Application of recombinant VP1 protein as coating antigen in preparation of avian encephalomyelitis virus antibody ELISA (enzyme-linked immuno sorbent assay) detection kit

Abstract

The invention provides an application of a recombinant VP1 protein as a coating antigen in preparation of an avian encephalomyelitis virus antibody ELISA detection kit, and belongs to the technical field of immunodetection. The invention provides an application of a recombinant VP1 protein as a coating antigen in preparation of an avian encephalomyelitis virus antibody ELISA (enzyme-linked immuno sorbent assay) detection kit. The conservative recombinant VP1 protein of the AEV is used as the coating antigen, has the characteristics of low cost, high yield, easiness in preparation and the like, is an optimal coating antigen for establishing AEV antibody ELISA (enzyme-linked immunosorbent assay) detection, and can meet the requirements of clinical large-batch detection. The recombinant VP1 protein provided by the invention can be used for ELISA detection of AEV antibody, does not generate cross reaction with other virus antibodies, is convenient and efficient, has strong operability, and has high specificity and sensitivity. Popularization and application of the method are beneficial to further guarantee of purity and safety of the avian virus live vaccines or virus seeds, and further vaccine quality is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and in particular relates to the application of recombinant VP1 protein as a coating antigen in the preparation of an avian encephalomyelitis virus antibody ELISA detection kit. Background Art

[0002] Avian encephalomyelitis (AE) is an infectious disease caused by avian encephalomyelitis virus (AEV) that mainly attacks the central nervous system of chickens.

[0003] The prevention and control of AE mainly relies on vaccination. AEV contamination in vaccines is a potential risk for the spread and epidemic of AEV. Therefore, it is urgent to establish a stable and efficient method for detecting AEV antibodies for the quality inspection of live poultry vaccines and the diagnosis of AEV diseases.

[0004] Methods for determining whether AEV infection is present in chickens, vaccines, or strains include ELISA, agar diffusion tests, and virus neutralization (VN) tests. ELISA is the most commonly used method for AEV antibody detection and is suitable for testing large flocks of chickens. ELISA determines the presence of AEV antibodies by preparing antigens that specifically bind to antibodies. These antigens can be crude viral extracts or eukaryotic or prokaryotic expressed protein products. These antigens are then coated onto the antigens to detect the presence of AEV antibodies in chickens.

[0005] Currently, commercial ELISA kits for detecting AEV antibodies use the whole virus of the AEVVR strain as the coating antigen, which is expensive and not suitable for large-scale clinical testing. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of recombinant VP1 protein as a coating antigen in the preparation of an avian encephalomyelitis virus antibody ELISA detection kit, which can meet the needs of clinical large-scale detection.

[0007] The present invention provides the use of a recombinant VP1 protein as a coating antigen in the preparation of an avian encephalomyelitis virus antibody ELISA detection kit, wherein the recombinant VP1 protein comprises any one of the following:

[0008] 1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;

[0009] 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having more than 80% identity with the protein described in 1) and having an antibody activity targeting avian encephalomyelitis virus;

[0010] 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

[0011] Preferably, the gene encoding the recombinant VP1 protein includes any one of the following:

[0012] (1) A gene comprising the nucleotide sequence shown in SEQ ID NO: 2;

[0013] (2) A gene encoding the recombinant VP1 protein according to claim 1 obtained by replacing, deleting and / or adding one or more nucleotides of the gene described in (1);

[0014] (3) A fusion gene obtained by connecting the 3' end and / or 5' end of the gene described in (1) or the gene described in (2) to a gene encoding a protein tag.

[0015] The present invention also provides an avian encephalomyelitis virus antibody ELISA detection kit, which uses recombinant VP1 protein as a coating antigen;

[0016] The recombinant VP1 protein includes any one of the following:

[0017] 1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;

[0018] 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having more than 80% identity with the protein described in 1) and having an antibody activity targeting avian encephalomyelitis virus;

[0019] 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

[0020] Preferably, the method further comprises 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 color developing solution and a reaction stopping solution.

[0021] Preferably, the coating solution is carbonate buffer; the washing solution is PBST; the enzyme-labeled secondary antibody is a donkey anti-chicken IgG antibody labeled with horseradish peroxide; the blocking solution is 10% rabbit serum; the sample diluent is PBST; the positive serum control is an SPF chicken serum sample obtained by immunization with AEV, which is positive when detected by a commercial ELISA antibody detection kit; the negative serum control is an SPF chicken serum sample not infected with AEV, which is negative when detected by a commercial ELISA antibody detection kit.

[0022] The present invention also provides the use of the avian encephalomyelitis virus antibody ELISA detection kit described in the above scheme in at least one of the following: 1) detecting avian encephalomyelitis virus antibodies; 2) screening SPF chickens; 3) detecting whether avian encephalomyelitis virus infection exists in vaccines and / or virus seeds.

[0023] The present invention also provides an ELISA method for detecting avian encephalomyelitis virus antibodies, which is performed using the avian encephalomyelitis virus antibody ELISA detection kit described in the above scheme.

[0024] Preferably, the method comprises the following steps:

[0025] S1. Dilute the recombinant VP1 protein with coating solution and coat the plate.

[0026] S2. Remove the coating solution, wash with detergent, pat dry, and add blocking solution to the ELISA plate for blocking.

