Recombinant antigen protein for detecting porcine circovirus type 3 as well as preparation method and application of recombinant antigen protein

Abstract

The invention provides a recombinant antigen protein for detecting porcine circovirus type 3 as well as a preparation method and application of the recombinant antigen protein. The recombinant antigen protein has an amino acid sequence as shown in SEQ ID NO.2; or the protein is derived protein which is obtained by replacing, increasing or truncating an amino acid sequence shown as SEQ ID NO.2 and has the capability of combining with a porcine circovirus type 3 antibody. The ELISA detection kit containing the recombinant antigen protein is simple and convenient to operate, good in specificity and high in sensitivity, can be popularized on a large scale and is used for detecting the recombinant antigen protein of PCV3 and a detection product with the antigen protein, such as an antibody detection kit and the like, so that real-time monitoring on the immune level of herd pigs PCV3 is realized; and a scientific and flexible pig herd epidemic disease immune program is established.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, and in particular to a recombinant antigen protein for detecting porcine circovirus type 3, its preparation method, and its application. Background Technology

[0002] Porcine circovirus (PCV) belongs to the family Circoviridae and the genus Circovirus. It is a non-enveloped, single-stranded, negative-stranded circular DNA virus and one of the smallest animal viruses. To date, three genotypes of porcine circovirus have been identified: porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2), and porcine circovirus type 3 (PCV3). PCV2 and PCV3 are pathogenic, while PCV1 is non-pathogenic.

[0003] PCV3 is a single-stranded circular DNA virus, approximately 2000 bp in size, encoding two main functional proteins: a capsid protein (Cap) and a replicase protein (Rep). The capsid protein is the only structural protein constituting the viral capsid and the most important immunogenic protein. Currently, there are many laboratory diagnostic methods for PCV3, including highly specific virological, immunological, and molecular biological diagnostic methods, such as virus isolation and identification, indirect immunofluorescence (IFA), immunoperoxidase monolayer assay (IPMA), in situ hybridization (ISH), and polymerase chain reaction (PCR). However, these techniques require high levels of experimental conditions and professional skills, are cumbersome to operate, and are not suitable for the needs of rapid and large-scale clinical testing in veterinary medicine. Moreover, existing technologies have no practical application.

[0004] Currently, there are many ELISA detection methods for detecting PCV3 antibodies in porcine serum both domestically and internationally. These ELISA methods are basically ELISA kits prepared using the full-length CAP protein antigen, with the Cap protein serving as the indirect ELISA coating antigen. However, the sensitivity of these ELISA kits is insufficient, making it difficult to effectively purify PCV3 in porcine herds. Therefore, there is an urgent need to develop a PCV3 antibody detection kit that is easy to operate, has good specificity, high sensitivity, and can be widely used. This kit would enable real-time monitoring of PCV3 immunity levels in porcine herds and the establishment of a scientific and flexible immunization program for porcine diseases, which is of great significance for the prevention and control of PCV3. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] The purpose of this invention is to provide a recombinant antigen protein for detecting porcine circovirus type 3 (PCV3), its preparation method, and its application. The aim is to develop a simple, specific, sensitive, and scalable recombinant antigen protein for detecting PCV3, as well as detection products containing this antigen protein, such as antibody detection kits, to achieve real-time monitoring of PCV3 immunity levels in swine herds and establish a scientific and flexible swine disease immunization program.

[0007] (II) Technical Solution

[0008] In a first aspect, the present invention provides a recombinant antigen protein for detecting porcine circovirus type 3, said recombinant antigen protein having the amino acid sequence shown in SEQ ID NO. 2; or having a derived protein obtained by replacing, adding or shortening the amino acid sequence shown in SEQ ID NO. 2, and having the ability to bind to porcine circovirus type 3 antibodies.

[0009] Secondly, the present invention further proposes a method for preparing the recombinant antigen protein as described above, comprising: constructing a recombinant expression vector for expressing the recombinant antigen protein as described in claim 1; homologously recombining the recombinant expression vector into the baculovirus genome and transfecting it into a host cell; culturing the host cell; and isolating and purifying the resulting exogenous protein to obtain the recombinant antigen protein.

[0010] Further, the preparation of the recombinant expression vector includes cloning the PCV3-Fu gene with the nucleotide sequence shown in SEQ ID NO:1 into a PUC vector to obtain the pUC-Fu plasmid vector; using the pUC-Fu plasmid vector as a template, and Fu-F and Fu-R as upstream and downstream primers, respectively, performing PCR amplification and purification to obtain the PCV3-Fu gene fragment; digesting and purifying the PCV3-Fu gene fragment and the expression plasmid with enzymes, and then ligating them with ligase to obtain the ligation product; transforming the ligation product into Escherichia coli DH5α competent cells for positive screening and amplification to construct a transfer vector containing the PCV3-Fu gene, thus obtaining the recombinant expression vector.

