Celine parvovirus VP2 protein, preparation method and application thereof, and kit

Abstract

The invention belongs to the technical field of biology, and discloses a feline parvovirus VP2 protein, and the amino acid sequence of the VP2 protein is as shown in SEQ ID NO. 1; the nucleotide sequence of the VP2 protein is as shown in SEQ ID NO. 2. A large amount of FPV-VP2 recombinant protein is expressed through prokaryotic expression, purified protein immunized mice obtain the polyclonal antibody, the titer can reach 1: 100000 after purification, and the polyclonal antibody can be used for identification of IFA, WB and the like; indirect ELISA established based on the protein is high in sensitivity which can reach 1: 551, good in specificity and repeatability, convenient to operate, free of a constant-temperature box, fast in overall process and short in time. The kit can be used for clinical early infection diagnosis of the feline plague virus, and has the characteristics of accuracy and high efficiency. Meanwhile, the invention further provides a preparation method and application of the protein and a detection kit adopting the protein.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a feline parvovirus VP2 protein, its preparation method and application, and a reagent kit. Background Technology

[0002] Feline parvovirus (FPV) is a member of the Parvoviridae family and the Parvovirus genus. Under normal environmental conditions, it can infect cats, lions, leopards, etc., but it is most susceptible to young felines under 6 months of age. Infection varies depending on the cat's age, living environment, immune status, and concurrent infections; not all infected individuals will develop characteristic symptoms. In recent years, it has become known for its high infectivity, high mortality rate, wide host range, and significant harm, with a mortality rate between 25% and 100%. The virus spreads rapidly and requires rapidly proliferating cells in the S phase of cell division to replicate. Therefore, viral replication mainly occurs in mitotically active tissues, particularly in cells with high mitotic activity, such as intestinal epithelial cells, lymphoid tissue, and intestinal crypt cells. In the bone marrow, viral replication occurs in early progenitor cells, significantly affecting almost all myeloid cell populations. Treatment must begin once clinical symptoms appear. The virus causes leukopenia and severe intestinal symptoms and can proliferate in tissues and cells of the cat's lungs, kidneys, and testes.

[0003] FPV is a single-stranded linear DNA virus, approximately 25 nm in diameter, with icosahedral symmetry. Its genome is about 5.2 kb in length, and its nucleic acid molecular weight is approximately 1.6 × 10⁶. Its unique palindromic structure forms a hairpin double helix, with the central sequence encoding two open reading frames (ORFs). The first ORF encodes the non-structural proteins NS1 and NS2, and the second ORF encodes the structural proteins VP1 and VP2. It can also utilize the host cell's own RNA polymerase II to transcribe the viral genes encoding VP1, VP2, NS1, and NS2. Furthermore, different splicing of the same mRNA can lead to the translation of VP1, VP2, NS1, and NS2.

[0004] The neutralizing antigenic site of FPV is mainly concentrated in the VP2 capsid protein, whose full-length gene is approximately 1.7 kb, encoding 584 amino acids (aa). Mutations at certain key amino acid sites on the VP2 gene using reverse genetics may alter its host range and antigenic properties, potentially leading to changes in the pathogenesis of FPV. Furthermore, crystallography has been used to determine the capsid structure of FPV, revealing that it is composed of 5–6 VP1 protein molecules and 54–55 VP2 protein molecules, which perform its function. Other studies have shown that virus-like particles composed of VP2 protein can induce humoral and cellular immunity.

[0005] Enzyme-linked immunosorbent assay (ELISA) can be widely used for the qualitative and quantitative detection of antigens and antibodies related to bacteria, viruses, tumors, and hematological diseases. Similarly, ELISA relies on the specificity of antigen-antibody binding. Specific antibodies against specific antigens are immobilized on a solid-phase carrier (such as microplates or nitrocellulose membranes), allowing the test antigen to bind and react with them. Currently, the main commercially available kit for feline parvovirus antibody detection is Immuno Comb Feline Vacci Check, a point-of-care ELISA kit for feline parvovirus, herpesvirus, and calicivirus. However, this method is relatively complex when the sample size is small. While the popular colloidal gold detection method is simple to operate and visually detectable, its sensitivity and accuracy often vary significantly, and it cannot accurately detect early infections. Serum neutralization assays are the gold standard serological test, exhibiting the highest viral serological response. However, due to the high cost and time-consuming nature of serum neutralization assays, HI clinical trials are often used as a simple alternative, although their sensitivity is slightly lower than that of serum neutralization assays.

