Characteristic protein of serotype I chicken Marek's disease virus and detection application

Abstract

The invention provides a characteristic protein of serotype I chicken Marek's disease virus and detection application, and belongs to the technical field of virus detection. The invention provides a recombinant gene derived from serum I type chicken Marek's disease virus. The nucleotide sequence of the recombinant gene is shown as SEQ ID No. 1. The recombinant gene disclosed by the invention is used for coding the MDV-1 recombinant PP38 protein, and the MDV-1 recombinant PP38 protein can be applied to enzyme-linked immuno sorbent assay (ELISA) detection. The invention also provides an ELISA detection kit constructed by using the MDV-1 recombinant PP38 protein, and the ELISA detection kit can be used for clinical MDV-1 serum antibody detection, SPF chicken screening, exogenous virus antibody detection of poultry live vaccines or virus seeds, epidemiological investigation and the like, so as to prevent and control transmission of MDV-1 infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and particularly relates to a characteristic protein of Marek's disease virus serotype I and its detection application. Background Art

[0002] Marek disease (MD) is a highly contagious and tumorous disease caused by Marek disease virus (MDV), characterized by lymphoid tissue hyperplasia and tumor formation. MDV can be divided into three serotypes, namely MDV-1, MDV-2, and MDV-3. MDV-1 includes all tumorigenic virulent strains (such as GA strain), artificially attenuated vaccine strains (such as CVI988, 814 strain), and MDV-1 vaccine strains lacking the meq gene. MDV-2 is a non-pathogenic natural attenuated strain (such as SB-1); MDV-3 is herpesvirus of turkeys (HVT). In recent years, due to the presence of immunosuppressive diseases such as chicken infectious anemia and avian leukosis in chicken flocks, and the continuous increase in the virulence of MDV with a tendency to evolve towards stronger virulence, although MD vaccines are widely inoculated in chicken flocks, MD still occurs, causing huge economic losses to the poultry industry.

[0003] The main methods for determining whether there is MDV-1 infection in chicken flocks, vaccines, and virus seeds are ELISA, agar diffusion test, and virus neutralization test (VN). Among them, the agar diffusion test is low-cost, but has low sensitivity and low accuracy of detection results, thus greatly limiting its application in production. In the inspection of finished products of live avian vaccines, the "Pharmacopoeia of the People's Republic of China" (Part III) 2020 edition stipulates the chicken inspection method to detect MDV-1 agar diffusion antibody to determine whether there is MDV-1 contamination in the vaccine. Contamination of the vaccine with MDV-1 is a potential risk for the spread and prevalence of MDV-1. Therefore, there is an urgent need to establish a stable and efficient method for detecting MDV-1 antibody for the quality detection of live avian vaccines and the diagnosis of MDV-1 diseases. Summary of the Invention

[0004] The present invention provides a characteristic protein of Marek's disease virus serotype I and its detection application, which can quickly, accurately, and highly sensitively detect MDV-1 and / or MDV-1 antibody.

[0005] The present invention provides a recombinant gene derived from Marek's disease virus serotype I, and the nucleotide sequence of the recombinant gene is shown in SEQ ID No.1.

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

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

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

[0009] The present invention also provides the use of the above recombinant gene, the above recombinant protein or the above biological material in the preparation of a reagent for detecting Marek's disease virus serotype 1 in serum.

[0010] In a preferred embodiment of the present invention, the detection method matching 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 Marek's disease virus serotype 1 in serum, comprising the above recombinant protein as a coating antigen or an antibody prepared from the above recombinant protein as a coating antibody.

[0012] The present invention also provides an ELISA kit for detecting Marek's disease virus serotype 1 in serum, comprising the above recombinant protein and / or an antibody prepared from the above recombinant protein.

[0013] The present invention also provides the use of the above reagent or the above ELISA kit in screening SPF-class poultry free from contamination with Marek's disease virus serotype 1.

[0014] The present invention also provides the use of the above reagent or the above ELISA kit in detecting whether live avian vaccines and / or virus seeds are contaminated with Marek's disease virus serotype 1.

