Small molecule peptides for detecting endogenous retroviral k capsid protein and applications thereof

Abstract

The application relates to a small molecule peptide for detecting an endogenous retrovirus K capsid protein and application thereof. The amino acid sequence of the small molecule peptide is shown as SEQ ID NO. 6. The designed HERV-K CA small molecule binding peptide is fused with nanoluciferase (Nanoluciferase, NLuc) for expression, can be specifically combined with the CA protein of the endogenous retrovirus K, and the affinity reaches 10 ‑7 M provides a solution for detecting the CA protein of a human serum or tissue sample, and has better detection efficiency compared with the existing HERV-K Gag antibody for immunodetection of the HERV-K CA protein of a human serum.

Description

Technical Field

[0001] This application relates to the field of endogenous retrovirus detection technology, and in particular to a small molecule peptide for detecting endogenous retrovirus K capsid protein (Capsid, CA) and its application. Background Technology

[0002] Endogenous retroviruses (ERVs) are a type of retrovirus widely found in nature. During species evolution, exogenous retroviruses infect and integrate the proviral sequence into the host's genome, passing it on to daughter cells during germ cell division, resulting in lifelong infection. HERV-K is the youngest, most structurally complete, and most potentially active type of endogenous retrovirus in the human genome. Its aberrant expression has significant specificity in various cancers, neurodegenerative diseases, and immune diseases. In 2023, Liu Guanghui's research group published data in Cell (Liu et al. 2023, DOI: 10.1016 / j.cell.2022.12.017) using HERV-K envelope protein Env antibody detection, finding significantly increased HERV-K Env levels in serum samples from elderly individuals, indicating that serum HERV-K levels are a potential candidate indicator of aging processes such as autoimmune diseases and neurodegenerative diseases. Therefore, HERV-K testing not only helps with early diagnosis, disease classification, and efficacy prediction, but may also become a new target for immunotherapy or targeted intervention.

[0003] The HERV-K molecular structure includes flanking long terminal repeat (LTR) sequences and a sequence encoding four retroviral functional genes. gag , pro , pol and env The open reading box. (Among them) gag The gene encodes the structural proteins of the virus, which are pro The gene-encoded protease hydrolyzes the protein into matrix (MA), ca (carboxylic acid), and nucleocapsid (NC). CA protein forms the outer shell of the retroviral capsid particle, homopolymerizing into fullerene structures to protect the viral RNA. CA protein includes an amino-terminal domain (CA-NTD) and a carboxyl-terminal domain (CA-CTD). The CA-NTD, located on the outer side of the viral capsid particle, is a key region involved in immune regulation and an ideal domain for candidate detection targets.

[0004] Currently, there are no commercially available detection products for the CA protein of HERV-K virus. The commercially available murine anti-HERVK-Gag monoclonal antibody from Austral Biologicals in the United States is generated by immunization with the precursor Gag protein of the MA, CA, and NC genes and is not used to detect the CA protein, which has a real spatial structure in HERV-K. Single envelope protein Env antibody detection can only reflect viral load. However, many pathological processes of HERV-K virus, such as activation, assembly, recognition, and immune escape, require more node proteins to coordinate with the Env system indicators. The CA protein is involved throughout the early stages of viral infection and the entire process of genome integration, and is a key protein in the interaction between the virus and the host. Therefore, the development of antibodies to detect it is of great significance for systematically characterizing changes in HERV-K activity.

[0005] Traditional hybridoma preparation methods for diagnostic reagents are characterized by long experimental cycles, high costs, and low efficiency. Denovo protein design technology generates complementary binding interfaces through large-scale protein data models, then recombinantly expresses the target protein, and finally performs in vitro validation. This technology allows for atomic-level precision customization of single-point mutant epitopes, eliminating the need for library construction and animal facilities. Targeting the complex spatial structure of the CA-NTD protein of HERV-K, it can rapidly obtain mg-level and pM-level affinity proteins for diagnostic reagent development.

