Polypeptide-based avian reticuloendotheliosis virus indirect ELISA antibody detection method

Abstract

The invention discloses a polypeptide-based avian reticuloendotheliosis virus indirect ELISA antibody detection method, and belongs to the technical field of biology. According to the kit, potential epitopes of REV gp90 protein are systematically screened to obtain three sections of specific polypeptide antigens for detecting the avian reticuloendotheliosis virus antibody, and a mixed product of the three sections of specific polypeptide antigens is used as a coating antigen to develop the indirect ELISA antibody detection kit for the avian reticuloendotheliosis virus. On the basis of the indirect ELISA antibody detection kit, the avian reticuloendotheliosis virus antibody detection method established by optimizing the coating concentration, the sealing condition, the serum incubation time and the like has the advantages of strong specificity, high sensitivity, good repeatability and convenience in operation; the kit can effectively and rapidly diagnose epidemic diseases caused by the avian reticuloendotheliosis virus, and is beneficial to popularization and application.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a polypeptide-based indirect ELISA antibody detection method for avian reticuloendotheliosis virus. Background Art

[0002] Reticuloendotheliosis (RE), a neoplastic immunosuppressive disease of poultry caused by infection with the reticuloendotheliosis virus (REV), has become a major global epidemic in the poultry industry. REV can cause lesions in the thymus, bursa of Fabricius, and spleen, leading to lymphocyte apoptosis and immune dysfunction, significantly increasing the risk of secondary infection in infected poultry and causing severe economic losses. REV can be transmitted through contact, insect vectors, or vertically via eggs. Notably, contaminated attenuated poultry vaccines can carry REV, becoming a significant mode of viral spread. Epidemiological studies have shown that while REV is not strictly seasonal, its incidence is higher in the summer and autumn. Symptoms of RE include dwarfism syndrome, chronic tumors, and acute reticulocyte tumors, with young chicks being particularly susceptible.

[0003] The REV genome is a linear, single-stranded, positive-sense RNA with a genome size of 8.3 kb. It contains three open reading frames (ORFs), encoding gag (capsid protein), pol (reverse transcriptase / integrase protein), and env (envelope protein). The gp90 protein, produced by hydrolysis of the env protein, is a key glycoprotein and the primary immunogenic protein that induces antibody production. It is often used as an important target for REV serological testing.

[0004] Because REV can be vertically transmitted through seed sources, pathogen monitoring and seed source purification have become the focus of blocking transmission and preventing and controlling the disease. Currently developed REV antibody ELISA methods mostly use artificially expressed recombinant gp90 protein or p30-gp90 fusion protein as the coating antigen. However, there are three problems with using recombinant protein as the coating antigen: first, the use of protein as the coating antigen is easily affected by changes in temperature and pH or protein degradation, which affects the repeatability of the test results; second, the expression and purification of recombinant protein requires complex processes, and there are quality fluctuations between different batches; third, the spatial folding of the recombinant protein may mask certain linear epitopes, resulting in a decrease in the sensitivity of the antibody ELISA test kit. Therefore, it is urgent to develop an indirect ELISA antibody detection method for avian reticuloendotheliosis virus with strong specificity, high sensitivity, good repeatability, and easy operation. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide an indirect ELISA antibody detection method for avian reticuloendotheliosis virus based on polypeptides, which provides technical support for REV serum epidemiological surveys and seed purification.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a specific polypeptide antigen, wherein the amino acid sequence of the specific polypeptide antigen is shown in any one of SEQ ID NO.1 to SEQ ID NO.3.

[0008] The second aspect of the present invention provides the use of the above-mentioned specific polypeptide antigen in the preparation of an avian reticuloendotheliosis virus detection product.

[0009] In a third aspect, the present invention provides an indirect ELISA antibody detection kit for avian reticuloendotheliosis virus, wherein the indirect ELISA antibody detection kit uses the above-mentioned specific polypeptide antigen as a coating antigen.

[0010] Preferably, the indirect ELISA antibody detection kit uses a mixed product of specific polypeptide antigens shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 as a coating antigen.

[0011] The specific polypeptide antigens represented by SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are mixed at a molar ratio of 1:1:1.

[0012] The indirect ELISA antibody detection kit further comprises: an enzyme labeling plate, a diluent, a horseradish peroxidase-labeled goat anti-chicken IgG enzyme-labeled antibody, a substrate color developing solution and a stop solution.

