Double-genotype goose astrovirus multi-epitope fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a double-genotype goose astrovirus multi-epitope fusion protein and a preparation method and application thereof, and relates to the technical field of chemistry. Screening of GAstV positive serum; designing and synthesizing a B cell epitope of the GAstV cap protein; screening of dominant B cell epitopes of the single genotype GAstV cap protein; expression and purification of the fusion epitope all-CAP; establishing a fusion epitope ELISA antibody detection method; detecting a clinical sample; and making a detection conclusion according to a detection result. According to the double-genotype goose astrovirus multi-epitope fusion protein as well as the preparation method and the application thereof, prepared GAstV single-genotype positive goose serum is used for screening to obtain cap protein B cell epitope polypeptide with good reactivity, and the fusion epitope polypeptide all-CAP is obtained after tandem expression and purification; a universal indirect ELISA antibody detection technology which is strong in specificity, high in sensitivity and good in repeatability and can be used for common detection of GAstV-1 and GAstV-2 is established by taking the GAstV-1 and GAstV-2 as a coating antigen.

Description

Technical Field

[0001] The present invention relates to the field of chemical technology, and in particular to a double-genotype goose astrovirus multi-epitope fusion protein, a preparation method and an application thereof. Background Art

[0002] Gout in goslings is a viral infectious disease caused by goose astrovirus infection, with urate deposition in the internal organs and joint cavities as the main autopsy lesion. The virus can be divided into two genotypes, GAstV-1 and GAstV-2. Currently, there are a large number of mixed infections of the two GAstV genotypes in clinical practice. The disease can be transmitted horizontally through the fecal-oral route and vertically through contaminated breeding eggs, posing a huge challenge to disease diagnosis and prevention.

[0003] At present, mixed infection of GAstV-1 and GAstV-2 is common in goose flocks in my country, and the epidemic form is complex, which brings huge challenges to the diagnosis and prevention and control of the disease. GAstV has various forms of transmission, and is mostly transmitted horizontally among gosling groups through the fecal-oral route. More importantly, it can also be transmitted vertically through eggs through infected breeding geese, causing some goslings to fail to hatch in the later stage of incubation and reducing the hatching rate. There is currently no commercial vaccine for the prevention of gout in goslings. Referring to the prevention and control methods of vertically transmitted diseases such as pullorum and avian leukemia, establishing a universal antibody detection technology for GAstV pathogens to eliminate positive breeding geese is an important scientific measure to control GAstV infection and epidemic.

[0004] Studies have found that there are multiple B cell epitopes on the surface of the viral cap protein, and the sequences related to these epitopes do not exist in other members of the avian astrovirus genus, and are only relatively conserved in GAstV. On this basis, indirect ELISA or competitive ELISA antibody detection methods have been developed to eliminate positive breeding geese. However, in view of the current co-epidemic of GAstV-1 and GAstV-2, there is a lack of serological diagnostic technology that can simultaneously detect the two genotypes, which seriously hinders the clinical diagnosis and prevention and control of GAstV. Therefore, it is urgent to accelerate corresponding research and establish ELISA antibody detection methods for the two genotypes of GAstV to provide technical support for the screening and elimination of positive breeding geese. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to solve the problems raised by the background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: a dual-genotype goose astrovirus multi-epitope fusion protein and a preparation method, comprising the following methods:

[0007] S1: Material preparation;

[0008] S2: Screening of GAstV-positive serum;

[0009] S3: Design and synthesis of B cell epitopes of GAstV cap protein;

[0010] S4: Screening of dominant B cell epitopes of the monogenic GAstV cap protein;

[0011] S5: Expression and purification of the fusion epitope all-CAP;

[0012] S6: Establishment of fusion epitope ELISA antibody detection method;

[0013] S7: clinical sample testing;

[0014] S8: Draw conclusions based on the test results.

[0015] Preferably, the materials based on S1 include strains, animals, serum, proteins and related reagents.

[0016] Preferably, the indirect ELISA method established based on S2 using the prokaryotically expressed cap proteins of GAstV-1 and GAstV-2 as coating antigens is used to detect goose serum collected from the clinic, and GAstV-positive serum is preliminarily screened and obtained.

[0017] Preferably, the B cell epitope prediction analysis of GAstV-2cap protein in S3 is based on the same method used to analyze and predict the B cell epitopes of GAstV-1cap protein.

