Characteristic protein of serum type i chicken marek's disease virus and detection application

Abstract

The application provides a characteristic protein of a serum type I chicken Marek's disease virus and a detection application, and belongs to the technical field of virus detection. The application provides a recombinant gene derived from the serum type I chicken Marek's disease virus, and the nucleotide sequence of the recombinant gene is shown as SEQ ID No. 1. The recombinant gene encodes MDV-1 recombinant PP38 protein, and the MDV-1 recombinant PP38 protein can be used for ELISA detection. The application also provides an ELISA detection kit constructed by using the MDV-1 recombinant PP38 protein, which can be used for clinical MDV-1 serum antibody detection, screening of SPF chickens, detection of exogenous virus antibodies of avian live vaccines or virus seeds and epidemiological investigation, so as to prevent and control the spread of MDV-1 infection.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to a characteristic protein of type I chicken Marek's disease virus and its detection application. Background Technology

[0002] Marek's disease (MD) is a highly contagious neoplastic disease caused by Marek's disease virus (MDV), characterized by lymphoid tissue proliferation and tumor formation. MDV can be divided into three serotypes: MDV-1, MDV-2, and MDV-3. MDV-1 includes all highly virulent tumorigenic strains (such as the GA strain), artificially attenuated vaccine strains (such as CVI988 and 814 strains), and MDV-1 vaccine strains with the meq gene deleted. MDV-2 consists of non-pathogenic naturally attenuated strains (such as SB-1); MDV-3 is turkey herpesvirus (HVT). In recent years, due to the presence of immunosuppressive diseases such as infectious anemia and avian leukosis in chicken flocks, and the increasing virulence of MDV with a trend towards stronger virulence, despite widespread MD vaccination, MD still occurs, causing significant economic losses to the poultry industry.

[0003] The main methods for determining the presence of MDV-1 infection in chicken flocks, vaccines, or virus strains include ELISA, agar diffusion assay, and virus neutralization (VN) assay. Among these, the agar diffusion assay is inexpensive but has low sensitivity and accuracy, thus significantly limiting its application in production. In the inspection of finished poultry live vaccines, the 2020 edition of the *Veterinary Pharmacopoeia of the People's Republic of China* (Volume III) stipulates the use of the chicken testing method to detect MDV-1 agar diffusion antibodies to determine the presence of MDV-1 contamination in the vaccine. MDV-1 contamination in vaccines poses a potential risk for MDV-1 transmission and epidemics; therefore, there is an urgent need to establish a stable and efficient method for detecting MDV-1 antibodies for the quality control of poultry live vaccines and the diagnosis of MDV-1 disease. Summary of the Invention

[0004] This invention provides a characteristic protein of serum type I chicken Marek's disease virus and its detection application, which can rapidly, accurately and with high sensitivity detect MDV-1 and / or MDV-1 antibodies.

[0005] This invention provides a recombinant gene derived from serum type I chicken Marek's disease virus, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0006] The present invention also provides a recombinant protein encoded by the above-mentioned recombinant gene.

[0007] The present invention also provides a biological material comprising the above-mentioned recombinant gene and expressing the above-mentioned recombinant protein.

[0008] In a preferred embodiment of the present invention, the type of biomaterial includes an expression vector or an expression host cell.

[0009] The present invention also provides the application of the above-mentioned recombinant gene, recombinant protein or biological material in the preparation of reagents for detecting serum type I chicken Marek's disease virus.

[0010] In a preferred embodiment of the present invention, the detection method matched with the reagent includes at least one of the following: indirect ELISA detection method, direct ELISA detection method, and competitive ELISA detection method.

[0011] The present invention also provides a reagent for detecting serum type I chicken Marek's disease virus, comprising using the above-mentioned recombinant protein as a coating antigen or an antibody prepared from the above-mentioned recombinant protein as a coating antibody.

[0012] The present invention also provides an ELISA kit for detecting serum type I chicken Marek's disease virus, comprising the above-mentioned recombinant protein and / or an antibody prepared using the above-mentioned recombinant protein.

[0013] The present invention also provides the application of the above-mentioned reagents or the above-mentioned ELISA kits in screening SPF-grade poultry that are free from serum type I Marek's disease virus contamination.

[0014] The present invention also provides the application of the above-mentioned reagents or the above-mentioned ELISA kits in detecting whether poultry live vaccines and / or strains are contaminated with serum type I chicken Marek's disease virus.