[0006] NLuc is a genetically engineered, small luminescent enzyme derived from deep-sea bioluminescent shrimp, with a molecular weight of only 19.1 kDa. It uses a novel substrate, furimazine, to generate a high-intensity, continuous luminescent signal without the need for ATP, with a brightness approximately 100 times that of traditional firefly or reniform luciferases, and extremely low background luminescence. This application fuses a designed HERV-K CA small-molecule binding peptide with NLuc for expression, providing a solution for the detection of CA proteins in human serum or tissue samples for the first time. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a small molecule peptide for detecting endogenous retroviruses and its applications. This application employs de novo protein design technology, using a computationally driven reverse design strategy to generate small molecule binding peptides targeting antigenic epitopes, which are then recombinantly expressed and validated in vitro. Targeting the complex spatial structure of the CA-NTD protein of HERV-K, mg-level and pM-level affinity proteins can be rapidly obtained for diagnostic reagent development.

[0008] The first objective of this invention is to provide a small molecule peptide.

[0009] A second objective of this invention is to provide a fusion protein.

[0010] A third objective of this invention is to provide a biomaterial.

[0011] A fourth object of the present invention is to provide the application of the fusion protein or the biomaterial.

[0012] The fifth objective of this invention is to provide a method for detecting endogenous retroviruses or endogenous retrovirus CA proteins for non-diagnostic purposes.

[0013] The sixth object of the present invention is to provide an immunological detection kit for endogenous retroviruses or endogenous retrovirus CA proteins.

[0014] The seventh object of this invention is to provide a recombinant protein.

[0015] The eighth object of the present invention is to provide a biomaterial.

[0016] A ninth object of the present invention is to provide the application of the recombinant protein or the biomaterial.

[0017] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a solution for detecting CA protein in human serum or tissue samples by fusing a designed HERV-K CA small molecule binding peptide with NLuc for expression.

[0018] Therefore, this invention claims protection for a small molecule peptide whose amino acid sequence is shown in SEQ ID NO.6.

[0019] And, a fusion protein, which is a luciferase expressed fused to the N-terminus of the small peptide.

[0020] Preferably, the luciferase is luciferase NLuc.

[0021] As a specific implementation, its amino acid sequence is shown in SEQ ID NO.4.

[0022] This invention also claims protection for a biological material, which is any one of the following: (1) A nucleic acid molecule encoding the small molecule peptide or the fusion protein; (2) An expression cassette containing the nucleic acid molecule described in (1), or the small molecule peptide or the fusion protein; (3) A recombinant vector containing the nucleic acid molecule described in (1), the expression cassette described in (2), or expressing the small molecule peptide or the fusion protein; (4) A recombinant microorganism containing the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or expressing the small molecule peptide or the fusion protein; (5) A cell line containing the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or expressing the small molecule peptide or the fusion protein.

[0023] This invention also claims protection for the following applications: The small molecule peptide, the fusion protein, or the biomaterial is used for the detection of endogenous retroviruses for non-diagnostic purposes.

[0024] The application of the small molecule peptide, the fusion protein, or the biomaterial in the preparation of products for detecting endogenous retroviruses.

[0025] The small molecule peptide, the fusion protein, or the biomaterial is used for the detection of endogenous retroviral CA protein for non-diagnostic purposes.

[0026] The application of the small molecule peptide, the fusion protein, or the biomaterial in the preparation of products for detecting endogenous retroviral CA proteins.

[0027] The present invention also claims a method for detecting endogenous retrovirus or endogenous retrovirus CA protein for non-diagnostic purposes, wherein the small molecule peptide is used as a detection antibody for immunoassay of the sample to be tested.

[0028] The present invention also claims an immunological detection kit for an endogenous retrovirus or endogenous retrovirus CA protein, containing the small molecule peptide as a detection antibody.

[0029] Preferably, the immunological detection kit is an ELISA detection kit.

[0030] More preferably, it also contains ELISA detection reagents.

[0031] Another object of the present invention is to claim protection for a recombinant protein having the amino acid sequence shown in SEQ ID NO.2.

[0032] And a biological material, which is any one of the following: (1) The nucleic acid molecule encoding the recombinant protein; (2) Contains the nucleic acid molecule described in (1), or an expression cassette expressing the recombinant protein of claim 8; (3) Contains the nucleic acid molecule described in (1), the expression cassette described in (2), or a recombinant vector expressing the recombinant protein; (4) Contains the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or a recombinant microorganism expressing the recombinant protein; (5) Contains the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or a cell line expressing the recombinant protein.

[0033] And the application of the recombinant protein or the biomaterial, wherein the application is any one or more of the following: Mimicking the CA protein of the HERV-K virus; Screening for antibodies against HERV-K virus CA protein; Screening for antibodies against HERV-K virus; To evaluate the activity of the protein against HERV-K virus CA protein; To evaluate the protein's activity against HERV-K virus.