[0011] Furthermore, the pUC plasmid is selected from any one of pUC17, pUC18 and pUC19 plasmids; and / or the expression vector is any one of pFastBac 1, pVL1393 and pFastBac dual; and / or the host cell is Sf9 insect cell.

[0012] Thirdly, the present invention further proposes the application of the recombinant antigen protein in a detection product for detecting porcine circovirus type 3.

[0013] Fourthly, the present invention provides an ELISA antibody detection kit for detecting porcine circovirus type 3, comprising an antigen-coated plate coated with the recombinant antigen protein; the kit contains porcine circovirus type 3 positive serum, enzyme-labeled secondary antibody, substrate chromogenic solution, sample diluent, washing solution and stop solution.

[0014] Furthermore, the substrate chromogenic solution of the ELISA antibody detection kit includes chromogenic solution A and chromogenic solution B mixed in equal proportions; chromogenic solution A is an ethanol solution of tetramethylbenzidine; chromogenic solution B includes citric acid, disodium hydrogen phosphate, and hydrogen peroxide combined with urea; and / or, the enzyme-labeled secondary antibody is a secondary antibody labeled with HRP (horseradish peroxidase); and / or, the washing buffer is a 20×PBST solution; and / or, the stop solution is a sulfuric acid solution.

[0015] Fifthly, the present invention also includes a method for detecting porcine circovirus type 3 for non-disease diagnostic purposes, which uses the recombinant antigen protein as an antigen and detects antibodies against porcine circovirus type 3 through antigen-antibody reaction to diagnose porcine circovirus type 3.

[0016] Further, the specific steps include: coating the recombinant antigen protein onto an ELISA plate, adding enzyme-labeled secondary antibody after a first incubation, performing a second incubation, conducting a colorimetric reaction in the dark, and finally, adding a stop solution to terminate the reaction and performing OD. 450nm The value was detected; the conditions for the first incubation and the second incubation were 35-38℃ for 0.5-1h.

[0017] (III) Beneficial Effects

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) The specificity of the ELISA detection kit provided by the present invention was verified. The results showed that the ELISA detection kit provided by the present invention was negative for positive serum of classical swine fever virus (CFSV), porcine reproductive and respiratory syndrome virus (PPRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), while positive serum of PCV3 was positive. This indicates that the ELISA detection kit of the present invention has no cross-reactivity with other pathogens and has good specificity.

[0020] (2) The sensitivity of the ELISA detection kit provided by the present invention was verified and compared with that of the ELISA kit prepared using the full-length CAP protein antigen. The results showed that the sensitivity of the ELISA detection kit provided by the present invention was significantly higher than that of the ELISA kit prepared using the full-length CAP protein antigen.

[0021] (3) In the kit components of the present invention, the antigen used to coat the enzyme-labeled plate is the recombinant PCV3-Fu protein expressed by Sf9 cells. The antigen has high purity and can be cultured in a large-scale serum-free suspension using a bioreactor. The ELISA antibody detection kit established by the coated plate coated with the antigen has high detection sensitivity, high specificity, and strong binding ability with PCV3 antibody, and is easy to promote and apply on a large scale.

[0022] (4) The recombinant antigen protein provided by the technical solution of the present invention replaces the capsid protein in the prior art as the detection antigen, thereby improving the detection sensitivity and having a good detection effect for low concentration samples. Attached Figure Description

[0023] Figure 1 The image shows the SDS-PAGE detection of recombinant baculovirus expression products in Example 2 of this invention; where 1 is the control and 12 is rBac-Fu.

[0024] Figure 2 This is the Western Blot detection of recombinant baculovirus expression products in Example 2 of the present invention; wherein, 1 is rBac-Fu; and 2 is the control group.

[0025] Figure 3 This is an electron micrograph of the PCV2-Fu protein in Example 4 of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0028] The present invention provides a recombinant antigen protein of porcine circovirus type 3, comprising a protein composed of the amino acid sequence shown in SEQ ID NO:2; or, a derived protein obtained by substituting, adding or truncating the amino acid sequence shown in SEQ ID NO:2, and having the ability to bind to porcine circovirus type 3 antibodies.