[0018] Preferably, the method is based on using the synthesized polypeptide as the coating antigen in S4 and utilizing the GAstV single-positive serum screened in S2 as the primary antibody.

[0019] Preferably, after the pET30a-all-CAP plasmid in S5 is transformed into Escherichia coli BL21 (DE3) competent cells, a single colony is picked and inoculated into LB medium containing kanamycin resistance, and shaken at 30°C until OD600 is 0.4-0.6, and IPTG with final concentrations of 0.4mM, 0.6mM, 0.8mM, and 1mM is added to induce expression respectively.

[0020] Preferably, the method based on S6 is divided into the following steps: optimization of optimal reaction conditions - determination of critical value - specificity test - sensitivity test - repeatability test.

[0021] Preferably, multiple serum samples are collected from breeding goose farms and slaughterhouses in different regions based on S7, and the fusion epitope indirect ELISA antibody detection method established in this experiment and the indirect ELISA antibody detection method for single detection of GAstV-1 or GAstV-2 established in the laboratory in the early stage are used for detection, and the compliance rates are calculated respectively.

[0022] Preferably, the serum obtained by ELISA screening is used as the primary antibody, and the reactivity of the target serum with GAstV-1 or GAstV-2 cap protein is further clarified by western blot.

[0023] Preferably, the dominant B cell epitope of the monogenotype GAstV cap protein is determined by comparing the differences in absorbance values ​​of the polypeptides after reaction, and then the determined dominant epitope is connected in series with a flexible linker (Gly-Gly-Gly-Gly-Ser).

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The dual-genotype goose astrovirus multi-epitope fusion protein, preparation method and application, the prepared GAstV single-genotype positive goose serum was used to screen the cap protein B cell epitope polypeptide with good reactivity, and the fusion epitope polypeptide all-CAP was obtained after serial expression and purification. It was used as the coating antigen to establish a universal indirect ELISA antibody detection technology with strong specificity, high sensitivity, good repeatability, and can be used for the co-detection of GAstV-1 and GAstV-2. The test results of 87 clinical serum samples showed that this method had a high consistency rate with the single-genotype GAstV antibody detection method established in the laboratory in the early stage, strong sensitivity, and low missed detection rate. In this study, the dual-gene universal indirect ELISA antibody detection method was established using the serially expressed GAstV-1 and GAstV-2 cap protein dominant B cell epitope polypeptides as the coating antigen. The method has good specificity, sensitivity and repeatability, and can be used for large-scale clinical antibody screening, providing technical support for the prevention and control of gout in goslings.

[0026] (2) The dual-genotype goose astrovirus multi-epitope fusion protein, preparation method and application of the dual-genotype goose astrovirus multi-epitope fusion protein were prepared by using bioinformatics to analyze the dominant antigenic epitopes of each genotype GAstV cap protein, and the epitope peptides were identified and synthesized through positive serum. The peptide epitope sequences with good immunogenicity were synthesized in series and inserted into the pET30a vector to induce expression to obtain a soluble recombinant protein containing two GAstV genotype fusion epitopes, which was used as the coating antigen. The reaction conditions were optimized by chessboard titration, and a dual-gene universal indirect ELISA antibody detection method was established. The positive critical value of this method was 0.37, the positive serum detection sensitivity was 1:3200, the intra-batch and inter-batch test coefficients of variation were both less than 10%, and there was no nonspecific reaction with other common goose viral pathogen positive sera. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0028] Table 1 is a schematic diagram of the structure of the GAstV-1 and GAstV-2 B cell epitope sequence information of the present invention;

[0029] Table 2 is a schematic diagram of the structure of the screening of B cell dominant epitopes of the GAstV cap protein of the present invention;

[0030] Table 3 is a schematic diagram of the structure of determining the optimal antigen coating amount and serum dilution of the present invention;

[0031] Table 4 is a schematic diagram of the optimized structure of the best sealing conditions of the present invention;

[0032] Table 5 is a schematic diagram of the optimized antigen coating conditions, serum reaction conditions, secondary antibody reaction conditions, and TMB color development conditions of the present invention;

[0033] Table 6 is a schematic diagram of the structure of the repeatability detection of the present invention;

[0034] Table 7 is a schematic diagram showing the structural comparison of the coincidence rate between the fusion epitope indirect ELISA of the present invention and the single genotype GAstV indirect ELISA;

