CN108680741B

CN108680741B - Chicken infectious bronchitis virus 5b ELISA antibody detection kit and its application

Info

Publication number: CN108680741B
Application number: CN201810265000.2A
Authority: CN
Inventors: 任涛; 戴旭; 吴耀棠; 游人荣; 林秋燕; 廖明
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-03-28
Filing date: 2018-03-28
Publication date: 2021-05-11
Anticipated expiration: 2038-03-28
Also published as: CN108680741A

Abstract

The invention belongs to the field of biotechnology, and relates to a chicken infectious bronchitis virus 5b ELISA antibody detection kit and application thereof. The kit includes an ELISA reaction plate coated with recombinantly expressed IBV non-structural protein 5b, and also contains diluted IgG enzyme-labeled antibody solution, blocking solution, washing solution, sample dilution solution, positive control sample, negative control sample, display Color solution and stop solution. In the present invention, the non-structural protein 5b is used as the coating antigen, and the cost is low. The use of this kit has the advantages of convenience, rapidity, high sensitivity and good specificity, and provides a new and effective detection method for IBV antibody monitoring, epidemiological investigation and future IBV disease purification.

Description

Infectious bronchitis virus 5b ELISA antibody detection kit and application thereof

Technical Field

The invention relates to an ELISA antibody detection kit using chicken infectious bronchitis virus 5b protein as a coating antigen and application thereof, belonging to the technical field of biology.

Background

Infectious bronchitis coronavirus (IBV): infectious bronchitis virus IBV is an acute highly-contacted respiratory disease caused by IBV of gammahonia coronavirus subgenus of coronaviridae of the order of the nested virales, the family coronaviridae, and is characterized mainly by mouth-opening respiration, tracheal rales, cough, glandular gastric enlargement, hemorrhage, and "piebald kidney". IBV is an unsprung single-stranded positive-strand RNA virus approximately 27.6kb in length, with a discontinuous transcriptional machinery, containing ten distinct open reading frame ORFs, 5 '1 a-1b-s-3a-3b-E-M-5a-5 b-N-3'. Encodes 4 structural proteins S, N, M, E and 15 non-structural proteins nsp2-nsp16 and 4 accessory proteins 3a, 3b, 5a, 5 b.

After IBV infects cells, negative strand RNA is transcribed by early RNA polymerase and 6 subgenomic mRNAs (l-6) are transcribed from the negative strand RNA by late RNA polymerase in a discontinuous transcription mechanism. These mrnas have a common 3' end, forming a nested structure. Wherein mRNA2, mRNA4 and mRNA6 encode three major structural proteins of IBV, Spike (S), Membrane (M) and nucleoprotein (N), respectively. The mRNA5 consisted of approximately 443 nucleic acids and contained two ORFs (ORF5a and ORF5b) encoding two polypeptides with molecular weights of approximately 7.4kDa and 9.5kDa, respectively.

Unlike the alpha and beta coronaviruses nsp1, which inhibit host protein synthesis, gamma coronavirus IBV has no homologous chromosome to nsp1 and 5b instead of nsp1 has been documented in gamma coronavirus IBV to inhibit host protein synthesis. The structure and function of 5b is conserved in coronaviruses. The non-structural protein is generated in the virus infection replication stage and does not participate in the composition of virus particles, the inactivated vaccine does not have the infection replication capacity, and the non-structural protein cannot be generated, and the non-structural protein 5b serving as a detection antigen has the potential of distinguishing the inactivated vaccine immunity from live virus infection.

Because the prior domestic prevention and control of IBV mainly takes the immunization of inactivated and attenuated vaccines as main factors, the immune effect of the inactivated vaccines is far less than that of the attenuated vaccines, and the immune effect is good. IBV has the property of being mutable, wherein the attenuated vaccine may have the risk of virus dispersion, which brings great challenges for the purification of IBV diseases in the future, so the attenuated vaccine must be eliminated along with the development of the technology.

