Duck circovirus ELISA antibody detection kit and application thereof

Abstract

The invention discloses a duck circovirus ELISA antibody detection kit and application thereof, and relates to the technical field of biological detection. The duck circovirus ELISA antibody detection kit comprises a detection plate, wherein the detection plate is coated with duck circovirus Cap protein; wherein the duck circovirus Cap protein is obtained through expression of a eukaryotic expression system. The Cap protein prepared by using an eukaryotic expression system is used as a coating antigen, has good antigenicity, can be specifically combined with a duck circovirus antibody in a serum sample, and ensures that the provided kit has very strong specificity and sensitivity to the duck circovirus. According to the duck circovirus ELISA antibody detection kit provided by the invention, the duck circovirus antibody can be rapidly and accurately detected, and the problems of poor antigen quality and unstable detection performance of the existing duck circovirus ELISA antibody detection method are solved.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a duck circovirus ELISA antibody detection kit and its application. Background Technology

[0002] Duck circovirus (DuCV) is a non-enveloped, icosahedral virus with a genome of single-stranded, negative-sense circular DNA. The DuCV genome encodes two large proteins (ORF-V1 and ORF-C1). ORF-V1, located on the positive strand, encodes the DuCV replication-associated protein (Rep), composed of 292 amino acids (aa), and is involved in viral replication. ORF-C1, located on the negative strand, encodes the DuCV nucleocapsid protein (Cap), composed of 257 amino acids, which is the virus's main antigenic region and is strongly associated with viral infection and immunity.

[0003] The Cap protein, as the main protective epitope of DuCV virus, possesses strong immunogenicity and can induce the host to produce specific antibodies, making it an important target for serological detection and vaccine development. Currently, domestic and international research on DuCV mainly focuses on PCR detection and whole-genome sequencing analysis. Existing studies have shown that DuCV is prevalent in duck flocks in my country, and its infection may increase the incidence and pathogenicity of common duck diseases such as duck plague, duck viral hepatitis, duck infectious serositis, duck pasteurellosis, and Escherichia coli infection, causing economic losses to the duck farming industry.

[0004] In animal disease prevention and control, serological detection technology is of great significance for understanding the epidemic trend of pathogens and assessing immune status. Enzyme-linked immunosorbent assay (ELISA) is widely used for antibody screening of animal pathogens due to its advantages such as simple operation, high-throughput detection, and easy standardization of results. However, the existing ELISA testing methods still have the following problems: (1) lack of mature commercial ELISA test kits; (2) existing studies mostly rely on prokaryotically expressed Cap protein, which has limited antigen sources and unstable quality; (3) poor detection stability, and antigen activity is prone to decrease during storage and transportation, resulting in large batch-to-batch variations and short shelf life of the kits, which is difficult to meet the needs of practical applications. Therefore, it is urgent to develop a duck circovirus ELISA antibody test kit based on high-quality antigens, with stable detection performance and suitable for large-scale production. Summary of the Invention

[0005] The main objective of this invention is to propose a duck circovirus ELISA antibody detection kit and its application, aiming to solve the problems of poor antigen quality and unstable detection performance in existing duck circovirus ELISA antibody detection methods.

[0006] To achieve the above objectives, the present invention provides a duck circovirus ELISA antibody detection kit, the kit comprising a detection plate coated with duck circovirus Cap protein; The duck circovirus Cap protein was obtained by expression using a eukaryotic expression system, and the duck circovirus Cap protein has the amino acid sequence shown in SEQ ID NO.1.

[0007] In one embodiment, the method for preparing the duck circovirus Cap protein includes the following steps: Using duck circovirus nucleic acid as a template, the ORF-C1 gene fragment encoding the Cap protein was obtained by PCR amplification; The ORF-C1 gene fragment was inserted into the multiple cloning site of the vector pFastBac Dual to construct the recombinant plasmid pFastBac Dual-DuCV-Cap. The recombinant plasmid was introduced into competent cells DH10Bac containing Bacmid, and the recombinant Bacmid was obtained by blue-white screening. The recombinant Bacmid was transfected into the insect cell line Sf9 to obtain recombinant baculovirus; The recombinant baculovirus was inoculated into Sf9 cells for passage and expression. The expression product was collected and purified to obtain the duck circovirus Cap protein.

[0008] In one embodiment, the nucleic acid encoding the duck circovirus Cap protein has a nucleotide sequence as shown in SEQ ID NO.2.

[0009] In one embodiment, the detection plate has multiple detection wells, and the duck circovirus Cap protein is coated in the multiple detection wells; The content of the duck circovirus Cap protein is 0.1~0.3 μg / well.

[0010] In one embodiment, the duck circovirus ELISA antibody detection kit further includes duck circovirus negative control serum, duck circovirus positive control serum, diluent, washing solution, horseradish peroxidase (HRP) labeled anti-duck secondary antibody, tetramethylbenzidine (TMB) chromogenic solution, and stop solution.