[0035] Figure 1 This is a schematic diagram of the structure of GAstV positive serum screening of the present invention;

[0036] Figure 2 Schematic diagram showing the predicted spatial structure of B cell epitopes of GAstV-1 and GAstV-2 cap proteins of the present invention;

[0037] Figure 3 Schematic diagram of the structure of prokaryotic expression and identification of the fusion epitope all-CAP of the present invention;

[0038] Figure 4 Schematic diagram of the purification structure of the fusion epitope all-CAP of the present invention;

[0039] Figure 5 Schematic diagram of the structure for reactive identification of the fusion epitope all-CAP of the present invention;

[0040] Figure 6 A schematic diagram of the structure for determining the critical value of the present invention;

[0041] Figure 7 Schematic diagram of the structure of the specific detection of the present invention;

[0042] Figure 8 Schematic diagram of the structure of the sensitivity detection of the present invention. DETAILED DESCRIPTION

[0043] This section will describe in detail the specific embodiments of the present invention. The preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the accompanying drawings is to supplement the description of the text part of the specification with graphics, so that people can intuitively and vividly understand each technical feature and the overall technical solution of the present invention, but it should not be understood as a limitation on the scope of protection of the present invention.

[0044] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the present invention.

[0045] In the description of this invention, terms such as "greater than," "less than," and "exceed" are understood to exclude the number itself, while terms such as "above," "below," and "within" are understood to include the number itself. The use of terms such as "first" and "second" is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0046] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, and connecting should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0047] Please refer to Table 1-7 and Figure 1-8 The present invention provides a technical solution: a dual-genotype goose astrovirus multi-epitope fusion protein, a preparation method and an application thereof, the method comprising the following steps:

[0048] ①. Material preparation: bacterial strains, animals, sera, proteins, and BL21 (DE3) competent Escherichia coli cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; GAstV-negative goose sera and goose parvovirus (GPV), avian influenza virus H5 and H7 subtypes (AIV), Newcastle disease virus (NDV), and Tembusu virus (TMUV)-positive goose sera were all preserved in our laboratory; GAstV-1 and GAstV-2 prokaryotic expressed cap proteins were also preserved in our laboratory.

[0049] Reagent selection is divided into:

[0050] DNA / RNA extraction kits were purchased from Tiangen Biotechnology Co., Ltd.; HiScript II 1st Strand cDNA Synthesis Kit reverse transcription kit was purchased from Novozymes; PAGE gel preparation kit, Omni-ECL TM Basic chemiluminescence detection kit, Omni-Easy TM Instant protein loading buffer and colored protein pre-stained marker were purchased from Shanghai Yazyme Biotechnology Co., Ltd.; HRP-labeled rabbit anti-goose IgY secondary antibody was purchased from Frdbio, mouse anti-His tag monoclonal antibody and HRP-labeled goat anti-mouse IgG secondary antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.; TMB colorimetric solution, BSA, etc. were purchased from Beijing Solebow Technology Co., Ltd.

[0051] ②. Screening of GAstV-positive serum: GAstV-1 and GAstV-2 prokaryotic expressed cap proteins were used as coating antigens, respectively. The indirect ELISA method established in the early stage was used to detect goose sera collected from the clinic, and GAstV-positive serum was initially screened. Subsequently, the serum obtained by ELISA screening was used as the primary antibody, and the reactivity of the target serum with GAstV-1 or GAstV-2 cap protein was further clarified by western blot.

[0052] ③. Design and synthesis of GAstV cap protein B cell epitopes: The prediction and analysis results of GAstV-2 cap protein B cell epitopes were used. The same method was used to analyze and predict the B cell epitopes of GAstV-1 cap protein;

[0053] The specific process is as follows: the full-length ORF2 sequences of all GAstV-1 isolates in the past five years were downloaded from the NCBI database, the characteristics of their encoded amino acid sequences were analyzed by multiple sequence alignment, and Clustal Omega software was used for multiple sequence alignment to analyze sequence homology and variation regions. The cap protein domain was analyzed using the CD-Search online software in NCBI, and the cap protein B cell epitopes were analyzed in combination with the Immune Epitope Database (IEDB) and BepiPred 2.0. Subsequently, the spatial structure of the cap protein was predicted using the online software Zhang Lab (https: / / zhanggroup.org / ), and the spatial structure of the screened B cell epitopes was displayed using PyMOL software.