In the prior art, the indirect ELISA method coated by whole virus protein or non-structural protein and structural protein can not distinguish vaccine immunity from live virus infection, because the prior IBV mainly controls the immunity of inactivated vaccine and attenuated vaccine, and the attenuated vaccine can generate non-structural protein in the immune process. Therefore, as attenuated vaccines are withdrawn from the market in the future, a new, alternative indirect ELISA method is provided for the decontamination of IBV disease.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects in the prior art, and provides an antibody detection kit for infectious bronchitis, which provides an economical, practical and reliable detection tool for diagnosis of clinical IB (infectious bronchitis disease), detection of antibody level and purification of IBV (infectious bronchitis Virus) diseases in the future.

In order to solve the technical problems, the invention provides a kit for detecting chicken infectious bronchitis virus 5b ELISA antibody, which comprises: IBV nonstructural protein 5b coated ELISA reaction plates.

Wherein, the nucleotide sequence of IBV non-structural protein 5b is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2.

As a preferred embodiment, the IBV non-structural protein 5b is produced by recombinant expression.

As an alternative embodiment of the present invention, the method for recombinant expression of IBV non-structural protein 5b is as follows:

s1, amplifying an IBV 5b gene by RT-PCR; as a preferred embodiment, a primer pair for amplifying the IBV 5b gene is shown as SEQ ID NO.3 and SEQ ID NO. 4;

s2, integrating the gene obtained in the step S1 into a plasmid vector to obtain a recombinant plasmid; in a preferred embodiment, the plasmid vector is pET-32 a;

s3, transforming the recombinant plasmid constructed in the step S2 into host bacteria and carrying out induced expression to obtain a bacterial liquid; in a preferred embodiment, the host bacterium is Escherichia coli;

s4, screening recombinant bacteria carrying IBV 5b genes, and expressing a large amount of 5b recombinant proteins fused with HIS under IPTG induction;

s5, purifying by using an HIS label nickel column affinity chromatography column to obtain the purified IBV 5b recombinant protein.

Further, the kit of the invention also comprises the following components:

(1) enzyme labeling reagent: the enzyme labeling reagent is a goat anti-chicken IgG antibody labeled by horseradish peroxidase.

(2) Sealing liquid: the blocking solution is PBS (PBST) containing BSA or skimmed milk powder; preferably, it is PBS containing 5% skim milk powder.

(3) Washing liquid: the washing solution is PBS (PBST) containing 0.01-0.1% Tween-20; preferably, it is PBS containing 0.05% Tween-20.

(4) Diluting liquid: the diluent is PBST containing BSA or skimmed milk powder; preferably, it is pbst containing 5% skim milk powder.

(5) Positive control sample: the positive control sample is anti-IBV 5b chicken serum.

(6) Negative control samples: the negative control sample mentioned above is negative SPF (Specific Pathologen Free No particular Pathogen)

(7) Color development liquid: the color developing liquid is TMB single-component color developing liquid.

(8) Stopping liquid: the stop solution is 1M to 2M H₂SO₄(ii) a Preferably, it is 2M H₂SO₄。

The use method of the kit comprises the following steps:

1) antigen coating;

and (3) after the concentration of the prepared recombinant 5b protein is measured by a BCA protein detection kit, diluting the protein by using a coating solution, adding the diluted protein into an enzyme-linked reaction plate, and incubating overnight to prepare the enzyme-linked reaction plate. Wherein the concentration of the antigen coating is 2-10 mug/mL, and the antigen coating is incubated overnight at 4 ℃; preferably, the concentration of antigen coating is 5 μ g/mL; preferably, the coating solution is a 0.05M carbonate buffer at pH 9.6.