[0006] Therefore, the technical problem to be solved in this case is: how to improve the detection accuracy, specificity, and sensitivity of the kit by developing a new VP2 protein. Summary of the Invention

[0007] The purpose of this invention is to provide a feline parvovirus VP2 protein, which, when applied to a kit, exhibits superior specificity, sensitivity, repeatability, and concordance rate against feline parvovirus.

[0008] In addition, the present invention also provides a method for preparing the protein and its applications, as well as a detection kit using the protein.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A feline parvovirus VP2 protein, the amino acid sequence of which is shown in SEQ ID NO.1; the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] Furthermore, this invention discloses a method for preparing the feline parvovirus VP2 protein as described above, comprising the following steps:

[0012] Step 1: Based on the FPV-VP2 sequence downloaded from GenBank, pCR amplification was performed using primers to obtain the gene encoding the VP2 protein. The nucleotide sequence of the gene is shown in SEQ ID NO.2; the FPV-VP2 sequence is numbered MK266784 in GenBank.

[0013] Step 2: Using homologous recombination, the gene encoding the VP2 protein was cloned into the vector PET-28a to obtain the PET-28a-FPV-VP2-HIS recombinant plasmid;

[0014] Step 3: Transform the PET-28a-FPV-VP2-HIS recombinant plasmid into BL21(DE3) competent cells to obtain the recombinant expression strain;

[0015] Step 4: The recombinant expression strain was induced by IPTG, lysed, and centrifuged. The supernatant and precipitate were collected. The feline parvovirus VP2 protein was obtained by Ni-NTAResin affinity chromatography.

[0016] Furthermore, this invention discloses the application of the above-mentioned feline parvovirus VP2 protein in the preparation of feline parvovirus VP2 protein antibody detection products.

[0017] Finally, this invention discloses an indirect ELISA detection kit for feline parvovirus VP2 protein antibody, comprising a pre-coated plate, wherein the protein coated in the pre-coated plate is the feline parvovirus VP2 protein as described above.

[0018] Preferably, the test kit further includes sample diluent, enzyme-labeled antibody, substrate solution, stop solution, concentrated washing solution, sealing film, dilution plate, negative control, and positive control.

[0019] Preferably, the positive control is cat positive serum diluted with the sample diluent; the negative control is cat negative serum diluted with the sample diluent.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention utilizes prokaryotic expression to highly express recombinant FPV-VP2 protein. The purified protein is used to immunize mice to obtain polyclonal antibodies, achieving a titer of 1:100,000 after purification. These antibodies can be used for identification using IFA and WB. The protein is also used to establish an indirect ELISA. The indirect ELISA established in this invention exhibits high sensitivity (up to 1:512.0), good specificity and reproducibility, is easy to operate, requires no incubator, and has a fast overall process. It can be used for early clinical diagnosis of feline panleukopenia virus infection, demonstrating precision and efficiency. Future applications in hospital clinical diagnosis will lay the foundation for the diagnosis and prevention of feline panleukopenia. Attached Figure Description

[0022] Figure 1 Image of PET-28a-FPV-VP2 plasmid;

[0023] Figure 2 SDS plot of the optimal expression conditions for FPV-VP2;

[0024] Figure 3 WB verification image of the sample under optimal conditions of 37℃;

[0025] Figure 4 Graph of SDS purified from VP2;

[0026] Figure 5 This is a standard curve for proteins.

[0027] Figure 6 SDS diagram for purification of FPV-VP2 polyclonal antibody;

[0028] Figure 7 SDS diagram for purification of FPV-VP2 polyclonal antibody;

[0029] Figure 8 A Western blot image showing the specificity of polyclonal antibodies;

[0030] Figure 9 The diagram shows the results of the exploration of the optimal sealing solution and sealing time;

[0031] Figure 10 The figure shows the results of the exploration of the optimal sample dilution and incubation time;

[0032] Figure 11 The figure shows the results of the exploration of the optimal dilution and incubation time for enzyme-labeled secondary antibodies;

[0033] Figure 12 The results of exploring the development time of TMB are shown in the figure;

[0034] Figure 13 The diagram shows the results of the search for the optimal reaction temperature;

[0035] Figure 14The image shows the results of the FPV-VP2 indirect ELISA specificity assay. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Information on the source of experimental materials:

[0038] PET-28a vector: stored at the Zhaoqing branch of the Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology.

[0039] BL21(DE3) competent cells: purchased from Shanghai Sangon Biotech Co., Ltd.

[0040] NaCO3, NaHCO3, urea, and imidazole were purchased from Maclean's Reagent Company.