[0015] Beneficial Effects: This invention screens for a specific protein unique to MDV-1 and highly conserved among different MDV-1 strains, and modifies it to obtain the recombinant gene shown in SEQ ID No. 1 of this invention. The recombinant gene of this invention encodes the MDV-1 recombinant PP38 protein. The MDV-1 recombinant PP38 protein can be used for ELISA detection; when the MDV-1 recombinant PP38 protein is used as the coating antigen, indirect ELISA detection can be performed; when the antibody prepared from the MDV-1 recombinant PP38 protein is used as the coating antibody, direct ELISA detection can be performed; and competitive ELISA can also be used to detect the corresponding antigen or antibody. In one embodiment of this invention, an indirect ELISA kit for detecting MDV-1 antibodies was constructed using the MDV-1 recombinant PP38 protein as the coating antigen. This kit exhibits good reactivity, specificity, sensitivity, and repeatability, and can be applied to clinical MDV-1 serum antibody detection, SPF chicken screening, detection of exogenous viral antibodies in poultry live vaccines or strains, and epidemiological surveys, thereby controlling the spread of MDV-1 infection. Attached Figure Description

[0016] Figure 1 This is a diagram showing the low-level expression of MDV-1PP38 protein in Example 2; where M represents Marker; 1 represents IPTG induction (BL21); and 2 represents the uninduced control (BL21).

[0017] Figure 2 This is a diagram showing the high expression of MDV-1PP38 protein in Example 2; M represents Marker; 1 represents supernatant after sonication; 2 represents precipitate after sonication.

[0018] Figure 3 This is a diagram of the purification of MDV-1PP38 protein in Example 2; M represents the marker; 1-3 represent the purified protein. Detailed Implementation

[0019] This invention provides a recombinant gene derived from MDV-1, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0020] The MDV-1 strain described in this invention contains a specific protein that can distinguish between MDV-2 and MDV-3, and this specific protein is highly conserved across different MDV-1 strains. In one embodiment of this invention, based on the PP38 sequence of MDV-1J-1 strain (Genbank accession number: AQN77164.1), a nucleotide fragment (486 bp) in the 60-221aa region was selected, codon-optimized, and the synthesized nucleotide sequence is shown in SEQ ID No. 1:

[0021] GATCGGGTCCAGAGGGACCGGTGGAGATTCAGTTCTCCGCCCTCACTCTGGAGTCACGGGGGAAGGGGGCTATTCCAATAAAGGGTGATGGGAAGGCGATAGAATGCCAGGAGCTAACCGGAGAGGGAGTGGCTGTCACAGTGGGGGGAGCTACCGCCTGAGCCCCGGAGGTCAGGGAATGAACATCTTGACGAAAGTCGGTATGCGAAACAAACCGAAAGGGGTAGCTCTACGGGGGAAA GAAGAGGGAGATGGTATGAAGCAGATGGGGGAGCTTGCCCAGCAGTGCGAAGGAGGAACATATGCGGACTTGCTTGTCGAAGCAGAGCAAGCTGTTGTACATTCCGTTCGCGCATTAATGCTGGCCGAAAGACAAAACCCAAATATATTGGGGGAGCATTTGAATAAAAAACGGGTTCTTGTACAACGACCCCGTACTATTCTATCCGTGGAGTCAGAGAATGCAACAATGCGTTCTTATATG.

[0022] The present invention also provides a recombinant protein encoded by the above-mentioned recombinant gene.

[0023] In one embodiment of the present invention, the amino acid sequence of the MDV-1PP38 recombinant protein encoded by SEQ ID No. 1 is shown in SEQ ID No. 2:

[0024] DRVQRDRWRFSSPPPHSGVTGKGAIPIKGDGKAIECQELTGEGEWLSQWGELPPEPRRSGNEHLDESRYAKQTERGSSTGKEEGDGMKQMGELAQQCEGGTYADLLVEAEQAVVHSVRALMLAERQNPNILGEHLNKKRVLVQRPRTILSVESENATMRSYM.

[0025] The present invention also provides a biological material comprising the above-mentioned recombinant gene and expressing the above-mentioned recombinant protein.

[0026] In a preferred embodiment of the present invention, the type of biological material includes an expression vector or an expression host cell. The present invention does not specifically limit the type and source of the expression vector and the expression host cell, as long as they are compatible enough to complete the expression of the recombinant protein. For example, a prokaryotic expression vector and prokaryotic expression bacteria can be used for matching, or a eukaryotic expression vector and eukaryotic expression cells can be used for matching. In one embodiment of the present invention, taking prokaryotic expression as an example, the nucleotide sequence shown in SEQ ID No. 1 is inserted into the EcoRI and Xho I restriction sites of pET-30a, and transformed into Escherichia coli BL21. After culture, the MDV-1PP38 recombinant protein is produced under IPTG induction.

[0027] The present invention also provides the application of the above-mentioned recombinant gene, recombinant protein or biological material in the preparation of reagents for detecting serum type I chicken Marek's disease virus.