[0027] S3. Remove the blocking solution, wash with a washing solution, pat dry, and add the sample to be tested diluted with a sample diluent to the ELISA plate for the first incubation;

[0028] S4. Remove the sample to be tested, wash it with detergent, pat it dry, and add the enzyme-labeled secondary antibody diluted with sample diluent to the ELISA plate for a second incubation;

[0029] S5. Remove the enzyme-labeled secondary antibody, wash with detergent, pat dry, add TMB colorimetric solution to the plate, perform color development, and then add stop solution to the plate to terminate the reaction.

[0030] S6. Measure the OD of the liquid in the microplate wells 450nm value, when the OD of the sample to be tested is 450nm When the OD value is ≥0.30, the test result is considered positive; when the OD value of the sample to be tested is 450nm When the value is less than 0.30, the test result is negative.

[0031] Preferably, the concentration of the recombinant VP1 protein after dilution in step S1 is 0.125-1 μg / mL.

[0032] Preferably, the ELISA comprises one or more of direct ELISA, indirect ELISA and competitive ELISA.

[0033] The present invention provides the use of a recombinant VP1 protein as a coating antigen in the preparation of an avian encephalomyelitis virus antibody ELISA detection kit, wherein the recombinant VP1 protein includes any of the following: 1) a protein including the amino acid sequence shown in SEQ ID NO: 1; 2) a protein obtained by replacing, deleting and / or adding one or several amino acid residues of the protein in 1) and having more than 80% identity with the protein shown in 1) and having the function of targeting avian encephalomyelitis virus antibodies; 3) a fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag. The present invention uses the conserved recombinant VP1 protein of AEV as a coating antigen, which has the characteristics of low cost, high yield and easy preparation. It is the preferred coating antigen for establishing AEV antibody ELISA detection and can meet the needs of large-scale clinical detection. The recombinant VP1 protein of the present invention can be used for ELISA detection of AEV antibodies without cross-reacting with other viral antibodies. It is convenient and efficient, highly operable, and has high specificity and sensitivity. The promotion and use of the method of the present invention is conducive to further ensuring the purity and safety of poultry virus live vaccines or virus strains, thereby improving the quality of vaccines, and can also be used for clinical detection and epidemiological investigation of AEV. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is a graph showing the small-scale expression of protein in Example 1; wherein, M: Marker; 1: uninduced control (BL21); 2-3: IPTG-induced (BL21);

[0036] Figure 2 This is a diagram showing the protein mass expression in Example 1; wherein, M: Marker; 1: whole bacteria after ultrasound; 2: supernatant after ultrasound; 3: precipitate after ultrasound;

[0037] Figure 3 This is a diagram of protein purification in Example 1; wherein, M: Marker; 1: 5-fold dilution of the purified protein; 2: 10-fold dilution of the purified protein. DETAILED DESCRIPTION

[0038] The present invention provides the use of a recombinant VP1 protein as a coating antigen in the preparation of an avian encephalomyelitis virus antibody ELISA detection kit, wherein the recombinant VP1 protein comprises any one of the following:

[0039] 1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, specifically: EFGKGDEGGFSSVPEVEQHVVEDKEPQGPLHVTPFGAVKAMEDPQLARKTPGTFPELAPGKPRHTVDHMDLYKFMGRAHYLWGHKFTKTDMQYTFQIPLSPIKEGFVTGTLRWFLSLFQLYRGSLDITMTFAGKTNVDGIVYFVPEGVAIETERKEQTPLLTLNYKTSVGAIRFNTGQTTNVQFRIPFYTPLEHIATHSKNAMDSVLGAITTQITNYSAQDEYLQVTYYISFNEDSQFSVPRAVPVVSSFTDTSSKTVMNTYWLDDDELVEELE;

[0040] 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having more than 80% identity with the protein described in 1) and having an antibody activity targeting avian encephalomyelitis virus;

[0041] 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

[0042] In the present invention, VP1 protein, as an important immunogenic protein of AEV, is highly conserved among different strains of AEV and has extremely low antigenic differences. It is an ideal antigen for establishing AEV antibody detection and can produce a good reaction with AEV-positive serum. As a coating antigen, it has the advantages of low cost, high yield and easy preparation.

[0043] As an embodiment, the present invention conducts comprehensive secondary structure, hydrophilicity, antigenicity and other analyses on the AEVVP1 protein sequence information, selects the full-length VP1 sequence and constructs it into a prokaryotic expression vector, and expresses and purifies the protein.

[0044] As an embodiment, the gene encoding the recombinant VP1 protein includes any one of the following:

[0045] (1) A gene comprising the nucleotide sequence shown in SEQ ID NO: 2, specifically:

[0046] (2) A gene encoding the recombinant VP1 protein according to claim 1 obtained by replacing, deleting and / or adding one or more nucleotides of the gene described in (1);

[0047] (3) A fusion gene obtained by connecting the 3' end and / or 5' end of the gene described in (1) or the gene described in (2) to a gene encoding a protein tag.

[0048] The present invention conducts comprehensive secondary structure, hydrophilicity, antigenicity and other analyses on the AEV VP1 protein sequence information, selects the full-length VP1 sequence and constructs it into a prokaryotic expression vector for protein expression and purification.

[0049] As one embodiment, the present invention uses the VP1 sequence of the AEV VR strain (Genbank accession number: AY466473.1) as a basis, based on protein sequence secondary structure analysis and antigenicity. A nucleotide fragment (822bp) of the full-length VP1 protein was selected and synthesized after codon optimization. EcoRI (GAATTC) and XhoI (CTCGAG) restriction sites were added to the 5' and 3' ends for vector cloning. The sequence with the added restriction sites was synthesized at Beijing Liuhe BGI Protein Research and Development Center Co., Ltd.

