Human bocavirus broad-spectrum recombinant antigen, test strip and preparation and application thereof

Abstract

The present application relates to a kind of human bocavirus broad-spectrum recombinant antigen, detection test paper and its preparation and application, to solve the technical problem of lack of simultaneous detection of multiple HBoV genotype antibody fast, simple diagnostic tool in prior art.The core of technical solution is: based on the design of broad-spectrum recombinant antigen (rVP1) of multiple types of common conserved epitope, and it is applied to the construction of colloidal gold immunochromatography test paper.The test paper will be marked in colloidal gold particles as detection probe, by indirect method reaction principle, can realize the synchronous detection of the antibody produced by four main genotypes of HBoV infection in 15-20 minutes.This scheme has the advantages of simple operation, without professional equipment, result is interpreted intuitively, while having good broad-spectrum, sensitivity and specificity, suitable for primary medical institutions rapid screening, field epidemiological investigation and vaccine immune effect evaluation and a variety of application scenarios.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to a human bocavirus broad-spectrum recombinant antigen, a test strip, and their preparation and application. Background Technology

[0002] Human Bocavirus ( Human Bocavirus HBoV (Hyperbovirus) was first discovered in 2005 in the nasopharyngeal secretions of children with lower respiratory tract infections and has since been detected globally. HBoV belongs to the family Parvoviridae, genus Bocavirus. It is a non-enveloped, single-stranded linear DNA virus whose genome encodes multiple proteins, including capsid proteins VP1–VP3. Currently, four HBoV genotypes (HBoV 1–4) have been identified. HBoV 1 is the main type causing respiratory infections (such as pneumonia and bronchiolitis) and its clinical importance is increasingly prominent; while HBoV 2–4 are more associated with gastrointestinal infections. Epidemiological studies show that HBoV infection is particularly common in infants and young children and can cause severe illness and even death. However, there are currently no clinically approved specific antiviral drugs or vaccines. Therefore, early and accurate diagnosis of HBoV infection is crucial.

[0003] Existing HBoV detection methods mainly include the following categories: (1) Morphological and culture methods: such as electron microscopy and virus isolation and culture. Although electron microscopy can directly observe the morphology of the virus, the equipment is expensive, the operation is complicated and the sensitivity is limited, so it is not suitable for clinical application. Virus isolation and culture is greatly limited due to the difficulty of in vitro culture of HBoV and the immaturity of animal models.

[0004] (2) Molecular biological detection method: Real-time quantitative PCR (qPCR) is the main method and is currently the most commonly used nucleic acid diagnostic method in clinical practice. However, HBoV viral DNA can persist in the body for a long time after infection and may also be detected in asymptomatic individuals. Therefore, it is difficult to distinguish between acute infection, past infection or asymptomatic carrier based solely on a positive PCR test result, which carries the risk of misdiagnosis and cannot be used as the sole basis for diagnosing acute infection.

[0005] (3) Serological testing: By detecting specific antibodies against HBoV (such as IgM and IgG) in the patient's serum, the immune response status of the body can be reflected, which helps to determine recent or past infection and is an important means to make up for the inadequacy of nucleic acid testing. However, existing serological methods (such as ELISA) are usually cumbersome and time-consuming, and most of them are for single genotypes. There is a lack of broad-spectrum and rapid detection products that can simultaneously cover antibodies against different HBoV genotypes (HBoV 1-4).

[0006] In summary, the existing technologies have the following main defects or problems to be solved: ① There is a lack of convenient and rapid on-site diagnostic tools to distinguish acute human bocavirus infection; ② Existing serological testing methods have limitations in coverage, making it difficult to achieve simultaneous and broad-spectrum detection of antibodies against different human bocavirus genotypes; ③ Existing rapid diagnostic technologies (such as some test strips) need to be improved in terms of broad-spectrum and specificity.

[0007] Therefore, developing a rapid detection product for human bocavirus antibodies that is easy to operate, fast, highly specific, and broad-spectrum is of urgent need and great significance for the early diagnosis of the virus, epidemiological investigation, screening of susceptible populations, and evaluation of vaccine efficacy.

[0008] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide a broad-spectrum recombinant antigen for human bocavirus, a test strip, and its preparation and application, to address the problem of the lack of diagnostic tools in the prior art that can rapidly, conveniently, and broadly detect human bocavirus antibodies. This test strip is a colloidal gold immunochromatographic test strip capable of simultaneously detecting different genotypes of human bocavirus, and has the advantages of being simple, rapid, sensitive, and accurate, enabling real-time on-site detection.