[0028] In a preferred embodiment of the present invention, the detection method compatible with the reagent includes at least one of the following: indirect ELISA, direct ELISA, and competitive ELISA. In indirect ELISA, a known antigen is adsorbed onto the surface of a solid-phase carrier. The sample to be tested (containing antibodies) is added, allowing the antibodies in the sample to specifically bind to the solid-phase antigen, forming an antigen-antibody complex. After washing, an enzyme-labeled anti-antibody (secondary antibody) is added. The secondary antibody binds to the antibody bound to the antigen, forming an antigen-antibody-enzyme-labeled secondary antibody complex. A substrate is then added for color development. The presence and content of antibodies in the sample are detected by the color reaction; the darker the color, the higher the antibody content in the sample. In direct ELISA testing, known antigens or antibodies are adsorbed onto the surface of a solid-phase support, allowing the enzyme-labeled antigen-antibody reaction to occur on the solid-phase surface. The amount of antigen or antibody is detected by the color development of the substrate. In direct detection, the antigen to be tested is directly immobilized on the solid-phase support, and then enzyme-labeled specific antibodies are added to bind with the antigen to form an antigen-enzyme-labeled antibody complex. After washing to remove unbound enzyme-labeled antibodies, substrate is added for color development, and the intensity of the color indicates the antigen content.

[0029] In this invention, detection can also be performed using a competitive ELISA method. When using a competitive ELISA to detect antigens, a known antibody is coated onto a solid-phase support, and the antigen to be tested and a certain amount of enzyme-labeled antigen are added. The two compete to bind to the solid-phase antibody. The higher the content of the antigen to be tested, the more opportunities it has to bind to the solid-phase antibody, and the less the enzyme-labeled antigen binds. Finally, color development is achieved through substrate, and the color intensity is inversely proportional to the content of the antigen to be tested. When using a competitive ELISA to detect antibodies, a known antigen is first coated onto a solid-phase support, and the antibody to be tested and a certain amount of enzyme-labeled antibody are added. The antibody to be tested and the enzyme-labeled antibody compete to bind to the solid-phase antigen. The higher the content of the antibody to be tested, the less the enzyme-labeled antibody binds to the solid-phase antigen. Similarly, color development is achieved through substrate, and the color intensity is inversely proportional to the content of the antibody to be tested.

[0030] The present invention also provides a reagent for detecting serum type I chicken Marek's disease virus, comprising using the above-mentioned recombinant protein as a coating antigen or an antibody prepared from the above-mentioned recombinant protein as a coating antibody.

[0031] The specific contents of the reagent described in this invention may be slightly adjusted based on the detection method. In one embodiment, the MDV-1PP38 recombinant protein is used as the coating antigen to detect the presence of antibodies, thereby determining the presence of MDV-1.

[0032] The present invention also provides an ELISA kit for detecting serum type I chicken Marek's disease virus, comprising the above-mentioned recombinant protein and / or an antibody prepared using the above-mentioned recombinant protein.

[0033] The ELISA kit described in this invention can be an ELISA kit for direct detection of antigens, an ELISA kit for indirect detection of antibodies, or an ELISA kit for competitive binding detection of antigens or antibodies.

[0034] In one embodiment of the present invention, taking an ELISA kit for indirect antibody detection as an example, the ELISA kit for detecting MDV-1 antibodies, using the MDV-1 PP38 recombinant protein as the coating antigen for detecting MDV-1 antibodies, may further include an enzyme-labeled plate, coating buffer, blocking buffer, positive serum control, negative serum control, enzyme-labeled secondary antibody, sample diluent, washing buffer, TMB chromogenic solution, and reaction termination solution. The coating buffer in the present invention can be carbonate buffer, the washing buffer can be PBST, the enzyme-labeled secondary antibody can be horseradish peroxidase-labeled donkey anti-chicken IgG antibody, the blocking buffer can be 10% rabbit serum, and the sample diluent can be PBST. The positive serum control in the present invention is an SPF chicken serum sample obtained from immunization with a live MDV-1 vaccine; the negative serum control is an SPF chicken serum sample not infected with MDV-1.

[0035] The method for detecting MDV-1 antibodies in serum samples using the indirect ELISA detection kit described in this invention includes the following steps:

[0036] S1: Dilute MDV-1PP38 protein to a concentration of 4 μg / mL with coating buffer, add 50 μL / well to the ELISA plate, and coat the ELISA plate at 37°C for 3 hours.

[0037] S2: Remove the coating solution from S1, wash 3 times with PBST washing solution, 250 μL / well, pat dry and add blocking solution, 50 μL / well, incubate at 37℃ for 60 min;

[0038] S3: Remove the blocking solution from S2, wash 3 times with PBST washing solution, 250 μL / well, pat dry, add the serum sample to be tested diluted 1:100 with sample dilution solution, 50 μL / well, and incubate at 37℃ for 60 min.