[0013] In a fourth aspect, the present invention provides an indirect ELISA method for detecting antibodies to avian reticuloendotheliosis virus. The indirect ELISA method for detecting antibodies to avian reticuloendotheliosis virus uses the above-mentioned indirect ELISA antibody detection kit.

[0014] The indirect ELISA antibody detection method for avian reticuloendotheliosis virus comprises the following steps:

[0015] (1) Coating an ELISA plate with a mixture of the specific polypeptide antigens represented by SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 at a rate of (125-126) ng / well;

[0016] (2) Dilute the primary antibody serum to 1:200 and incubate at 37°C for 25-30 min. Dilute the secondary antibody horseradish peroxidase-labeled goat anti-chicken IgG to 1:5000 and incubate at 37°C for 25-30 min.

[0017] (3) TMB substrate was added and color was developed at room temperature in the dark for 15-20 min. 5% skim milk powder was used to block the reaction at 37°C for 2 h to terminate the reaction and interpret the results.

[0018] In step (3), the result interpretation standard is: using a microplate reader to measure the OD of each well 450nm The absorbance value is 450nm ≥0.18 was considered positive. 450nm When <0.18, it was considered negative.

[0019] The minimum detection limit of the indirect ELISA antibody detection method for avian reticuloendotheliosis virus is 1:25600.

[0020] Beneficial effects of the present invention:

[0021] 1. The present invention systematically screens potential antigenic epitopes of REV gp90 protein to obtain three specific polypeptide antigens for detecting avian reticuloendotheliosis virus antibodies. The three specific polypeptide antigens are mixed in a molar ratio of 1:1:1 as coating antigens to develop an indirect ELISA antibody detection kit for avian reticuloendotheliosis virus. The kit can effectively improve the binding efficiency between the coating antigen and the target antibody, significantly improving the detection sensitivity and specificity. At the same time, compared with recombinant proteins, the polypeptide antigen is not easily affected by temperature, pH changes or protein degradation, has high stability, can significantly reduce production costs and batch differences, and has broad market application prospects.

[0022] 2. Furthermore, the present invention establishes an avian reticuloendotheliosis virus antibody detection method based on the indirect ELISA antibody detection kit by optimizing the coating concentration, blocking conditions, serum incubation time, etc., which has the advantages of strong specificity, high sensitivity, good repeatability and easy operation. It can effectively and rapidly diagnose diseases caused by avian reticuloendotheliosis virus, and is conducive to its popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a conservation analysis of B cell epitopes of REV gp90 protein.

[0024] Figure 2 The sensitivity of the indirect ELISA antibody detection method provided by the present invention and the commercial kit based on the indirect immunofluorescence method for avian reticuloendotheliosis virus is compared. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0026] The accession numbers of the different REV isolates used in the examples of the present invention in GenBank are as follows:

[0027] REV-T (GenBank ID: GU222419), 3337-05 (GenBank ID: FJ439120.1), MD-2 (GenBank ID: JX912710), 3410-06 (GenBank ID: FJ439119), APC-566 (GenBank ID: DQ387450), BJ1503 (GenBank ID: MG471384), HA9901 (GenBank ID: NC006934), FPVintegrated REV (GenBank ID: AF246698), HLJ071 (GenBank ID: GQ375848), HLJR0901 (GenBank ID: GQ415646), LN1210 (GenBank ID: KU641115), SD2101 (GenBank ID: OM864267).

[0028] In the examples of the present invention, the sources of the strains used are as follows:

[0029] ALV-A: SDAU14A1 (GenBank ID: KU375453.1) is deposited in our laboratory. Reference: Xu Shuzhen, Wang Yixin, Li Yang, et al. Genomic analysis of a subgroup A avian leukosis virus isolated from Longshengfeng chicken and its pathogenicity to SPF chickens [J]. Chinese Journal of Preventive Veterinary Medicine, 2016, 038(009): 705-710.

[0030] ALV-B: SDAU09C2 (GenBank ID: HM446005.1) is deposited in our laboratory. Reference: Zhao Dongmin, Zhang Qingchan, Cui Zhizhong. Isolation and identification of subgroup B avian leukosis virus in reed chickens[J]. Acta Virologica Sinica, 2010(1): 53-57.

[0031] ALV-J: SDAU1005 (GenBank ID: KT156668.1) is deposited in our laboratory. Reference: Wang Y, Li J, Li Y, et al. Identification of ALV-J associated acutely transforming virus Fu-J carrying complete v-fps oncogene[J]. Virus Genes, 2016, 52(3): 365-371.