[0050] The present invention also provides an avian encephalomyelitis virus antibody ELISA detection kit, which uses recombinant VP1 protein as a coating antigen;

[0051] The recombinant VP1 protein includes any one of the following:

[0052] 1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;

[0053] 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having more than 80% identity with the protein described in 1) and having an antibody activity targeting avian encephalomyelitis virus;

[0054] 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

[0055] In one embodiment, the present invention uses recombinant VP1 protein as a coating antigen coated onto an ELISA plate. The ELISA detection kit for avian encephalomyelitis virus antibodies can be used for clinical AEV serum antibody testing, screening of SPF chickens, detection of exogenous viral antibodies in live poultry vaccines or strains, and epidemiological surveys, thereby preventing and controlling the spread of AEV infection. Furthermore, the ELISA detection kit for avian encephalomyelitis virus antibodies exhibits excellent reactogenicity, specificity, sensitivity, and reproducibility.

[0056] As an embodiment, the kit further includes an ELISA 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 color developing solution, and a reaction stopping solution.

[0057] As an embodiment, the coating solution is carbonate buffer; the washing solution is PBST; the enzyme-labeled secondary antibody is a donkey anti-chicken IgG antibody labeled with horseradish peroxide; the blocking solution is 5% (v / v) skim milk, 1% (v / v) gelatin, 10% (v / v) horse serum, 1% (v / v) BSA, 10% (v / v) rabbit serum or 10% (v / v) sheep serum, and further 10% (v / v) rabbit serum, and the P / N value is the largest when blocked with 10% rabbit serum; the sample diluent is PBST; the positive serum control is an SPF chicken serum sample obtained by immunizing AEV, which is positive when detected by a commercial ELISA antibody detection kit; the negative serum control is an SPF chicken serum sample not infected with AEV, which is negative when detected by a commercial ELISA antibody detection kit.

[0058] The present invention also provides the use of the avian encephalomyelitis virus antibody ELISA detection kit described in the above scheme in at least one of the following:

[0059] 1) Detection of avian encephalomyelitis virus antibodies;

[0060] 2) Screening SPF chickens;

[0061] 3) Detection of vaccines and / or virus strains for the presence of avian encephalomyelitis virus infection.

[0062] As an embodiment, the screening of SPF chickens includes screening SPF chickens that are not contaminated with AEV; the screening step includes: collecting blood from the SPF chicken flock and separating the serum, and using the serum as the test sample to detect avian encephalomyelitis virus antibodies; the serum to be tested should be negative after AEV antibody detection, otherwise it indicates that the SPF chicken flock is contaminated with AEV exogenous virus and must not be used for vaccine preparation and serum preparation, etc.

[0063] In one embodiment, the vaccine is a live vaccine for poultry; and the detection of whether the vaccine and / or virus seed contains avian encephalomyelitis virus infection comprises:

[0064] Take several SPF chickens, inoculate each of them with 10 doses of vaccine or virus by eye drop and nasal drop at the same time, and inject 100 doses of vaccine or virus into the muscle. After 21 days, repeat the vaccination once according to the above method and dosage. 42 days after the first vaccination, collect blood and separate the serum, and use the serum as the sample to be tested for avian encephalomyelitis virus antibodies; the serum to be tested should be negative for AEV antibody test, otherwise it means that the vaccine or virus is contaminated with AEV exogenous virus, which will pose a threat to the safety of the vaccine or virus.

[0065] The present invention also provides an ELISA method for detecting avian encephalomyelitis virus antibodies, which is performed using the avian encephalomyelitis virus antibody ELISA detection kit described in the above scheme.

[0066] As an embodiment, the method comprises the following steps:

[0067] S1. Dilute the recombinant VP1 protein with coating solution and coat the plate.

[0068] S2. Remove the coating solution, wash with detergent, pat dry, and add blocking solution to the ELISA plate for blocking.

[0069] S3. Remove the blocking solution, wash with a washing solution, pat dry, and add the sample to be tested diluted with a sample diluent to the ELISA plate for the first incubation;

[0070] S4. Remove the sample to be tested, wash it with detergent, pat it dry, and add the enzyme-labeled secondary antibody diluted with sample diluent to the ELISA plate for a second incubation;

[0071] S5. Remove the enzyme-labeled secondary antibody, wash with detergent, pat dry, add TMB colorimetric solution to the plate, perform color development, and then add stop solution to the plate to terminate the reaction.

[0072] S6. Measure the OD of the liquid in the microplate wells 450nm value, when the OD of the sample to be tested is 450nm When the OD value is ≥0.30, the test result is considered positive; when the OD value of the sample to be tested is 450nm When the value is less than 0.30, the test result is negative.

[0073] As an embodiment, the ELISA includes one or more of direct ELISA, indirect ELISA and competitive ELISA, and is further an indirect ELISA.

[0074] The present invention first uses a coating solution to dilute the recombinant VP1 protein and then coat it on an ELISA plate. In one embodiment, the concentration of the diluted recombinant VP1 protein is 0.125-1 μg / mL, further 0.125, 0.25, 0.5, and 1 μg / mL, and the amount added to the ELISA plate is 50 μL / well. The coating conditions are any of the following: incubation at 37°C for 3 hours; coating at 4°C overnight; incubation at 37°C for 1 hour, coating at 4°C overnight, and further incubation at 37°C for 3 hours.