[0010] The first aspect of the present invention discloses a capture antigen rVP1, which is a recombinant human bocavirus (HBoV) VP1 fusion protein, the amino acid sequence of which includes at least two conserved B-cell epitope regions of human bocavirus HBoV1-4 VP1 proteins linked by flexible linker peptides, wherein the conserved B-cell epitope regions are regions with ≥80% sequence identity in HBoV1-4 as determined by multiple sequence alignment.

[0011] In some embodiments disclosed in this invention, the flexible linker peptide is (GGGGS). n Where n≥1; and / or, the C-terminus or N-terminus of the fusion protein further comprises a tag protein.

[0012] In some embodiments disclosed in this invention, the amino acid sequence of the capture antigen rVP1 is shown in SEQ ID NO.1, or the coding gene sequence of the capture antigen rVP1 is shown in SEQ ID NO.2.

[0013] A second aspect of this invention discloses a broad-spectrum antibody test strip for human bocavirus, comprising a support substrate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially laminated and fixed on the support substrate. The nitrocellulose membrane pad is provided with a detection line and a control line. The detection line is coated with a protein capable of specifically binding to the human IgG Fc fragment, and the control line is coated with a monoclonal antibody against the capture antigen. The conjugate pad is loaded with colloidal gold-labeled capture antigen rVP1.

[0014] In some embodiments disclosed in this invention, the protein that can specifically bind to the human IgG Fc fragment is staphylococcal protein A, streptococcal protein G, or a functional fragment thereof.

[0015] A third aspect of this invention relates to a method for preparing the capture antigen rVP1 according to any one of the claims, comprising the following steps: (1) Based on multiple sequence alignment of HBoV1-4 type VP1 protein amino acid sequences, conserved B cell epitope regions were screened, and rVP1 protein amino acid sequences were designed by tandem flexible linker peptides and adding purification tags. (2) Optimize the nucleotide sequence encoding the rVP1 protein according to the codon preference of eukaryotic cells, and construct a eukaryotic expression vector; (3) The eukaryotic expression vector is transfected into mammalian cells for expression, and purified by ion exchange chromatography and affinity chromatography to obtain the final product.

[0016] The fourth aspect disclosed in this application relates to a method for preparing the aforementioned human bocavirus broad-spectrum antibody test strip, comprising the following steps: (1) The captured antigen rVP1 was coupled with colloidal gold and sprayed onto a glass fiber membrane to prepare a binding pad; (2) Prepare monoclonal antibodies by immunizing animals with the captured antigen rVP1, and spray the monoclonal antibodies and proteins that can specifically bind to the human IgG Fc fragment onto nitrocellulose membranes to form control lines and detection lines; (3) Assemble the nitrocellulose membrane, conjugate pad, sample pad and absorbent pad in the order of lamination on the support base plate and cut them into test strips.

[0017] The fifth aspect of this invention discloses a kit for diagnosing human bocavirus (HBoV) infection, comprising the capture antigen rVP1 as described in any one of the claims, or the human bocavirus broad-spectrum antibody test strip as described in any one of the claims.

[0018] The sixth aspect of this invention relates to the use of the capture antigen rVP1 as described in any one of the claims in the preparation of products for the detection of human bocavirus antibodies.

[0019] In some embodiments disclosed in this invention, the human bocavirus antibody detection includes: auxiliary diagnosis of acute human bocavirus infection, serological screening of susceptible populations, or evaluation of the immunization effect of human bocavirus vaccine.

[0020] One or more technical solutions provided in the embodiments of the present invention have at least one of the following technical effects or advantages: 1. Broad-spectrum and efficient detection capability: A modified rVP1 protein, incorporating conserved VP1 sequences from different HBoV genotypes, was creatively designed and expressed as the core capture antigen. This recombinant antigen retains the common specific B-cell epitopes of HBoV 1–4 to the maximum extent, enabling the test strip based on it to simultaneously recognize and bind to specific antibodies against different HBoV genotypes. This achieves broad-spectrum detection of HBoV antibodies, solving the problem of narrow genotype coverage in existing methods and avoiding the cumbersome process of separate detection for each genotype. Detection efficiency is significantly improved while reducing reagent consumption and labor costs. Furthermore, the specific binding of antigen and antibody, along with the use of a eukaryotic system to ensure the correct spatial conformation of the protein, guarantees high detection specificity.