[0032] MDV: GX0101 (GenBank ID: JX844666.1) is deposited in our laboratory. Reference: Sun AJ, Lawrence P, Zhao YG, et al. A BAC clone of MDV strain GX0101 with REV-LTR integration retained its pathogenicity[J]. Chinese Science Bulletin, 2009(15):7.

[0033] CIAV: SD2102 (GenBank ID: OL448856.1) is deposited by our laboratory. References: [1] Liu L, Li Y, Yin M, et al. Genomic Characterization of Chicken Anemia Virus in Broilers in Shandong Province, China, 2020–2021[J]. Frontiers in Veterinary Science, 2022, 9.

[0034] ARV: live vaccine for chicken viral arthritis (ZJS strain), purchased from Qingdao Yibang Bioengineering Co., Ltd.; NDV: live vaccine for Newcastle disease (La Sota strain), purchased from Qingdao Yibang Bioengineering Co., Ltd.; AIV: recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine, purchased from Qingdao Yibang Bioengineering Co., Ltd.; IBDV: live vaccine for infectious bursal disease (B87 strain), purchased from Qingdao Yibang Bioengineering Co., Ltd.; IBV: live vaccine for infectious bronchitis (LDT3-A strain), purchased from Harbin Weike Biotechnology Co., Ltd.; ILTV: live vaccine for infectious laryngotracheitis (K317 strain), purchased from Qingdao Yibang Bioengineering Co., Ltd.

[0035] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0036] Example 1: Screening of specific polypeptide antigens

[0037] The potential antigenic epitopes of gp90 protein were systematically screened by integrating epitope prediction algorithms (BepiPred-2.0, SEMA 2.0, IEDB). The hydrophilicity parameter (reflecting the tendency of epitope exposure), accessibility parameter (assessing spatial structural accessibility), polarity parameter (indicating the distribution of surface hydrophilic regions) and flexibility parameter (characterizing the conformational freedom of the peptide chain) were evaluated to screen out three regions with the highest probability of B cell antigenic epitopes (Table 1). To verify the conservation of the epitope, multiple sequence alignment and phylogenetic analysis were performed on the gp90 gene sequences of different REV isolates in GenBank. The results showed that the amino acid sequence homology of the three epitope regions was high ( Figure 1 ). Based on this, the above polypeptide was selected as the ELISA coating antigen.

[0038] Table 1. Prediction results of B cell epitopes of REV gp90 protein

[0039]

[0040] Example 2: Indirect ELISA method for detecting avian reticuloendotheliosis virus antibodies based on polypeptides

[0041] (1) Determination of the optimal reaction conditions for indirect ELISA

[0042] The indirect ELISA reaction system was optimized using the checkerboard titration method. The three specific polypeptide antigens screened in Example 1 were mixed at a molar ratio of 1:1:1. The results showed that when the coating concentration was 125 ng / well (0.05 M carbonate buffer, pH 9.6) and the optimal working dilution of the primary antibody serum was 1:200, the P / N value was the highest (Table 2). The same method was used to optimize other conditions, and the optimal conditions were finally determined: incubation at 37°C for 30 min; the optimal dilution of the secondary antibody horseradish peroxidase-labeled goat anti-chicken IgG was 1:5000, incubation at 37°C for 30 min; TMB substrate was used for color development at room temperature in the dark for 15 min; 5% skim milk powder was used at 37°C for blocking for 2 h.

[0043] Table 2 Determination of optimal coating antigen concentration and serum primary antibody dilution by checkerboard method

[0044]

[0045] (2) Determination of the critical value of the indirect ELISA method

[0046] According to the best reaction conditions found, OD values ​​of 150 negative serum samples were measured. 450nm The absorbance value at OD was measured. After calculation, the average value was 0.1048 and the standard deviation s was 0.0247. Therefore, the critical value of indirect ELISA was determined to be 0.1048 + 3 × 0.0247 = 0.1789, that is, when OD 450 ≥0.18 was considered positive, OD 450 When <0.18, it was considered negative.

[0047] Example 3: Specificity Detection

[0048] The polypeptide-based indirect ELISA antibody detection method for avian reticuloendotheliosis virus provided by the present invention was used to detect antibodies to other poultry diseases. The results showed that this method was effective for the OD detection of ALV-A, ALV-B, ALV-J, MDV, CIAV, ARV, NDV, AIV, IBDV, IBV and ILTV of subgroup A. 450 The average values ​​were 0.098, 0.092, 0.086, 0.107, 0.094, 0.103, 0.088, 0.094, 0.086, 0.107 and 0.115 respectively. The detection results showed that the method had no cross-reaction and had strong specificity.