[0039] S4: Remove the serum sample from S3, wash 4 times with PBST washing buffer, 250 μL / well, pat dry, add enzyme-labeled secondary antibody diluted 1:10000 with sample dilution buffer, 50 μL / well, incubate at 37℃ for 60 min;

[0040] S5: Remove the enzyme-labeled secondary antibody from S4, wash 4 times with PBST washing buffer (250 μL / well), pat dry, add TMB colorimetric solution (100 μL / well), incubate at room temperature in the dark for 10 min, then add stop solution (50 μL / well) to stop the reaction.

[0041] S6: Measure the OD of the liquid in the wells of the ELISA plate using a microplate reader. 450 Value, when the OD of the sample to be tested 450 A test result is considered positive when the OD value is ≥0.31; when the OD value of the sample to be tested is ≥0.31, the result is considered positive. 450 When the value is <0.31, the test result is negative.

[0042] The present invention also provides the application of the above-mentioned reagents or the above-mentioned ELISA kits in screening SPF-grade poultry that are free from serum type I Marek's disease virus contamination.

[0043] In one embodiment of the present invention, an indirect ELISA detection kit is constructed to screen SPF chickens free from MDV-1 contamination. The specific method includes the following steps: collecting blood from the SPF chicken flock and separating serum, and performing MDV-1 antibody detection according to the usage instructions of the kit. The serum should be negative for MDV-1 antibody; otherwise, it indicates that the SPF chicken flock is contaminated with exogenous MDV-1 virus and should not be used for vaccine preparation or serum preparation.

[0044] The present invention also provides the application of the above-mentioned reagents or the above-mentioned ELISA kits in detecting whether avian live vaccines and / or strains are contaminated with MDV-1.

[0045] In one embodiment of the present invention, a constructed indirect ELISA detection kit is used to screen whether poultry live vaccines and / or strains have been contaminated with exogenous MDV-1. The specific method includes the following steps: Twenty SPF chickens suitable for inoculation with poultry live vaccines or strains are simultaneously inoculated with 10 doses of vaccine or strain via eye drops and 100 doses via intramuscular injection. Twenty-one days later, the inoculation is repeated once using the same method and dosage. Blood is collected 42 days after the first inoculation, and serum is separated. MDV-1 antibody detection is performed according to the instructions for use of the kit. The serum from all 20 SPF chickens should be negative for MDV-1 antibodies; otherwise, it indicates the presence of exogenous MDV-1 virus contamination in the poultry live vaccine or strain, which may threaten the safety of the vaccine or strain.

[0046] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the characteristic proteins and detection applications of a type I chicken Marek's disease virus provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1: MDV-1PP38 protein sequence analysis

[0048] Based on the PP38 sequence of MDV-1J-1 strain (Genbank accession number: AQN77164.1), a nucleotide fragment (486 bp) in the 60-221aa region was selected. After codon optimization, the sequence shown in SEQ ID No. 1 was synthesized by Beijing Liuhe Huada Protein R&D Center Co., Ltd., and EcoRI (GAATTC) and XhoI (CTCGAG) restriction sites were added to the 5' and 3' ends for vector cloning.

[0049] Example 2: Construction of recombinant expression plasmid and expression and purification of PP38 protein

[0050] 1. Construction of recombinant expression plasmids

[0051] EcoRI and Xho I restriction sites were introduced at the 5' and 3' ends of SEQ ID No. 1 in Example 1, respectively, using a sequence synthesis method for the preparation of recombinant expression plasmids.

[0052] The sequence shown in SEQ ID No. 2 after the addition of EcoRI and XhoI restriction sites and the plasmid pET-30a were double-digested with restriction endonucleases EcoRI and XhoI, respectively. The purified and recovered fragments and the digested products of the expression vector were ligated with a DNALigation Kit to obtain the recombinant expression plasmid, which was then transformed into competent cells (BL21).

[0053] 2. Low-level expression of MDV-1 PP38 recombinant protein

[0054] BL21 obtained in step (1) and identified as positive by PCR were selected and cloned into 1.5 mL of LB liquid medium containing kanamycin resistance (50 μg / mL). The medium was cultured at 37°C and 200 r / min. The culture was continued until OD200 was reached. 600 The value was 0.6–0.8. IPTG was added to the cultured bacterial solution to a final concentration of 0.5 mM, and the mixture was induced for 2 h at 37 °C and 200 r / min. 1 mL of the induced bacterial solution was taken and centrifuged at 12000 r / min for 1 min. The supernatant was discarded, and the precipitate was dispersed with 100 μL of Tris-HCl (pH 8.0) buffer. An equal volume of 2× loading buffer was added, and the mixture was kept at 100 °C for 5 min before electrophoresis detection.

[0055] Electrophoresis results as follows Figure 1 As shown, a specific target band of recombinant PP38 protein appears at a size of approximately 30 kDa, indicating that PP38 protein expression was successful.