[0034] Compared with the prior art, the present invention has the following beneficial effects: This invention fuses a designed HERV-K CA small molecule binding peptide with NLuc for expression, enabling it to specifically bind to CA proteins of endogenous retroviruses. This provides a solution for the detection of CA proteins in human serum or tissue samples, offering higher sensitivity compared to existing antibodies used for the immunoassay of CA proteins. (1) The protein de novo design technology used in this invention is a purely computationally driven reverse design strategy. First, the target epitope is defined on a silicon wafer, and then the algorithm generates a complementary binding interface to recombinantly express the target protein. The results show that the HERV-KCA small molecule binding peptide has good affinity. This technology bypasses the immune system and library screening, can perform atomic-level precise customization, requires no library construction or animal facility, and has a short cycle.

[0035] (2) The antibody obtained by this invention is fused with Nluc for expression, which enables the new product to leverage the characteristics of Nluc, such as low molecular weight, high signal intensity (fluorescence signal is 100 times that of traditional luciferase (firefly)) and low detection background. A better signal-to-noise ratio for the detection protein is achieved through the fusion expression of Nluc.

[0036] (3) The present invention omits the chemical coupling step of antibody protein and labeling molecule, and directly solves the labeling problem by fusion expression of Nluc protein, which simplifies the process and reduces costs. Attached Figure Description

[0037] Figure 1 for Figure 1 HERV-K CA rec -NTD structural analysis; A: HERV-K CA rec -NTD's cartoon structure diagram; B:CA recCartoon diagram comparing the simulated AlphaFold3 structure of -NTD with its native protein PDB structure (pdb_00006sa9) (the copper-red part is the native protein structure).

[0038] Figure 2 For HERV-K CA rec Results of Coomassie Brilliant Blue staining and SDS-PAGE of NTD protein induced expression and affinity purification.

[0039] Figure 3 To design and generate small molecule binding peptides and Nluc fusion proteins from scratch; A: CA rec - NTD antigenic epitope prediction map (orange part is candidate antigenic epitopes); B: Coomassie Brilliant Blue SDS-PAGE results showing the induced expression and affinity purification results of the designed protein Nluc-Binder382; C: Nluc-Binder382 small molecule binding protein homoantigen HERV-KCA obtained by RosettaFold AI large model design and screening. rec NTD molecular docking diagram.

[0040] Figure 4 Affinity assay for the small molecule binding protein Nluc-Binder 382; A: ELISA serial dilution assay for Nluc-Binder 382 and HERV-K CA rec -NTD antigen protein binding efficacy; B: MST assay to determine Nluc-Binder382 and HERV-K CA rec - Affinity of NTD antigen proteins; C: BLI assay to determine the affinity of Nluc-Binder382 for HERV-K CA rec - Affinity constant of NTD antigen protein.

[0041] Figure 5 The results are for testing clinical samples; A: Test results of Example 5; B: Test results of Comparative Example 1; C: Test results of Comparative Example 2. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0043] Example 1 HERV-K CA rec Expression and purification of NTD sequences I. Design of recombinant substitutes for the native CA-NTD protein of HERV-K 1. The full-length sequence of the HERV-K CA protein precursor Gag protein (https: / / www.uniprot.org / uniprotkb / P63128) was used to extract the NTD (N-terminal domain) amino acid sequence of the CA protein, which is 153 amino acid residues long. AlphaFold3 (https: / / alphafoldserver.com / ) was used for structural prediction to obtain the CA protein sequence. rec NTD structural image (see) Figure 1 (A in the middle).

[0044] 2. CA rec The AlphaFold3 simulated structure of -NTD was compared with its native protein PDB structure (pdb_00006sa9). The structural deviation RMSD of 0.253 Å is within the acceptable range. AlphaFold3 also completes the missing amino acid terminal β-sheet secondary structure in the PDB structure file (see...). Figure 1 (B in the original text). It was used as a recombinant substitute for the native CA-NTD protein of HERV-K (HERV-K CA). rec The NTD (Non-Digital Transformer) is used for protein design and subsequent screening. Its nucleotide sequence is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2.