[0011] In one embodiment, the duck circovirus negative control serum is serum from healthy ducks that have not been infected with duck circovirus; and / or, The duck circovirus positive control serum is duck serum immunized with duck circovirus Cap protein; and / or, The diluent is a mixture of phosphate buffer and bovine serum albumin; and / or, The washing solution is a phosphate buffer containing Tween-20; and / or, The terminating solution is a sulfuric acid solution.

[0012] In one embodiment, the duck circovirus ELISA antibody detection kit further includes a detection plate stabilizer comprising 0.01 mol / L phosphate buffer, 3%–5% sucrose, 0.04%–0.06% Tween-20, and 0.01%–0.03% Proclin-300.

[0013] This invention also proposes the application of the duck circovirus ELISA antibody detection kit described in the foregoing technical solution in the detection of duck circovirus antibodies, comprising the following steps: S10. Dilute the test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody with diluent to obtain the diluted test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody solutions respectively. S20. Add the diluted serum to be tested, duck circovirus negative control serum and duck circovirus positive control serum to the test plate respectively. After the first incubation, washing with washing solution and drying, add the diluted HRP-labeled anti-duck secondary antibody solution to each plate. After the second incubation, washing with washing solution and drying, add TMB colorimetric solution to each plate and react in the dark. Then add the stop solution to each plate to obtain the test plate. S30. Measure the absorbance of the test plate to be tested, calculate the S / P value based on the absorbance value and determine the test result.

[0014] In one embodiment, in step S10: The serum to be tested was diluted 1:100 with the diluent; and / or, The duck circular negative control serum was diluted 1:100 with diluent; and / or, The duck porcine circovirus-positive control serum was diluted 1:100 with diluent; and / or, The HRP-labeled anti-duck secondary antibody was diluted at a dilution ratio of 10,000 to 20,000 times.

[0015] In one embodiment, in step S20: The incubation temperature for the first incubation is 36~38℃, and the incubation time is 30~60 min; and / or, The second incubation is carried out at a temperature of 36-38°C for 30-60 minutes; and / or, The light-avoidance reaction takes 5 to 15 minutes.

[0016] The technical solution of this invention provides a duck circovirus ELISA antibody detection kit comprising a detection plate coated with duck circovirus Cap protein. The duck circovirus Cap protein is prepared using a eukaryotic expression system and possesses good antigenicity, specifically binding to duck circovirus antibodies in serum samples. This ensures that the kit provided by this invention has strong specificity and sensitivity for duck circovirus. The duck circovirus ELISA antibody detection kit provided by this invention can be applied to the detection of duck circovirus antibodies, achieving rapid and accurate detection of duck circovirus antibodies and solving the problems of poor antigen quality and unstable detection performance in existing duck circovirus ELISA antibody detection methods. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0018] Figure 1 The image shows the PCR identification results of the recombinant plasmid in Example 1, where M is the marker and 1-8 represent the recombinant plasmid. Figure 2 The image shows the identification results of recombinant Bacmid in Example 1, where M is the marker, 1 is the recombinant Bacmid, and 2 is the blank. Figure 3 The image shows the SDS-PAGE analysis results of the recombinant duck circular Cap protein in Example 1, where M is the marker and 1 is the recombinant protein. Figure 4 The image shows the Western blot results of recombinant duck circular Cap protein in Example 1, where M is the marker, 1 is the blank, and 2 is the recombinant protein.

[0019] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.

[0021] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0022] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0023] In animal disease prevention and control, serological detection technology is of great significance for understanding the epidemic trend of pathogens and assessing immune status. Enzyme-linked immunosorbent assay (ELISA) is widely used for antibody screening of animal pathogens due to its advantages such as simple operation, high-throughput detection, and easy standardization of results. However, the following problems still exist in the existing technology: (1) there is a lack of mature commercial ELISA detection kits; (2) existing studies mostly rely on prokaryotically expressed Cap protein, which has limited antigen sources and unstable quality; (3) the detection stability is poor, and antigen activity is prone to decrease during storage and transportation, resulting in large batch-to-batch differences and short shelf life of the kits, which is difficult to meet the needs of practical applications. Therefore, there is an urgent need to develop a duck circovirus ELISA antibody detection kit based on high-quality antigens, with stable detection performance and suitable for large-scale production.

[0024] In view of this, the present invention proposes a duck circovirus ELISA antibody detection kit, the kit comprising a detection plate coated with duck circovirus Cap protein; The duck circovirus Cap protein was obtained by expression using a eukaryotic expression system, and the duck circovirus Cap protein has the amino acid sequence shown in SEQ ID NO.1.

[0025] The present invention provides a duck circovirus ELISA antibody detection kit, comprising a detection plate coated with duck circovirus Cap protein. The duck circovirus Cap protein, prepared using a eukaryotic expression system, possesses excellent antigenicity and can specifically bind to duck circovirus antibodies in serum samples, thereby ensuring that the kit provided by the present invention has strong specificity and sensitivity for duck circovirus. The duck circovirus ELISA antibody detection kit provided by the present invention enables rapid and accurate detection of duck circovirus antibodies, solving the problems of poor antigen quality and unstable detection performance in existing duck circovirus ELISA antibody detection methods.