[0029] The present invention also provides a gene sequence encoding the above-mentioned recombinant antigen protein, the gene sequence having a nucleotide sequence as shown in SEQ ID NO:1; or a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:2; or a nucleotide sequence encoding a derived protein having the ability to bind to a PCV3 antibody, the derived protein being obtained by substitution, addition or truncation of the amino acid sequence as shown in SEQ ID NO:2.

[0030] As a preferred embodiment, the present invention further provides an expression vector carrying a gene with the above-mentioned nucleotide sequence as shown in SEQ ID NO:1.

[0031] Specifically, the preparation of the recombinant expression vector includes cloning the PCV3-Fu gene with the nucleotide sequence shown in SEQ ID NO:1 into a PUC vector to obtain the pUC-Fu plasmid vector; using the pUC-Fu plasmid vector as a template, and Fu-F and Fu-R as upstream and downstream primers, respectively, performing PCR amplification and purification to obtain the PCV3-Fu gene fragment; using an enzyme digestion and ligation method to digest and purify the PCV3-Fu gene fragment and the digested plasmid, and ligating them with a ligase to obtain the ligation product; transforming, screening, and amplifying the ligation product using microbial cells to construct a transfer vector containing the PCV3-Fu gene, thus obtaining the recombinant expression vector.

[0032] The present invention also provides a method for preparing recombinant antigen protein, the specific steps of which include: homologously recombining the above-mentioned recombinant expression vector into the baculovirus genome and transfecting insect cells to construct a recombinant baculovirus.

[0033] Specifically, the recombinant expression vector is homologously recombined into the baculovirus genome to obtain a recombinant baculovirus genome; after transfecting insect cells with the recombinant baculovirus genome, the infected insect cells are cultured, and the exogenous protein produced after isolation and purification is the recombinant antigen protein; wherein, the insect cells include Sf9 insect cells.

[0034] A method for diagnosing porcine circovirus type 3 (PCV3) by detecting antibodies against PCV3 through antigen-antibody reaction, based on the recombinant antigen protein provided by the above technical solution.

[0035] Specifically, the detection method includes the following steps: coating the recombinant antigen protein onto an ELISA plate to obtain an antigen-coated plate; adding enzyme-labeled secondary antibody after a first incubation; performing a second incubation; conducting a colorimetric reaction in the dark; finally, adding a stop solution to terminate the reaction; and finally performing OD. 450nm Value detection.

[0036] Preferably, the conditions for the first incubation and the second incubation are: incubation at 35-38°C for 0.5-1 hour.

[0037] As a preferred embodiment, the present invention also provides a PCV3 ELISA antibody detection kit, comprising porcine circovirus type 3 positive serum, antigen-coated plate, enzyme-labeled secondary antibody, substrate chromogenic solution, sample diluent, washing solution and stop solution; wherein, the antigen-coated plate is coated with recombinant antigen protein (PCV3-Fu).

[0038] As a preferred embodiment, the preparation method of the antigen-coated plate includes the following steps: adding recombinant antigen protein PCV3-Fu to the enzyme-labeled plate, incubating overnight, adding blocking solution, washing after incubation to obtain the coated reaction plate, sealing and packaging it to obtain the antigen-coated plate, and storing it at 0-4°C.

[0039] As a typical implementation method, the preparation of the antigen-coated plate includes: taking a 96-well ELISA plate, adding 100 μL of purified PCV3-Fu protein (1 mg / L) to each well, and incubating overnight at 2–8°C; discarding the liquid, adding 250 μL of PBST (0.01 mol / L, pH 7.2) to each well, letting it stand for 1 minute, and washing 5 times; adding 200 μL of 1% BSA to each well for blocking, incubating at 37°C for 1 hour, and discarding the blocking solution; adding 250 μL of PBST to each well, letting it stand for 1 minute, and washing 5 times.

[0040] Seal the coated reaction plates and store them at 4°C.

[0041] In a typical implementation, the enzyme-labeled secondary antibody is a secondary antibody labeled with HRP (horseradish peroxidase).

[0042] As a typical implementation method, the preparation of the substrate colorimetric solution includes dissolving 0.2 g of tetramethylbenzidine (TMB) solution in 100 mL of anhydrous ethanol and making up to 1 L to obtain colorimetric solution A; adding 9.33 g of citric acid and 14.6 g of disodium hydrogen phosphate to 6.4 mL of 0.75% hydrogen peroxide urea, adjusting the pH to 5.0, and making up to 1 L to obtain colorimetric solution B; mixing equal volumes of colorimetric solutions A and B, measuring 111.2 mL of 18 mol / L concentrated sulfuric acid, and making up to 1000 mL with the mixed colorimetric solution.

[0043] As a typical implementation, the preparation of the sample diluent involves preparing a 0.1 mol / L phosphate buffer (PBS) at pH 7.4.