[0056] The primers used for PCR identification include primer pair 1, which amplifies a 285 bp fragment, and primer pair 2, which amplifies a 141 bp fragment. Their sequences are shown below:

[0057] Primer pair 1:

[0058] F1 (SEQ ID No. 3): 5'-CAGGGAATGAACATCTTGAC-3';

[0059] R1 (SEQ ID No. 4): 5'-CTGACTCCACGGATAGAATAG-3';

[0060] Primer pair 2:

[0061] F1 (SEQ ID No. 5): 5'-GGCTATTCCAATAAAGGGTG-3';

[0062] R1 (SEQ ID No. 6): 5'-GCATACCGACTTTCGTCAA-3'.

[0063] 3. High expression of MDV-1PP38 recombinant protein

[0064] The transformed BL21 obtained in step 1 was identified by PCR, and the positive BL21 samples were cultured. The resulting bacterial culture was transferred to 250 mL of kanamycin-resistant (50 μg / mL) LB liquid medium at a volume ratio of 1:50 and cultured with shaking at 37°C and 200 rpm until OD500 was reached. 600 =0.6~0.8. Add IPTG to the culture medium to a final concentration of 0.5mM and induce at 37℃ for 3h.

[0065] Centrifuge at 8000 r / min for 6 min, discard the supernatant to obtain bacterial cells; perform ultrasonic disruption on the obtained bacterial cells, the specific process is as follows: after blowing the obtained bacterial cells with 30 mL of 10 mM Tris-HCl (pH 8.0) solution, perform ultrasonic disruption, the ultrasonic disruption power is 500 W, ultrasonic disruption is performed 180 times, each time for 5 seconds, and the next ultrasonic disruption is performed after a 5-second interval.

[0066] The product obtained from ultrasonic disruption was analyzed by electrophoresis. The specific procedure was as follows: 100 μL of the ultrasonically disrupted bacterial suspension was centrifuged at 12000 r / min for 10 min. After centrifugation, 50 μL of supernatant and the resulting precipitate were retained. The precipitate was dispersed with 50 μL of 10 mM Tris-HCl (pH 8.0) solution. SDS-PAGE analysis was performed on the supernatant and the solution obtained after precipitate dispersion. The results showed a large amount of the target protein detected in the precipitate, indicating that the recombinant bacterial expression mode was inclusion body expression. Figure 2 ).

[0067] The well-expressed MDV-1 PP38 protein in the above bacterial precipitate was purified as follows: The precipitate obtained by ultrasonic centrifugation was resuspended in 20–30 mL of Tris-HCl (10 mM, pH 8.0) solution and allowed to stand for 10 min; then centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to another tube for storage. For the precipitate, it was resuspended in 20–30 mL of Tris-HCl (10 mM, pH 8.0) solution and allowed to stand for 10 min; then centrifuged at 12000 rpm for 10 min, and the supernatant was discarded to obtain the first precipitate; the resuspension and centrifugation steps were repeated for the first precipitate to obtain the second precipitate. The precipitate was first resuspended in a small amount of 10 mM Tris-HCl (pH 8.0) solution, then the protein was dissolved in 5–10 mL of Tris-HCl (10 mM, pH 8.0) solution containing 8 M urea. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. 50 μL of the sample was then analyzed by SDS-PAGE electrophoresis. Figure 3 Using BSA (bovine serum albumin) as a standard, the purified protein concentration was estimated to be >0.5 mg / mL and the purity >85% by SDS-PAGE gel scanning analysis, and it was named MDV-1PP38.

[0068] Example 3: Determination of Optimal Reaction Conditions for the ELISA Method

[0069] 1. Determination of optimal PP38 coating concentration and optimal working antibody concentration

[0070] The purified PP38 protein was diluted with coating buffer to 4 μg / mL, 2 μg / mL, 1 μg / mL and 0.5 μg / mL, and added to ELISA plates at 50 μL / well. The plates were incubated at 37°C for 3 h. The coating buffer was discarded, and the plates were washed three times with PBST and patted dry. The plates were then blocked with 50 μL / well of 10% rabbit serum at 37°C for 1 h. Discard the blocking solution, wash three times with PBST, and pat dry. Dilute the standard positive control serum and negative control serum serially at 1:20, 1:50, 1:100, and 1:200, add 50 μL / well to the ELISA plate, incubate at 37°C for 1 h, and perform a checkerboard assay. Discard the serum, wash four times with PBST, and pat dry. Add 1:10000 ELISA-labeled secondary antibody and incubate at 37°C for 1 h. Discard the ELISA-labeled secondary antibody, wash four times with PBST, and pat dry. Add 100 μL TMB chromogenic buffer to each well and incubate at room temperature in the dark for 10 min. Add 50 μL of stop solution to terminate the reaction, and immediately measure the OD using an ELISA reader. 450 Value. According to OD 450 The P / N value and the P / N value determine the optimal working concentration of the coating antigen and antibody.

