CN110850084A

CN110850084A - Polypeptide sequence for typing and identifying avian adenovirus serotype 4 group I and application

Info

Publication number: CN110850084A
Application number: CN201911250502.9A
Authority: CN
Inventors: 李守军; 牛登云; 马利芳; 陈家锃; 冯敬敬; 段宝敏
Original assignee: Tianjin Bohai Agricultural And Animal Husbandry Joint Research Institute Co Ltd
Current assignee: Tianjin Bohai Agricultural And Animal Husbandry Joint Research Institute Co Ltd
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2020-02-28

Abstract

The invention provides a polypeptide sequence for typing and identifying serotype 4 group I avian adenovirus, which can generate biological reaction with FAdV-4 positive serum through immunoblotting experiment verification, has biological activity and does not have cross reaction with the FAdV positive serum of other serotypes. In addition, the indirect ELISA detection method established by the polypeptide can rapidly and sensitively identify the avian adenovirus type 4 serum antibody, reduce false positive data caused by non-specific reaction, has better specificity compared with a kit coated by whole virus or Hexon, and can effectively perform immune evaluation on the vaccine type 4 and identify the FAdV-4 antibody.

Description

Polypeptide sequence for typing and identifying avian adenovirus serotype 4 group I and application

Technical Field

The invention belongs to the field of biological products for livestock, and particularly relates to a polypeptide sequence for typing and identifying avian adenovirus serotype 4 group I and application thereof.

Background

Fowl adenovirus (FAdV) belongs to the family of Fowl adenovirus and the genus Fowl adenovirus, and is the etiological agent of acute infectious disease which causes sudden death, severe anemia and jaundice of sick chickens as main clinical symptoms, and the Fowl adenovirus is often mixed with pathogenic microorganisms which can cause respiratory diseases of the fowls to infect or cause diseases during immunosuppression, thereby bringing huge loss to the Fowl breeding industry.

Research shows that the group I of the avian adenovirus is divided into 12 serotypes (serotypes 1-7, 8a, 8b and 9-11), structural proteins of the avian adenovirus are mainly Hexon genes, are target of neutralizing antibodies and main protective antigen genes and are closely related to pathogenicity, and molecular epidemiological investigation of the avian adenovirus in 2015-2018 shows that the avian adenovirus strains circulating in recent two years do not change greatly in a conserved region of the Hexon genes.

According to investigation, several avian adenoviruses of different serotypes exist in the same farm, the FAdV-4 type is the most common, but FAdV-11, FAdV-8a and FAdV-8b have a gradual diffusion trend, so that the pathogeny is complex, researchers are difficult to determine the infection pathogeny, and vaccines of adenovirus in the market are relatively disordered at present, so that the typing of antibodies and the antibody level of the immunized FAdV type 4 serum are difficult to monitor.

Disclosure of Invention

The invention aims to provide a polypeptide sequence for typing and identifying serotype 4 avian adenovirus group I, so as to solve the problem that the serotype 4 avian adenovirus group I is difficult to identify effectively.

The invention aims to provide application for typing and identifying the serotype 4 avian adenovirus group I, and an ELISA detection method established by utilizing the polypeptide identified by the serotype 4 avian adenovirus group I can sensitively and effectively detect the antibody condition of the serotype 4 FAdV-I.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

the polypeptide sequence for typing and identifying the serotype 4 group I avian adenovirus is shown as SEQ ID NO. 1.

Further, the polypeptide sequence is coupled with a BSA molecule.

The polypeptide sequence is applied to identifying serotype 4 group I avian adenovirus.

An ELISA kit for typing and identifying group I avian adenovirus serotype 4 comprising the sequences of claims 1 and 2 comprising: coating antigen, antigen coating liquid, confining liquid, sample diluent, developing liquid, secondary antibody, washing liquid, stop solution, negative standard substance and positive standard substance.

Further, the coating antigen is the BSA conjugated polypeptide sequence of claim 2.

Further, the antigen coating solution is phosphate buffer solution, pH 6.0.

Further, the sample diluent was phosphate buffer containing 0.5% BSA, pH 7.4.

Further, the secondary antibody is a goat anti-chicken IgG enzyme-labeled secondary antibody.

Further, the stop solution is 2M H₂SO₄And (3) solution.

Further, the wash solution was 0.05% PBST, pH 7.4.

Compared with the prior art, the invention has the following advantages:

the invention provides a polypeptide capable of identifying FAdV-4 type serum antibodies, which can generate biological reaction with FAdV-4 type positive serum through immunoblotting experiment verification, has biological activity and does not have cross reaction with FAdV positive serum of other serotypes.