[0045] II. Expression of recombinant substitutes for the native CA-NTD protein of HERV-K 1. Experimental Methods HERV-K CA rec The DNA sequence of NTD (nucleotide sequence as shown in SEQ ID NO:1, and the amino acid sequence it encodes as shown in SEQ ID NO:2) was recombined into the NdeⅠ and HindⅢ sites of the multip cloning site (MCS) region of the pET-28a prokaryotic expression vector to obtain the recombinant expression vector.

[0046] The protein was further expressed in the E. coli system, purified by affinity chromatography using a nickel column, and then identified by SDS-PAGE. rec The IPTG induction and soluble expression of NTD proteins are shown in the following steps: (1) Transfer to E. coli Subsequently, 1 μl of the recombinant expression vector was transformed into 100 μl of BL21 competent cells, incubated at 4°C on ice for 20 min, heat-shocked at 45°C for 90 s, incubated at 4°C on ice for 2 min, and then 200 μl of LB liquid medium was added. After incubation at 220 rpm for 30 min, the culture was plated onto LB agar plates containing kanamycin and incubated overnight at 37°C. After incubation, single colonies were picked and added to 25 ml of LB liquid medium containing kanamycin, and amplified at 200 rpm. 200 μl of the bacterial culture was added to a 96-well plate, and the OD value was measured at 600 nm.

[0047] (2) IPTG induction When the OD value reached 0.6–0.8, IPTG was added to a final concentration of 0.3 mM to induce protein expression, and the induction was performed overnight at 25°C and 170 rpm. After induction, the bacterial culture was centrifuged at 4000 rpm for 40 min, and the bacterial weight was measured. The cells were resuspended in non-denaturing lysis buffer at a volume ratio of 1:50, and then sonicated on ice under the following conditions: 3 s sonication followed by 5 s rest for 30 min. After sonication, the entire solution was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. The supernatant was then filtered through a 0.2 μm filter membrane, and the filtrate was collected.

[0048] (3) His purification 4 mL of His-tag purification resin was loaded into a 50 mL centrifuge tube with an inner nested tube. The tube was first equilibrated with 2 column volumes of non-denaturing wash buffer (50 mM Tris, 500 mM NaCl, 10 mM imidazole). Then, the filtrate obtained in step (2) was added through the sample loading channel. The flow-through buffer was collected by adding the aforementioned non-denaturing buffer. Finally, 5 mL of elution buffer (50 mM Tris, 500 mM NaCl, 250 mM imidazole) was added for elution. The elution product was collected and dialyzed into PBS. After dialysis, the elution product was concentrated using a 3 kDa ultrafiltration tube to obtain the purified four candidate target small molecule binding peptides. The protein concentration of each fusion protein, Nluc-Binder, was determined using the BCA method and stored for later use.

[0049] The four purified fusion proteins Nluc-Binder were identified by SDS-PAGE and Coomassie Brilliant Blue staining.

[0050] 2. Experimental Results See results Figure 2 From left to right: Marker, HERV-K CA rec -NTD induction before induction, induction after induction, sonication supernatant, sonication precipitation, flow-through collection, elution collection (i.e., the final purified HERV-KCA)rec NTD protein). Results showed HERV-KCA rec The NTD sequence was successfully induced to be expressed and purified.

[0051] Example 2: De novo design and generation of small molecule binding peptides and Nluc fusion proteins I. Experimental Methods 1. Design of small molecule binding peptides Predict CA using the SEPPA3.0 database (http: / / www.badd-cao.net / seppa3 / submission.html) rec -NTD is a possible antibody-binding epitope; orange indicates the most likely amino acid residues (>0.2) (see...) Figure 3 (A in the middle).

[0052] For HERV-K CA rec NTD spatial epitopes were used to design 1,000 small molecule binding protein backbones and sequences using de novo protein design technology. Then, the spatial structures were simulated using AlphaFold3 and four candidate target small molecule binding peptides were obtained by screening using parameters such as pLDDT.

[0053] 2. Fusion expression and purification of four candidate target small molecule binding peptides with the Nluc gene The four candidate target small molecule binding peptides were fused with the Nluc gene and expressed to obtain the fusion protein Nluc-Binder.

[0054] (1) pET-28a-E.coli expression Using the pET-28a vector as a backbone vector, the coding gene of the fusion protein Nluc-Binder was ligated between the NdeⅠ and HindⅢ sites of the pET-28a vector to obtain four recombinant expression vectors.