2) Sealing;

removing the coating solution of the enzyme-linked reaction plate, adding a sealing solution for incubation, and washing the plate once by using PBST; wherein the blocking solution is skimmed milk powder or PBS of BSA, and the blocking incubation time is 1-3 h; preferably, the blocking solution is 0.2% BSA or 5% or 7.5% skimmed milk powder, and the blocking time is 2 h; more preferably, the blocking solution is 5% skim milk powder in PBS.

3) Primary antibody incubation;

adding a serum (primary antibody) to be detected diluted by a diluent at a ratio of 1:400, and setting positive and negative serum controls at the same time; incubate, wash plate 5 times with PBST. Wherein the dilution concentration of the serum to be detected is 1: 200-1: 600, and the primary antibody is incubated at 35-39 ℃ for 45-90 min; preferably, the serum is diluted at a concentration of 1: 400; primary antibody was incubated at 37 ℃ for 1 h. Wherein, the diluent is PBST containing BSA or skimmed milk powder; preferably, it is PBST with 5% skim milk powder.

4) Incubating a second antibody;

adding a secondary goat anti-chicken IgG antibody marked by HRP diluted by a diluent at a ratio of 1:10000, incubating, and washing the plate for 5 times by PBST. Wherein the dilution ratio of the enzyme-labeled secondary antibody is 1: 5000-1: 10000, incubating the secondary antibody at 35-39 ℃ for 30-90 min; preferably, the enzyme-labeled secondary antibody is diluted 1:10000, and the secondary antibody is incubated at 37 ℃ for 45 min.

5) Developing color;

to add color reagent into the reaction hole, and develop color in dark place. Wherein the color developing agent is TMB, and the color development is carried out for 8-15 min at room temperature in a dark place; preferably, the color is developed for 10 min.

6) Terminating;

the adopted stop solution is 1M-2M H2SO₄(ii) a Preferably, it is 2M H₂SO₄。

7) And (6) judging the result.

OD measurement by enzyme-linked immunosorbent assay₄₅₀The determination standard of the absorbance value at nm is as follows: for sample OD₄₅₀nm value of not less than

The IBV is judged to be positive when the number of IBV is 0.336, and is judged to be negative when the number of IBV is not.

The kit is applied to detecting the infectious bronchitis virus specific antibody induced by the infectious bronchitis inactivated vaccine immunized chicken and the chicken infected with infectious bronchitis.

Compared with the prior art, the invention has the beneficial effects that:

the IBV ELISA antibody detection kit provided by the invention takes the non-structural protein 5b as the coating antigen.

The non-structural protein is not involved in the composition of virus particles, and the non-structural protein 5b serving as a detection antigen has the potential of distinguishing inactivated vaccine immunity from live virus infection. The kit has the advantages of convenience, rapidness, high sensitivity, strong specificity and the like, and provides a new effective detection means for IBV antibody monitoring, epidemiological investigation and future disease purification of IBV.

Drawings

FIG. 1 expression of recombinant IBV 5b protein;

FIG. 25 b purification of Western Blot; …

FIG. 3 shows the test results of 5b-ELISA of the strain group of the challenge LX 4;

FIG. 4 challenge QXIBV2 strain group 5b-ELISA test results.

Detailed Description

Chicken infectious bronchitis virus 5b ELISA antibody detection kit

The kit used in the invention comprises the following components:

(1) recombinant expressed IBV nonstructural protein 5b and ELISA reaction plate;

(2) and (3) diluting the IgG enzyme-labeled antibody solution with a sample diluent. The enzyme-labeled antibody is a goat anti-chicken IgG antibody labeled by Horse Radish Peroxidase (HRP);

(3) sealing liquid: PBS containing 5% skim milk powder;

(4) washing liquid: PBS containing 0.05% Tween-20, PBST;

(5) sample diluent: PBST with 5% skim milk powder;

(6) positive control sample: anti-IBV 5b chicken serum;

(7) negative control samples: negative SPF chicken serum;

(8) color development liquid: TMB Single component color developing solution (biohao)

(9) Stopping liquid: 2M H2SO 4.