[0054] ④. Screening of dominant B cell epitopes of monogenotype GAstV cap protein: using the synthetic peptide as the coating antigen and the GAstV single positive serum screened above as the primary antibody, the dominant B cell epitopes of monogenotype GAstV cap protein were screened according to the following method: 2 μg of synthetic peptide was added to each well, coated overnight at 4°C, the coating solution was discarded, 150 μl of PBST was added to each well and washed 5 times, each wash for 3 minutes, 100 μl of 5% skim milk was added to each well and blocked at 37°C for 2 hours, the blocking solution was discarded, and the cells were washed 5 times with PBST using the same method, and the cells were blocked with PBS according to the following method: Dilute the GAstV positive serum of the corresponding genotype and the collected negative serum at a ratio of 1:500, add 100ul to each well, incubate at 37℃ for 1h, discard the primary antibody, wash 5 times with PBST in the same way, dilute HRP-labeled rabbit anti-goose IgY secondary antibody at a ratio of 1:10000 with PBS, add 100ul to each well, incubate at 37℃ for 1h, discard the secondary antibody, wash 5 times with PBST in the same way, add 100ul TMB color development solution to each well in a dark environment, develop at 37℃ in the dark for 5min, and add 50ul 1M The reaction was terminated with H2SO4, and the absorbance of each well was measured at OD450 using a microplate reader. By comparing the differences in absorbance values ​​after the reaction of each peptide, the dominant B cell epitope of the monogenic GAstV cap protein was determined. The identified dominant epitope was then concatenated with a flexible linker (Gly-Gly-Gly-Gly-Ser) and sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis and ligation into the prokaryotic expression vector pET30a.

[0055] ----Named it pET30a-all-CAP for subsequent experimental research.

[0056] ⑤. Expression and purification of the fusion epitope all-CAP: After the pET30a-all-CAP plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, a single colony was picked and inoculated into LB medium containing kanamycin resistance. The cells were shaken at 30°C until OD600 was 0.4-0.6, and IPTG at final concentrations of 0.4mM, 0.6mM, 0.8mM, and 1mM was added to induce expression, respectively. The bacteria were collected by centrifugation, and after ultrasonic disruption, the supernatant and precipitate were taken for SDS-PAGE gel electrophoresis analysis, respectively. The soluble protein was purified by Ni column and eluted with 10mM imidazole. The flow-through and eluate were collected for SDS-PAGE analysis. At the same time, western blot analysis was performed using mouse anti-His tag monoclonal antibody, single-positive GAstV goose serum, double-positive GAstV goose serum and corresponding secondary antibodies.

[0057] ⑥. Establishment of fusion epitope ELISA antibody detection method:

[0058] 1. Optimization of optimal reaction conditions

[0059] An indirect ELISA antibody detection method was established using the purified fusion epitope all-CAP as the coating antigen and the double-genotype co-detection goose serum as the primary antibody. The checkerboard titration method was used to gradually determine the antigen coating amount and coating conditions, serum dilution and incubation time, blocking conditions, secondary antibody dilution and reaction time, and TMB development time to determine the optimal reaction conditions.

[0060] 2. Determination of critical value

[0061] The optimized ELISA method was used to test 20 negative sera, determine the OD450 value, and calculate the mean (X) and standard deviation (SD). When the sample OD450 value ≥ X + 3SD, it was judged as positive; when the sample OD450 ≤ X + 2SD, it was judged as negative; when it was between the two, it was judged as suspected and the test needed to be repeated.

[0062] 3. Specificity test

[0063] According to the established method, GAstV-1, GAstV-2, GPV, H5+H7 AIV, NDV, and TMUV-positive goose sera were tested, and the OD450 values ​​were measured to evaluate the specificity of the method.

[0064] Four: sensitivity test

[0065] GAstV-1 and GAstV-2 positive sera were diluted at ratios of 1:400, 1:800, 1:1600, 1:3200, and 1:6400, with three replicates for each dilution. The established ELISA method was used for detection, and the sensitivity of the method was evaluated by measuring the OD450 value.