[0055] The protein was then expressed in the E. coli system following the method described in Example 1, and purified by affinity chromatography using a nickel column. CA was then identified by SDS-PAGE. rec -IPTG-induced and soluble expression of NTD proteins.

[0056] II. Experimental Results SDS-PAGE and Coomassie Brilliant Blue staining results showed that all four candidate target small molecule binding peptides were successfully fused with Nluc for expression. Among them, the fusion protein named Nluc-Binder382 was expressed efficiently in a soluble form (see...). Figure 3(B) is used for subsequent experiments; its nucleotide sequence is shown in SEQ ID NO:3, and its encoded amino acid sequence is shown in SEQ ID NO:4. Nluc-Binder382 small molecule binding protein is an antigen HERV-KCA. rec See NTD molecular docking diagram. Figure 3 C in the sequence represents the nucleotide sequence encoding the small molecule binding peptide, as shown in SEQ ID NO:5, and its amino acid sequence is shown in SEQ ID NO:6.

[0057] Example 3: Fusion protein Nluc-Binder and HERV-KCA rec - Affinity for NTD antigen protein binding I. Luciferase Binding Analysis 1. Experimental Methods The antigen protein HERV-KCA prepared in Example 1 rec -NTD was coated into the wells of a 96-well white opaque microplate with high adsorption (4 μg / ml), 100 μl / well, and coated at 4°C for 24 hours. Then, serially diluted N-terminal fusion-expressing recombinant protein Nluc-Binder was added and incubated at 10 concentration gradients (40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.31 μM, 0.16 μM, 0.08 μM). Finally, the luciferase substrate Furimazine was added, and the fluorescence intensity was measured using a fluorescence spectrophotometer.

[0058] 2. Experimental Results The results show that ( Figure 4 (A) The fusion protein Nluc-Binder382 and HERV-K CA rec -NTD protein ELISA has a minimum detection concentration of less than 0.08 μM for serial dilutions.

[0059] II. MST Detection 1. Experimental Methods Using Monolith TM The RED-NHS second-generation protein labeling kit (NanoTemper, MO-L011) was used to label the antigen protein HERV-KCA prepared in Example 1 at a dye:protein molar ratio (MCR) of 3:1. rec -NTD is used for marking.

[0060] HERV-K CA tagged rec-NTD concentration was 40 nM, and the Nluc-Binder382 concentration prepared in Example 2 was 4 μM; 80 μl of HERV-K CA was taken. rec - Dilute the NTD to 20 nM and add 10 μl of 20 nM labeled HERV-KCA to PCR tubes 2 through 16. rec -NTD, add 10 μl of 40 nM HERV-K CA to PCR tube #1. rec -NTD and 10 μl of 4 μM Nluc-Binder382 were serially diluted at a 1:1 ratio. Finally, 20 μl samples were drawn by capillary tube and placed into a Monolith X Biomolecular Interaction Detector (NanoTemper) in order of concentration for analysis. The results were fitted with the target protein concentration (M) on the X-axis and the ratio of fluorescence after heating to fluorescence before heating (Fnorm) on the Y-axis to obtain the affinity constant (Kd) value.

[0061] 2. Experimental Results The results show that ( Figure 4 (B in the text), the fusion protein Nluc-Binder382 and HERV-K CA rec The kD value of the -NTD protein is 0.69 μM.

[0062] III. BLI Experiment 1. Experimental Methods The HERV-K CA prepared in Example 1 was labeled at room temperature using a biotinylation kit (Genemore, G-MM-IGT) at a molar ratio of NHS-biotin to target protein (MCR) of 1:1. rec -NTD 60 min, ultrafiltration was performed using a 3kD ultrafiltration tube to remove free biotin, and then the protein was quantified using the BCA method.

[0063] 96-well plate layout: Columns 1 and 3 contained 0.02% PBST buffer (for baseline and dissociation); column 2 contained 0.28 mg / ml biotinylated target protein (loading); column 4 contained the test protein Nluc-Binder382 (concentration gradient: 60 μM, 40 μM, 20 μM, 5 μM). The SSA sensor was hydrated in 0.02% PBST kinetic buffer for ≥20 min. Samples were analyzed using the Octet R8 label-free protein analysis system (Sartorius), and the data were processed to calculate the binding rate (ka), dissociation rate (kd), and affinity constant (Kd). Finally, graphs were generated using GraphPad Prism.