[0075] After coating, the present invention removes the coating solution, washes with a wash solution, pats dry, and then adds a blocking solution to the ELISA plate for blocking. In one embodiment, the wash solution is used three times, with 250 μL of wash solution added per well for each wash; the blocking solution is added in an amount of 50 μL per well; the blocking temperature is 37°C, and the blocking time is 60 minutes.

[0076] After blocking, the present invention removes the blocking solution, washes with a washing solution, pats dry, and then adds the sample to be tested diluted with a sample diluent to the ELISA plate for a first incubation. As an embodiment, the washing solution is used for three washes, and the amount of washing solution added for each wash is 250 μL / well; the sample to be tested is diluted with the sample diluent at a ratio of 1: (100-800), and further, the sample to be tested is diluted with the sample diluent at a ratio of 1:100, 1:200, 1:400, or 1:800; the amount of the diluted sample to be tested is 50 μL / well; the temperature of the first incubation is 37°C; and the time of the first incubation is 60 minutes.

[0077] After the first incubation, the present invention removes the sample to be tested, washes it with a washing solution, pats it dry, and then adds an enzyme-labeled secondary antibody diluted with a sample diluent to the ELISA plate for a second incubation. As an embodiment, the washing solution is used for four washings, and the amount of washing solution added for each washing is 250 μL / well; the enzyme-labeled secondary antibody is diluted with the sample diluent at a ratio of 1: (8000 to 20000), and further, the enzyme-labeled secondary antibody is diluted with the sample diluent at a ratio of 1: 8000, 1: 10000, 1: 16000, or 1: 20000; the amount of the diluted enzyme-labeled secondary antibody added is 50 μL / well; the temperature of the second incubation is 37° C.; and the time of the second incubation is 60 minutes.

[0078] After the second incubation, the present invention removes the enzyme-labeled secondary antibody, washes with a washing solution, pats dry, and then adds TMB color development solution to the ELISA plate for a color development reaction. A stop solution is then added to the ELISA plate to terminate the reaction. In one embodiment, the washing solution is used for four washes, with 250 μL / well added for each wash; the TMB color development solution is added in an amount of 100 μL / well; the color development reaction is performed at room temperature in the dark; the color development reaction lasts for 10 to 30 minutes, further 10, 20, or 30 minutes, wherein the P / N value is maximized when the color development lasts for 10 minutes; and the stop solution is added in an amount of 50 μL / well.

[0079] After the termination reaction, the present invention measures the OD of the liquid in the ELISA plate well. 450nm value, when the OD of the sample to be tested is 450nm When the OD value is ≥0.30, the test result is considered positive; when the OD value of the sample to be tested is450nm When the value is less than 0.30, the test result is negative. As an embodiment, the OD value of the liquid in the well of the microplate is measured on a microplate reader. 450nm value.

[0080] To further illustrate the present invention, the application of the recombinant VP1 protein provided by the present invention as a coating antigen in the preparation of an avian encephalomyelitis virus antibody ELISA detection kit is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0081] Example 1

[0082] 1. AEVVP1 protein sequence analysis

[0083] Different AEV strains have only one serotype. Reference sequences for different AEV strains were downloaded from NCBI and analyzed, revealing high homology between the AEV VP1 protein. Comprehensive secondary and antigenicity analyses of the VP1 protein from the AEV VR strain revealed that the AEV VP1 protein is highly conserved, with minimal antigenic variation, making it a suitable candidate for protein expression. Based on protein sequence secondary structure analysis and antigenicity, a codon-optimized nucleotide fragment (822 bp) of the full-length VP1 protein was synthesized using the VP1 sequence of the AEV VR strain (Genbank accession number: AY466473.1). EcoRI (GAATTC) and XhoI (CTCGAG) restriction sites were added at the 5' and 3' ends for vector cloning. The encoded amino acid sequence is shown in SEQ ID NO. 1. The sequence with the restriction sites added was synthesized at Beijing Liuhe BGI Protein Research and Development Center Co., Ltd. (see SEQ ID NO. 2).

[0084] 2. Construction of recombinant expression plasmid and purification of VP1 protein

[0085] 1. Construction of recombinant expression plasmid

[0086] EcoRI and Xho I restriction sites were introduced into the 5' and 3' ends of the coding gene of SEQ ID NO. 2, respectively (added by sequence synthesis) for the preparation of recombinant expression plasmids.

[0087] The SEQ ID NO. 2 sequence and plasmid pET-30a after adding EcoRI and XhoI restriction endonucleases were double-digested with EcoRI and XhoI, respectively. The purified fragment and the expression vector digestion product were ligated with DNA Ligation Kit to obtain a recombinant expression plasmid, which was then transformed into competent cells (BL21).