[0021] 2. Enhanced convenience, speed, and applicability of testing: Utilizing colloidal gold immunochromatography technology, the system systematically integrates a broad-spectrum antigen (colloidal gold-rVP1) with SPA (T-line) and rVP1 monoclonal antibody (C-line) on the detection membrane. This design eliminates the need for specialized equipment and complex operations, enabling testing to be completed within 15-20 minutes. Results are interpreted intuitively and clearly, significantly lowering the barrier to entry and making it suitable for diverse scenarios such as primary healthcare institutions, on-site screening, and home self-testing.

[0022] 3. Ensures the accuracy and reliability of detection: rVP1 is easily purified by a two-step chromatography method of cation exchange and nickel affinity, thereby improving high sensitivity and reducing non-specific reactions; the quality control line (C line) composed of rVP1 monoclonal antibody is set on the NC membrane, which can effectively indicate whether the test strip itself is effective and whether the chromatography process is normal, avoiding false negative judgments caused by test strip failure or improper operation, and greatly improving the accuracy and reliability of the test results.

[0023] 4. Excellent application value and economic benefits: By combining broad-spectrum detection capabilities with a rapid chromatography platform, a single sample can be tested to screen for multiple HBoV genotypes, improving detection efficiency and saving testing costs and time. This test strip can not only be used for early auxiliary diagnosis of HBoV infection and screening of susceptible populations, but also provides a simple technical means for evaluating the immunization efficacy of HBoV vaccines, demonstrating broad clinical application prospects and promotional value. Attached Figure Description

[0024] Figure 1 This is the result of amino acid sequence alignment of VP1 protein of four different genotypes of human bocavirus in one embodiment of the present invention.

[0025] Figure 2 This is a structural diagram of the modified rVP1 protein in one embodiment of the present invention.

[0026] Figure 3 This is a schematic diagram of a human bocavirus broad-spectrum antibody test strip in one embodiment of the present invention, wherein A: test strip structure diagram, 1-sample pad, 2-conjugation pad, 3-nitrocellulose membrane, 4-absorbent pad, 5-support base plate, 6-control line, 7-detection line; B: schematic diagram of the results of different sample detection. Detailed Implementation

[0027] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0028] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0029] Example 1: Expression and purification of HBoV rVP1 protein

[0030] HBoV capsid protein VP1 contains multiple B-cell epitopes, which can be used as detection targets in the development of HBoV detection reagents. Therefore, this invention selects to express recombinant VP1 (rVP1) protein in HEK293F cells. Specifically, the preparation of rVP1 includes the following steps: 1. Bioinformatics analysis and modification of HBoV VP1 protein

[0031] The VP1 amino acid sequences of 52 different HBoV genotypes published in NCBI were compared and analyzed. The results were as follows: Figure 1 The N-terminal amino acid sequences from positions 1 to 130 and from positions 340 to 671 were relatively conserved. Therefore, the major amino acid sequences from positions 1 to 130 and from positions 340 to 671 (i.e., the amino acids with the highest frequency in the alignment results) were selected, tandem with a flexible peptide (GGGGS)2, and an HIS tag (8×His) was added to the C-terminus. This yielded the modified capture antigen rVP1 amino acid sequence, as shown in SEQ ID NO. 1. The structure of the modified capture antigen rVP1 is as follows. Figure 2As shown in Table 1, the physicochemical properties of the modified capture antigen rVP1 were analyzed using bioinformatics. The results show that the isoelectric point of the modified capture antigen rVP1 was 9.06, the molecular weight was 53616.51, and the extinction coefficient (M) was [missing information]. -1 cm -1 The molecular formula is C66665, the instability index (II) is 39.10, the fatty acid index is 60.19, the total average hydrophilicity is -0.649, and the molecular formula is C66665. 2394 H 3640 N 670 O 698 S 20 .

[0032] Table 1 Physicochemical properties analysis of HBoV rVP1 protein

[0033] 2. Expression and purification of rVP1 protein

[0034] (1) Construction of recombinant expression vector: Based on the codon preference of HEK293F cells, the amino acid sequence of the modified capture antigen rVP1 was reverse transcribed into the gene sequence of the modified capture antigen rVP1, as shown in SEQ ID NO. 2. The gene sequence of the modified capture antigen rVP1 was constructed into the pcDNA3.1 eukaryotic expression vector using a double enzyme digestion method, with EcoRI as the upstream restriction enzyme site and XhoI as the downstream restriction enzyme site. The constructed recombinant expression vector pcDNA3.1-rVP1 was transformed into DH5α competent cells, single clones were picked, expanded cultured, and the recombinant expression plasmid pcDNA3.1-rVP1 was extracted at a concentration of 826 ng / μL.