[0026] In an embodiment of the present invention, the sequence of SEQ ID NO.1 is specifically as follows: MRGRTYRRAYRGRRKRRGLRRRFRRRRLRIARPRRRFSVVTYKVTRNTVFGFFGSQTGPTAAGKWQSLSLEDGAQYTDPPARGNNICGLNMRWAMFGDTNSYMTGSTPFYHYPYDYYMIKGVAITLRPA YNIYQKSKTQGSTVIDKDGQIVKTSTTGWSIDPYGSTSSRRTWDPSRVHRRYFIPKPIIQGAGEGTKHSTFFLGGKNFTWINCTQDQVVHYGMGMSLRKPDNTTGVNAQYDIEAQFTFYIKFGQFTGF.

[0027] In an embodiment of the present invention, the method for preparing the duck circovirus Cap protein includes the following steps: Using duck circovirus nucleic acid as a template, the ORF-C1 gene fragment encoding the Cap protein was obtained by PCR amplification; The ORF-C1 gene fragment was inserted into the multiple cloning site of the vector pFastBac Dual to construct the recombinant plasmid pFastBac Dual-DuCV-Cap. The recombinant plasmid was introduced into competent cells DH10Bac containing Bacmid, and the recombinant Bacmid was obtained by blue-white screening. The recombinant Bacmid was transfected into the insect cell line Sf9 to obtain recombinant baculovirus; The recombinant baculovirus was inoculated into Sf9 cells for passage and expression. The expression product was collected and purified to obtain the duck circovirus Cap protein.

[0028] This invention expresses Cap protein using a eukaryotic expression system, specifically a baculovirus-insect cell expression system. The antigen prepared using the baculovirus expression system offers several advantages, such as high yield, various post-translational modifications, and the ability to mimic viral characteristics. Furthermore, the large capsid and genome of baculoviruses can accommodate approximately 10kb of exogenous DNA without affecting replication, and multiple exogenous fragments can be expressed simultaneously. The use of late-stage protein promoters ensures that even if the exogenous gene product is toxic to cells, it does not affect the expression level, resulting in high yield of the target protein. Insect cells, as eukaryotic cells, can complete a series of transcriptional and translational processes followed by modification of the exogenous protein. The physicochemical and biological characteristics of the expressed product are similar to those of the natural product, maintaining good antigenicity. This technical solution facilitates the production of stable Cap protein and is simple to operate under standard experimental conditions, making it suitable for large-scale production of duck circovirus Cap protein.

[0029] It should be noted that the technical solution of this invention is not limited to the pFastBac Dual vector; other vectors can also be used to construct recombinant plasmids. The technical solution of this invention is also not limited to the Sf9 insect cell line; other insect cells, such as Sf21 insect cells, can also be used. Furthermore, this invention does not limit the method of protein purification; any conventional method in the art capable of purifying proteins can be used in this invention to purify the duck circovirus Cap protein. Preferably, molecular sieve purification methods can be used.

[0030] In an embodiment of the present invention, the nucleic acid encoding the duck circovirus Cap protein has the nucleotide sequence shown in SEQ ID NO.2. The sequence of SEQ ID NO.2 is as follows: .

[0031] In an embodiment of the present invention, the detection plate is provided with a plurality of detection wells, and the duck circovirus Cap protein is coated in the plurality of detection wells; The content of the anti-duck circovirus Cap protein is 0.1~0.3 μg / well.

[0032] Anti-duck circotropic cap protein is coated into multiple test wells, allowing different samples to be added to different wells during detection. The duck circotropic antibody can be immobilized in the test wells via antigen-antibody specific binding, thus determining the presence or absence of the antibody in the sample based on the signal intensity within the wells.

[0033] If the concentration of anti-duck circocap protein is below 0.1 μg / well, the binding rate of the antibody to the duck circocap protein antigen decreases, the signal intensity decreases, and the effect of improving sensitivity is not achieved. If the concentration of duck circocap protein is above 0.3 μg / well, the binding rate of the antibody to the duck circocap protein antigen does not significantly improve further. The technical solution of this invention, by setting the concentration of duck circocap protein to 0.1~0.3 μg / well, can improve the binding rate of duck circocap protein antigen to antibody, thereby improving the binding rate with enzyme-labeled secondary antibody and enhancing detection sensitivity and specificity.

[0034] In an embodiment of the present invention, the method for preparing the detection plate includes the following steps: The purified duck circovirus Cap protein prepared by eukaryotic expression was used as the coating antigen. It was diluted with 0.05M, pH 9.6 carbonate buffer and added to a 96-well ELISA plate, 100 μL per well, and coated overnight at 4°C. Excess antigen in the coated wells was discarded, and each well was washed three times with 300 μL of PBST buffer (i.e., phosphate buffer containing Tween-20). After washing, the plate was blotted dry. 150 μL of PBST buffer containing 2% bovine serum albumin (BSA) (i.e., 2% BSA-PBST) was added to each well as the blocking solution and blocked at 37°C for 1 h. The blocking solution was discarded, and the plate was washed once with PBST and blotted dry.

[0035] In an embodiment of the present invention, the duck circovirus ELISA antibody detection kit further includes duck circovirus negative control serum, duck circovirus positive control serum, diluent, washing solution, HRP-labeled anti-duck secondary antibody, TMB chromogenic solution, and stop solution.