[0066] 5. Repeatability test

[0067] Intra-batch repeat test: Using the fusion epitope all-CAP prepared in the same batch as the coating antigen, 4 sera (including 2 positive sera and 2 negative sera) were tested, and 3 replicate wells were set for each serum. Inter-batch repeat test: Using the fusion epitope all-CAP prepared in different batches as the coating antigen, the above 4 sera were tested, and 3 replicate wells were set for each serum. The repeatability of the method was evaluated by measuring the OD450 value and calculating the coefficient of variation.

[0068] ⑦. Clinical sample testing: 87 serum samples were collected from goose breeding farms and slaughterhouses in Yuanyang, Xuchang, Xinyang, Jiaozuo, and Hebi, Henan Province. The serum samples were tested using the indirect ELISA antibody detection method for the fusion epitope established in this study and the indirect ELISA antibody detection method for single GAstV-1 or GAstV-2 established in the laboratory earlier. The compliance rates were calculated for each method.

[0069] Draw conclusions based on the above results;

[0070] 1. Screening of GAstV-positive serum: GAstV-1 and GAstV-2 prokaryotic expressed cap proteins were used as coating antigens, and the goose serum collected from the clinic was tested using the indirect ELISA antibody detection method established in the laboratory in the early stage, and the test results were reviewed by western blot, such as Figure 1 As shown, single-positive sera that can detect GAstV-1 and GAstV-2 respectively, as well as double-positive sera that can detect both genotypes, were screened, and their reactivity was good and can be used for subsequent tests.

[0071] 2. Prediction and identification of GAstV cap protein B cell epitopes: Based on the laboratory's previous prediction method for the dominant B cell epitopes of GAstV-2 cap protein, the online software IEDB and BepiPred 2.0 were used to predict the B cell epitopes of GAstV-1 cap protein after sequence alignment analysis. A total of 5 GAstV-1 cap protein antigenic dominant epitopes and 5 GAstV-2 cap protein antigenic dominant epitopes were screened. The specific sequence information is shown in Table 1. Subsequently, the online software Zhanglab (https: / / zhanggroup.org / ) was used to predict the spatial structure of the cap protein, and the predicted B cell epitopes were displayed using PyMOL. Figure 2 It can be seen that the predicted B cell epitopes are all located on the surface of the cap protein head region and can react directly with antibodies.

[0072] GAstV-1 and GAstV-2 B cell epitope sequence information

[0073]

[0074] Table 1

[0075] 3. Evaluation of the reactivity of GAstV cap protein B cell epitope peptides: Ten synthesized GAstV cap protein B cell epitope peptides were used as coating antigens, and GAstV single-positive goose serum was used as the primary antibody. The dominant antigenic epitopes of the monogenotype viral cap protein were screened by ELISA antibody detection method. The results are shown in Table 2. GAstV-1 had better reactivity with peptides 2 and 5, with P / N values ​​of 4.93 and 4.97, respectively; GAstV-2 had better reactivity with peptides 7 and 9, with P / N values ​​of 4.7 and 4.52, respectively. Therefore, these four peptides were synthesized in series with a flexible linker into the pET30a vector to construct the pET30a-all-CAP plasmid.

[0076] Screening of B cell dominant epitopes of GAstV cap protein

[0077]

[0078] Table 2

[0079] 4. Expression and purification of fusion epitope all-CAP: pET30a-all-CAP plasmid was transformed into BL21 (DE3) competent cells, induced by different concentrations of IPTG, and the supernatant and precipitate were collected after breaking the cells and analyzed by SDS-PAGE. Figure 3 As shown, the recombinant all-CAP protein is approximately 20 kDa in size and is expressed in a soluble form in the supernatant. The expression levels induced by different IPTG concentrations are not much different. The bacterial culture is expanded and induced with 0.6 mM IPTG, and the supernatant is collected. The recombinant protein is purified using a Ni column and analyzed by SDS-PAGE. The results show that the purified protein has the correct size, high purity, and no impurity bands ( Figure 4 ), and its concentration was determined by BCA kit to be 1300 μg / ml. Subsequently, the reactivity of the purified protein with positive goose serum was detected by western blot. The results were as follows Figure 5 As shown, the control group used mouse anti-His tag monoclonal antibody as the primary antibody, and the recombinant protein was successfully detected at 20 kDa. Similarly, GAstV single-positive serum and double-positive serum also showed specific bands at the corresponding positions, indicating that the fusion epitope all-CAP can be recognized by goose anti-GAstV-1 and GAstV-2 sera at the same time, and can be used as a coating antigen for the establishment of subsequent ELISA antibody detection methods.