[0015] Beneficial effects: The present invention screens a specific protein that is unique to MDV-1 and highly conserved among different MDV-1 strains, and after modification, obtains the recombinant gene shown in SEQ ID No.1 of the present invention. The recombinant gene of the present 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 a coating antigen, indirect ELISA detection can be performed; when an antibody prepared from the MDV-1 recombinant PP38 protein is used as a coating antibody, direct ELISA detection can be performed; and the corresponding antigen or antibody can also be detected by competitive ELISA. In an embodiment of the present invention, an indirect ELISA detection kit for detecting MDV-1 antibodies is constructed with the MDV-1 recombinant PP38 protein as a coating antigen, which has good reactogenicity, specificity, sensitivity and repeatability. It can be applied to clinical MDV-1 serum antibody detection, screening of SPF chickens, detection of exogenous virus antibodies in live avian vaccines or virus seeds and epidemiological investigations, etc., so as to prevent and control the spread of MDV-1 infection. Brief Description of the Drawings

[0016] Figure 1 It is a diagram of the small-scale expression of MDV-1 PP38 protein in Example 2; among them, M represents Marker; 1 represents IPTG induction (BL21); 2 represents the non-induced control (BL21);

[0017] Figure 2 It is a diagram of the large-scale expression of MDV-1 PP38 protein in Example 2; M represents Marker; 1 represents the supernatant after sonication; 2 represents the precipitate after sonication;

[0018] Figure 3 It is a diagram of the purification of MDV-1 PP38 protein in Example 2; M represents Marker; 1-3 represent the purified protein. Detailed Description of the Invention

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

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

[0021] GATCGGGTCCAGAGGGACCGGTGGAGATTCAGTTCTCCGCCCCCTCACTCTGGAGTCACGGGGAAGGGGGCTATTCCAATAAAGGGTGATGGGAAGGCGATAGAATGCCAGGAGCTAACCGGAGAGGGAGAGTGGCTGTCACAGTGGGGGGAGCTACCGCCTGAGCCCCGGAGGTCAGGGAATGAACATCTTGACGAAAGTCGGTATGCGAAACAAACCGAAAGGGGTAGCTCTACGGGGAAAGAAGAGGGAGATGGTATGAAGCAGATGGGGGAGCTTGCCCAGCAGTGCGAAGGAGGAACATATGCGGACTTGCTTGTCGAAGCAGAGCAAGCTGTTGTACATTCCGTTCGCGCATTAATGCTGGCCGAAAGACAAAACCCAAATATATTGGGGGAGCATTTGAATAAAAAACGGGTTCTTGTACAACGACCCCGTACTATTCTATCCGTGGAGTCAGAGAATGCAACAATGCGTTCTTATATG。

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

[0023] In one embodiment of the present invention, the amino acid sequence of the MDV-1 PP38 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 recombinant gene and expressing the above recombinant protein.

[0026] In a preferred embodiment of the present invention, the type of the biomaterial includes an expression vector or an expression host cell. The present invention does not particularly limit the types and sources of the expression vector and the expression host cell, as long as they can match each other to complete the expression of the recombinant protein. For example, a prokaryotic expression vector and a prokaryotic expression bacterium can be matched, or a eukaryotic expression vector and a eukaryotic expression cell can be matched. 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 enzyme sites of pET-30a, and then transformed into Escherichia coli BL21. After cultivation, the MDV-1 PP38 recombinant protein is produced under the induction of IPTG.

[0027] The present invention also provides the use of the above-mentioned recombinant gene, the above-mentioned recombinant protein or the above-mentioned biomaterial in the preparation of a reagent for detecting Marek's disease virus serotype I in serum.