[0071] The results are shown in Tables 1 and 2. When the antigen coating concentration was 4 μg / mL and the antibody dilution was 1:100, the OD of the positive control serum was... 450 The value was close to 1.0, and the P / N value was the highest. Therefore, the optimal coating concentration of the ELISA antigen was determined to be 4 μg / mL, and the optimal serum dilution was determined to be 1:100.

[0072] Table 1. Detection results of optimal working concentrations (OD) of coated PP38 antigen and antibody. 450 value)

[0073]

[0074] Table 2. Detection results of optimal working concentrations of coated PP38 antigen and antibody (P / N values)

[0075]

[0076] 2. Determination of the optimal dilution of enzyme-labeled secondary antibodies

[0077] The ELISA plate was coated with the optimal concentration of PP38 antigen. Positive and negative sera were diluted to the optimal concentration. The ELISA-labeled secondary antibody was diluted 1:8000, 1:10000, 1:16000, and 1:20000. Each dilution was performed in duplicate, and the average value was taken. The results were analyzed by OD... 450 The optimal working concentration of the enzyme-labeled secondary antibody was determined using the P / N ratio. The results are shown in Tables 3 and 4. The P / N ratio was highest when the enzyme-labeled secondary antibody concentration was 1:10000.

[0078] Table 3 Results of detection at optimal dilution of enzyme-labeled secondary antibody (OD) 450 value)

[0079]

[0080] Table 4. Results of Detection of Optimal Dilution of Enzyme-Labeled Secondary Antibody (P / N Value)

[0081] enzyme-labeled secondary antibody dilution 1:8000 1:10000 1:16000 1:20000

[0082] 3. Determination of optimal coating conditions and time

[0083] ELISA plates were coated with PP38 protein at the optimal concentration under three coating conditions: incubation at 37°C for 3 hours; overnight at 4°C; and incubation at 37°C for 1 hour followed by overnight at 4°C. Two replicates were performed for each coating condition, and the average value was used. Positive and negative sera and the ELISA-labeled secondary antibody were diluted to the optimal dilution and analyzed by OD... 450 The P / N ratio and P value were used to determine the optimal coating conditions and time for the antigen. The results are shown in Tables 5 and 6. The optimal coating condition was 37℃ for 3 hours.

[0084] Table 5 Detection results of optimal coating conditions (OD) 450 value)

[0085] Envelope conditions 37℃3h 37℃ for 1 hour, 4℃ overnight wrapped in a blanket 4℃ overnight wrapped in a blanket Positive serum 1.2013 1.0114 0.9314 negative serum 0.1131 0.1191 0.1039

[0086] Table 6. Detection results of optimal coating conditions (P / N values)

[0087]

[0088] 4. Selection of the optimal sealing solution

[0089] The microplate was coated with PP38 protein at the optimal concentration and coated at 37°C for 3 hours. After washing, blocking buffers were prepared using 5% skim milk, 1% gelatin, 10% horse serum, 1% BSA, 10% rabbit serum, and 10% sheep serum, respectively. Two replicates were performed for each well, and the average value was taken. Positive and negative sera and enzyme-labeled secondary antibodies were diluted to the optimal dilution. The buffer was then analyzed by OD... 450 The optimal blocking solution was determined using the P / N ratio and the P value. The results are shown in Tables 7 and 8. The P / N ratio was the highest when using 10% rabbit serum for blocking, therefore 10% rabbit serum was selected as the optimal blocking solution.

[0090] Table 7 Determination of Optimal Sealing Solution (OD) 450 value)

[0091] Sealing liquid 5% skim milk 1% gelatin 10% horse serum 1% BSA 10% rabbit serum 10% sheep serum Positive serum 0.1458 1.2058 0.8813 1.0149 1.1786 1.1943 negative serum 0.1514 1.0901 0.1169 0.1391 0.1068 0.1304

[0092] Table 8 Determination of the optimal sealing solution (OD) 450 value)

[0093] Sealing liquid 5% skim milk 1% gelatin 10% horse serum 1% BSA 10% rabbit serum 10% sheep serum P / N value 0.9630 1.1061 7.5389 7.2962 11.0356 9.1587

[0094] 5. Determining the color development time

[0095] Add TMB chromogenic solution and incubate at room temperature in the dark for 10 min, 20 min, and 30 min, respectively. For each chromogenic time point, set up two replicates for both positive and negative sera and take the average value. Analyze the OD... 450 The optimal color development time was determined using the P / N value and the P / N ratio. The results are shown in Tables 9 and 10. The P / N ratio was the highest when the color development time was 10 min at room temperature, therefore the optimal color development time for TMB was 10 min.