[0080] 5. Determination of the optimal reaction conditions for indirect ELISA: Using the fusion epitope all-CAP as the coating antigen, the positive serum for GAstV-1 and GAstV-2 dual genotype co-detection as the primary antibody, and HRP-labeled rabbit anti-goose IgY as the secondary antibody, the ELISA reaction conditions were optimized one by one by the checkerboard titration method. The results showed that the protein coating amount was 60 ng / well, and the coating was carried out at 37°C for 1 hour; 5% skim milk was used for blocking overnight at 4°C; the positive serum was diluted at 1:800 and incubated at 37°C for 1.5 hours; the HRP-labeled rabbit anti-goose IgY secondary antibody was diluted at 1:15000 and incubated at 37°C for 1.5 hours; TMB color development was carried out at 37°C in the dark for 5 minutes, which was the optimal reaction condition (see Tables 3, 4, and 5).

[0081] Determination of optimal antigen coating amount and serum dilution

[0082]

[0083] Table 3

[0084] Optimization of optimal sealing conditions

[0085]

[0086] Table 4

[0087] Optimization of antigen coating conditions, serum reaction conditions, secondary antibody reaction conditions, and TMB color development conditions

[0088]

[0089] Table 5

[0090] 6. Determination of critical value: 20 negative goose sera were tested using the ELISA method established in this study. The statistical results showed that the average value (X) of the OD450 of the samples was 0.319 and the standard deviation (SD) was 0.017. When the OD450 of the sample was ≥ 0.37, it was judged as positive; when the OD450 was ≤ 0.353, it was judged as negative; and when it was between the two values, it was judged as suspicious (see Figure 6 ).

[0091] 7. Specificity test: The established ELISA method was used to detect positive goose sera for GAstV-1, GAstV-2, GPV, H5+H7AIV, NDV, and TMUV. The results showed that this method could only specifically identify positive sera for GAstV-1 and GAstV-2, and had no cross-reaction with positive sera for common goose viral pathogens, indicating that it has good specificity (see Figure 7 ).

[0092] Sensitivity test: GAstV-1 and GAstV-2 positive serum were diluted at a ratio of 1:800, 1:1600, 1:3200, and 1:6400, and then tested using the established ELISA method. The results showed that when the GAstV-1 and GAstV-2 positive serum was diluted to a maximum of 1:3200, the OD450 value was still greater than 0.37, indicating that the method had good sensitivity (see Figure 8 ).

[0093] Repeatability test: Two positive sera and two negative sera were tested using the ELISA method established in this study, and the intra-assay and inter-assay coefficients of variation were calculated. The results showed that both the intra-assay and inter-assay coefficients of variation were less than 10%, indicating that this method has good repeatability (see Table 6).

[0094] Repeatability testing

[0095]

[0096] Table 6

[0097] Clinical sample testing: 87 sera collected from goose breeding farms and slaughterhouses in Henan Province were tested using the fusion epitope indirect ELISA antibody detection method established in this experiment and the indirect ELISA antibody detection method for single GAstV-1 or GAstV-2 established in the laboratory in the early stage. The results are shown in Table 7. The concordance rates of the fusion epitope indirect ELISA and the indirect ELISA for single GAstV-1 or GAstV-2 were 96.6% and 90.8%, respectively, and the missed detection rate was 0, indicating that the method established in this experiment can be used for clinical diagnosis and serological surveys.

[0098] Comparison of coincidence rates between fusion epitope indirect ELISA and single genotype GAstV indirect ELISA

[0099]

[0100] Table 7

[0101] In summary, faced with the complex situation of co-epidemic of GAstV-1 and GAstV-2, the risk of missed detection is high, leading to inaccurate clinical diagnostic results. Based on this, this study first used the prepared GAstV single genotype positive goose serum to screen for well-reactive cap protein B cell epitope peptides. After tandem expression and purification, the fusion epitope peptide all-CAP was obtained. This was used as the coating antigen to establish a universal indirect ELISA antibody detection technology with strong specificity, high sensitivity, good reproducibility, and can be used for the co-detection of GAstV-1 and GAstV-2. The test results of 87 clinical serum samples showed that this method had a high concordance rate, strong sensitivity, and low missed detection rate with the single genotype GAstV antibody detection method established in the laboratory earlier. In this study, using the tandemly expressed GAstV-1 and GAstV-2 cap protein dominant B cell epitope peptides as the coating antigen, a dual-gene universal indirect ELISA antibody detection method was established. This method has good specificity, sensitivity, and reproducibility, can be used for large-scale clinical antibody screening, and provides technical support for the prevention and control of gout in goslings.