[0028] In a preferred embodiment of the present invention, the detection method matching the reagent includes at least one of the following: indirect ELISA, direct ELISA, and competitive ELISA. Among them, in indirect ELISA, a known antigen is adsorbed on the surface of a solid-phase carrier, and a test sample (containing antibodies) is added. The antibodies in the sample specifically bind to the solid-phase antigen to form 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 to form an antigen-antibody-enzyme-labeled secondary antibody complex. Then, a substrate is added for color development. The presence and content of antibodies in the sample are detected through the color reaction. The darker the color, the higher the antibody content in the sample. In direct ELISA, a known antigen or antibody is adsorbed on the surface of a solid-phase carrier, and the enzyme-labeled antigen-antibody reaction is carried out on the solid surface. The amount of antigen or antibody is detected through substrate color development. In direct detection, the test antigen is directly immobilized on the solid-phase carrier, and then an enzyme-labeled specific antibody is added to bind to the antigen to form an antigen-enzyme-labeled antibody complex. After washing to remove the unbound enzyme-labeled antibody, a substrate is added for color development, and the antigen content is judged by the depth of the color.

[0029] In the present invention, detection can also be carried out by the competitive ELISA method. When detecting an antigen by competitive ELISA, a known antibody is coated on a solid-phase carrier, and the antigen to be detected and a certain amount of enzyme-labeled antigen are added. The two compete for binding to the solid-phase antibody. The higher the content of the antigen to be detected, the more opportunities it has to bind to the solid-phase antibody, and the less the enzyme-labeled antigen binds. Finally, through substrate color development, the depth of the color is inversely proportional to the content of the antigen to be detected. When detecting an antibody by competitive ELISA, a known antigen is first coated on a solid-phase carrier, and the antibody to be detected and a certain amount of enzyme-labeled antibody are added. The antibody to be detected and the enzyme-labeled antibody compete for binding to the solid-phase antigen. The higher the content of the antibody to be detected, the less the amount of the enzyme-labeled antibody that binds to the solid-phase antigen. Similarly, through substrate color development, the depth of the color is inversely proportional to the content of the antibody to be detected.

[0030] The present invention also provides a reagent for detecting Marek's disease virus serotype 1 in serum, including the above-mentioned recombinant protein as the coated antigen or the antibody prepared from the above-mentioned recombinant protein as the coated antibody.

[0031] The specific content of the reagent of the present invention can be slightly adjusted based on the detection method. In one embodiment, the MDV-1 PP38 recombinant protein is used as the coated antigen to detect the presence of the antibody, so as to determine whether MDV-1 exists.

[0032] The present invention also provides an ELISA kit for detecting Marek's disease virus serotype 1 in serum, including the above-mentioned recombinant protein and / or the antibody prepared from the above-mentioned recombinant protein.

[0033] The ELISA kit of the present invention can be either an ELISA kit for directly detecting an antigen, or an ELISA kit for indirectly detecting an antibody, or an ELISA kit for detecting an antigen or an antibody through competitive binding.

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

[0035] Method for detecting MDV-1 antibody in serum samples using the indirect ELISA detection kit of the present invention, comprising the following steps:

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

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

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

[0039] S4: Remove the serum sample in S3, wash 4 times with PBST washing solution, 250 μL / well, pat dry, then add the enzyme-labeled secondary antibody diluted 1:10000 with sample diluent, 50 μL / well, and incubate at 37 °C for 60 min;

[0040] S5: Remove the enzyme-labeled secondary antibody in S4, wash 4 times with PBST washing solution, 250 μL / well, pat dry, then add TMB chromogenic solution, 100 μL / well, place at room temperature in the dark for reaction for 10 min, and then add the termination solution, 50 μL / well to terminate the reaction.

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

[0042] The present invention also provides the application of the above reagent or the above ELISA kit in screening SPF-class poultry free from contamination of serotype I Marek's disease virus without serum.

[0043] In one embodiment of the present invention, an indirect ELISA detection kit is used to screen SPF chickens free from MDV-1 contamination. The specific method comprises the following steps: Collect blood from the SPF chicken flock and separate the serum, and perform MDV-1 antibody detection with reference to the usage method of the kit. The serum should be negative after MDV-1 antibody detection. Otherwise, it indicates that the SPF chicken flock is contaminated with exogenous MDV-1 virus and cannot be used for vaccine preparation and serum preparation, etc.

[0044] The present invention also provides the use of the above-mentioned reagent or the above-mentioned ELISA kit in detecting whether avian live vaccines and / or virus seeds are contaminated with MDV-1.