The indirect ELISA detection method established by the polypeptide can quickly and sensitively identify the avian adenovirus type 4 serum antibody, reduce false positive data caused by non-specific reaction, has better specificity compared with a kit coated by whole virus or Hexon, and can effectively perform immune evaluation on the vaccine type 4 and identify the FAdV-4 antibody.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 SDS-PAGE electrophoresis result of polypeptide sequence for typing and identifying avian adenovirus group I serotype 4

Detailed Description

In the examples, unless otherwise noted, the biological materials such as kits, vectors, enzymes, host bacteria and the like used are all derived from commercial products.

Example 1: synthesis of polypeptide sequence for typing and identifying serotype 4 avian adenovirus group I

Molecular epidemiological investigation and biosynthesis of group I avian adenoviruses

The research carries out separation and identification on group I avian adenovirus which is epidemic in 2015-2018 and is in China, 9 serotype epidemic strains are separated, the serotypes are FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10 and FAdV-11 respectively, molecular biological analysis is carried out through hexon gene sequencing, and the comparison with related references and reference strains searched on NCBI shows that the 9 serotype hexon genes have obvious difference parts in antigenic site regions, and FAdV-4 type field isolates have no time and regional difference and belong to conserved regions.

The FAdV-4 type polypeptide is biosynthesized, and the length of the polypeptide is 31 amino acids, and the isoelectric point of the polypeptide is 5.99.

2. Purity, specificity and biological activity identification

The artificially synthesized polypeptide is white solid powder and can be diluted and dissolved by using sterile deionized water. The polypeptide is identified by the immunoblotting method for biological activity and specificity, and the result shows that the polypeptide can specifically react with FAdV-4 type positive serum, but can cross-react with the positive serum of the rest serotypes. Thus, the present study conjugated the polypeptide to BSA, followed by elimination of non-specific reactions, and the polypeptide did not cross-react with positive sera from the remaining serotypes, and we named the product of the conjugation of the polypeptide to BSA as C-138.

In this study, C-138 was diluted to 25mg/ml, stored at-20 ℃ and identified by SDS-PAGE, and the vertical electrophoresis (see FIG. 1) showed a single pure band with a size consistent with the expected result.

3. Stability characterization of the polypeptide (C-138)

Performing immunoblotting verification after repeatedly freezing and thawing the polypeptide (C-138) for three times at-20 ℃, wherein the result shows that the polypeptide has biological activity and does not have inactivation after repeatedly freezing and thawing for three times.

Example 2 establishment of indirect ELISA detection method for FAdV synthetic polypeptide

1. Selection of optimal reaction conditions for indirect ELISA

1.1 selection of optimal coating solution, coating amount and optimal dilution of antigen

Respectively preparing three coating solutions of citrate, carbonate and phosphate according to the isoelectric points of the synthetic peptides, diluting the standard antigen into 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60ug/ml by using the coating solutions, coating 96 empty enzyme-labeled plates (100 ul/hole), coating one column at each dilution, and coating overnight at 4 ℃; diluting FAdV-4 negative and positive serum at a multiple ratio (1:50,1:100,1:200,1:400), coating one line (100 ul/well) for each dilution, incubating at 37 ℃ for 1h, washing with PBST for 5 times, and patting to dry; adding 1:1000 times diluted goat anti-chicken IgG-HRP (immunoglobulin G-peroxidase) with the concentration of 100 ul/hole, incubating for 1h at 37 ℃, washing for 4 times by PBST (basic-particle system test), and patting dry; adding a newly configured TMB chromogenic substrate, developing for 15min in a dark place at a rate of 100 ul/hole. Finally adding 2mol/L H₂SO₄The reaction was stopped, 50 ul/well, OD was measured with microplate reader₄₅₀. The experiment was repeated 3 times and the average OD of the samples was determined₄₅₀Selection of positive serum OD₄₅₀About 1.0, negative serum OD₄₅₀A dilution of the coating antigen of less than 0.1 was taken as the working concentration. The results show that the absorbance values obtained after the polypeptide is coated with phosphate buffer solution at pH6.0 are higher than those of the other two buffers. The data show that antigen coating is at 35ug/ml, 400-fold optimal for serum dilution (see table 1).

TABLE 1 determination of optimal coating concentration and optimal dilution of antigen

1.2 selection of optimal blocking solution and blocking time

Selecting optimal antigen coating concentration and coating condition to coat antigen, respectively blocking ELISA plate with 5% skimmed milk, 2.5% skimmed milk, 5% BSA, and 1% BSA as blocking solution for 45min, 60min, 90min, and 120min, repeating the experiment for three times, and determining OD of FAdV-4 negative and positive detection serum₄₅₀And (4) determining the optimal sealing liquid and sealing time. According to the data, the maximum P/N value is shown when 5% BSA blocks for 90min (see Table 2 and Table 3).