[0088] 2. Small-scale expression of AEV-VP1 recombinant protein

[0089] Pick the BL21 obtained in step 1 that was positive by PCR, clone the BL21 into 1.5 mL of LB liquid medium containing kanamycin resistance, and culture at 37°C and 200 rpm. Culture until OD 600 The value is 0.6-0.8. The mass concentration of kanamycin in LB liquid culture medium is 50μg / mL. IPTG is added to the bacterial solution obtained by accurate culture for induction. The final concentration of IPTG in the bacterial solution obtained by culture is 0.5mM, the induction temperature is 37℃, the induction speed is 200r / min, and the induction time is 2h. Take 1mL of the induced bacterial solution, centrifuge it at a speed of 12000r / min for 1min, discard the supernatant, blow off the precipitate with 100μLTris-HCl (pH 8.0) buffer, add 2× loading buffer with the same volume as the buffer, keep it at a temperature of 100℃ for 5min, and then perform electrophoresis detection. The results of electrophoresis detection are shown in Figure 1 ,according to Figure 1 It can be seen that a specific target band of the recombinant VP1 protein appeared at a size of about 43KD (the actual molecular weight of this protein is larger than the theoretical value, which was correct after plasmid gene sequencing), indicating that the VP1 protein was successfully expressed.

[0090] 3. Large-scale expression of AEV-VP1 recombinant protein

[0091] The transformed BL21 obtained in step 1 was identified by PCR, and the positive BL21 was cultured. The cultured bacterial liquid was transferred to 250 mL of kanamycin-resistant LB liquid medium at a volume ratio of 1:50, and cultured at a temperature of 37°C and a speed of 200 r / min until the OD of the cultured LB liquid medium reached 0. 600 =0.6~0.8. The mass concentration of kanamycin in the LB liquid medium is 50μg / mL. IPTG is added to the LB liquid medium obtained by culture for induction. The final concentration of IPTG in the LB liquid medium is 0.5mM. The IPTG induction temperature is 37℃, and the induction time is 3h. The induced product is centrifuged to collect the bacteria at a centrifugal speed of 8000r / min and a centrifugal time of 6min. After centrifugation, the supernatant is discarded to obtain the bacterial cells; the obtained bacterial cells are ultrasonically disrupted, and the specific process is as follows: the obtained bacterial cells are blown away with 30mL of 10mM Tris-HCl (pH8.0) solution and then ultrasonically disrupted at a power of 500W, and ultrasonically disrupted 180 times, each for 5s, with the next ultrasonic disruption after an interval of 5s.

[0092] The ultrasonic fragments were subjected to electrophoresis. The specific process was as follows: 100 μL of the sonicated bacterial suspension was taken and centrifuged at 12,000 r / min for 10 minutes. After centrifugation, 50 μL of the supernatant and the resulting precipitate were retained. The resulting precipitate was then blown off with 50 μL of 10 mM Tris-HCl (pH 8.0) solution. The supernatant and the resulting solution after the precipitate was blown off were respectively taken for SDS-PAGE detection. The results showed that a large amount of target protein was detected in the precipitate, indicating that the recombinant bacteria expressed the protein in inclusion bodies ( Figure 2 ).

[0093] The AEV-VP1 protein expressed in the bacterial pellet was purified as follows: the pellet obtained by ultrasonic centrifugation was resuspended in 20-30 mL of 10 mM Tris-HCl (pH 8.0) solution and allowed to stand for 10 minutes; the pellet was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was transferred to another tube for storage. The pellet was resuspended in 20-30 mL of 10 mM Tris-HCl (pH 8.0) solution and allowed to stand for 10 minutes; the pellet was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was discarded to obtain a first pellet. The resuspension and centrifugation steps were repeated once for the first pellet to obtain a second pellet. In the second precipitate, a small amount of 10mM Tris-HCl (pH 8.0) solution was added to resuspend the precipitate, and then 5-10mL of 10mM Tris-HCl (pH 8.0) solution containing 8M urea was added to dissolve the protein. The precipitate was centrifuged at 12000r / min for 10min, and the supernatant was collected. 50μL of the sample was taken for SDS-PAGE electrophoresis detection ( Figure 3 Using BSA (bovine serum albumin) as the standard, the purified protein concentration was estimated to be >0.5 mg / mL and the purity was >85% by SDS-PAGE gel scanning analysis, and was named AEV-VP1.

[0094] Example 2 A method for detecting avian encephalomyelitis virus antibodies by ELISA

[0095] Here are the steps:

[0096] S1: Dilute the purified recombinant VP1 protein prepared in Example 1 to a concentration of 0.125 μg / mL with coating solution (carbonate buffer), add 50 μL / well to the ELISA plate, and place the ELISA plate at 37°C for coating for 3 hours.

[0097] S2: Remove the coating solution in S1, wash three times with PBST washing solution, 250 μL / well, pat dry, add blocking solution (10% rabbit serum), 50 μL / well, and incubate at 37°C for 60 min;

[0098] S3: Remove the blocking solution in S2, wash three times with PBST washing solution, 250 μL / well, pat dry, add standard positive control serum and negative control serum diluted 1:200 with sample diluent, 50 μL / well, perform two replicates, and incubate at 37°C for 60 min;

[0099] S4: Remove the serum sample from S3 and wash four times with PBST washing solution (250 μL / well). Pat dry and add enzyme-labeled secondary antibody diluted 1:10,000 with sample diluent (50 μL / well). Repeat two times and incubate at 37°C for 60 min.

[0100] S5: Remove the enzyme-labeled secondary antibody (horseradish peroxide-labeled donkey anti-chicken IgG antibody) in S4, wash four times with PBST washing solution, 250 μL / well, pat dry, add TMB color development solution, 100 μL / well, incubate at room temperature in the dark for 10 minutes, and then add stop solution, 50 μL / well to terminate the reaction.