[0045] In one embodiment of the present invention, an indirect ELISA detection kit constructed is used to screen whether avian live vaccines and / or virus seeds have been contaminated with exogenous MDV-1. The specific method includes the following steps: 20 SPF chickens suitable for inoculating avian live vaccines or virus seeds are each inoculated with 10 doses of vaccines or virus seeds by eye dropping and nasal dripping simultaneously, and 100 doses of vaccines or virus seeds by intramuscular injection. After 21 days, inoculation is repeated once according to the above method and dose. Blood is collected 42 days after the first inoculation, and serum is separated. MDV-1 antibody detection is carried out with reference to the use method of the kit. The sera of 20 SPF chickens should all be negative after MDV-1 antibody detection. Otherwise, it indicates that there is contamination of exogenous MDV-1 virus in the avian live vaccine or virus seed, which will pose a threat to the safety of the vaccine or virus seed.

[0046] In order to further illustrate the present invention, the following describes in detail a characteristic protein of Marek's disease virus serotype I in serum and its detection application provided by the present invention in combination with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1 Sequence analysis of MDV-1 PP38 protein

[0048] Based on the PP38 sequence of MDV-1 J-1 strain (Genbank accession number: AQN77164.1), a nucleotide fragment (486 bp) in the 60-221 aa region was selected, codon-optimized, and then entrusted to Beijing Liuhua Huada Protein R & D Center Co., Ltd. to synthesize the sequence shown in SEQ ID No.1, and EcoR I (GAATTC) and Xho I (CTCGAG) restriction sites were added at the 5' and 3' ends for cloning into the vector.

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

[0050] 1. Construction of recombinant expression plasmid

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

[0052] The sequence shown in SEQ ID No.2 after adding EcoRⅠ and Xho I restriction sites and the plasmid pET-30a were double digested with the restriction enzymes EcoRⅠ and Xho I respectively. After ligating the purified and recovered fragments with the enzyme digestion products of the expression vector using the DNA Ligation Kit, a recombinant expression plasmid was obtained and transformed into competent cells (BL21).

[0053] 2. Small-scale expression of MDV-1 PP38 recombinant protein

[0054] Pick the BL21 obtained in step (1) identified as positive by PCR and clone it into 1.5 mL of LB liquid medium containing kanamycin resistance (50 μg / mL), and culture it at a temperature of 37 °C and a rotation speed of 200 r / min. Culture until the OD 600 value is 0.6 - 0.8. Add IPTG with a final concentration of 0.5 mM to the cultured bacterial solution and induce for 2 h at 37 °C and 200 r / min. Take 1 mL of the induced bacterial solution, centrifuge it at a rotation speed of 12,000 r / min for 1 min, discard the supernatant, disperse the precipitate with 100 μL of Tris-HCl (pH 8.0) buffer, add 2×loading buffer equal in volume to the buffer, and keep it at 100 °C for 5 min before performing electrophoresis detection.

[0055] The electrophoresis detection results are as Figure 1 shown. A specific target band of the recombinant PP38 protein appears at approximately 30 KD in size, indicating the successful expression of the PP38 protein.

[0056] The primers for PCR identification include primer pair 1 with an amplified fragment of 285 bp and primer pair 2 with an amplified fragment of 141 bp, and their sequences are as follows:

[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. Large-scale expression of MDV-1 PP38 recombinant protein

[0064] The transformed BL21 obtained in Step 1 was identified by PCR, and the positive BL21 was cultured. The obtained bacterial liquid 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 a temperature of 37 °C and a rotation speed of 200 r / min until OD 600 = 0.6 - 0.8. IPTG with a final concentration of 0.5 mM was added to the culture solution, and induced at 37 °C for 3 h.

[0065] Centrifuge at 8000 r / min for 6 min, discard the supernatant to obtain the bacterial cells; the obtained bacterial cells were lysed by ultrasonic wave, and the specific process was as follows: the obtained bacterial cells were dispersed with 30 mL of 10 mM Tris-HCl (pH 8.0) solution and then subjected to ultrasonic wave disruption. The ultrasonic wave disruption power was 500 W, and the ultrasonic wave disruption was performed 180 times, 5 s each time, and the next ultrasonic wave disruption was carried out after an interval of 5 s.