[0036] In an embodiment of the present invention, the duck circovirus negative control serum is serum from healthy ducks that have not been infected with duck circovirus.

[0037] In an embodiment of the present invention, the duck circovirus positive control serum is duck serum immunized with duck circovirus Cap protein.

[0038] In an embodiment of the present invention, the diluent is a mixture of phosphate-buffered saline (PBS) and bovine serum albumin. Adding BSA to commonly used PBS buffer can effectively reduce background noise and improve detection specificity. In one embodiment of the present invention, the diluent is a PBS buffer (pH 7.4) containing 1% BSA.

[0039] In an embodiment of the present invention, the washing solution is a phosphate buffer containing Tween-20. In one embodiment of the present invention, 0.05% Tween-20 is added to the phosphate buffer (pH 7.4), which can effectively reduce the background of the test and improve the detection specificity.

[0040] In an embodiment of the present invention, the terminating solution is a sulfuric acid solution. In one embodiment of the present invention, the terminating solution is a 0.3M sulfuric acid solution.

[0041] In an embodiment of the present invention, the HRP-labeled anti-duck secondary antibody was purchased from Luoyang Baitong Experimental Materials Center.

[0042] The anti-duck enzyme-labeled secondary antibody uses horseradish peroxidase (HRP). HRP is widely distributed in the plant kingdom and has the characteristics of high specific activity, stability, small molecular weight, and easy preparation of pure enzymes. Choosing horseradish peroxidase as the labeled enzyme not only ensures the quality of the kit, but also makes the preparation simple and inexpensive.

[0043] In an embodiment of the present invention, the duck circovirus ELISA antibody detection kit further includes a detection plate stabilizer comprising 0.01 mol / L phosphate buffer, 3% to 5% sucrose, 0.04% to 0.06% Tween-20, and 0.01% to 0.03% Proclin-300.

[0044] The duck circovirus ELISA antibody detection kit provided by this invention uses Cap protein prepared by a eukaryotic expression system as the coating antigen, which has good conformational integrity and high antigenicity. At the same time, with the addition of a specific stabilizer, it can significantly improve the sensitivity, specificity, stability and shelf life of the detection kit, meeting the needs of large-scale clinical screening and epidemiological monitoring.

[0045] This invention also proposes the application of the duck circovirus ELISA antibody detection kit as described in the foregoing technical solution in the detection of duck circovirus antibodies, comprising the following steps: S10. Dilute the test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody with diluent to obtain the diluted test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody solutions respectively. S20. Add the diluted serum to be tested, duck circovirus negative control serum and duck circovirus positive control serum to the test plate respectively. After the first incubation, washing with washing solution and drying, add the diluted HRP-labeled anti-duck secondary antibody solution to each plate. After the second incubation, washing with washing solution and drying, add TMB colorimetric solution to each plate and react in the dark. Then add the stop solution to each plate to obtain the test plate. S30. Measure the absorbance of the test plate to be tested, calculate the S / P value based on the absorbance value and determine the test result.

[0046] The effectiveness of the detection can be determined by setting up a positive control group and a negative control group, based on the signal intensity measured in each group.

[0047] In an embodiment of the present invention, in step S10, the serum to be tested is diluted with a diluent at a ratio of 1:100.

[0048] In an embodiment of the present invention, in step S10, the duck circular negative control serum is diluted with diluent at a ratio of 1:100.

[0049] In an embodiment of the present invention, in step S10, the duck circular positive control serum is diluted with a diluent at a ratio of 1:100.

[0050] In an embodiment of the present invention, in step S10, the HRP-labeled anti-duck secondary antibody is diluted to a concentration of 10,000 to 20,000 times. By setting the dilution of the HRP-labeled anti-duck secondary antibody to 10,000 to 20,000 times, the binding rate between the antibody and the anti-duck enzyme-labeled secondary antibody can be increased, thus enhancing the signal intensity. If the dilution of the HRP-labeled anti-duck secondary antibody is higher than 20,000, the binding rate between the enzyme-labeled secondary antibody and the antibody decreases, the signal intensity decreases, and the effect of improving sensitivity is not achieved; if the dilution of the HRP-labeled anti-duck secondary antibody is lower than 10,000, there is no significant further improvement in the binding rate between the enzyme-labeled secondary antibody and the antibody.

[0051] In an embodiment of the present invention, in step S20, the incubation temperature for the first incubation is 36~38℃, and the incubation time is 30~60min. Exemplarily, the incubation temperature for the first incubation can be 36℃, 37℃, or 38℃, and the incubation time for the first incubation can be 30min, 40min, 50min, or 60min.

[0052] In an embodiment of the present invention, in step S20, the incubation temperature for the second incubation is 36~38℃, and the incubation time is 30~60min. Exemplarily, the incubation temperature for the second incubation can be 36℃, 37℃, or 38℃, and the incubation time for the second incubation can be 30min, 40min, 50min, or 60min.