[0101] S6: Measure the OD value of the liquid in the microplate wells on a microplate reader 450nm value, when the OD of the sample to be tested is 450nm When the OD value is ≥0.30, the test result is considered positive; when the OD value of the sample to be tested is 450nm When the value is less than 0.30, the test result is negative.

[0102] Example 3

[0103] The same procedures as in Example 2 were followed except that the purified recombinant VP1 protein was diluted to 1 μg / mL with the coating solution.

[0104] Example 4

[0105] The same procedures as in Example 2 were followed except that the purified recombinant VP1 protein was diluted to 0.5 μg / mL with the coating solution.

[0106] Example 5

[0107] The same procedures as in Example 2 were followed except that the purified recombinant VP1 protein was diluted to 0.25 μg / mL with the coating solution.

[0108] Example 6

[0109] The same procedures as in Example 2 were followed except that the standard positive control serum and negative control serum were diluted 1:100.

[0110] Example 7

[0111] The same procedures as in Example 2 were followed except that the standard positive control serum and negative control serum were diluted 1:400.

[0112] Example 8

[0113] The same procedures as in Example 2 were followed except that the standard positive control serum and negative control serum were diluted 1:800.

[0114] The results of Examples 1 to 8 are shown in Tables 1 and 2. The results show that when the antigen coating concentration is 0.125 μg / mL and the antibody dilution is 1:200, the OD of the positive control serum is 450nm The value is close to 1.0, and the P / N value is the largest. Therefore, the optimal coating concentration of ELISA antigen is determined to be 0.125 μg / mL, and the optimal serum dilution is determined to be 1:200.

[0115] Table 1 Detection results of the optimal working concentrations of coated VP2 antigen and antibody (OD 450nm value)

[0116]

[0117]

[0118] Table 2 Optimal working concentration test results of coated VP2 antigen and antibody (P / N value)

[0119]

[0120] Example 9

[0121] Except for the enzyme-labeled secondary antibody diluted at 1:8000, the rest was the same as in Example 2.

[0122] Example 10

[0123] Except for the enzyme-labeled secondary antibody diluted at 1:16000, the rest was the same as in Example 2.

[0124] Example 11

[0125] Except for the enzyme-labeled secondary antibody diluted at 1:20,000, the rest was the same as in Example 2.

[0126] The results of Example 1 and Examples 9 to 11 are shown in Tables 3 and 4. The results show that when the concentration of the enzyme-labeled secondary antibody is 1:10,000, the P / N is the largest.

[0127] Table 3 Optimal dilution test results of enzyme-labeled secondary antibody (OD 450nm value)

[0128]

[0129] Table 4 Optimal dilution test results of enzyme-labeled secondary antibodies (P / N value)

[0130] Enzyme-labeled secondary antibody dilution 1：8000 1：10000 1：16000 1：20000 P / N value 15.4956 17.1871 12.7263 11.8879

[0131] Example 12

[0132] The same procedures as in Example 2 were followed except that the ELISA plate was placed at 4°C for coating overnight.

[0133] Example 13

[0134] The same procedures as in Example 2 were followed except that the ELISA plate was incubated at 37°C for 1 hour and coated at 4°C overnight.

[0135] The results of Example 1 and Examples 12-13 are shown in Tables 5 and 6. The results show that the optimal coating condition is 37° C. for 3 h.

[0136] Table 5 Optimal coating conditions test results (OD 450nm value)

[0137]

[0138] Table 6 Optimal coating conditions test results (P / N value)

[0139] Coating conditions 37℃3h 37℃ for 1h, 4℃ overnight Coat overnight at 4°C P / N value 18.1609 12.7559 16.3653

[0140] Example 14

[0141] The procedure was the same as in Example 2 except that the blocking solution was replaced with 5% (v / v) skim milk.

[0142] Example 15

[0143] The procedure was the same as in Example 2 except that the blocking solution was replaced with 1% (v / v) gelatin.

[0144] Example 16

[0145] The same procedures as in Example 2 were followed except that the blocking solution was replaced with 10% (v / v) horse serum.

[0146] Example 17

[0147] The procedure was the same as in Example 2 except that the blocking solution was replaced with 1% (v / v) BSA.

[0148] Example 18

[0149] The procedure was the same as in Example 2 except that the blocking solution was replaced with 10% (v / v) goat serum.

[0150] The results of Example 1 and Examples 14 to 18 are shown in Tables 7 and 8. The results show that when 10% rabbit serum is used for blocking, the P / N value is the largest, so 10% rabbit serum is selected as the optimal blocking solution.

[0151] Table 7 Determination of the best blocking solution (OD 450nm value)

[0152]

[0153] Table 8 Determination of the best blocking solution (OD 450nm value)

[0154]

[0155]

[0156] Example 19

[0157] The reaction was carried out in the dark at room temperature for 20 min, and the rest was the same as in Example 2.

[0158] Example 20

[0159] The reaction was carried out at room temperature in the dark for 30 min, and the rest was the same as in Example 2.

[0160] The results of Example 1 and Examples 19 to 20 are shown in Tables 9 and 10. The results show that when the color development is carried out at room temperature for 10 minutes, the P / N value is the largest, so the optimal color development time of TMB is 10 minutes.