TABLE 2 determination of optimal blocking solution

TABLE 3 determination of optimal seal time

1.3 selection of optimal serum dilutions

Diluting the serum to be detected with PBS, PBST, PBS containing 0.5% BSA and PBST as serum diluent under the determined optimal conditions, and determining the OD of the FAdV-4 negative and positive detection serum₄₅₀Value, the experiment was repeated three times to determine the optimal serum dilution. The data show that the P/N values are maximal after dilution of the sera with PBS containing 0.5% BSA by indirect ELISA procedure (see Table 4).

TABLE 4 determination of optimal serum dilutions

1.4 selection of optimal serum response time

Using the above determinationsAdding FAdV-4 negative and positive serum, incubating in 37 deg.C incubator for 30min, 60min, 90min and 120min, performing experiment according to indirect ELISA method, and determining OD of FAdV-4 negative and positive detection serum₄₅₀Value, experiments were repeated three times to determine optimal serum reaction conditions. The data show that the serum was incubated at 37 ℃ for 60min with the maximum P/N value (see Table 5).

TABLE 5 determination of optimal reaction time of sera

1.5 selection of optimal working concentration of enzyme-labeled Secondary antibody

Using the optimal conditions determined above, the enzyme-labeled secondary antibody was serially diluted with PBS (1:1000, 1:1500, 1:2000, 1:2500, 1:3000) and tested by indirect ELISA protocol to determine the OD of the FAdV-4 negative and positive test sera₄₅₀The experiment is repeated three times respectively to determine the optimal working concentration of the enzyme-labeled secondary antibody. According to data, enzyme-labeled secondary antibody is subjected to 1:2500 ℃ dilution, the maximum P/N value experimentally determined (see Table 6).

TABLE 6 determination of optimal working concentration of enzyme-labeled secondary antibody

1.6 selection of optimal reaction time for enzyme-labeled Secondary antibodies

Searching the optimal action time of the secondary antibody under the determined optimal conditions, incubating the ELISA plate added with the secondary antibody in a 37 ℃ incubator for 30min, 60min, 90min and 120min, performing experiments according to an indirect ELISA operation method, and determining the OD of the FAdV-4 negative and positive detection serum₄₅₀The experiment is repeated three times respectively to determine the optimal action time of the enzyme-labeled secondary antibody. According to the data, the enzyme-labeled secondary antibody is incubated at 37 ℃ for 120min, and the experimentally measured P/N value is the largest (see Table 7).

TABLE 7 determination of optimal working time of enzyme-labeled secondary antibody

1.7 selection of optimal action time of the substrate

Adding TMB substrate solution with new configuration under the above determined optimum conditions, incubating at 37 deg.C for 5min, 10min, 15min, and 20min, performing experiment according to indirect ELISA operation method, adding stop solution, and determining FAdV-4 negative and positive detection serum OD₄₅₀Values, experiments were performed in triplicate to determine the optimal time of action of the substrate. The data show that TMB substrate was reacted at 37 ℃ for 15min with the largest experimentally measured P/N value (see Table 8).

TABLE 8 determination of the time of action of the TMB substrate

1.8 determination of the Critical value

ELISA was performed using 30 laboratory-stored FAdV-4 negative serum samples, and OD of 30 FAdV-4 negative serum samples was measured₄₅₀Average value of (2)

Sum standard deviation s, upper limit of signal interval

And lower limit

The value of (1) is OD₄₅₀Value of

Is positive, OD₄₅₀Value of

Is negative, between the two is suspected, andthe standard negative control serum is less than or equal to 0.15, and the standard positive control serum OD₄₅₀The result is true for values greater than or equal to 0.5. Displaying OD according to data₄₅₀Mean value of

0.128 and a standard deviation(s) of 0.091, so that if the OD of the sample is detected₄₅₀Greater than or equal to 0.401 is positive, OD₄₅₀Negative when the ratio is less than or equal to 0.310, OD is less than or equal to 0.401₄₅₀Less than or equal to 0.310 is a suspicious sample.

2. Indirect ELISA stability test

2.1 specificity test of the detection method

Detecting positive sera of H9, ND, H5, FAdV-1, FAdV-2, FAdV-3, FAdV-8a, FAdV-8b, FAdV-10 and FAdV-11 stored in a laboratory by using an established indirect ELISA detection method, repeating the detection steps for 5 times, simultaneously setting up negative and positive controls, and calculating OD of the detected sera₄₅₀Value, test the specificity of the established method. The results show that OD₄₅₀All values were less than 0.310 and were negative, indicating that the ELISA assay using synthetic peptide C-138 had better specificity (Table 9).

TABLE 9 results of specificity test

2.2 sensitivity test

After the FAdV-4 positive serum fold ratio (1:10,1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560 and 1:5120) is diluted, sensitivity test is carried out by using an established detection method, and the result shows that when the positive serum fold is 10-1280, the detection result is positive, and when the fold is more than 1280, the detection result is negative (Table 10).