Method for using the above kit

Sequentially comprises the following steps:

(1) preparation of ELISA antigen:

the IBV 5b specific primer is used for amplifying IBV 5b genes by using nucleic acid extracted from allantoic fluid of chicken embryos infected by IBV LX4 strain as a template through an RT-PCR method, the IBV 5b genes are cloned into a prokaryotic expression vector pET-32a, escherichia coli is transformed, recombinant bacteria carrying the IBV 5b genes are screened, a large amount of 5b recombinant proteins fused with HIS are expressed under IPTG induction, and the HIS-labeled nickel column affinity chromatography column is used for purification, so that the high-purity IBV 5b recombinant proteins are obtained and stored at the temperature of minus 80 ℃ for later use. The nucleotide sequence of the IBV 5b gene is shown as SEQ ID NO: 1 is shown in the specification; the amino acid sequence coded by the IBV 5b gene is shown as SEQ ID NO: 2, respectively.

SEQ ID NO: 1IBV 5b Gene

atgaataatagtaaagataatccttttcgcggagcaatagcacgaaaagcgcgaatttatctgagaggaggattagattgtgtttactttcttaacaaagcaggacaagcagagccttgccccgcgtgcacatcactagtattccaggggaaaacttgtgaagagcacatacgtaataataacttgctatcatggcgagcggtaaagcagctggaaagtcagactcccccgcgccaatcatcaaactag

SEQ ID NO：2

Amino acid sequence of IBV 5b gene

MNNSKDNPFRGAIARKARIYLRGGLDCVYFLNKAGQAEPCPACTSLVFQGKTCEEHIRNNNLLSWRAVKQLESQTPPRQSSN

The primer pair for amplifying the IBV 5b gene is shown as SEQ ID NO.3 and SEQ ID NO. 4:

SEQIDNO.3:5b-F CGCGGATCCATGAATAATAGTAAAGATA

SEQIDNO.4:5b-R CCCAAGCTTCTAGTTTGATGATTGGCGC

(2) preparation of IBV 5b coated enzyme-linked reaction plates:

after the concentration of the prepared recombinant 5b protein is measured by a BCA protein detection kit, the recombinant 5b protein is diluted to 5 mu g/ml by using a coating solution, added into an enzyme-linked reaction plate according to 200 mu l/hole, and incubated overnight at 4 ℃, thus preparing the enzyme-linked reaction plate;

(3) and (3) sealing: after the coating liquid is removed from the prepared enzyme-linked reaction plate, washing the plate for 5 times by PBST, adding 200 mul/hole of sealing liquid, incubating for 2 hours at 37 ℃, and washing the plate once by PBST;

(4) primary antibody incubation: a100. mu.l/well dilution of the test serum (primary antibody) with a diluent at a rate of 1:400 was added, and positive and negative serum controls were set. Incubation for 1 hour at 37 ℃ and PBST washing of the plates 5 times;

(5) and (3) secondary antibody incubation: adding 100 μ l/well HRP-labeled goat anti-chicken IgG secondary antibody diluted with diluent at a ratio of 1:10000, incubating for 45 minutes at 37 ℃, and washing the plate for 5 times by PBST;

(6) color development: adding 100 mul/hole into the reaction hole, and developing for 10 minutes in dark;

(7) and (4) terminating: add 100. mu.l/well stop solution.

(8) And (4) judging a result: and (4) determining OD450 readings by using an enzyme-labeling instrument, and judging that the IBV is positive if the serum sample value is more than 0.336, otherwise, judging that the IBV is negative.