[0161] Table 9 Determination of TMB color development (OD 450nm value)

[0162]

[0163] Table 10 Determination of optimal color development time (P / N value)

[0164] Room temperature display time 10min 20min 30min P / N value 18.0401 12.9939 9.5319

[0165] Example 21 Indirect ELISA kit for detecting AEV antibodies

[0166] The method for detecting AEV antibodies using the ELISA kit of Example 2 comprises the following steps:

[0167] S1: Dilute the AEVVP1 protein to a concentration of 0.125 μg / mL with coating solution, add 50 μL / well into the ELISA plate, and place the ELISA plate at 37°C for 3 hours.

[0168] S2: Remove the coating solution in S1, wash three times with PBST washing solution, 250 μL / well, pat dry, add blocking solution, 50 μL / well, and incubate at 37°C for 60 min;

[0169] S3: Remove the blocking solution in S2, wash three times with PBST washing solution, 250 μL / well, pat dry, add the serum sample to be tested diluted 1:200 with sample diluent, 50 μL / well, and incubate at 37°C for 60 min;

[0170] S4: Remove the serum sample from S3, wash four times with PBST washing solution (250 μL / well), pat dry, add enzyme-labeled secondary antibody diluted 1:10,000 with sample diluent (50 μL / well), and incubate at 37°C for 60 min;

[0171] S5: Remove the enzyme-labeled secondary antibody in S4, wash four times with PBST washing solution, 250μL / well, pat dry and add TMB color development solution, 100μL / well, incubate at room temperature in the dark for 10 minutes, then add stop solution, 50μL / well to terminate the reaction.

[0172] S6: Measure the OD value of the liquid in the microplate wells on a microplate reader 450nm value, when the OD of the sample to be tested is 450nm When the OD value is ≥0.30, the test result is considered positive; when the OD value of the sample to be tested is 450nm When the value is less than 0.30, the test result is negative.

[0173] Determination of negative and positive cut-off values: 49 negative serum samples were tested, with 2 replicates for each sample. The test results are shown in Table 11. Calculate the OD of negative serum samples. 450nm The mean (X) and standard deviation (SD) of the values ​​were calculated as threshold = X + 3SD to obtain the OD of ELISA. 450nm The threshold is 0.30. 450nm When the OD value is greater than 0.30, the result is considered positive; when the OD value of the sample to be tested is greater than 0.30, the result is considered positive. 450nm When the value is ≤0.30, the result is considered negative.

[0174] Table 11 Test results of 49 negative sera and determination of critical values

[0175]

[0176] Example 22 Specificity test of indirect ELISA for AEV antibody detection

[0177] The indirect ELISA method established in Example 2 was used to detect known positive sera of 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. At the same time, CIAV positive and negative control sera were set up to determine whether there was a cross reaction to analyze the specificity of this kit. The results showed that OD 450nm The values ​​were all less than 0.30, which was judged as negative, indicating that the VP1 protein had no cross-reaction with the positive serum of the above viruses and the indirect ELISA kit had good specificity.

[0178] Table 12 Specificity test results of AEV indirect ELISA antibody detection method

[0179]

[0180]

[0181] Example 23 Sensitivity test of indirect ELISA for AEV antibody detection

[0182] Three positive serum samples were taken, and the serum was diluted from 1:200 to 6400, and tested according to the detection method of Example 2. The results showed that for different positive sera, after being diluted to 1:1600, they were still tested positive, indicating that the sensitivity of the VP1 coating antigen is high.

[0183] Example 24: Compliance test of indirect ELISA for AEV antibody detection

[0184] With reference to Example 2, 134 clinical serum samples were detected using the indirect ELISA kit of the present invention and compared with a commercial kit to analyze the sensitivity, specificity and coincidence rate of the kit and the commercial kit.

[0185] Relative sensitivity (%) = number of positive results / (number of positive results + number of false negative results) × 100%

[0186] Relative specificity (%) = number of negative results / (number of negative results + number of false positive results) × 100%

[0187] Total compliance rate (%) = (number of positives + number of negatives) / total number of tests × 100%

[0188] The results showed that the indirect ELISA kit of the present invention tested 134 clinical serum samples, of which 31 were positive and 103 were negative. A commercial kit was used to test 134 clinical serum samples, of which 33 were positive and 101 were negative (Table 13). It can be seen that the relative sensitivity of the indirect ELISA method of the present invention was 93.9%, the relative specificity was 100%, and the total compliance rate was 98.5% (Tables 13 and 14). The indirect ELISA kit of the present invention has high sensitivity and specificity, and has a higher compliance rate than existing commercial kits.

[0189] Table 13 Test results of AEV indirect ELISA antibody detection kit and commercial kit

[0190]

[0191]

[0192]

[0193] Table 14: Concordance between the results of the AEV indirect ELISA antibody detection kit and the commercial kit

[0194]

[0195] Example 25 Repeatability test of indirect ELISA for AEV antibody detection

[0196] According to the detection method of Example 2, the reproducibility test within and between batches was carried out. The ELISA plate was coated with the VP1 protein of the same batch, and 4 positive sera and negative sera with different antibody titers were tested respectively. Each sample was repeated in 3 wells in parallel, and the OD of each well was measured. 450nm The standard deviation of each sample was calculated, and then the coefficient of variation (CV) within each batch was calculated. CV = (standard deviation SD / mean) × 100%. Under the same experimental environment, VP1 protein was used to coat the microplate at three different time periods. Four positive and negative sera with different antibody titers were tested respectively. Each sample was repeated in three parallel wells, and the OD value of each well was measured. 450nm The standard deviation of each sample was calculated, and then the inter-batch coefficient of variation (CV) of each sample was calculated: CV = (standard deviation SD / 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.