[0066] The product obtained by ultrasonic wave disruption was detected by electrophoresis. The specific process was as follows: take 100 μL of the ultrasonicated bacterial suspension, centrifuge at a rotation speed of 12000 r / min for 10 min, and retain 50 μL of the supernatant and the obtained precipitate after centrifugation. The obtained precipitate was dispersed with 50 μL of 10 mM Tris-HCl (pH 8.0) solution. The solutions obtained after dispersing the supernatant and the precipitate were respectively subjected to SDS-PAGE detection. A large amount of target protein was detected in the precipitate, indicating that the expression form of this recombinant bacterium was inclusion body expression ( Figure 2 ).

[0067] The well-expressed MDV-1 PP38 protein in the above-mentioned bacterial cell precipitate was purified by the following method: resuspend the precipitate obtained by ultrasonic centrifugation with 20 - 30 mL of Tris-HCl (10 mM, pH 8.0) solution, and let it stand for 10 min; centrifuge at 12000 r / min for 10 min, and transfer the supernatant to another tube for storage. For the precipitate, resuspend the precipitate with 20 - 30 mL of Tris-HCl (10 mM, pH 8.0) solution, and let it stand for 10 min; centrifuge at 12000 r / min for 10 min, discard the supernatant to obtain the first precipitate; for the first precipitate, repeat the above-mentioned resuspension and centrifugation steps to obtain the second precipitate. First, add a small amount of 10 mM Tris-HCl (pH value 8.0) solution to resuspend the second precipitate, then add 5 - 10 mL of Tris-HCl (10 mM, pH 8.0) solution containing 8 M urea to dissolve the protein, centrifuge at 12000 r / min for 10 min, collect the supernatant, and take 50 μL of the sample for SDS-PAGE electrophoresis detection (Figure 3 )。Using BSA (bovine serum albumin) as a standard, the concentration of the purified protein was estimated to be >0.5 mg / mL and the purity >85% by SDS-PAGE gel scanning analysis, and it was named MDV-1 PP38.

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

[0069] 1. Determination of the Optimal Coating Concentration of PP38 and the Optimal Working Concentration of the Antibody

[0070] The purified PP38 protein was diluted with the coating solution to 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, added to the ELISA plates, 50 μL per well, and incubated at 37°C for 3 h; the coating solution was discarded, and the plates were washed 3 times with PBST and patted dry; 50 μL of 10% rabbit serum per well was added and incubated at 37°C for 1 h to block. The blocking solution was discarded, and the plates were washed 3 times with PBST and patted dry; the standard positive control serum and negative control serum were serially diluted at 1:20, 1:50, 1:100, and 1:200, 50 μL per well was added to the ELISA plates, and reacted at 37°C for 1 h for checkerboard assays; the serum was discarded, and the plates were washed 4 times with PBST and patted dry; 1:10000 enzyme-labeled secondary antibody was added and incubated at 37°C for 1 h; the enzyme-labeled secondary antibody was discarded, and the plates were washed 4 times with PBST and patted dry; 100 μL of TMB chromogenic solution was added to each well and developed at room temperature in the dark for 10 min; 50 μL of the termination solution was added to terminate the reaction, and the OD 450 value was immediately measured using an ELISA reader. Based on the OD 450 value and the P / N value, the optimal working concentrations of the coated antigen and antibody were determined.

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

[0072] Table 1 Detection Results of the Optimal Working Concentrations of Coated PP38 Antigen and Antibody (OD 450 value)

[0073]

[0074] Table 2 Detection Results of the Optimal Working Concentrations of Coated PP38 Antigen and Antibody (P / N value)

[0075]

[0076] 2. Determination of the Optimal Dilution of the Enzyme-Labeled Secondary Antibody

[0077] Coat an ELISA plate with the PP38 antigen at the optimal concentration, dilute the negative and positive sera at the optimal dilution, dilute the enzyme-labeled secondary antibody at 1:8000, 1:10000, 1:16000, and 1:20000, perform two replicates for each dilution and take the average value. Determine the optimal working concentration of the enzyme-labeled secondary antibody based on the OD 450 value and P / N value. The results are shown in Tables 3 and 4. When the concentration of the enzyme-labeled secondary antibody is 1:10000, the P / N value is the largest.