[0096] Table 9 Determination of TMB color development (OD) 450 value)

[0097] Room temperature display time 10min 20min 30min Positive serum 1.1131 1.2390 1.3031 negative serum 0.0983 0.1210 0.1440

[0098] Table 10 Determination of Optimal Color Development Time (P / N Value)

[0099] Room temperature display time 10min 20min 30min P / N value 11.3234 10.2397 9.0493

[0100] Example 4: Indirect ELISA kit for detecting MDV-1 antibodies

[0101] The method for detecting MDV-1 antibodies using the ELISA kit of Example 3 includes the following steps:

[0102] S1: Dilute MDV-1PP38 protein to a concentration of 4 μg / mL with coating buffer, add 50 μL / well to the ELISA plate, and coat the ELISA plate at 37℃ for 3 h.

[0103] S2: Remove the coating solution from S1, wash 3 times with PBST washing solution, 250 μL / well, pat dry and add blocking solution, 50 μL / well, incubate at 37℃ for 60 min;

[0104] S3: Remove the blocking solution from S2, wash 3 times with PBST washing solution, 250 μL / well, pat dry, add the serum sample to be tested diluted 1:100 with sample dilution solution, 50 μL / well, and incubate at 37℃ for 60 min.

[0105] S4: Remove the serum sample from S3, wash 4 times with PBST washing buffer, 250 μL / well, pat dry, add enzyme-labeled secondary antibody diluted 1:10000 with sample dilution buffer, 50 μL / well, incubate at 37℃ for 60 min;

[0106] S5: Remove the enzyme-labeled secondary antibody from S4, wash 4 times with PBST washing buffer (250 μL / well), pat dry, add TMB colorimetric solution (100 μL / well), incubate at room temperature in the dark for 10 min, then add stop solution (50 μL / well) to stop the reaction.

[0107] S6: Measure the OD of the liquid in the wells of the ELISA plate using a microplate reader. 450 Value, when the OD of the sample to be tested 450 A test result is considered positive when the OD value is ≥0.31; when the OD value of the sample to be tested is ≥0.31, the result is considered positive. 450 When the value is <0.31, the test result is negative.

[0108] Determination of positive and negative cutoff values: 49 negative serum samples were tested, with each sample tested in duplicate in two wells. The results are shown in Table 11. The OD values ​​of the negative serum samples were calculated. 450 The mean (X) and standard deviation (SD) of the values ​​are calculated using the formula: threshold = X + 3SD, to obtain the OD of the ELISA. 450 The threshold is 0.31. When the OD of the sample to be tested... 450 A result is considered positive when the OD value is ≥0.31; when the OD value of the sample to be tested is ≥0.31, the result is considered positive. 450 When the value is <0.31, the result is considered negative.

[0109] Table 11. Detection results and cutoff values ​​of 49 negative serum samples.

[0110]

[0111] Example 5: Specificity assay for MDV-1 antibody detection using indirect ELISA

[0112] The indirect ELISA method established in Example 3 was used to detect positive sera for known pathogens such as infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), egg drop syndrome virus (EDSV), Newcastle disease virus (NDV), Marek's disease virus (MDV), and avian reovirus. Simultaneously, positive and negative control sera for MDV-1 were established to determine the presence of cross-reactivity and to analyze the specificity of the kit described in Example 4. The results showed that OD... 450 The values ​​were all less than 0.31, which was considered negative, indicating that the PP38 protein did not cross-react with the positive serum of the above-mentioned virus, and that the indirect ELISA kit had good specificity.

[0113] Table 12 Specificity results of the MDV-1 indirect ELISA antibody detection method

[0114] sample <![CDATA[OD 450 Value determination Positive for infectious bronchitis virus in chickens 0.1140 Negative Positive for infectious bursal disease virus in chickens 0.1891 Negative Positive for infectious laryngotracheitis virus in chickens 0.1043 Negative Decreased egg production in chickens, positive for viral infection. 0.1631 Negative Newcastle disease virus clear serum in chickens 0.1390 Negative Chicken Marek's disease virus positive serum 0.1101 Negative Avian reovirus positive serum 0.0943 Negative

[0115] Example 6: Sensitivity test of MDV-1 antibody detection by indirect ELISA

[0116] Three positive serum samples were taken, and the serum was serially diluted from 1:100 to 3200. The tests were performed according to the detection method in Example 3. The results showed that for different positive sera, after dilution to 1:400, the OD... 450 The value was 0.4313, which was still a positive result, indicating that the PP38-coated antigen has high sensitivity.

[0117] Example 7: Concordance test of MDV-1 antibody detection by indirect ELISA

[0118] The main method for detecting MDV-1 antibodies is the agar diffusion assay, but this method has low sensitivity and is prone to failing to detect positive samples. Of the 134 samples selected, negative and positive samples were detected using the agar diffusion assay. The results were compared with the ELISA method established in this invention to compare the relative sensitivity and specificity of the two methods. The results showed that the kit described in this invention detected more positive samples, indicating high sensitivity.