[0053] In an embodiment of the present invention, in step S20, the reaction time in the dark is 5-15 min. Exemplarily, the reaction time in the dark can be 5 min, 10 min, or 15 min. In one embodiment of the present invention, after the second incubation, washing with washing solution, and drying, 100 μL of TMB colorimetric solution is added to the detection plate, and the plate is incubated at room temperature in the dark for 10 min, then 100 μL of stop solution is added to terminate the reaction.

[0054] In an embodiment of the present invention, in step S30, the absorbance of the test plate is measured at a wavelength of 450 nm. Specifically, the OD of each well is measured using a microplate reader. 450nm The value is calculated, and the S / P value is determined. S / P value = (OD value of the serum sample to be tested) 450nm Value - Negative control OD 450nm (mean) / (positive control OD) 450nm Mean - Negative Control OD 450nm (Average value). The criteria for determining the effectiveness of the kit in this invention are: an S / P value ≥ 0.3 indicates a positive result for duck circovirus antibody; an S / P value < 0.3 indicates a negative result for duck circovirus antibody.

[0055] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0056] In the following examples, the ELISA plates were purchased from Corning. BSA was acquired from Sangon Biotech (Shanghai) Co., Ltd. HRP-labeled anti-duck secondary antibody was purchased from Luoyang Baitong Experimental Materials Center; TMB developer was purchased from Seracare. Concentrated sulfuric acid, sucrose, and Tween-20 were purchased from Sinopharm Chemical Reagent Co., Ltd., and Proclin-300 was purchased from Sigma-Aldrich.

[0057] Example 1 A method for preparing duck circovirus Cap protein includes the following steps: (1) Construction of recombinant plasmid pFastBac Dual-DuCV-Cap: ①PCR amplification of the Cap gene fragment: Using duck circovirus nucleic acid as a template (the nucleic acid encoding the duck circovirus Cap protein has the nucleotide sequence shown in SEQ ID NO. 2), a pair of specific primers containing BamHI and HindIII restriction sites were designed: Upstream primer (PF): 5'-TCTGGATCC atgcgaggccgcacctatcggcgtgc-3' (underlined is the BamHI restriction site); Downstream primer (PR): 5'-GCG AAGCTT ttagaatccagtgaactgtccaaatttg-3' (underlined characters indicate HindIII restriction sites). Ununderlined uppercase letters represent protective bases, underlined uppercase letters represent restriction enzyme sites, and lowercase letters represent the gene sequence used as a primer.

[0058] Prepare the amplification system: 5×PS Buffer 5μL, dNTP Mixture (2.5mM each) 2μL, PF (10μm) 1μL, PR (10μm) 1μL, PrimeStar HS DNA Polymerase (2.5U / μL) 0.5μL, template 2μL, and add nuclease-free water to a final volume of 25μL.

[0059] The amplification program was as follows: pre-denaturation at 98℃ for 30 seconds; 35 cycles (denaturation at 98℃ for 10 seconds, annealing at 58℃ for 15 seconds, extension at 72℃ for 1 minute); and a final extension at 72℃ for 10 minutes. The ORF-C1 gene fragment encoding the Cap protein was obtained through this PCR amplification process. ② Double enzyme digestion and ligation reaction: The PCR amplification products from step ① were double-digested with BamHI (15 U / μL) and HindIII (15 U / μL) purchased from Takara (37℃, 2 h), respectively. Then, they were ligated with the baculovirus vector pFastBac Dual, which had been digested with the same enzymes, to construct the recombinant plasmid pFastBac Dual-DuCV-Cap. T4 DNA ligase (350 U / μL) was used for ligation, the molar ratio of insert fragment to vector was 3:1, and ligation was carried out at 16℃ for 30 minutes. ③ Transformation, screening, and identification: The recombinant plasmid was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 100 μg / mL ampicillin and 7 μg / mL gentamicin, and incubated overnight at 37°C. The next day, multiple single colonies were randomly picked and inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and 7 μg / mL gentamicin, respectively, and incubated overnight at 37°C with shaking. The plasmid was then extracted using the alkaline lysis method.

[0060] The extracted plasmid was identified by colony PCR using the amplification primers and conditions described in step ①. Recombinant plasmids that tested positive by PCR were then confirmed by sequencing using universal sequencing primers pFastbac-F (5'-TATTCCGGATTATTCATACC-3').

[0061] (2) Obtaining recombinant baculovirus: ① Competent cells DH10Bac containing Bacmid, whose recombinant plasmid was correctly sequenced, were activated at 37°C for 4 hours. Then, they were plated on LB agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, Bluo-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) 100 μg / mL, and IPTG (isopropyl-β-D-thiogalactopyranoside) 40 μg / mL, and incubated at 37°C for 48 hours. White colonies grown on the plates were picked and inoculated into LB liquid medium (containing kanamycin, gentamicin, and tetracycline), and cultured overnight at 37°C with shaking. PCR identification using the universal identification primers M13 upstream primer (-40) and M13 downstream primer pair yielded positive bacteria containing recombinant Bacmid.

[0062] ② Recombinant Bacmid was transfected into the insect cell line Sf9 using the transfection reagent CellfectionII. The supernatant was harvested after 4-5 days to obtain the recombinant baculovirus.