[0041] TRIS and glycine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0042] NaCl, PBS powder, and dialysis bags (8k-14k) were purchased from Solarbio Biotechnology Co., Ltd.

[0043] The DNA gel recovery kit and Endo-Free Plasmid Mini Kit were purchased from OMEGA Bio Tek.

[0044] The homologous recombinase was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0045] The 250 kDa protein Maker was purchased from Shanghai Yamei Biomedical Technology Co., Ltd. (item number WJ03);

[0046] The BCA protein concentration assay kit, ultrasensitive chemiluminescence solution, 50×TAE buffer, Tween, Coomassie brilliant blue ultrafast staining solution, 5×SDS-PAGE protein loading buffer, and single-component TMB chromogenic solution were purchased from Beyotime Biotechnology Co., Ltd.

[0047] The nitrocellulose membrane (NC membrane) was purchased from Merck Millipore Ltd.

[0048] Proteinlso Ni-NTA Resin was purchased from Beijing TransGen Biotech Co., Ltd.

[0049] IPTG powder and kanamycin were purchased from Sangon Biotech; Anti-His murine monoclonal antibody and HRP-labeled goat anti-mouse IgG were purchased from Suzhou Bio-Long Immunotechnology Co., Ltd.

[0050] HRP-labeled goat anti-cat IgG was purchased from Abcam.

[0051] Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma.

[0052] HRP enzyme-labeled dilution solution was purchased from Guangdong Dahua Agricultural Biotechnology Co., Ltd.

[0053] Isopropanol and anhydrous ethanol were purchased from Zhejiang Ningbo Cuiying Chemical Technology Co., Ltd.

[0054] BamHI, XhoI restriction endonuclease, and Ex Premier DNA polymerase were purchased from TaKaRa Biotechnology Co., Ltd.

[0055] Example 1: Preparation of recombinant VP2 protein

[0056] 1.1 Based on the FPV-VP2 sequence downloaded from GenBank, PCR amplification was performed using primers to obtain the gene encoding the VP2 protein. The nucleotide sequence of the gene is shown in SEQ ID NO.2; the FPV-VP2 sequence is numbered MK266784 in GenBank. Using homologous recombination, the gene encoding the VP2 protein was cloned into the vector PET-28a to obtain the PET-28a-FPV-VP2-HIS recombinant plasmid, the map of which is shown below. Figure 1 As shown.

[0057] 1.2 The PET-28a-FPV-VP2-HIS recombinant plasmid was transformed into BL21(DE3) competent cells. The transformed bacterial culture was spread on kanamycin-resistant agar medium and incubated at 37°C for 10 h. Positive single colonies were picked and added to kanamycin-resistant and 0.1% glucose-containing broth medium. The culture was then expanded at 37°C and 220 rpm in a shaker until the OD600 nm = 0.6–0.8. The bacterial culture was centrifuged at 5000 rpm for 10 min, the glucose-containing medium was removed, and the medium was replaced with normal sugar-free LB broth medium. A portion of the bacterial culture served as an uninduced control, and the remaining bacterial culture was added with IPTG inducer to a final concentration of 0.5 mmol / L. The bacterial cultures that reached the target OD600 were then incubated at 16°C, 28°C, and 37°C in a shaker at 220 rpm for 12 h, 8 h, and 4 h, respectively. After induction, the culture was centrifuged at 12,000 rpm for 15 min at 4°C, resuspended in TBS at a 1:9 ratio, and 1 ml of ACE *Cytomyces cerevisiae* broth was added to every 9 ml of TBS. Lysis was performed for 1.5 h, followed by sonication for 20 min, centrifugation at 12,000 rpm for 15 min at 4°C, and the supernatant and precipitate were collected separately. Denatured protein buffer was added, and the mixture was incubated in a 98°C metal bath for 10 min for SDS-PAGE analysis to observe the protein expression pattern. Results are as follows: Figure 2 As shown.

[0058] Figure 2 The SDS plot shows the optimal expression conditions for FPV-VP2.

[0059] Lane 1 was SD induced by IPTG at 16℃; Lane 2 was SD without IPTG induction at 16℃; Lane 3 was SP induced by IPTG at 16℃; Lane 4 was SP without IPTG induction at 16℃; Lane 5 was SP induced by IPTG at 37℃; Lane 6 was SP without IPTG induction at 37℃; Lane 7 was SD induced by IPTG at 37℃; Lane 8 was SD without PTG induction at 37℃; Lane 9 was SD induced by IPTG at 30℃; Lane 10 was SD without IPTG induction at 30℃; Lane 11 was SP induced by IPTG at 30℃; Lane 12 was SP without IPTG induction at 30℃; Lane M was YARNWJ03.