[0044] As a typical implementation method, the preparation of the washing solution includes 20×PBST solution, 160g of sodium chloride (NaCl), 4g of potassium chloride (KCl), 57.8g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 4g of potassium dihydrogen phosphate (KH2PO4), dissolved in 800mL of ultrapure water, then 10mL of Tween-20 is added, and the solution is brought to a final volume in a 1000mL volumetric flask.

[0045] As a typical implementation method, the terminating solution includes a 2 mol / L sulfuric acid solution.

[0046] As a typical implementation method, the preparation of PCV3 positive serum involves immunizing test animals with PCV3-Fu protein to obtain PCV3 antibody positive serum.

[0047] Transfection medium T1 was purchased from Mirus, Cellfectin transfection reagent was purchased from Thermo Fisher Scientific, and fresh SF-SFM medium was purchased from Thermo Fisher Scientific.

[0048] Commercial plasmid pFastBac 1 and E. coli DH10Bac competent cells were purchased from Thermo Fisher Scientific.

[0049] Example 1: Construction and Identification of Transfer Vector pF-Fu

[0050] 1. PCV3-Fu gene amplification and purification: In this example, the codon-optimized PCV3-Fu gene (SEQ ID NO:1) synthesized by Nanjing Genscript Biotech Co., Ltd. was cloned into the pUC17 vector to obtain the pUC-Fu plasmid vector.

[0051] Using pUC-Fu plasmid as a template, Fu-F and Fu-R were used as upstream and downstream primers for PCR amplification (the gene sequences of Fu-F and Fu-R are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively). The amplification system is shown in Table 1.

[0052] Table 1. PCV3-Fu gene amplification system

[0053]

[0054]

[0055] The reaction conditions were: 95℃ pre-denaturation for 5 minutes; 94℃ denaturation for 45 seconds, 54℃ annealing for 45 seconds, 72℃ extension for 1 minute, 35 cycles; 72℃ extension for 10 minutes.

[0056] The PCR product was subjected to gel electrophoresis to verify the size of the target gene. The target band appeared at the 0.9kbp position, indicating that the PCV3-Fu gene fragment was successfully amplified. The fragment was then recovered and purified using a gel recovery and purification kit.

[0057] 2. Enzyme digestion and purification: pFastBac 1 plasmid and the PCV3-Fu gene fragment purified in step 1 were digested with BamHⅠ and HindⅢ at 37℃ for 3 hours. The specific enzyme digestion reaction system is shown in Table 2 and Table 3.

[0058] The enzyme digestion products were subjected to gel electrophoresis, and the digested pFastBac 1 plasmid and PCV3-Fu gene fragment were purified using a gel recovery and purification kit, respectively.

[0059] Table 2 PCV3-Fu gene enzyme digestion reaction system

[0060]

[0061] Table 3 pFastBac 1 plasmid digestion reaction system

[0062]

[0063]

[0064] 3. Ligation: The pFastBac 1 plasmid digested in step 2 and the PCV3-Fu gene fragment were ligated using T4 DNA ligase and incubated overnight at 16°C to obtain the ligation product. The ligation system is shown in Table 4.

[0065] Table 4. Ligation system of PCV3-Fu gene and pFastBac 1 plasmid

[0066]

[0067] 4. Transformation: Add 10 μL of the ligation product to 100 μL of DH5α competent cells, mix well, incubate at 42°C for 90 seconds under heat shock, then on ice for 2 minutes. Add 900 μL of LB medium without Amp and incubate at 37°C for 1 hour. Centrifuge 1.0 mL of the bacterial culture to concentrate it to 100 μL, spread it on LB solid medium containing Amp (100 μg / mL), and incubate at 37°C for 16 hours.

[0068] 5. Colony PCR and sequencing identification: Single colonies from the plates were inoculated into LB liquid medium and incubated at 37°C for 2 hours. Colony PCR was performed using the bacterial culture as a template and Fu-F and Fu-R as primers.

[0069] The PCR products were subjected to gel electrophoresis to verify the size of the target gene; samples showing a band around 0.9 kbp were considered positive. The bacterial cultures that tested positive by PCR were sequenced, and those with correct sequencing results were selected for preservation.

[0070] The transfer vector pF-Fu containing the target gene was constructed using the above steps.