[0197] Table 15 Results of intra-batch repeatability tests for different positive and negative serum samples

[0198]

[0199]

[0200] Table 16 Results of repeatability test between batches of serum samples with different positive and negative characteristics

[0201]

[0202] Initial application of the kit:

[0203] This kit was applied to the detection of exogenous viruses in live vaccines of avian viruses. In this experiment, key varieties of live vaccines for poultry produced by 10 domestic manufacturers were selected, and AEV antibody detection was performed according to the kit of this patent. Serological tests for AEV contamination were performed on the selected live vaccines for poultry diseases according to Part III of the 2020 edition of the Chinese Pharmacopoeia of Veterinary Medicine. At the same time, the PBS group was set as a negative control, and the group infected with AEV (YBF02 strain) was set as a positive control. According to Table 17, the results of the serological test and the test using this kit were consistent. The selected live vaccines for poultry were not contaminated with AEV, the negative control was AEV negative, and the positive control was AEV positive.

[0204] Table 17: Results of testing vaccines from domestic companies using the kit of the present invention

[0205]

[0206]

[0207] In summary, the AEV ELISA antibody detection kit described in the present invention is based on the fact that the AEV VP1 protein is a key immunogenic protein of AEV, which is highly conserved among different AEV strains. It can simultaneously identify different AEV strains without cross-reacting with viruses such as egg drop syndrome virus (EDSV). The kit of the present invention has good specificity. This kit can be used not only for the detection of exogenous AEV viruses in live avian viral vaccines (chicken test method), but also for clinical testing and epidemiological surveys of AEV.

[0208] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of recombinant VP1 protein as a coating antigen in the preparation of an ELISA kit for detecting antibodies against avian encephalomyelitis virus, characterized in that: The recombinant VP1 protein includes any one of the following: 1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1; 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having more than 80% identity with the protein described in 1) and having an antibody activity targeting avian encephalomyelitis virus; 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

2. The use according to claim 1, characterized in that The gene encoding the recombinant VP1 protein includes any one of the following: (1) A gene comprising the nucleotide sequence shown in SEQ ID NO: 2; (2) A gene encoding the recombinant VP1 protein according to claim 1 obtained by replacing, deleting and / or adding one or more nucleotides of the gene described in (1); (3) A fusion gene obtained by connecting the 3' end and / or 5' end of the gene described in (1) or the gene described in (2) to a gene encoding a protein tag.

3. An avian encephalomyelitis virus antibody ELISA detection kit, characterized in that: Recombinant VP1 protein was used as the coating antigen; The recombinant VP1 protein includes any one of the following: 1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1; 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having more than 80% identity with the protein described in 1) and having an antibody activity targeting avian encephalomyelitis virus; 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

4. The avian encephalomyelitis virus antibody ELISA detection kit according to claim 3, characterized in that It also includes 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 color developing solution, and a reaction stopping solution.

5. The avian encephalomyelitis virus antibody ELISA detection kit according to claim 4, characterized in that The coating solution is a carbonate buffer; the washing solution is PBST; the enzyme-labeled secondary antibody is a donkey anti-chicken IgG antibody labeled with horseradish peroxide; the blocking solution is 10% rabbit serum; the sample diluent is PBST; the positive serum control is an SPF chicken serum sample obtained by immunizing AEV, which is positive when detected by a commercial ELISA antibody detection kit; the negative serum control is an SPF chicken serum sample not infected with AEV, which is negative when detected by a commercial ELISA antibody detection kit.

6. Use of the avian encephalomyelitis virus antibody ELISA detection kit according to any one of claims 3 to 5 in at least one of the following: 1) Detection of avian encephalomyelitis virus antibodies; 2) Screening SPF chickens; 3) Detection of vaccines and / or virus strains for the presence of avian encephalomyelitis virus infection.

7. A method for detecting avian encephalomyelitis virus antibodies by ELISA, characterized in that: The method is carried out using the avian encephalomyelitis virus antibody ELISA detection kit according to any one of claims 3 to 5.

8. The method according to claim 7, characterized in that The method comprises the following steps: S1. Dilute the recombinant VP1 protein with coating solution and coat the plate. S2. Remove the coating solution, wash with detergent, pat dry, and add blocking solution to the ELISA plate for blocking. S3. Remove the blocking solution, wash with a washing solution, pat dry, and add the sample to be tested diluted with a sample diluent to the ELISA plate for the first incubation; S4. Remove the sample to be tested, wash it with detergent, pat it dry, and add the enzyme-labeled secondary antibody diluted with sample diluent to the ELISA plate for a second incubation; S5. Remove the enzyme-labeled secondary antibody, wash with detergent, pat dry, add TMB colorimetric solution to the plate, perform color development, and then add stop solution to the plate to terminate the reaction. S6. Measure the OD of the liquid in the microplate wells 450nm value, when the OD of the sample to be tested is 450nm When the OD value is ≥0.30, the test result is considered positive; when the OD value of the sample to be tested is 450nm When the value is less than 0.30, the test result is negative.

9. The method according to claim 8, characterized in that The concentration of the recombinant VP1 protein after dilution in step S1 is 0.125-1 μg / mL.

10. The method according to claim 7, characterized in that The ELISA includes one or more of direct ELISA, indirect ELISA and competitive ELISA.

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