[0035] (2) Expression of rVP1 protein: HEK293F cells were transfected with the recombinant expression plasmid pcDNA3.1-rVP1 to induce the expression of the capture antigen rVP1 in vitro. After culturing for 72 h, HEK293F cells were collected by centrifugation, resuspended in PBS, and sonicated. The supernatant was then centrifuged and Western blot was performed for identification.

[0036] (3) Purification of rVP1 protein: The supernatant after sonication was subjected to SP Sepharose FF cation exchange chromatography and High Affinity Ni-Charged Resin FF nickel affinity chromatography, respectively. The purification results were analyzed by 12% SDS-PAGE. The purified rVP1 protein was stored at -20℃.

[0037] Example 2: Preparation of Monoclonal Antibodies 1. Animal immunization

[0038] (1) Add Freund's complete adjuvant to the immunogen rVP1 protein, emulsify it and use it for the first immunization; (2) Two female BALB / c mice aged 4-8 weeks were immunized by subcutaneous injection at multiple points on the back, with an immunization dose of 10 μg / mouse; (3) Every two weeks, BALB / c mice were immunized with Freund's incomplete adjuvant and the immunogen was emulsified, and then immunized with the same method and dosage. A total of 4 immunizations were performed. (4) After the fourth immunization, blood was collected from the tail vein to determine the titer of the specific antibody against rVP1 protein. Mice with high titers were selected and BALB / c mice were given a super-immunization with an adjuvant-free immunogen by tail vein injection 3 to 4 days before cell fusion. The immunization dose was 50 μg / mouse.

[0039] 2. Cell fusion

[0040] Using polyethylene glycol, spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 at a ratio of 7:1. The fused cells were then screened using HAT selective medium. Ten days after fusion, positive hybridoma cells were initially screened using indirect ELISA with rVP1 protein as the coating antigen.

[0041] 3. Subcloning

[0042] Subcloning of positive wells was performed using the limiting dilution method, as follows: Dilute the above-mentioned positive hybridoma cells to approximately 10 cells / mL with complete culture medium. Add 100 μL to each well of a 96-well plate pre-coated with 100 μL of feeder cells and incubate at 37°C in a 5% CO2 incubator for 6–8 days. Further screen for positive hybridoma cells using indirect ELISA. Perform 2–3 subcloning cycles until a hybridoma cell line stably secreting anti-rVP1 monoclonal antibody is obtained. Measure the cell supernatant titer using indirect ELISA. Select the positive monoclonal hybridoma cell line with the highest titer (1A9) for expansion culture at 1–2 × 10⁻⁶ cells / mL. 6 Cells / tubes are cryopreserved.

[0043] 4. Preparation of monoclonal antibodies

[0044] Monoclonal antibodies were prepared using an in vivo ascites induction method, and the procedure is as follows: Multiparous female BALB / c mice were selected and injected intraperitoneally with 500 μL of Freund's incomplete adjuvant. One week later, they were injected intraperitoneally again with 2 × 10⁻⁶ 1A9 hybridoma cells. 5 One cell was used to extract ascites fluid after the mouse abdomen swelled. The supernatant was collected after centrifugation and was the ascites-type monoclonal antibody.

[0045] 5. Purification of monoclonal antibodies

[0046] The ascites fluid was purified using the ammonium octanoate-sulfate method, and the procedure is as follows: (1) Take 5 mL of monoclonal antibody ascites fluid, add 5 mL of PBS buffer, and then add 2.5 mL of saturated ammonium sulfate solution dropwise to make an ammonium sulfate solution with a final concentration of 20%. Stir while adding the solution and mix thoroughly. Let stand for 30 min.

[0047] (2) Centrifuge at 8000 r / min for 20 min and discard the precipitate to remove fibrin.

[0048] (3) Add 12.5 mL of saturated ammonium sulfate solution to the supernatant, mix thoroughly, and let stand for 30 min.

[0049] (4) Centrifuge at 8000 r / min for 20 min and discard the supernatant.