[0071] Example 2: Construction of recombinant baculovirus genome Bac-Fu and transfection with recombinant baculovirus

[0072] This embodiment constructs the recombinant baculovirus genome Bac-Fu and transfects it with recombinant baculovirus. The specific steps include:

[0073] 1. Transformation of DH10Bac bacteria: Take 1 μL of the transfer vector pF-Fu obtained in Example 1 and add it to 100 μL of DH10Bac competent cells. Mix well, incubate on ice for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, then incubate on ice for 2 minutes. Add 900 μL of LB liquid medium without Amp and incubate at 37°C for 5 hours. Take 100 μL of the bacterial solution and dilute it 81 times. Take another 100 μL of the diluted bacterial solution and spread it on LB solid medium containing kanamycin 50 μg / mL, gentamicin 7 μg / mL, tetracycline 10 μg / mL, X-gal 100 μg / mL, and IPTG 40 μg / mL. Incubate at 37°C for 48 hours.

[0074] 2. Selection of single colonies: Large white colonies were picked using an inoculation needle and streaked onto LB solid medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL X-gal, and 40 μg / mL IPTG. The culture was incubated at 37°C for 48 hours. Then, single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL X-gal, and 40 μg / mL IPTG. The bacterial strain was preserved, and plasmids were extracted to obtain the recombinant plasmid Bacmid-Fu.

[0075] 3. Preparation of recombinant baculovirus rBac-Fu:

[0076] 1) Inoculate each well of the six-well plate with 0.8 × 10⁸ g of styrax. 6 Each Sf9 cell had a confluence of 50-70%.

[0077] 2) Prepare the following complex for each well: Dilute 4 μL of Cellfectin transfection reagent with 100 μL of transfection medium T1 and vortex briefly; dilute 3 μg of recombinant plasmid Bacmid-Fu from step 2 with 100 μL of transfection medium T1, mix the diluted transfection reagent and plasmid, and gently blow to prepare the transfection mixture.

[0078] 3) After the cells adhere to the wall, add the transfection complex from step 2), incubate at 27°C for 5 hours, remove the supernatant, add 2 mL of fresh SF-SFM medium, incubate at 27°C for 4-5 days, harvest the supernatant, and obtain recombinant baculovirus rBac-Fu.

[0079] The viral load of the harvested F1 generation recombinant baculovirus was determined using indirect immunofluorescence assay. The viral load of the rBac-Fu generation was 1.81 × 10⁻⁶. 8 TCID 50 .

[0080] The recombinant baculovirus rBac-Fu was amplified and used as a seed virus.

[0081] 4) SDS-PAGE and Western Blot Detection: The supernatant harvested in step 3 was subjected to SDS-PAGE detection. Western blot identification was performed using rabbit anti-PCV3 positive serum as the primary antibody and HRP-labeled goat anti-rabbit IgG as the secondary antibody.

[0082] like Figure 1 The figure shows the SDS-PAGE detection results. As can be seen from the figure, rBac-Fu showed the target band at a molecular weight of approximately 25 kDa, while the negative control did not show a band at the corresponding position. The size of the bands in the electrophoresis results were consistent with the theoretical molecular weight of the target protein, proving that the protein expression was successful.

[0083] like Figure 2 The results of Western blotting are shown. As can be seen from the figure, the recombinant baculovirus expression samples all have the target band, while the negative control does not have the target band, indicating that the target protein is correctly expressed in Sf9 cells.

[0084] Example 3: Serum-free suspension culture of insect cells in a bioreactor

[0085] In this embodiment, the rBac-Fu culture obtained in Example 2 was obtained by serum-free suspension culture of insect cells in a bioreactor.

[0086] Sf9 insect cells were aseptically cultured for 3-4 days in a 1000 mL shake flask containing 300 mL of SF-SFM medium until the concentration reached 3-5 × 10⁻⁵. 6When the cell count is 3.8 × 10⁶ cells / mL and the viability is greater than 95%, the cells are seeded into a 5L bioreactor containing 3000mL of SF-SFM medium at a seeding concentration of 3-8 × 10⁶ cells / mL. 5 cell / mL. When the cell concentration reaches 3-5 × 10⁻⁵ 6 When the cell density reaches 3.5 × 10⁶ cells / mL, the cells are seeded into a 50L bioreactor containing 30,000 mL of SF-SFM medium. The cells are allowed to grow to a concentration of 3-5.5 × 10⁶ cells / mL. 6 Cells were seeded at concentrations of 2.5 × 10⁻⁶ cells / mL into a 500L bioreactor containing 300,000 mL of SF-SFM medium until the cell concentration reached 2-8.5 × 10⁻⁶ cells / mL. 6 At a cell / mL concentration, rBac-Fu obtained in Example 2 was inoculated at a 1% inoculum. The reactor culture conditions were pH 6.0-6.5, temperature 25-27℃, dissolved oxygen 30-80%, and stirring speed 100-180 rpm. After culturing for 5-9 days following infection, a final concentration of diethyleneimine (BEI) was added as an inactivating agent. After incubation at 37℃ for 48 hours, a final concentration of Na2S2O3 was added to terminate inactivation, and the rBac-Fu cell culture was harvested.