[0063] (3) Expression and purification of recombinant baculovirus: The harvested recombinant baculovirus was inoculated into Sf9 insect cells for amplification culture, and the supernatant was harvested as the seed virus. The amplified seed virus was inoculated into Sf9 insect cells at a volume ratio of 1:100 and cultured with shaking at 110-120 rpm and 27°C for 5-7 days. The expression supernatant sample was harvested after 5-7 days. The expression supernatant sample was purified by molecular sieve method, and the target protein was eluted stepwise and the eluent was collected to obtain purified recombinant duck circotropic cap protein. The purified recombinant duck circotropic cap protein was detected by SDS-PAGE.

[0064] (4) Identification of recombinant proteins: The purified recombinant duck circular Cap protein was subjected to SDS-PAGE electrophoresis and membrane transfer, followed by blocking with 5% (m / v) skim milk powder overnight at 4°C. Western blot identification was performed using DuCV-positive serum as the primary antibody and HRP-labeled anti-duck IgG as the secondary antibody.

[0065] PCR identification results of recombinant plasmids are as follows Figure 1 As shown, the identification results of recombinant Bacmid are as follows: Figure 2 As shown, the SDS-PAGE detection results are as follows: Figure 3As shown, the Western blot test results are as follows: Figure 4 As shown in SEQ ID NO.1, the amino acid sequence of the prepared duck circovirus Cap protein has been identified. The above results indicate that the duck circovirus Cap protein has been successfully prepared.

[0066] Example 2 A duck circovirus ELISA antibody detection kit includes a detection plate, duck circovirus negative control serum, duck circovirus positive control serum, diluent, washing buffer, HRP-labeled anti-duck secondary antibody, TMB chromogenic solution, and stop solution; wherein: The detection plate is coated with duck circovirus Cap protein prepared in Example 1. The duck circovirus negative control serum is serum from healthy ducks that are not infected with duck circovirus. The duck circovirus positive control serum is serum from ducks immunized with duck circovirus Cap protein. The diluent is 1% BSA-PBS. The washing solution is PBST. The stop solution is 0.3M sulfuric acid solution. The duck porcine circovirus ELISA antibody detection kit was prepared using the following method: (1) Preparation of negative control serum: Blood was collected from healthy ducks that were not infected with duck circovirus and serum was separated; (2) Preparation of positive control serum: Blood was collected from ducks immunized with duck circocap protein and serum was separated; (3) Preparation of dilution buffer (1% BSA-PBS): Prepare a phosphate buffer with NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4·12H2O 3.6 g / L, and KH2PO4 0.24 g / L, with a pH of 7.4, and then add BSA to make the concentration of BSA 1% (g / v); (4) Preparation of washing buffer (PBST): Prepare a phosphate buffer with NaCl 80 g / L, KCl 2 g / L, Na2HPO4·12H2O 36 g / L and KH2PO4 2.4 g / L, with a pH of 7.4, and then add Tween-20 to make the concentration of Tween-20 0.5% (v / v); (5) Preparation of detection plate: The purified duck circovirus Cap protein was used as the coating antigen and diluted to 2 μg / mL with 0.05M pH9.6 carbonate buffer. 100 μL was added to each well of a 96-well ELISA plate and coated overnight at 4°C. The excess antigen in the wells after coating was discarded. 300 μL of PBST was added to each well and the plate was washed 3 times. After washing, the plate was dried. 150 μL of blocking buffer (2% BSA-PBST) was added to each well of the plate and the plate was blocked at 37°C for 1 h. The blocking buffer was discarded and the plate was washed once with PBST. After washing, the plate was dried. (6) Preparation of the stop solution: Take 16.3 mL of 98% concentrated sulfuric acid and add distilled water to 1000 mL; (7) Assembly: Provide commercially available enzyme-labeled secondary antibody and TMB chromogenic solution, and assemble the prepared detection plate, duck circovirus negative control serum, duck circovirus positive control serum, diluent, washing solution, HRP-labeled anti-duck secondary antibody, TMB chromogenic solution and stop solution to obtain the duck circovirus ELISA antibody detection kit.

[0067] Example 3 Compared with Example 2, the difference is that the detection plate of the duck circovirus ELISA antibody detection kit also includes an ELISA detection plate stabilizer.

[0068] The steps for preparing the detection plate include: ① Preparation of ELISA plate stabilizer: The ELISA plate stabilizer consists of PBS (0.01 mol / L, pH 7.4), 4% sucrose, 0.05% Tween-20, and 0.02% Proclin-300; ② The purified duck circovirus Cap protein was used as the coating antigen and diluted to 2 μg / mL with 0.05 M pH 9.6 carbonate buffer. 100 μL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. Excess antigen in the wells was discarded, and each well was washed three times with 300 μL of PBST. After washing, the plate was dried. 150 μL of blocking buffer (2% BSA-PBST) was added to each well of the ELISA plate and incubated at 37°C for 1 h. The blocking buffer was discarded, and the plate was washed once with PBST. After washing, the plate was dried to obtain the detection plate. Add 200 μL of the ELISA plate stabilizer from step ① to the test plate.