[0050] (5) Add 10 mL of PBS buffer to the precipitate to dissolve it, then add 5 mL of saturated ammonium sulfate solution to make a 33% ammonium sulfate solution. Mix well and let stand for 30 min.

[0051] (6) Centrifuge at 8000 r / min for 20 min, discard the supernatant to remove albumin.

[0052] (7) Repeat step 5, 2 to 3 times.

[0053] (8) Dissolve the precipitate with 5 mL of PBS buffer, put it into a dialysis bag, dialyze with PBS buffer at 4°C, and change the buffer 4 times.

[0054] (9) Centrifuge at 8000 r / min for 20 min, discard the precipitate, and the supernatant is the purified antibody. After measuring the antibody concentration, store at -20℃.

[0055] Example 3: Preparation and application of a broad-spectrum antibody test strip for human bocavirus.

[0056] This embodiment provides a broad-spectrum antibody test strip for human bocavirus, which contains the human bocavirus rVP1 protein and its 1A9 monoclonal antibody provided by the present invention.

[0057] 1. Preparation of colloidal gold

[0058] This example uses the trisodium citrate reduction method to prepare colloidal gold. The specific steps are as follows: (1) Measure 190 mL of ultrapure water and pour it into a 200 mL clean Erlenmeyer flask. Place it on a magnetic stirrer and heat and stir at the same time.

[0059] (2) Add 2 mL of 1% chloroauric acid solution to the above-mentioned Erlenmeyer flask, and after heating and boiling, quickly add 3.2 mL of 1% trisodium citrate solution.

[0060] (3) Continue heating and stirring, and observe the color change of the solution. When the color changes from light yellow to wine red and no longer changes, start timing for 5 minutes. After the time is up, stop heating.

[0061] (4) After the solution returns to room temperature, dilute it to 200 mL with ultrapure water and store it at 4°C for later use.

[0062] 2. Colloidal gold labeling of rVP1 protein

[0063] (1) Take 10 mL of colloidal gold solution, add 120 μL of 0.2 M K2CO3 solution, and mix well.

[0064] (2) The rVP1 protein developed in this invention was serially diluted in a dilution cup using ddw, with a total volume of 30 μL.

[0065] (3) Add 125 μL of colloidal gold solution to each well and react at room temperature for 5 min.

[0066] (4) Add 125 μL of 10% NaCl solution to each well and observe the color change of the solution.

[0067] (5) When the color changes from wine red to purplish red and then to blue, select the rVP1 dilution ratio corresponding to the previous well where the color changes to purplish red, which is the optimal labeling dilution ratio for rVP1.

[0068] (6) Dilute the rVP1 protein with ddw in a 1.5 mL sterile centrifuge tube according to the optimal labeling dilution ratio determined above. The total system is then expanded to 240 μL.

[0069] (7) Add 1 mL of the above pH-adjusted colloidal gold solution to the diluted rVP1 protein, vortex mix, place on a horizontal shaker, and label at room temperature for 0.5 h.

[0070] (8) Add 137 μL of 10% BSA solution to the above reaction mixture to make the final BSA concentration 1%. After mixing, place it on a horizontal shaker and seal it at room temperature for 0.5 h.

[0071] (9) After centrifuging at 12,000 rpm for 30 min at 4℃, discard the supernatant and resuspend the precipitate with 100 μL of borate buffer to obtain a 10-fold concentrated gold-labeled monoclonal antibody, which is stored at 4℃ for later use.

[0072] 3. Development of antibody test strips

[0073] First, colloidal gold-labeled rVP1 was sprayed onto the conjugation pad at a speed of 7 μL / cm. Then, 1 mg / mL SPA solution and 1 mg / mL 1A9 monoclonal antibody were streaked onto the detection line (T line) and control line (C line) of the NC membrane at a speed of 1 μL / cm. The above materials were then assembled into a test strip, as shown below. Figure 3 As shown in Figure A, first attach the NC membrane to the center of the support base plate. Then, attach the bonding pad and sample pad coated with gold label rVP1 to the sample end of the NC membrane in sequence, with each layer overlapping by 1-2 mm. Next, attach the absorbent pad to the other end of the NC membrane, overlapping it by 1-2 mm. Finally, use a strip cutter to cut it into 4 mm wide test strips, dry and seal them for storage.

[0074] Result Interpretation: Add the sample to the sample pad and perform chromatography at room temperature for 10 minutes. If both the C and T lines show color, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the interpretation is invalid (e.g., ...). Figure 3 As shown in B).