[0087] Example 4 Protein purification and electron microscopy detection

[0088] Preparation of recombinant antigen protein (PCV3-Fu protein), including protein purification and electron microscopy analysis of the rBac-Fu cell culture from Example 3, specifically including:

[0089] 1. Sucrose density gradient centrifugation: The rBac-Fu cell culture harvested in Example 3 was sonicated and centrifuged at 12000 rpm for 30 minutes. The supernatant was collected, filtered through a 0.22 μm filter to remove impurities, and concentrated 10-fold using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. 10 mL of 40% sucrose solution was added to each centrifuge tube, followed by 2.0 mL of the ultrafiltration concentrated sample. The tube was ultracentrifuged at 29000 rpm for 2 hours, the supernatant was discarded, and the precipitate was resuspended in 2.0 mL of PBS. The suspension was then centrifuged at gradient concentrations of sucrose (50%, 60%, 70%, and 80%), with 2.0 mL of the suspension added, and centrifuged at 38000 rpm for 2 hours. After centrifugation, a white band appeared at the 50%–60% concentration boundary. The white band was carefully aspirated, yielding the purified PCV3-Fu protein. The purified PCV3-Fu protein was analyzed for protein content using the BCA total protein quantification method. The concentration was determined by SDS-PAGE electrophoresis combined with grayscale scanning. The PCV3-Fu protein concentration was 2.2 g / L, and the purity was 98.4%.

[0090] 2. Electron Microscopy Observation: The PCV3-Fu protein collected in step 1 was dropped onto a copper grid with a carbon film. After air drying, 2% sodium phosphotungstenate solution was added for negative staining, followed by electron microscopy observation. Virus-like particles with similar size and morphology to PCV3 virus particles were observed under the electron microscope. See below for details. Figure 3 .

[0091] Example 5: Preparation of PCV3 ELISA Antibody Detection Kit

[0092] 1. Preparation of antigen-coated plates: Take a 96-well ELISA plate and add 100 μL of purified PCV3-Fu protein (1 mg / L) from Example 4 to each well. Incubate overnight at 2–8°C. Discard the liquid. Add 250 μL of PBST (0.01 mol / L, pH 7.2) to each well, let stand for 1 minute, and wash 5 times. Add 200 μL of 1% BSA to each well for blocking, incubate at 37°C for 1 hour, and discard the blocking solution. Add 250 μL of PBST to each well, let stand for 1 minute, and wash 5 times. Seal the coated reaction plate and store at 4°C.

[0093] 2. Preparation of enzyme-labeled secondary antibody: HRP-labeled enzyme-labeled secondary antibody (goat anti-pig IgG) was purchased from Bioworld.

[0094] 3. Preparation of substrate colorimetric solution: Dissolve 0.2 g of tetramethylbenzidine (TMB) solution in 100 mL of anhydrous ethanol, and bring the volume to 1 L to obtain colorimetric solution A. Add 9.33 g of citric acid and 14.6 g of disodium hydrogen phosphate to 6.4 mL of 0.75% hydrogen peroxide urea, adjust the pH to 5.0, and bring the volume to 1 L to obtain colorimetric solution B. Mix equal volumes of colorimetric solutions A and B, measure 111.2 mL of 18 mol / L concentrated sulfuric acid, and use the mixed colorimetric solution to bring the volume to 1000 mL.

[0095] 4. Preparation of sample dilution buffer: Prepare 0.1 mol / L phosphate buffer (PBS) at pH 7.4.

[0096] 5. Preparation of washing solution: 20×PBST solution: 160g sodium chloride (NaCl), 4g potassium chloride (KCl), 57.8g disodium hydrogen phosphate·12 water (Na2HPO4·12H2O), 4g potassium dihydrogen phosphate (KH2PO4), dissolved in 800mL ultrapure water, then add 10mL Tween-20, and dilute to volume in a 1000mL volumetric flask.

[0097] 6. Preparation of the stop solution: Prepare a 2 mol / L sulfuric acid solution.

[0098] 7. Preparation of positive serum: PCV3 type antibody positive serum was obtained by immunizing experimental animals with PCV3-Fu protein.