[0078] Table 3 Detection results of the optimal dilution of the enzyme-labeled secondary antibody (OD 450 value)

[0079]

[0080] Table 4 Detection results of the optimal dilution of the 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 the optimal coating conditions and time

[0083] Coat an ELISA plate with the PP38 protein at the optimal concentration, set three coating conditions: incubate at 37°C for 3 h; coat overnight at 4°C; incubate at 37°C for 1 h and then coat overnight at 4°C. Set two replicates for each coating condition and take the average value. Dilute the negative and positive sera and the enzyme-labeled secondary antibody at the optimal dilution, and determine the optimal coating conditions and time of the antigen based on the OD 450 value and P / N value. The results are shown in Tables 5 and 6. The optimal coating condition is 37°C for 3 h.

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

[0085] Coating conditions 37℃3h Coat at 37°C for 1 h and then overnight at 4°C Coat overnight at 4°C Positive serum 1.2013 1.0114 0.9314 Negative serum 0.1131 0.1191 0.1039

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

[0087]

[0088] 4. Selection of the optimal blocking solution

[0089] Coat an ELISA plate with the PP38 protein at the optimal concentration, coat at 37°C for 3 h, after washing, use 5% skim milk, 1% gelatin, 10% horse serum, 1% BSA, 10% rabbit serum, and 10% sheep serum as blocking solutions respectively, perform two replicates for each well and take the average value. Dilute the negative and positive sera and the enzyme-labeled secondary antibody at the optimal dilution, and determine the optimal blocking solution based on the OD 450 value and P / N value. The results are shown in Tables 7 and 8. When blocking with 10% rabbit serum, the P / N value is the largest. Therefore, 10% rabbit serum is selected as the optimal blocking solution.

[0090] Table 7 Determination of the best blocking solution (OD 450 value)

[0091] Blocking solution 5% Skim milk 1% Gelatin 10% Horse serum 1% BSA 10% Rabbit serum 10% Goat 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 best blocking solution (OD 450 value)

[0093] Blocking solution 5% Skim milk 1% Gelatin 10% Horse serum 1% BSA 10% Rabbit serum 10% Goat serum P / N value 0.9630 1.1061 7.5389 7.2962 11.0356 9.1587

[0094] 5. Determination of the color development time

[0095] Add TMB color development solution and incubate in the dark at room temperature for 10 min, 20 min, and 30 min respectively. Set 2 replicates for each color development time for negative and positive sera and take the average value. Determine the best color development time by OD 450 value and P / N value. The results are shown in Table 9 and Table 10. When the color development is carried out at room temperature for 10 min, the P / N value is the largest. Therefore, the best color development time of TMB is 10 min.

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

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

[0098] Table 10 Determination of the best color development time (P / N value)

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

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

[0101] Using the method for detecting MDV-1 antibody with the ELISA kit in Example 3, it includes the following steps:

[0102] S1: Dilute MDV-1 PP38 protein with coating buffer to a concentration of 4 μg / mL, add 50 μL / well into the enzyme-linked immunosorbent assay (ELISA) plate, and place the ELISA plate at 37 °C for coating for 3 h.

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

[0104] S3: Remove the blocking solution in S2, wash 3 times with PBST washing solution, 250 μL / well, pat dry, then add the serum sample to be detected diluted 1:100 with sample diluent, 50 μL / well, and incubate at 37 °C for 60 min;

[0105] S4: Remove the serum sample in S3, wash it 4 times with PBST washing solution, 250 μL per well. After patting dry, add the enzyme-labeled secondary antibody diluted 1:10,000 with sample diluent, 50 μL per well, and incubate at 37 °C for 60 min;

[0106] S5: Remove the enzyme-labeled secondary antibody in S4, wash it 4 times with PBST washing solution, 250 μL per well. After patting dry, add TMB chromogenic solution, 100 μL per well, place it in the dark at room temperature for 10 min, and then add the stop solution, 50 μL per well to terminate the reaction.