[0049] Example 4: Sensitivity Detection

[0050] The REV standard positive serum was serially diluted from 1:100 to 1:102400, and the ELISA method based on the polypeptide antigen established in this experiment and the commercial kit based on the indirect immunofluorescence method (IFA) were used for detection. The results showed that the minimum detection limit of the ELISA method established in this experiment was 1:25600, while the minimum detection limit of IFA was 1:12800. The sensitivity of the ELISA detection method provided by the present invention is twice that of the IFA method ( Figure 2 ).

[0051] Application examples:

[0052] The indirect ELISA method established in this protocol was used in parallel with IFA to analyze 500 serum samples collected from commercial broiler farms in Shandong Province (Table 3). The results showed that the indirect ELISA detected 126 positive samples (a positive rate of 25.2%, 126 / 500), of which 112 were consistent with the IFA test results; the remaining 374 samples were seronegative by both methods. Discrepant sample analysis identified 14 ELISA-positive / IFA-negative samples, suggesting early seroconversion or low-titer infection below the IFA detection threshold, while no ELISA-negative / IFA-positive cases occurred. The experimental results showed that the indirect ELISA and IFA results established in this protocol had an 88.88% concordance rate, demonstrating that the indirect ELISA had higher sensitivity. In practice, since REV infection requires 7-10 days to induce antibody production, the indirect ELISA is more suitable for early diagnosis of the disease. Early culling of infected chickens after antibody positivity can minimize the risk of horizontal transmission caused by REV.

[0053] Table 3 Detection of REV antibodies in clinical samples using indirect ELISA and IFA

[0054]

[0055] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A specific polypeptide antigen, characterized in that: The amino acid sequence of the specific polypeptide antigen is shown in any one of SEQ ID NO.1 to SEQ ID NO.

3.

2. Use of the specific polypeptide antigen according to claim 1 in the preparation of a product for detecting avian reticuloendotheliosis virus.

3. An indirect ELISA antibody detection kit for avian reticuloendotheliosis virus, characterized in that: The indirect ELISA antibody detection kit uses the specific polypeptide antigen according to claim 1 as a coating antigen.

4. The indirect ELISA antibody detection kit according to claim 3, characterized in that The indirect ELISA antibody detection kit uses a mixed product of specific polypeptide antigens shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 as a coating antigen.

5. The indirect ELISA antibody detection kit according to claim 4, characterized in that The specific polypeptide antigens represented by SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are mixed at a molar ratio of 1:1:

1.

6. The indirect ELISA antibody detection kit according to claim 5, characterized in that The indirect ELISA antibody detection kit further comprises: an enzyme labeling plate, a diluent, a horseradish peroxidase-labeled goat anti-chicken IgG enzyme-labeled antibody, a substrate color developing solution and a stop solution.

7. An indirect ELISA method for detecting antibodies to avian reticuloendotheliosis virus, characterized in that: The indirect ELISA antibody detection method for avian reticuloendotheliosis virus uses the indirect ELISA antibody detection kit according to claim 6.

8. The indirect ELISA method for detecting antibodies to avian reticuloendotheliosis virus according to claim 7, wherein: The indirect ELISA antibody detection method for avian reticuloendotheliosis virus comprises the following steps: (1) Coating an ELISA plate with a mixture of the specific polypeptide antigens represented by SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 at a rate of (125-126) ng / well; (2) Dilute the primary antibody serum to 1:200 and incubate at 37°C for 25-30 min. Dilute the secondary antibody horseradish peroxidase-labeled goat anti-chicken IgG to 1:5000 and incubate at 37°C for 25-30 min. (3) TMB substrate was added and color was developed at room temperature in the dark for 15-20 min. 5% skim milk powder was used to block the reaction at 37°C for 2 h to terminate the reaction and interpret the results.

9. The indirect ELISA method for detecting antibodies to avian reticuloendotheliosis virus according to claim 8, characterized in that: In step (3), the result interpretation standard is: using a microplate reader to measure the OD of each well 450nm The absorbance value is 450nm ≥0.18 was considered positive, and when OD 450nm When <0.18, it was judged as negative.

10. The indirect ELISA method for detecting antibodies to avian reticuloendotheliosis virus according to claim 8, characterized in that: The minimum detection limit of the indirect ELISA antibody detection method for avian reticuloendotheliosis virus is 1:25600.

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