TABLE 10 results of sensitivity test

2.3 repeatability test

2.3.1 in-batch repeatability test

Using the same batch of prepared synthetic peptide C-138, taking 20 parts of FAdV-4 positive serum with different antibody levels, determining according to the optimal coating concentration and optimal reaction condition determined by the test, determining at three different time points (0h, 24h and 48h) according to the program of an indirect ELISA detection method, making 4 holes in parallel for each sample, and calculating the coefficient of variation (coefficient of variation)

) The test results show that the coefficient of variation is 0.5-5.2%, and the coefficient of variation is less than 5.2%, which indicates that the synthetic peptide prepared in the same batch has better batch-to-batch repeatability (Table 11).

TABLE 11 test results of in-batch reproducibility test

2.3.2 batch-to-batch repeatability test

Preparing synthetic peptide C-138 from 3 different batches, coating an ELISA plate according to the optimal coating concentration and the optimal reaction condition, detecting 20 parts of FAdV-4 positive serum under the same time and the same condition, repeating 4 holes for each sample, and calculating the coefficient of variation. The results of the batch-to-batch repeated tests show that the coefficient of variation is 0.7-4.7% and less than 4.7%, which indicates that the synthetic peptides of different batches have better batch-to-batch repeatability.

TABLE 12 test results of the repeatability test between batches

2.4 clinical sample testing

200 parts of chicken serum of a certain chicken farm are randomly collected, the chicken serum is numbered according to a collection house, an established indirect ELISA detection method is used for detection, and simultaneously, the FAdV-I whole virus ELISA is used for comparative detection, according to the result of the ELISA detection method, 113 parts of FAdV-4 positive serum and 87 parts of negative serum are displayed, and the FAdV-I whole virus ELISA result displays 120 parts of FAdV positive serum and 80 parts of negative serum. The result obtained by the ELISA detection method accords with the FAdV-4 vaccine immunity condition of the farm, while the FAdV-I whole virus ELISA result can not specifically show the FAdV-4 vaccine immunity condition.

Example 3 comparison of the FAdV-4 type identification ELISA kit with the commercially available FAdV-I antibody detection kit

A FAdV-I antibody detection kit which is sold in the market and has a good using effect is selected and purchased, 50 FAdV-I positive serums (comprising 8 serotypes including FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-8a, FAdV-8b, FAdV-10 and FAdV-11) stored in the experiment are detected, the FAdV-I positive serums are compared and analyzed with the ELISA detection method, the experiment is repeated for three times, and the average value is taken. The detection result shows that the ELISA detection method can sensitively distinguish the FAdV-4 type serum, and 3 commercially available FAdV-I antibody detection kits cannot distinguish the FAdV-4 type serum. The determination standards of the three FAdV-I ELISA antibody detection kits are shown in the following table 13, and the specific detection results are shown in the following table 14:

TABLE 13 different kit criteria

TABLE 14 results of different kits

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The polypeptide sequence for typing and identifying avian adenovirus serotype 4 group I is characterized in that: the sequence is shown in SEQ ID NO. 1.

2. The polypeptide sequence for serogroup I avian adenovirus group 4 typing according to claim 1, wherein the polypeptide sequence is conjugated to a BSA molecule.

3. Use of a polypeptide sequence as claimed in claims 1 and 2 for identifying serotype 4 group I avian adenoviruses.

4. An ELISA kit for typing and identifying group I avian adenovirus serotype 4 comprising the sequences according to claims 1 and 2, characterized in that: the kit comprises: coating antigen, antigen coating liquid, confining liquid, sample diluent, developing liquid, secondary antibody, washing liquid, stop solution, negative standard substance and positive standard substance.

5. The ELISA kit for typing and identifying avian group I serotype 4 virus according to claim 4 wherein: the coating antigen is the BSA-conjugated polypeptide sequence of claim 2.

6. The ELISA kit for typing and identifying avian group I serotype 4 virus according to claim 4 wherein: the antigen coating solution is phosphate buffer solution with pH of 6.0.

7. The ELISA kit for typing and identifying avian group I serotype 4 virus according to claim 4 wherein: the sample diluent was phosphate buffer containing 0.5% BSA, pH 7.4.

8. The ELISA kit for typing and identifying avian group I serotype 4 virus according to claim 4 wherein: the second antibody is goat anti-chicken IgG enzyme-labeled second antibody.

9. The ELISA kit for typing and identifying avian group I serotype 4 virus according to claim 4 wherein: the stop solution is 2M H₂SO₄And (3) solution.

10. The ELISA kit for typing and identifying avian group I serotype 4 virus according to claim 4 wherein: the wash was 0.05% PBST, pH 7.4.