[0060] according to Figure 2 As a result, lane 7 (37℃) showed the clearest target band in IPTG-induced SD expression, with fewer impurities and a thickest band. Therefore, 37℃ is the optimal expression condition. To confirm that it is the target protein of VP2, further Western blotting analysis was performed using protein solutions under these conditions. The results are referenced below. Figure 3 .

[0061] Figure 3The image shows the Western blot (WB) validation of the sample at the optimal condition of 37℃. The protein expression solution at 37℃ was identified using an Anti-His mouse monoclonal antibody. The SP band in the image is thick but not uniform, while the SD band, although lighter, is uniform and clear. Therefore, inclusion body expression at 37℃ was determined to be the optimal expression condition.

[0062] 1.3 The optimal expression conditions were determined to be 37℃ and 0.5 mm IPTG induction for 5 h. Although WB and SDS-PAGE showed a higher protein concentration in the supernatant, it also contained more contaminants and could not directly bind to the nickel column during purification, requiring denaturation with 8 M urea. Therefore, considering all factors, inclusion body protein was chosen for large-scale expression and purification. Following the above method, 4 ml of bacterial culture was added to 400 ml of 0.1% glucose broth at a 1:100 ratio and incubated at 37℃ and 220 rpm until OD600nm = 0.7. IPTG was then added to achieve a final concentration of 0.5 mm / ml, and the culture was induced in a constant temperature shaking incubator at 37℃ for 5 h. After induction, the bacterial culture was aliquoted into 50 mL centrifuge tubes and balanced. The tubes were centrifuged at 12000 rpm for 105 min at 4℃, the supernatant was discarded, and the precipitate was washed 2-3 times with PBS and resuspended. The bacterial cell weight was measured, and 1 ml of TBS was added per 9 ml of TBS. ACE *Cytoplasmic mantle* culture was lysed for 1–2 h, sonicated for 20 min, centrifuged at 12,000 rpm for 15 min at 4 °C, the supernatant was discarded, the precipitate was washed twice with 2 M urea, and an equal volume of denaturing lysis buffer containing 8 M urea (8 M urea, 300 mM NaCl, 20 mM Tris, pH = 8) was added to resuspend the precipitate. The mixture was denatured at 4 °C and 360 °C on a shaker for 2 h, centrifuged at 10,000 rpm at 4 °C for 5 min, and the supernatant was collected.

[0063] 1.4 Proteins were purified by Ni-affinity chromatography using a full-gold Proteinlso Ni-NTA Resin. 2 mL of gel was added to a blank column at a ratio of 1:10. The protective solution in the gel was washed away, and the nickel column was equilibrated with 10 column volumes of denaturing lysis buffer containing 20 mM imidazole. The supernatant collected in step 1.3 was filtered through a 45 mm filter and added to the nickel column. The column was then incubated overnight at 4°C on a shaker at 360°C.

[0064] The flow buffer was flowed through the column three times, followed by three washes with 10 column volumes of 20 mm, 30 mm, and 40 mm imidazole eluent, and then six washes with 10 column volumes of 500 mm imidazole eluent (8M urea, 300 mm NaCl, 20 mm Tris, 500 mm imidazole pH=8). The recombinant protein bound to the column was eluted. The collected flow buffer, wash buffer, and eluent were analyzed by SDS-PAGE. The results are shown below. Figure 4As shown, the total supernatant contained many impurities and the target band was not obvious, while the total precipitate contained fewer impurities and the target band was clear. There was not much protein loss from the flow through to the three washes. The first wash with 500 Mmimidazole had the highest concentration and fewer impurities, so E1 was chosen for renaturation.

[0065] Figure 4 The image shows the SDS chromatogram of VP2 purification. In the image, lane 1 is the total supernatant; lane 2 is the total precipitate; lane 3 is the through-flow buffer; lanes 4-6 are 20mm, 30mm, and 40mm imidazole wash buffers, respectively; and lanes 7-12 are the first to sixth 500mm imidazole elution buffers, respectively.