[0102] Note: (Serological testing is a commonly used method in epidemic monitoring, disease diagnosis, and vaccine immune efficacy evaluation. It has the advantages of simple operation, short time consumption, and accurate results. At present, many articles have reported the establishment of serological detection methods for GAstV. Gao Wei et al. used prokaryotic cap protein as the coating antigen to establish an indirect ELISA antibody detection method for GAstV. Ye Jianqiang et al. used the 627-646 amino acids of cap protein as the polypeptide antigen to establish an indirect ELISA antibody detection method for GAstV-1. Tang Yi et al. used a cap protein monoclonal antibody as the competitive To compete for antibodies, a GAstV antibody competition ELISA detection method was established. Li Yin et al. used GAstV-1 purified virus and GAstV-2 cap protein as coating antigens to establish indirect ELISA antibody detection methods for GAstV-1 and GAstV-2 respectively. These results indicate that the cap protein of GAstV has good immunogenicity and can stimulate the body to produce specific protective antibodies. Therefore, the cap protein or its epitope can be used as a coating antigen to establish an antibody detection method). The above-mentioned indirect ELISA antibody detection methods for GAstV are all supported by literature.

Claims

1. A dual-genotype goose astrovirus multi-epitope fusion protein and a preparation method thereof, characterized in that: This includes the following methods: S1: Material preparation; S2: Screening of GAstV-positive serum; S3: Design and synthesis of B cell epitopes of GAstV cap protein; S4: Screening of dominant B cell epitopes of the monogenic GAstV cap protein; S5: Expression and purification of the fusion epitope all-CAP; S6: Establishment of fusion epitope ELISA antibody detection method; S7: clinical sample testing; S8: Draw conclusions based on the test results.

2. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 1, characterized in that: The materials based on S1 include strains, animals, serum, proteins and related reagents.

3. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 1, characterized in that: The indirect ELISA method established based on S2 using the prokaryotic cap proteins of GAstV-1 and GAstV-2 as coating antigens was used to detect goose serum collected from the clinic, and GAstV-positive serum was preliminarily screened.

4. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 1, characterized in that: Based on the prediction and analysis of the B cell epitopes of the GAstV-2cap protein in S3, the B cell epitopes of the GAstV-1cap protein were analyzed and predicted using the same method.

5. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 1, characterized in that: The method is based on using the synthesized polypeptide as the coating antigen in S4 and the GAstV single-positive serum screened in S2 as the primary antibody.

6. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 1, characterized in that: After the pET30a-all-CAP plasmid in S5 was transformed into Escherichia coli BL21 (DE3) competent cells, a single colony was picked and inoculated into LB medium containing kanamycin resistance, and shaken at 30°C until the OD600 was 0.4-0.6, and IPTG with final concentrations of 0.4mM, 0.6mM, 0.8mM, and 1mM was added to induce expression, respectively.

7. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 1, characterized in that: The method based on S6 is divided into the following steps: optimization of optimal reaction conditions - determination of critical value - specificity test - sensitivity test - repeatability test.

8. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 1, characterized in that: Based on S7, multiple serum samples were collected from breeding goose farms and slaughterhouses in different regions, and the fusion epitope indirect ELISA antibody detection method established in this experiment and the indirect ELISA antibody detection method for single detection of GAstV-1 or GAstV-2 established in the laboratory in the early stage were used for detection, and the compliance rates were calculated respectively.

9. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 3, characterized in that: The serum obtained by ELISA screening was used as the primary antibody, and the reactivity of the target serum with GAstV-1 or GAstV-2 cap protein was further clarified by western blot.

10. The dual-genotype goose astrovirus multi-epitope fusion protein and preparation method according to claim 5, characterized in that: The dominant B cell epitope of the monogenotype GAstV cap protein was determined by comparing the differences in absorbance values ​​after reaction of each polypeptide, and then the determined dominant epitope was connected in series with a flexible linker (Gly-Gly-Gly-Gly-Ser).

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