[0069] Example 4 An application of a duck circovirus ELISA antibody detection kit, using the duck circovirus ELISA antibody detection kit of Example 2, includes the following steps: (1) The test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody were diluted with diluent to obtain the diluted test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody solutions respectively; wherein, the test serum was diluted 100 times with diluent, the duck circular negative control serum was diluted 100 times with diluent, the duck circular positive control serum was diluted 100 times with diluent, and the HRP-labeled anti-duck secondary antibody was diluted 15000 times; (2) Take the duck circovirus antibody detection plate and add 100 μL of diluted serum to be tested, duck circovirus negative control serum and duck circovirus positive control serum to different detection wells respectively, with 2 wells for each of the duck circovirus negative control serum and duck circovirus positive control serum.

[0070] (3) After the sample is added, the first incubation is carried out at 37℃. After incubation for 45 min, the excess serum in the well is discarded and the well is washed 3 times with diluted washing solution. After washing, the well is patted dry. The washing solution is diluted 10 times with purified water. (4) Add 100 μL of diluted HRP-labeled anti-duck secondary antibody to each well and incubate for the second time at 37°C. After incubation for 45 min, discard the excess secondary antibody in the wells and wash three times with diluted washing buffer. After washing, pat dry. The washing buffer should be diluted 10 times with purified water. (5) Add 100 μL of TMB colorimetric solution to each well and develop the color for 10 min in the dark; (6) Add 100 μL of stop solution to each well to terminate the reaction; place it in an ELISA reader and measure the absorbance at a wavelength of 450 nm. (7) Result calculation and judgment: Result calculation: S / P value = (OD of serum sample) 450nm Value - Negative control OD 450nm (mean) / (positive control OD) 450nm Mean - Negative Control OD 450nm average value); Result interpretation: S / P value ≥ 0.3 indicates positive duck circovirus antibody; S / P value < 0.3 indicates negative duck circovirus antibody.

[0071] Example 5 Compared with Example 4, the difference is that the duck circovirus ELISA antibody detection reagent of Example 3 is used.

[0072] Performance testing 1. Determination of critical values: Following the application method of the duck circovirus ELISA antibody detection kit in Example 4, 90 duck circovirus-negative serum samples were tested, and the OD values ​​of each sample and control were measured. 450nm The S / P value of the sample was calculated. See Table 1 for the results.

[0073] Table 1 Test results of critical values

[0074] The mean and standard deviation of the S / P values ​​were calculated, and the result of the mean + 3 × standard deviation was used as the cutoff value for judging positive and negative serological samples. According to the results in Table 1, the mean S / P value of the 90 negative serological samples was 0.079, and the standard deviation was 0.073. The formula is: mean + 3 × standard deviation = 0.079 + 3 × 0.073 = 0.3. Therefore, an S / P value ≥ 0.3 is considered positive, and an S / P value < 0.3 is considered negative.

[0075] 2. Specific detection experiment: According to the application method of the duck circovirus ELISA antibody detection kit in Example 4, positive sera for duck hepatitis virus type 1, duck hepatitis virus type 3, duck astrovirus, duck hepatitis B virus, duck parvovirus, duck egg drop syndrome, avian influenza virus, duck plague virus, reovirus, and duck Tembusu virus were detected.

[0076] OD was measured in two wells for each of the above samples and the control. 450nm Value, calculate control OD 450nm The average value and sample S / P value are shown in Table 2.

[0077] Table 2 Specificity test results

[0078] Table 2 shows that the S / P values ​​of positive sera for duck hepatitis virus type 1, duck hepatitis virus type 3, duck astrovirus, duck hepatitis B virus, duck parvovirus, duck egg drop syndrome, avian influenza virus, duck plague virus, reovirus, and duck Tembusu virus were all less than 0.3, and were therefore considered negative. This indicates that there was no cross-reaction with other pathogen antigens, demonstrating high specificity.

[0079] 3. Sensitivity detection experiment: Following the application method of the duck circovirus ELISA antibody detection kit in Example 4, the duck circovirus positive control serum was serially diluted with diluents at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, and added to the wells of the duck circovirus antibody detection plate, 100 μL per well. Two wells were set up for negative control and two wells for positive control.

[0080] OD was measured in two wells for each sample and control in parallel. 450nm Value, calculate control OD 450nm The average value and sample S / P value are recorded in Table 3 below.

[0081] Table 3 Sensitivity Test Results

[0082] As can be seen from the test results in Table 3, the duck circovirus positive control serum was positive at concentrations from 1:100 to 1:6400, indicating that the method has good sensitivity.

[0083] 4. Repeatability test: Following the application method of the duck circovirus ELISA antibody detection kit in Example 4, three duck circovirus positive samples and three duck circovirus negative samples were tested, with the tests repeated three times. The results are shown in Table 4.

[0084] Table 4 Repeatability Test Results

[0085] As shown in Table 4, the CV values ​​of the three duck circovirus positive samples and the three duck circovirus negative samples were all <10%, indicating that the detection results of the kit of the present invention are stable and have good repeatability.