[0066] Renaturation: 8-14 kDa dry dialysis bags were boiled for 10 minutes with 1 mm EDTA (pH=8) and 2% sodium bicarbonate, then boiled for 10 minutes with pure water. The dialysis buffer was prepared as follows: 6 M urea, 20 mm Tris, 300 mm NaCl, 500 mm L-arginine, 2 mm DTT, and 1 mm EDTA. Dialysis was performed at 4°C, with the buffer changed three times every 12 hours, decreasing the urea and NaCl concentration by 2 M and 100 mm each time, maintaining a pH of 9. After dialysis, sucrose was added and the solution was concentrated to 1 ml at 4°C. Protein concentration was then determined using the BCA protein concentration assay kit from Beyotime Biotechnology Co., Ltd. A protein standard curve was constructed according to the BCA protein concentration assay kit instructions. Figure 5 As shown; the formula for calculating protein concentration is obtained, VP2 protein A. 562nm =1.94. According to the formula y = 0.8987x - 0.1028, the concentration of S1 protein is calculated to be 1.641 mg / mL. There is a total of 1.2 mL, which means 1.9692 mg of protein is obtained.

[0067] Example 2: Preparation of Polyclonal Antibodies

[0068] 2.1 Immunizing mice

[0069] Recombinant FPV-VP2 protein was thoroughly emulsified with an equal volume of Freund's complete adjuvant. Mice were initially immunized with 100 μg / mouse via subcutaneous injection at multiple sites on the back. Two weeks later, a booster immunization was performed, with the protein emulsified using Freund's incomplete adjuvant and injected subcutaneously at multiple sites on the back (100 μg / mouse). Three immunizations were administered, each two weeks apart. Seven days after the third immunization, a second pulse immunization with 100 μg / mouse of protein was given. Seven days later, blood was collected from the orbital sinus of mice, centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was collected. The purified FPV protein (1 μg / mL) was used as the antigen to coat ELISA plates. The serum from immunized mice was serially diluted 10-fold (1×10⁻⁶). 2 ~5×10 2The primary antibody was IgG with peroxidase-affinity, and the secondary antibody was Fcγ fragment specific. The serum titer of mice after immunization was determined by indirect ELISA. The results are shown in Table 1.

[0070] Table 1. Determination of polyclonal antibody titers in immunized mice.

[0071]

[0072] Note: A P / N ratio greater than 2 is considered positive, and the serum titer is higher than 1:100000.

[0073] 2.2 Purification of Antibodies

[0074] 2.2.1 Crude Ammonium Sulfate

[0075] The serum was centrifuged at 10,000 x g for 20 min at 4 °C. The supernatant was collected and mixed with 50% saturated ammonium sulfate at a 1:1 ratio with the serum. The mixture was sealed and incubated at 4 °C for 16 h. After centrifugation at 12,000 x g for 30 min at 4 °C, the supernatant was discarded. The precipitate was resuspended in pre-cooled PBS (1 / 5 of the initial volume) to dissolve it. The mixture was dialyzed against PBS buffer for 36 h, with the buffer changed three times, to obtain the crude purified antibody.

[0076] 2.2.2 Passing through a Protein A affinity chromatography column

[0077] The crude purified antibody was filtered through a 0.45 μm filter and mounted on a Protein A affinity chromatography column. 1 mL of resin was added to the column and allowed to settle naturally. 5 mL of equilibration buffer (20 M Na₂HPO₄, 0.15 M NaCl, pH 7) was added to the column to equilibrate the resin. The equilibration buffer was eluent at a flow rate of approximately 1 mL / min. The sample was loaded onto the column at a flow rate of approximately 1 mL / min, and the eluent was collected. The resin was washed with 30 mL of equilibration buffer at a flow rate of approximately 2 mL / min. The antibody was eluted with 10–15 mL of elution buffer (0.1 M Mglycine, pH 3) at a flow rate of approximately 1 mL / min. 500 μL of the eluent containing the target immunoglobulin was collected per tube, and the pH was immediately adjusted to 7.4 by adding 1 / 10 volume of neutralization buffer (1 M Tris-HCl, pH 8.5). 20 μL of each collected solution was analyzed by SDS-PAGE. Purification yielded two clear bands, representing the dissociation of IgG: a 60 kDa heavy chain and a 250 kDa light chain. SDS-PAGE identification results are as follows: Figures 6-7 As shown.

[0078] Figure 6 The image shows the SDS of purified FPV-VP2 polyclonal antibody in the flow-through washing buffer. In the image, lane 1 is the flow-through buffer; lane 2 is the first washing buffer; and lane M is YARNWJ03.

[0079] It is evident that the cross-flow band is single and shallow, indicating good binding effect of the affinity chromatography column; the wash solution contains many impurities, indicating significant impurity removal effect.

[0080] Figure 7 The image shows the SDS-PAGE of the purified FPV-VP2 polyclonal antibody in the elution buffer. Lanes 1-5 represent the first to fifth elution buffers, and lane M represents the YARN-WJ03 enzyme.