[0064] 2. Experimental Results The results show that ( Figure 4 (C) The fusion protein Nluc-Binder382 and HERV-K CA rec The kD value of the -NTD protein is 0.17 μM.

[0065] In summary, a large-scale AI model based on RFdifussion was designed for HERV-K CA. rec -NTD 10 -7 Small molecule binding proteins with M affinity, combined with the high signal and low background characteristics of the amino-terminal fusion protein Nluc, can achieve better detection efficiency for HERV-K CA proteins.

[0066] Example 4: A method for detecting endogenous retroviruses Serum sample coating of 96-well plate: Add serum sample to 96-well plate, 100 μl / well, incubate at 37°C for 2 hours, discard supernatant, wash 3 times with PBS, add blocking buffer, 200 μl / well, incubate at 37°C for 1 hour, then wash 3 times with PBS, discard supernatant, aspirate dry, seal and place at 4°C overnight for coating. Detection: After the overnight coated 96-well plates returned to room temperature, the fusion protein Nluc-Binder382 prepared in Example 2 was added to each well at 50 ng / well, and incubated at 37°C for 1 hour. The supernatant was discarded, and the plates were washed three times with PBS. Furimazine working solution (Nano-Glo® Luciferase Assay, Promega, USA) was added at 200 μl / well, and the plates were incubated at 37°C in the dark for 20 min. Fluorescence intensity was detected using a GloMax® Discover instrument (Promega, USA). The experimental data were then processed and standardized, and plotted using GraphPad Prism.

[0067] Example 5: Detection of Clinical Samples 1. Sample Source Serum samples were collected from 60 adults at the Physical Examination Center of the Second Affiliated Hospital of Guangzhou Medical University. Specific inclusion criteria were: 30 individuals aged 19–25 years and 30 individuals aged 65–90 years. Patients with malignant tumors, hyperthyroidism, undergoing thyroid hormone replacement therapy, severe infections, or burns / trauma were excluded from both groups. The patients were divided into a young adult group (15 males, 15 females) and an elderly group (15 males, 15 females).

[0068] 2. Sample testing The sample was tested according to the method in Example 4.

[0069] 3. Test Results The results showed that, compared with the younger group, the serum HERV-K CA protein content in the elderly group was significantly increased ( Figure 5 (A in the middle).

[0070] Comparative Example 1: Detection of Clinical Samples 1. Sample Source Same as Example 5.

[0071] 2. Sample testing HERV-K Env protein in clinical blood samples was detected using the Human HERV-K 7p22.1 (ERVK6) ELISA kit (Huamei Biotechnology, China). Standards were diluted (concentration gradient: 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, 0.625 ng / ml, 0.312 ng / ml, 0.156 ng / ml and 0 ng / ml).

[0072] Standards and test samples were coated onto 96-well plates, 30 wells for young adults and 30 wells for older adults. 100 μl of standard and test sample were applied to each well. The plates were mixed, covered, and incubated at 37°C for 2 hours. The liquid in the wells was discarded, the plates were dried, and no washing was performed. 100 μl of biotin-labeled antibody working solution was added to each well, and a new plate was applied. The plates were incubated at 37°C for 1 hour. The liquid in the wells was discarded, the plates were dried, and the plates were washed three times with 200 μl of washing buffer, soaking for 2 minutes each time. The plates were then dried. 100 μl of horseradish peroxidase-labeled avidin working solution was added, and a new plate was applied. The plates were incubated at 37°C for 1 hour. The liquid in the wells was discarded, the plates were dried, and the plates were washed five times, soaking for 2 minutes each time with 200 μl of washing buffer. The plates were then dried. 90 μl of substrate solution was added to each well sequentially, and the plates were incubated at 37°C in the dark for 15-30 minutes. Finally, 50 μl of reaction stop solution was added to each well to terminate the reaction. The absorbance (OD value) of each well at a wavelength of 450 nm was measured using an ELISA reader. The data were statistically analyzed and standardized, and plotted using GraphPad Prism.

[0073] 3. Test Results The results showed that, compared with the younger group, the serum HERV-K Env protein level in the elderly group was significantly increased ( Figure 5 The result (B) indicates an increase in HERV-K viral load in the subject's serum. However, Env antibodies do not reflect the assembly of capsid particles within the viral envelope or the interaction between capsid proteins and host factors.