[0086] 5. Stability testing experiment: Following the application methods of the duck circovirus ELISA antibody detection kits in Examples 4 and 5, duck circovirus positive control sera were tested at different time points. The results are shown in Table 5 below.

[0087] Table 5. Stability test results of the reagent kit of the present invention

[0088] As shown in Table 5, the positive control detection value of the kit prepared in Example 4 decreased after 6 months of storage, while the positive control detection value of the kit in Example 5 remained stable after 18 months of storage. This indicates that the kit in Example 5 of the present invention has better stability, demonstrating that the addition of an ELISA plate stabilizer can improve the stability and shelf life of the prepared ELISA kit.

[0089] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A duck circovirus ELISA antibody detection kit, characterized in that, The kit includes a detection plate coated with duck porcine circovirus Cap protein; The duck circovirus Cap protein was obtained by expression using a eukaryotic expression system, and the duck circovirus Cap protein has the amino acid sequence shown in SEQ ID NO.

1.

2. The duck circovirus ELISA antibody detection kit as described in claim 1, characterized in that, The method for preparing the duck circovirus Cap protein includes the following steps: Using duck circovirus nucleic acid as a template, the ORF-C1 gene fragment encoding the Cap protein was obtained by PCR amplification; The ORF-C1 gene fragment was inserted into the multiple cloning site of the vector pFastBac Dual to construct the recombinant plasmid pFastBac Dual-DuCV-Cap. The recombinant plasmid was introduced into competent cells DH10Bac containing Bacmid, and the recombinant Bacmid was obtained by blue-white screening. The recombinant Bacmid was transfected into the insect cell line Sf9 to obtain recombinant baculovirus; The recombinant baculovirus was inoculated into Sf9 cells for passage and expression. The expression product was collected and purified to obtain the duck circovirus Cap protein.

3. The duck circovirus ELISA antibody detection kit as described in claim 2, characterized in that, The nucleic acid encoding the duck circovirus Cap protein has the nucleotide sequence shown in SEQ ID NO.

2.

4. The duck circovirus ELISA antibody detection kit as described in claim 1, characterized in that, The detection plate has multiple detection wells, and the duck circovirus Cap protein is coated in the multiple detection wells; The content of the duck circovirus Cap protein is 0.1~0.3 μg / well.

5. The duck circovirus ELISA antibody detection kit as described in claim 1, characterized in that, The duck circovirus ELISA antibody detection kit also includes duck circovirus negative control serum, duck circovirus positive control serum, diluent, washing solution, HRP-labeled anti-duck secondary antibody, TMB chromogenic solution, and stop solution.

6. The duck circovirus ELISA antibody detection kit as described in claim 5, characterized in that, The duck circovirus negative control serum is serum from healthy ducks that have not been infected with duck circovirus; and / or, The duck circovirus positive control serum is duck serum immunized with duck circovirus Cap protein; and / or, The diluent is a mixture of phosphate buffer and bovine serum albumin; and / or, The washing solution is a phosphate buffer containing Tween-20; and / or, The terminating solution is a sulfuric acid solution.

7. The duck circovirus ELISA antibody detection kit as described in claim 5, characterized in that, The duck circovirus ELISA antibody detection kit also includes a detection plate stabilizer, which comprises 0.01 mol / L phosphate buffer, 3%–5% sucrose, 0.04%–0.06% Tween-20, and 0.01%–0.03% Proclin-300.

8. The application of the duck circovirus ELISA antibody detection kit as described in any one of claims 1 to 7 in the detection of duck circovirus antibodies, characterized in that, Includes the following steps: S10. Dilute the test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody with diluent to obtain the diluted test serum, duck circular negative control serum, duck circular positive control serum and HRP-labeled anti-duck secondary antibody solutions respectively. S20. Add the diluted serum to be tested, duck circovirus negative control serum and duck circovirus positive control serum to the test plate respectively. After the first incubation, washing with washing solution and drying, add the diluted HRP-labeled anti-duck secondary antibody solution to each plate. After the second incubation, washing with washing solution and drying, add TMB colorimetric solution to each plate and react in the dark. Then add the stop solution to each plate to obtain the test plate. S30. Measure the absorbance of the test plate to be tested, calculate the S / P value based on the absorbance value and determine the test result.

9. The application of the duck circovirus ELISA antibody detection kit as described in claim 8 in the detection of duck circovirus antibodies, characterized in that, In step S10: The serum to be tested was diluted 1:100 with the diluent; and / or, The duck circular negative control serum was diluted 1:100 with diluent; and / or, The duck porcine circovirus-positive control serum was diluted 1:100 with diluent; and / or, The HRP-labeled anti-duck secondary antibody was diluted at a dilution ratio of 10,000 to 20,000 times.

10. The application of the duck circovirus ELISA antibody detection kit as described in claim 8 in the detection of duck circovirus antibodies, characterized in that, In step S20: The incubation temperature for the first incubation is 36~38℃, and the incubation time is 30~60 min; and / or, The second incubation is carried out at a temperature of 36-38°C for 30-60 minutes; and / or, The light-avoidance reaction takes 5 to 15 minutes.

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