[0107] S6: Measure the OD 450 value of the liquid in the enzyme-labeled plate wells on the enzyme-labeling instrument. When the OD 450 value of the sample to be tested ≥ 0.31, the test result is judged as positive; when the OD 450 value of the sample to be tested < 0.31, the test result is negative.

[0108] Determination of the positive and negative critical value: Detect 49 negative serum samples, repeat each sample in 2 wells, and the test results are shown in Table 11. Calculate the average value (X) and standard deviation (SD) of the OD 450 values of the negative serum samples. The calculation formula is threshold = X + 3SD. The OD 450 threshold of ELISA is calculated to be 0.31. When the OD 450 value of the sample to be tested ≥ 0.31, the result is judged as positive; when the OD 450 value of the sample to be tested < 0.31, the result is judged as negative.

[0109] Table 11 Determination of the test results of 49 negative serum samples and the critical value

[0110]

[0111] Example 5 Specificity test of the indirect ELISA for detecting MDV-1 antibody

[0112] Use the indirect ELISA method established in Example 3 to detect the positive sera of known pathogens such as avian 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), avian reovirus, etc. at the same time. Set up MDV-1 positive and negative control sera to determine whether there is cross-reaction, so as to analyze the specificity of the kit described in Example 4. The results show that the OD 450 values are all less than 0.31, judged as negative, indicating that there is no cross-reaction between the PP38 protein and the positive sera of the above viruses, and the indirect ELISA kit has good specificity.

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

[0114] Sample <![CDATA[OD 450 value]]> Judgment Positive for infectious bronchitis virus of chicken 0.1140 Negative Positive for infectious bursal disease virus of chicken 0.1891 Negative Positive for infectious laryngotracheitis virus of chicken 0.1043 Negative Positive for chicken egg drop syndrome virus 0.1631 Negative Positive serum for Newcastle disease virus of chicken 0.1390 Negative Positive serum for Marek's disease virus of chicken 0.1101 Negative Positive serum for avian reovirus 0.0943 Negative

[0115] Example 6 Sensitivity test of the indirect ELISA for MDV-1 antibody detection

[0116] Take 3 positive serum samples, serially dilute the serum from 1:100 to 3200, and perform the detection according to the detection method in Example 3. The results show that for different positive sera, after dilution to 1:400, the OD 450 value is 0.4313 and it is still detected as positive, indicating that the sensitivity of the PP38-coated antigen is high.

[0117] Example 7 Coincidence rate test of the indirect ELISA for MDV-1 antibody detection

[0118] The main method for MDV-1 antibody detection is the agar diffusion test, but this method has low sensitivity and is prone to the defect of failing to detect positive samples. For the 134 selected samples, negative and positive samples were detected by the agar diffusion test, and compared with the ELISA method established in the present invention to compare the relative sensitivity and specificity of the two methods. It was found that more positive samples were detected by the kit described in the present invention, indicating high sensitivity.

[0119] Use the indirect ELISA kit shown in Example 4 to detect 134 clinical serum samples, and compare with the agar diffusion test to analyze the sensitivity, specificity and coincidence rate of this kit and the agar diffusion test.

[0120] Relative sensitivity (%) = number of positives / (number of positives + number of false negatives) × 100%;

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

[0122] Total coincidence rate (%) = (number of positives + number of negatives) / total number of detections × 100%.

[0123] Use the indirect ELISA kit described in the present invention to detect 134 clinical serum samples, among which there are 35 positive samples and 99 negative samples. Use the agar diffusion test to detect 134 clinical serum samples, among which there are 30 positive samples and 108 negative samples (Table 13). It can be seen that the relative sensitivity of the indirect ELISA method of the present invention is 85.7%, the relative specificity is 95.2%, and the total coincidence rate is 96.3% (Table 13 and Table 14). The sensitivity and specificity of the indirect ELISA kit of the present invention are relatively high, and the sensitivity is higher than that of the agar diffusion test method.