[0099] Example 6: Initial Program Setup of the PCV3 ELISA Antibody Detection Kit

[0100] 1. Procedure: Serially dilute the samples to be tested and add 50 μL / well to the antigen-coated plate, with two replicates per sample. Add negative and positive controls simultaneously. Incubate at 37°C for 0.5 hours, discard the serum incubator, add 250 μL PBST to each well, let stand for 1 minute, discard the washing buffer, and wash 5 times. Add 100 μL of 1:2000 diluted enzyme-labeled secondary antibody to each well and incubate at 37°C for 0.5 hours. Add 250 μL PBST to each well, let stand for 1 minute, and wash 5 times. Add 100 μL of TMB chromogenic buffer to each well and incubate at 37°C in the dark for 15 minutes. Terminate the reaction by adding 100 μL of 2 mol / L sulfuric acid solution to each well. Detect OD using a microplate reader. 450nm value.

[0101] 2. Result Interpretation: Negative control serum OD 450nm When the value is <0.2, the test result is considered valid. Serum OD to be tested 450nm >2.1× negative control serum OD 450nm When the antibody titer is reached, it is considered positive. The highest dilution at which the sample tests positive is the antibody titer.

[0102] Example 7: Determination of reaction conditions for the PCV3 ELISA antibody detection kit

[0103] 1. Determination of antigen concentration in the antigen-coated plate: The purified PCV3-Fu protein obtained in Example 4 was diluted to different concentrations (0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L) using carbonate buffer. The 96-well plates were coated with PCV3-Fu protein at each dilution. The antibody titer of the test samples was detected according to the method in Example 6 to determine the optimal antigen coating concentration as 1 mg / L.

[0104] 2. Determination of coating temperature and time in antigen-coated plates: The purified PCV3-Fu protein obtained in Example 4 was diluted to 1 mg / L and coated onto 96-well microplates. Different incubation temperatures and times were set (2–8℃ for 8 h, 2–8℃ for 16 h, 2–8℃ for 24 h, and 2–8℃ for 32 h). The antibody titer of the samples was detected according to the method in Example 6 to determine that the antigen coating temperature and time were determined to be overnight incubation at 2–8℃.

[0105] 3. Determination of blocking substance concentration in antigen-coated plates: The purified PCV3-Fu protein obtained in Example 4 was diluted to 1 mg / L and coated onto 96-well ELISA plates, then incubated overnight at 2–8°C. Different concentrations of BSA (0.5%, 0.8%, 1%, 1.2%, and 1.5%) were used for blocking, 200 μL / well. The antibody titers of the samples were then measured according to the method in Example 6 to determine the blocking substance concentration as 1% BSA.

[0106] 4. Determination of blocking time in antigen-coated plates: The purified PCV3-Fu protein was diluted to 1 mg / L and coated onto 96-well ELISA plates, then incubated overnight at 2–8 °C. Blocking was performed using 1% BSA (200 μL / well) at 37 °C for different reaction times (20 min, 30 min, 40 min, 50 min, 60 min). The antibody titers of the samples were then measured according to the method in Example 6 to determine the blocking time as 50 min.

[0107] 5. Determination of secondary antibody dilution factor: The enzyme-labeled secondary antibody was diluted with PBST at different ratios (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600). The antibody titer of the sample was detected according to the optimized detection conditions and the method in Example 6 to determine the secondary antibody dilution factor as 1:12800.

[0108] 6. Determination of the reaction time of the chromogenic solution: TMB was allowed to recover to room temperature for 30 minutes. After adding TMB, the solution was incubated at 37℃ or room temperature for 15 and 30 minutes respectively. The antibody titer of the sample was then measured according to the optimized conditions to determine the reaction time of the chromogenic solution to be 15 minutes.

[0109] Example 8: Specificity and Sensitivity Detection

[0110] 1. Specificity Detection: The PCV3 ELISA antibody detection kit prepared in this invention was used to detect positive sera for classical swine fever virus (CFSV), porcine reproductive and respiratory syndrome virus (PPRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV). PCV3 positive and negative sera were used as controls. 100 μL / well of each sample was added to the reaction plate, and the detection was performed according to the detection conditions determined in Example 7. The results are shown in Table 5. Except for PCV3 positive sera, which was positive, all other detection results were negative, indicating that the ELISA antibody detection kit of this invention has no cross-reactivity with antibodies against other pathogens and exhibits good specificity.