[0075] 4. Testing of clinical samples

[0076] The human bocavirus broad-spectrum antibody test strip prepared in this example was applied to the detection of clinical samples. Positive sera of human bocavirus types 1-4, as well as positive sera of influenza virus, respiratory syncytial virus, rhinovirus, parainfluenza virus, adenovirus, coronavirus, metapneumovirus, enterovirus 71, coxsackievirus A16, coxsackievirus A10, coxsackievirus A6, coxsackievirus A21, rotavirus, and norovirus were detected. The test results showed that the human bocavirus broad-spectrum antibody test strip of this invention has high sensitivity, specificity, and broad spectrum. The test strip can detect positive sera of four different genotypes of bocavirus and does not cross-react with positive sera of other respiratory and enteric viruses.

[0077] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of its inventive concept. Therefore, if these modifications and variations of the invention fall within the scope of the claims of this application and their equivalents, the invention is also intended to include these modifications and variations.

Claims

1. A method for capturing antigen rVP1, characterized in that, It is a recombinant human bocavirus (HBoV) VP1 fusion protein, whose amino acid sequence contains at least two conserved B-cell epitope regions of the human bocavirus HBoV1-4 VP1 protein linked by a flexible linker peptide. The conserved B-cell epitope regions are regions with ≥80% sequence identity in HBoV1-4 as determined by multiple sequence alignment.

2. The capture antigen rVP1 according to claim 1, characterized in that, The flexible linker peptide is (GGGGS). n Where n≥1; and / or, the C-terminus or N-terminus of the fusion protein further comprises a tag protein.

3. The capture antigen rVP1 according to claim 1 or 2, characterized in that, The amino acid sequence of the capture antigen rVP1 is shown in SEQ ID NO.1, or the gene sequence encoding the capture antigen rVP1 is shown in SEQ ID NO.

2.

4. A broad-spectrum antibody test strip for human bocavirus, comprising a support base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially laminated and fixed on the support base plate, wherein the nitrocellulose membrane pad is provided with a detection line and a control line, characterized in that, The detection line is coated with a protein capable of specifically binding to the human IgG Fc fragment, the control line is coated with a monoclonal antibody against the capture antigen of claim 1, and the binding pad is loaded with colloidal gold-labeled capture antigen rVP1 of claim 1.

5. The human bocavirus broad-spectrum antibody test strip according to claim 4, characterized in that, The protein that can specifically bind to the human IgG Fc fragment is staphylococcal protein A, streptococcal protein G, or a functional fragment thereof.

6. The method for preparing the capture antigen rVP1 according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Based on multiple sequence alignment of HBoV1-4 type VP1 protein amino acid sequences, conserved B cell epitope regions were screened, and rVP1 protein amino acid sequences were designed by tandem flexible linker peptides and adding purification tags. (2) Optimize the nucleotide sequence encoding the rVP1 protein according to the codon preference of eukaryotic cells, and construct a eukaryotic expression vector; (3) The eukaryotic expression vector is transfected into mammalian cells for expression, and purified by ion exchange chromatography and affinity chromatography to obtain the final product.

7. The method for preparing the human bocavirus broad-spectrum antibody test strip according to claim 4 or 5, characterized in that, Includes the following steps: (1) The captured antigen rVP1 was coupled with colloidal gold and sprayed onto a glass fiber membrane to prepare a binding pad; (2) Prepare monoclonal antibodies by immunizing animals with the captured antigen rVP1, and spray the monoclonal antibodies and proteins that can specifically bind to the human IgG Fc fragment onto nitrocellulose membranes to form control lines and detection lines; (3) Assemble the nitrocellulose membrane, conjugate pad, sample pad and absorbent pad in the order of lamination on the support base plate and cut them into test strips.

8. A kit for diagnosing human bocavirus (HBoV) infection, characterized in that, The test strip contains the capture antigen rVP1 as described in any one of claims 1-3, or the human bocavirus broad-spectrum antibody test strip as described in any one of claims 4-5.

9. The use of the capture antigen rVP1 according to any one of claims 1-3 in the preparation of a product for the detection of human bocavirus antibodies.

10. The application according to claim 9, characterized in that, The human bocavirus antibody test includes: auxiliary diagnosis of acute human bocavirus infection, serological screening of susceptible populations, or evaluation of the immunization effect of human bocavirus vaccine.

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