[0081] It is evident that lanes 1 and 4 are shallow, lanes 2 and 3 are deep, and lane 5 is almost nonexistent, proving that the antibody elution was thorough.

[0082] 2.2.3 Western blot identification of polyclonal antibodies

[0083] (1) Sample collection: F81 cells were seeded in 12 wells until they reached 50% growth. The culture medium was replaced with serum-free DMEM. After 18 hours, the supernatant was discarded, and the cells were washed three times with PBS. 200 μL of strong lysis buffer was added and the cells were lysed on ice for 10 min. Then, the cells were centrifuged at 12,000 rpm for 10 min. The precipitate was lysed with 200 μL of 8M urea.

[0084] (2) Sample processing: Take 40 μL of cell supernatant and cell precipitate lysis buffer into 1.5 mL centrifuge tubes respectively, add 10 μL of 5 × SDS-PAGE protein loading buffer to each tube, and boil in a metal bath at 98℃ for 8 min.

[0085] (3) Agarose gel electrophoresis: Place the protein gel into the electrophoresis tank, add electrophoresis buffer to cover the comb, carefully remove the comb, add the protein marker and the prepared sample in sequence, connect the positive and negative terminals of the power supply, adjust the voltage to 160V and run for 50 minutes. Observe that the bromophenol blue has reached the bottom of the gel plate to complete the electrophoresis.

[0086] (4) Transfer: Extract the clamps, sponge pad, and filter paper used for transfer and immerse them in transfer buffer. After electrophoresis, remove the SDS-PAGE gel, cut off the top stacking gel, and place it in buffer. Cut the NC membrane to the appropriate size according to the gel size and immerse it in buffer as well. Place the clamp horizontally in the buffer, place the black electrode plate at the bottom, and place the sponge pad, filter paper, SDS-PAGE gel, NC membrane, filter paper, and sponge pad in sequence. Use a glass rod to slowly squeeze and remove air bubbles, then cover with the white electrode plate and clamp it tightly. Place the transfer clamp into the tank, pour in an appropriate amount of transfer buffer, cover the tank, connect the positive and negative terminals of the power supply, and transfer at 80V for about 2 hours. Place the transfer tank in an ice bath.

[0087] (5) Blocking: Block the NC membrane after transfer with a biocompatible, rapid, low-background blocking buffer at room temperature for 20 min. Discard the blocking buffer and wash the membrane 5 times with TBST, each time for 5 min.

[0088] (6) Primary antibody: Discard the washing solution, dilute the purified mouse polyclonal antibody with TBST at a ratio of 1:2000, and ensure the membrane is completely immersed in the primary antibody. Incubate overnight on a shaker at 4°C. Recover the primary antibody, wash the membrane 5 times with TBST, 5 min each time.

[0089] (7) Secondary antibody: Discard the washing solution, dilute goat anti-mouse HRP-IgG with TBST at 1:5000, incubate on a shaker at room temperature for 1 h, then recover the secondary antibody, wash the membrane 5 times with TBST, each time for 5 min.

[0090] (8) Color development: The ultrasensitive chemiluminescent liquid was uniformly dropped onto the NC membrane, and the light was luminescently imaged using the Amersham ImageQuant800 bio-imager.

[0091] The results are as follows Figure 8 As shown, the VP2 polyclonal antibody was verified to bind to the protein expressed by FPV-infected F81 cells with high specificity, showing only a single band.

[0092] Figure 8 This is a Western blotting image showing the specificity of the polyclonal antibody. In the image, lane M represents YARN-WJ03; lanes 1-2 are F81 cell negative controls; and lanes 3-4 represent FPV-VP2 protein. The negative control showed no band, while the positive sample showed clear and correctly sized bands, demonstrating that the antibody can specifically bind to the identified protein.

[0093] Example 3: Establishment of an Indirect ELISA Method

[0094] 3.1 The checkerboard method was used to determine the optimal protein coating concentration and the optimal serum dilution.

[0095] The purified and concentrated VP2 protein was diluted with carbonate solution at pH 9.6 to 4, 2, 1, and 0.5 μg / mL, 100 μl per well, and coated at 4℃ for 16 h. Large quantities of feline serum collected after immunization with Myco-Pulver (MPP) were concentrated using a sucrose coating method. The hemagglutination inhibition titer was measured to be 1:512 using the HI assay, and this was used as the positive serum. Feline negative serum (purchased from Shanghai Hengyuan Biotechnology) was used as the negative serum. The serum was serially diluted at 1:50, 1:100, 1:200, and 1:400, with three replicates for each dilution. The P / N ratio was calculated for different protein and serum dilutions. The optimal protein coating concentration and sample dilution were determined by the highest P / N ratio. The results are shown in Table 2. The optimal recombinant FPV-VP2 protein coating concentration was 0.5 μg / mL, and the optimal sample dilution was 1:100.