[0111] Table 5. ELISA kit test results

[0112]

[0113] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0114] 2. Sensitivity Detection: PCV3 antibody-positive sera were diluted at ratios of 1:100, 1:300, 1:1000, 1:3000, 1:10000, 1:30000, 1:50000, and 1:100000, and detected using the ELISA antibody detection kit of this invention. A control group was also set up using full-length PCV3Cap protein (NCBI Reference Sequence: OL956951) plated on the same substrate. The results showed that the kit of this invention could detect positive results at a maximum serum dilution of 100,000, while the control group could only detect positive results at a maximum serum dilution of 1:10,000. This indicates that the sensitivity of the ELISA antibody detection kit of this invention is significantly higher than that of the detection antigen prepared using full-length CAP protein.

[0115] Table 6. ELISA kit detection results (OD) 450nm

[0116]

[0117] In summary, the PCV3 kit provided by the present invention has significant specificity. The kit can detect positive results at serum dilutions as low as 100,000 times, and has higher detection sensitivity compared with existing kits prepared from the full-length PCV3Cap protein antigen.

[0118] Obviously, based on the above technical solution, the present invention provides an ELISA antibody detection kit or colloidal gold test strip that does not require CAP protein as an antigen in the prior art, and can detect PCV3 more quickly and accurately, with good specificity and detection sensitivity. It solves the technical problem in the prior art that the sensitivity is insufficient for detecting low-concentration samples when using capsid protein as the detection antigen.

[0119] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant antigen protein for detecting porcine circovirus type 3, characterized in that, The recombinant antigen protein has the amino acid sequence shown in SEQ ID NO.2; or it is a derived protein obtained by replacing, adding or shortening the amino acid sequence shown in SEQ ID NO.2 and having the ability to bind to porcine circovirus type 3 antibody.

2. A method for preparing the recombinant antigen protein as described in claim 1, characterized in that, A recombinant expression vector for expressing the recombinant antigen protein as described in claim 1 is constructed, the recombinant expression vector is homologously recombined into the baculovirus genome and transfected into host cells, the host cells are cultured, and the resulting exogenous protein is isolated and purified to obtain the recombinant antigen protein.

3. The preparation method according to claim 2, characterized in that, The preparation of the recombinant expression vector includes cloning the PCV3-Fu gene, whose nucleotide sequence is shown in SEQ ID NO:1, into a PUC vector to obtain the pUC-Fu plasmid vector; using the pUC-Fu plasmid vector as a template, and Fu-F and Fu-R as upstream and downstream primers, respectively, performing PCR amplification and purification to obtain the PCV3-Fu gene fragment; digesting and purifying the PCV3-Fu gene fragment and the expression plasmid with enzymes, and then ligating them with ligase to obtain the ligation product; transforming the ligation product into Escherichia coli DH5α competent cells for positive screening and amplification to construct a transfer vector containing the PCV3-Fu gene, thus obtaining the recombinant expression vector.

4. The preparation method according to claim 3, characterized in that, The pUC plasmid is selected from any one of pUC17, pUC18 and pUC19 plasmids; and / or the expression vector is any one of pFastBac1, pVL1393 and pFastBac dual; and / or the host cell is Sf9 insect cell.

5. The use of the recombinant antigen protein as described in claim 1 in a detection product for detecting porcine circovirus type 3.

6. An ELISA antibody detection kit for detecting porcine circovirus type 3, characterized in that, The kit includes an antigen-coated plate coated with the recombinant antigen protein as described in claim 1; the kit contains porcine circovirus type 3 positive serum, enzyme-labeled secondary antibody, substrate chromogenic solution, sample diluent, washing solution and stop solution.

7. The ELISA antibody detection kit as described in claim 6, characterized in that, The substrate colorimetric solution comprises colorimetric solution A and colorimetric solution B mixed in equal proportions; colorimetric solution A is an ethanol solution of tetramethylbenzidine; colorimetric solution B comprises citric acid, disodium hydrogen phosphate, and hydrogen peroxide combined with urea. And / or, the enzyme-labeled secondary antibody is an HRP horseradish peroxidase-labeled secondary antibody; and / or, the washing solution is a 20×PBST solution; And / or, the terminating solution is a sulfuric acid solution.

8. A method for detecting porcine circovirus type 3 for non-disease diagnostic purposes, characterized in that, Using the recombinant antigen protein described in claim 1 as the antigen, porcine circovirus type 3 (PCV3) can be diagnosed by detecting antibodies against PCV3 through antigen-antibody reaction.

9. The method as described in claim 8, characterized in that, The specific steps include: coating the recombinant antigen protein of claim 1 onto an ELISA plate, adding enzyme-labeled secondary antibody after a first incubation, performing a second incubation, conducting a colorimetric reaction in the dark, and finally adding a stop solution to terminate the reaction and perform OD. 450nm The value was detected; the conditions for the first incubation and the second incubation were 35-38℃ for 0.5-1h.

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