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

[0125]

[0126]

[0127] Table 14 Coincidence rate of detection results between MDV-1 indirect ELISA antibody detection kit and commercial agar diffusion reagent

[0128]

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

[0130] According to the detection method in Example 3, perform within-batch and between-batch repeatability tests. Coat the ELISA plate with the same batch of PP38 protein, and detect 4 positive sera and negative sera with different antibody titers respectively. Each sample is repeated 3 wells, and the OD value of each well is measured. 450 Calculate the standard deviation of each sample, and then calculate the within-batch coefficient of variation (CV) of each sample. CV = (standard deviation SD / mean Mean) × 100%. Under the same test environment, coat the ELISA plate with VP1 protein at 3 different time periods, and detect 4 positive sera and negative sera with different antibody titers respectively. Each sample is repeated 3 wells in parallel, and the OD value of each well is measured. 450 Calculate the standard deviation of each sample, and then calculate the between-batch coefficient of variation (CV) of each sample. CV = (standard deviation SD / mean Mean) × 100%. The results are shown in Table 15 and Table 16. The coefficients of variation of within-batch and between-batch repeatability tests are both less than 10%, indicating that the detection method has good repeatability and stable results.

[0131] Table 15 Results of within-batch repeatability test of different positive and negative serum samples

[0132]

[0133] Table 16 Results of between-batch repeatability test of different positive and negative serum samples

[0134]

[0135] Preliminary application of the kit:

[0136] The kit was applied to the detection of exogenous viruses in live avian vaccines. Ten key varieties of live avian vaccines produced by domestic manufacturers were selected. The MDV-1 antibody was detected using the kit described in Example 4, and the serological test for MDV-1 contamination of the selected live avian vaccines was carried out according to the third part of the Chinese Veterinary Pharmacopoeia 2020 Edition. At the same time, a PBS group was set as the negative control, and a group infected with MDV-1 (CVI988 live vaccine) was set as the positive control. According to Table 17, the results of the agar diffusion test were consistent with those detected using this kit. None of the selected live avian vaccines were contaminated with MDV-1. The negative control was negative for MDV-1 detection, and the positive control was positive for MDV-1 detection.

[0137] Table 17 Detection results of vaccines from domestic enterprises using the kit of the present invention

[0138]

[0139] In summary, the MDV-1 ELISA antibody detection kit of the present invention is based on the MDV-1 PP38 protein, which is an important immunogenic protein of MDV-1, highly conserved among different MDV-1 strains, can simultaneously recognize different MDV-1 strains, and does not cross-react with viruses such as egg drop syndrome virus (EDSV) of chickens. The kit of the present invention has good specificity and can be used not only for the detection of exogenous virus MDV-1 in live avian vaccines (chicken inspection method), but also for the clinical detection and epidemiological investigation of MDV-1.

[0140] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant gene derived from Marek's disease virus serotype I, characterized in that, The nucleotide sequence of the recombinant gene is as shown in SEQ ID No.

1.

2. The recombinant protein encoded by the recombinant gene according to claim 1.

3. A biological material comprising the recombinant gene according to claim 1 and expressing the recombinant protein according to claim 2.

4. The biomaterial according to claim 3, wherein The type of the biological material includes an expression vector or an expression host cell.

5. Use of the recombinant gene according to claim 1, the recombinant protein according to claim 2, or the biological material according to claim 3 or 4 in the preparation of a reagent for detecting Marek's disease virus serotype I in serum.

6. The application according to claim 5, wherein The detection method matching the reagent includes at least one of the following: indirect ELISA detection method, direct ELISA detection method, and competitive ELISA detection method.

7. A reagent for detecting Marek's disease virus serotype I in serum, characterized in that, It includes using the recombinant protein according to claim 2 as the coating antigen or using the antibody prepared from the recombinant protein according to claim 2 as the coating antibody.

8. An ELISA kit for detecting Marek's disease virus serotype I in serum, characterized in that, It includes the recombinant protein according to claim 2 and / or the antibody prepared using the recombinant protein according to claim 2.

9. Use of the reagent according to claim 7 or the ELISA kit according to claim 8 in screening SPF-class poultry free from contamination with Marek's disease virus serotype I in serum.

10. Use of the reagent according to claim 7 or the ELISA kit according to claim 8 in detecting whether live avian vaccines and / or virus seeds are contaminated with Marek's disease virus serotype I in serum.

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