[0096] Table 2. Exploration of Optimal Antigen Coating Concentration and Serum Dilution

[0097]

[0098] 3.2 Exploration of the optimal sealing solution and sealing time

[0099] The optimal VP2 protein coating concentration and serum dilution were used to coat the ELISA plate and dilute the serum sample. 5% skim milk diluted with 2% BSA, 4% BSA, 5% BSA, and PBST were used as blocking solutions. 100 μL was added to each well of the ELISA plate, and the plates were incubated at 37°C for 30 min, 60 min, and 90 min, respectively. The OD450 value was read using a microplate reader, and the P / N ratio was compared and analyzed. The results are as follows: Figure 9 As shown, the P / N value is the highest when 4% BSA is used as the blocking solution for 0.5 hours, which is the optimal blocking condition for ELISA.

[0100] 3.3 Exploration of the optimal incubation time for sample serum

[0101] The ELISA plates were coated and blocked using the optimal conditions established above. Sample diluents were prepared using PBS, PBST, and 15% FBS + 1% BSA, respectively, and serum samples were incubated at 37°C for 30 min, 45 min, and 60 min, respectively. Results are as follows: Figure 10 As shown in the figure, the P / N value was the highest when the sample dilution solution was 15% FBS + 1% BSA and the incubation time was 45 min.

[0102] 3.4 Exploration of optimal secondary antibody dilution and incubation time

[0103] Keeping the above optimal conditions unchanged, HRP-labeled goat anti-cat IgG was diluted with enzyme-labeled antibody dilution buffer to 1:10000, 1:25000, and 1:50000, respectively, and incubated at 37°C for 30 min, 45 min, and 60 min. The results are as follows: Figure 11 As shown in the figure, the P / N ratio was highest when the secondary antibody dilution was 1:50000 and the incubation time was 45 min.

[0104] 3.5 Exploring the Optimal Color Development Time

[0105] Under the same optimal conditions, 100 μL of chromogenic solution was added to each well, and the mixture was incubated at 37°C for 5 min, 10 min, and 15 min. The results are as follows: Figure 12 As shown in the figure, the P / N value is the largest when the color development time is 10 min.

[0106] 3.6 Exploration of the optimal incubation temperature

[0107] Under the same optimal conditions, incubation with primary antibody, secondary antibody, and chromogenic agent was performed at 37°C and room temperature, respectively. The results are as follows: Figure 13As shown, a comparative analysis of the P / N values ​​revealed that the P / N value was highest at 37℃. Observations showed that the P / N values ​​at room temperature and 37℃ were not significantly different. A significant difference analysis showed that there was no significant difference in P / N values ​​between incubation at room temperature and 37℃. For the convenience of clinical operation, incubation at room temperature was adopted.

[0108] 3.7 Determination of the critical value for determining positive or negative sex

[0109] Thirty IBV-negative serum samples were tested under the optimized ELISA conditions described above, and OD was measured. 450 Value. Based on statistical analysis, the OD values ​​of 30 negative serum samples were calculated. 450 average value And standard deviation (SD). According to and The critical value for determining positive or negative and the doubtful value, i.e., OD, are calculated. 450 A value ≥0.348 is considered positive; OD 450 <0.326, considered negative; 0.348>OD 450 Samples with nm ≥ 0.326 were deemed suspicious. The results are shown in Table 3.

[0110] Table 3. OD450 values ​​of FPV-negative serum

[0111] Sample number OD value Sample number OD value Sample number OD value Sample number OD value 1 0.268 9 0.290 17 0.267 25 0.294 2 0.249 10 0.280 18 0.275 26 0.274 3 0.261 11 0.265 19 0.272 27 0.293 4 0.283 12 0.288 20 0.276 28 0.300 5 0.232 13 0.280 21 0.279 29 0.321 6 0.281 14 0.228 22 0.298 30 0.308 7 0.320 15 0.290 23 0.310 8 0.287 16 0.307 24 0.262

[0112] 3.8 Specificity test

[0113] Using FPV-positive and negative sera as controls, the optimized ELISA method described above was used to detect feline calicivirus (FCV), feline herpesvirus (FHV), feline parvovirus (FPV)-positive sera, and feline parvovirus (FPV)-negative sera of clearly identified sources. The assay was repeated three times, and the specificity of the kit was analyzed. The test results are as follows: Figure 14 As shown, except for FPV serum which was positive, all other pathogen-positive sera were negative, indicating that the kit has good specificity and no cross-reactivity with positive sera from other common feline infectious diseases.