[0119] 134 clinical serum samples were tested using the indirect ELISA kit shown in Example 4. The sensitivity, specificity, and concordance rate of the kit with the agar diffusion assay were analyzed.

[0120] Relative sensitivity (%) = (Number of positive results / (Number of positive results + Number of false negative results)) × 100%;

[0121] Relative specificity (%) = (Number of negatives / (Number of negatives + Number of false positives)) × 100%;

[0122] Overall compliance rate (%) = (Number of positive cases + Number of negative cases) / Total number of tests × 100%.

[0123] 134 clinical serum samples were tested using the indirect ELISA kit described in this invention, of which 35 were positive and 99 were negative. The same 134 clinical serum samples were also tested using an agar diffusion assay, of which 30 were positive and 108 were negative (Table 13). Therefore, the relative sensitivity of the indirect ELISA method of this invention is 85.7%, the relative specificity is 95.2%, and the overall concordance rate is 96.3% (Tables 13 and 14). The indirect ELISA kit of this invention has high sensitivity and specificity, and its sensitivity is higher than that of the agar diffusion assay method.

[0124] Table 13. Detection results of the MDV-1 indirect ELISA antibody detection kit and the commercial kit.

[0125]

[0126]

[0127] Table 14 shows the concordance rate between the MDV-1 indirect ELISA antibody detection kit and the commercial agar diffusion reagent.

[0128]

[0129] Example 8: Repeatability test of MDV-1 antibody detection by indirect ELISA

[0130] Following the detection method in Example 3, intra-batch and inter-batch repeatability tests were performed. The same batch of PP38 protein was used to coat the ELISA plate, and four positive and negative sera with different antibody titers were tested. Each sample was tested in triplicate, and the OD value of each well was measured. 450 The standard deviation of each sample was calculated, and then the intra-batch coefficient of variation (CV) for each sample was calculated: CV = (standard deviation SD / mean Mean) × 100%. Under the same experimental conditions, VP1 protein-coated ELISA plates were used at three different time points to detect four positive and negative sera with different antibody titers. Each sample was tested in triplicate, and the OD of each well was measured. 450 The standard deviation of each sample was calculated, and then the coefficient of variation (CV) between batches for each sample was calculated. CV = (standard deviation SD / mean Mean) × 100%. The results are shown in Tables 15 and 16. The coefficients of variation for both intra-batch and inter-batch repeatability tests were less than 10%, indicating that the detection method has good repeatability and stable results.

[0131] Table 15 Results of intra-batch repeatability tests for serum samples with different positive and negative positivity.

[0132]

[0133] Table 16 Results of batch-to-batch repeatability tests for serum samples with different positive and negative positivity.

[0134]

[0135] Preliminary applications of the reagent kit:

[0136] This kit was applied to the detection of exogenous viruses in avian viral live vaccines. Ten key avian live vaccines produced by domestic manufacturers were selected, and MDV-1 antibody detection was performed using the kit described in Example 4. Serological testing for MDV-1 contamination was also conducted on the selected avian live vaccines according to the 2020 edition of the Chinese Veterinary Pharmacopoeia, Part III. A PBS group was set as a negative control, and an MDV-1-infected group (CVI988 live vaccine) was set as a positive control. As shown in Table 17, the results of the agar diffusion test were consistent with the results obtained using this kit; all selected avian live vaccines were free of MDV-1 contamination. The negative control was MDV-1 negative, and the positive control was MDV-1 positive.

[0137] Table 17 shows the test results of vaccines from domestic companies using the kit of this invention.

[0138]

[0139] In summary, the MDV-1 ELISA antibody detection kit of this invention is based on the fact that the MDV-1 PP38 protein is an important immunogenic protein of MDV-1 and is highly conserved among different MDV-1 strains. It can simultaneously recognize different MDV-1 strains without cross-reacting with viruses such as Egg Drop Syndrome Virus (EDSV). This kit exhibits good specificity and can be used not only for the detection of exogenous MDV-1 in avian viral live vaccines (chicken test method) but also for clinical detection and epidemiological investigations of MDV-1.

[0140] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a reagent for detecting serum type I Marek's disease virus in the screening of SPF-grade poultry free from serum type I Marek's disease virus contamination for vaccine preparation and serum preparation, characterized in that, The reagent is a reagent for detecting MDV-1 antibody using an indirect antibody detection method, and a recombinant protein is used as the coating antigen for detecting MDV-1 antibody; the amino acid sequence of the recombinant protein is shown in SEQ ID No. 2; The screening of SPF-grade poultry that are free from serum type I Marek's disease virus contamination is for non-disease diagnostic purposes.

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