[0074] Comparative Example 2: Examination of Clinical Samples 1. Sample Source Same as Example 5.

[0075] 2. Sample testing Following the method in Example 4, serum samples from the young adult group and the elderly group were coated into 96-well plates, respectively.

[0076] After overnight coating, the 96-well plates were brought to room temperature. Then, 100 μl of commercially available HERV-K Gag protein (CA precursor protein) IgG monoclonal antibody (Austral Biologicals, USA) was added to each well (1:2000 dilution). The plates were incubated at 37°C for 1 hour, the supernatant was discarded, and the plates were washed three times with PBS. Next, 200 μl of horseradish peroxidase-labeled secondary antibody (APExBIO, USA) was added to each well, and the plates were incubated at 37°C for 1 hour. The supernatant was discarded, and the plates were washed three times with PBS. Finally, 90 μl of TMB chromogenic buffer (ImiJet Scientific, China) was added to each well, and the plates were incubated at room temperature for 3 minutes. Stop solution (ImiJet Scientific, China) was then added to each well (50 μl). The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The data were statistically analyzed and standardized, and plotted using GraphPadPrism.

[0077] 3. Test Results The results showed no significant difference in serum HERV-K Gag protein levels between the young adult group and the elderly group. Figure 5 (C in the text). The above data indicate that monoclonal antibodies prepared using Gag protein, the precursor of HERV-K CA protein, as an immunogen cannot reflect the differences in serum HERV-K CA protein levels between young and elderly groups.

Claims

1. A small molecule peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

6.

2. A fusion protein, characterized in that, It involves fusing luciferase to the N-terminus of the small molecule peptide described in claim 1.

3. A biomaterial, characterized in that, It is any one of the following: (1) A nucleic acid molecule encoding the small molecule peptide of claim 1 or the fusion protein of claim 2; (2) An expression cassette containing the nucleic acid molecule described in (1), or expressing the small molecule peptide of claim 1 or the fusion protein of claim 2; (3) A recombinant vector containing the nucleic acid molecule described in (1), the expression cassette described in (2), or expressing the small molecule peptide described in claim 1 or the fusion protein described in claim 2; (4) A recombinant microorganism containing the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or expressing the small molecule peptide of claim 1 or the fusion protein of claim 2; (5) A cell line containing the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or expressing the small molecule peptide of claim 1 or the fusion protein of claim 2.

4. The application of the small molecule peptide of claim 1, the fusion protein of claim 2, or the biomaterial of claim 3, characterized in that, The application is any one or more of the following: Detection of endogenous retroviruses for non-diagnostic purposes; Applications in the preparation of products for detecting endogenous retroviruses; Detection of endogenous retroviral CA protein for non-diagnostic purposes; Application in the preparation of products for detecting endogenous retroviral CA proteins.

5. A method for detecting endogenous retroviruses or endogenous retroviral CA proteins for non-diagnostic purposes, characterized in that, The small molecule peptide described in claim 1 was used as a detection antibody for immunoassay of the sample to be tested.

6. An immunological detection kit for endogenous retrovirus or endogenous retrovirus CA protein, characterized in that, The small molecule peptide described in claim 1 is used as a detection antibody.

7. The immunological detection kit according to claim 6, characterized in that, The immunological detection kit is an ELISA detection kit.

8. A recombinant protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

9. A biomaterial, characterized in that, It is any one of the following: (1) A nucleic acid molecule encoding the recombinant protein of claim 8; (2) Containing the nucleic acid molecule described in (1), or an expression cassette expressing the recombinant protein of claim 8; (3) Contains the nucleic acid molecule described in (1), the expression cassette described in (2), or a recombinant vector expressing the recombinant protein of claim 8; (4) Containing the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or a recombinant microorganism expressing the recombinant protein of claim 8; (5) Contains the nucleic acid molecule described in (1), the expression cassette described in (2), the recombinant vector described in (3), or a cell line expressing the recombinant protein of claim 8.

10. The application of the recombinant protein of claim 8 or the biomaterial of claim 9, characterized in that, The application is any one or more of the following: Mimicking the CA protein of the HERV-K virus; Screening for antibodies against HERV-K virus CA protein; Screening for antibodies against HERV-K virus; To evaluate the activity of the protein against HERV-K virus CA protein; To evaluate the protein's activity against HERV-K virus.

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