[0114] 3.9 Sensitivity Experiment

[0115] Using the established FPV indirect ELISA method, FPV-positive serum at different dilutions was detected under the premise of positive and negative results. The results are shown in Table 4. The method reached the critical value for FPV-positive serum at a dilution of 1:5120, indicating that the established detection method has high sensitivity.

[0116] Table 4. Sensitivity test of FPV-VP2 indirect ELISA

[0117]

[0118] 3.10 Repeatability Experiment

[0119] Three ELISA plates prepared at the same time were used for intra-batch repeatability tests. Eight serum samples were tested according to the established indirect ELISA method, with three replicates for each serum sample. The results are shown in Table 5. The results show that the coefficients of variation for intra-batch repeatability tests ranged from 0.94% to 4.56%, all less than 10%, indicating that the method has a certain degree of reproducibility within the batch.

[0120] Table 5. Intra-assay repeatability test of FPV-VP2 indirect ELISA

[0121]

[0122]

[0123] Three ELISA plates coated with different batches were used for inter-batch repeatability tests, and eight serum samples were tested according to the established indirect ELISA method. The results are shown in Table 6. The results show that the CV values ​​of the inter-batch repeatability tests ranged from 1.56% to 9.15%, all less than 10%, proving that the method has a certain degree of reproducibility between batches.

[0124] Table 6. Inter-batch repeatability test of FPV-VP2 indirect ELISA

[0125]

[0126] 3.11 Compliance Rate Evaluation

[0127] The established FPV indirect ELISA antibody detection method and HI assay were used to detect 56 clinical serum samples collected from an animal hospital, and the sensitivity, specificity and concordance rate were calculated.

[0128] Relative sensitivity (%) = {number of positives / (number of positives + number of false negatives)} × 100%;

[0129] Relative specificity (%) = {number of negatives / (number of negatives + number of false positives)} × 100%;

[0130] Overall compliance rate (%) = {(number of positive tests + number of negative tests) / total number of tests} × 100%;

[0131] The results are shown in Table 7. The results show that the established ELISA detection method has a relative sensitivity of 100% (51 / 50), a relative specificity of 80% (4 / 5), and an overall concordance rate of 98.21% (55 / 56) compared with the HI experiment. This indicates that the established ELISA detection method can accurately and effectively detect the antibodies produced by the body against FPV.

[0132] Table 7 Compliance Rate Evaluation

[0133]

[0134] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A feline parvovirus VP2 protein, characterized in that, The amino acid sequence of the VP2 protein is shown in SEQ ID NO.1; the nucleotide sequence of the VP2 protein is shown in SEQ ID NO.

2.

2. The method for preparing feline parvovirus VP2 protein as described in claim 1, characterized in that, Includes the following steps: Step 1: Based on the FPV-VP2 sequence downloaded from GenBank, pCR amplification was performed using primers to obtain the gene encoding the VP2 protein. The nucleotide sequence of the gene is shown in SEQ ID NO.2; the FPV-VP2 sequence is numbered MK266784 in GenBank. Step 2: Using homologous recombination, the gene encoding the VP2 protein was cloned into the vector PET-28a to obtain the PET-28a-FPV-VP2-HIS recombinant plasmid; Step 3: Transform the PET-28a-FPV-VP2-HIS recombinant plasmid into BL21(DE3) competent cells to obtain the recombinant expression strain; Step 4: The recombinant expression strain was induced by IPTG, lysed, and centrifuged. The supernatant and precipitate were collected. The feline parvovirus VP2 protein was obtained by Ni-NTA Resin affinity chromatography.

3. The application of the feline parvovirus VP2 protein as described in claim 1 in the preparation of feline parvovirus VP2 protein antibody detection products.

4. A feline parvovirus VP2 protein antibody indirect ELISA detection kit, characterized in that, Includes a pre-coated plate, wherein the protein coated in the pre-coated plate is the feline parvovirus VP2 protein as described in claim 1.

5. The detection kit according to claim 4, characterized in that, It also includes sample diluent, enzyme-labeled antibody, substrate solution, stop solution, concentrated washing solution, as well as sealing film, dilution plate, negative control, and positive control.

6. The detection kit according to claim 5, characterized in that, The positive control is cat positive serum diluted with the sample diluent; the negative control is cat negative serum diluted with the sample diluent.

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