 1 mg / mL, containing 0.1% NaN3 preservative).

[0068] Auxiliary reagents (optional): including but not limited to HRP-labeled goat anti-rabbit IgG (secondary antibody), DAB chromogenic solution or TMB substrate solution, antigen retrieval solution (for IHC), ELISA coating buffer and washing solution, etc.

[0069] Instructions for use: This document describes the steps for detecting HERVH capsid protein levels in samples using IHC or ELISA methods with this antibody, as well as the criteria for determining a positive result.

[0070] This kit can be used in clinical pathology departments for staining and diagnosis of tissue sections from patients suspected of having colorectal cancer, or in research settings for detecting viral protein levels in patient serum / exosomes.

[0071] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. An antigenic polypeptide for preparing polyclonal antibodies that specifically bind to HERVH capsid proteins, characterized in that, The amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO:

2.

2. An immunogen for preparing polyclonal antibodies that specifically bind to the HERVH capsid protein, characterized in that, It is prepared using the antigenic polypeptide described in claim 1 as a hapten.

3. The immunogen according to claim 2, characterized in that, The immunogen is prepared by mixing the antigenic peptide described in claim 1 with a pre-maleylated carrier protein in a buffer solution at pH 6.5 to 7.5, wherein the molar ratio of the antigenic peptide to the carrier protein is 20:1 to 50:1; reacting at room temperature for 1 to 3 hours; and removing uncoupled antigenic peptides by dialysis or gel filtration after the reaction to obtain the immunogen.

4. A polyclonal antibody for the specific detection of HERVH capsid protein, characterized in that, It is prepared by immunizing non-human mammals with the immunogen described in claim 2 or 3.

5. The method for preparing the polyclonal antibody according to claim 4, characterized in that, The steps are as follows: S1. Immunize non-human mammals with the immunogen described in claim 2 or 3, and detect and screen immunized animals with high titers; S2. Collect whole blood from the selected immunized animals, separate serum, and use a solid-phase medium conjugated with the polypeptide shown in SEQ ID NO:2 for antigen affinity purification to obtain polyclonal antibodies.

6. The preparation method according to claim 5, characterized in that, The immunized animal is an immunized rabbit, the number of immunizations is 3 to 5, and the immunized animal selected for detection and screening refers to an immunized rabbit whose antigen polypeptide detection titer reaches 1:10000 or higher using the ELISA method.

7. The use of the polyclonal antibody of claim 4 in the preparation of products for the diagnosis, auxiliary diagnosis, prognosis assessment or monitoring of colorectal cancer.

8. The application according to claim 7, characterized in that, The product is used to detect the expression level of HERVH capsid protein in biological samples via immunological reactions, including ELISA, Western blot, or immunohistochemistry.

9. The application according to claim 8, characterized in that, The biological samples are selected from tumor tissue, adjacent normal tissue, blood, serum, plasma, exosomes, or cell culture supernatant.

10. A colorectal cancer diagnostic kit, characterized in that, The kit contains the polyclonal antibody as described in claim 4.

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