The following is a further description with reference to the examples and the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLE 15 b preparation of recombinant protein antigens

An expression host bacterium E.coli BL21(DE3) carrying a pET-32a-5b vector is activated by an LB liquid culture medium and is transferred into the LB liquid culture medium containing ampicillin resistance according to the ratio of 1:100 for mass culture and propagation. When OD600nm of the bacterial liquid reaches 0.4-0.6, IPTG with a final concentration of 1mM is added to induce expression for 6h, and then the bacterial cells are collected by centrifugation at 10,000rpm at 4 ℃ for 10min, and the target protein is purified by using a nickel column affinity chromatography column according to a product manual. Protein samples obtained after elution were analyzed by SDS-PAGE. As shown in FIG. 2, the purified recombinant protein pET-32a-5b has a molecular weight of 27KD and high purity. Purified pET-32a-5b was stored at-80 ℃ until use.

EXAMPLE 2 preparation and screening of negative and positive sera

The positive serum used by the kit can be used as IBV 5b positive serum by immunizing SPF chickens three times with 1 mg/SPF chicken recombinant pET-32a-5b protein purified in example 1, collecting and separating the immune chicken serum, verifying that the serum contains high-titer anti-5 b antibody by using indirect immunofluorescence assay (IFA), and then subpackaging at-80 ℃ for storage. And verifying the nonimmune SPF chicken serum as IBV 5b antibody negative by IFA, and subpackaging at-80 ℃ for storage to obtain the IBV 5b negative serum.

EXAMPLE 3 preparation of IBV-infected and non-infected serum

40 SPF chickens aged 3 weeks were divided into 4 groups, which were infected with the attenuated QXIBV2 and the virulent LX4, the immune M41 inactivated vaccine and the PBS control group, respectively.

TABLE 1

Example 45 b optimization of ELISA method

1.5b steps of ELISA method

(1) Coating: diluting the antigen to a working concentration by using an ELISA coating solution, coating an ELISA plate according to 200 mu l/hole, coating overnight at 4 ℃, and washing the plate for 5 times by using PBST;

(2) and (3) sealing: adding a sealing solution in an amount of 200 mu l/hole, sealing at 37 ℃ for 2h, and washing the plate for 5 times by PBST;

(3) incubation of primary antibody: adding diluted serum to be detected in an amount of 100 mul/hole, incubating for 1h in a constant temperature box at 37 ℃, and washing the plate for 5 times by PBST;

(4) and (3) incubation of the secondary antibody: adding diluted goat anti-chicken IgG labeled with HRP in an amount of 100 mu l/hole, incubating for 1h (time before optimization) in a 37 ℃ incubator, and washing the plate for 5 times by PBST;

(5) color development: adding TMB substrate in an amount of 100 μ l/hole, and developing in a 37 deg.C incubator for 10-15min in dark;

(6) and (4) terminating: adding 2M H2SO4 stop solution in an amount of 100 mu l/hole to terminate the reaction;

(7) reading: the absorbance at OD450nm was determined for each well using a microplate reader.

(8) And judging a result.

Based on the basic procedure described above, the reaction conditions of the 5b ELISA were optimized.

Optimization of ELISA reaction conditions

(1) Determination of optimal working concentrations of antigen and serum

According to the concentration of the recombinant protein, the protein is diluted into 0.5ug/ml, 1ug/ml, 2ug/ml, 5ug/ml, 10ug/ml and 20ug/ml by using an ELISA coating solution in sequence, each well is coated with 100ul of an ELISA plate, and the plate is coated overnight at 4 degrees. And (3) optimizing on a 96-well enzyme-linked reaction plate by adopting a square matrix method, and diluting positive serum and negative serum at a ratio of 1:20, 1:100, 1:200, 1:400 and 1: 600. After ELISA reaction, OD450nm value was determined, and the maximum P/N value (mean value of positive serum OD450 nm/mean value of negative serum OD450 nm) was selected as the optimal final condition. Through analysis, when the antigen coating concentration is 2-10 ug/ml, the serum dilution multiple is 1: 200-1: 600, and the P/N value is large, the detection function can be realized. Optimally, when the antigen coating concentration is 5 mu g/ml and the dilution multiple of the serum to be detected is 1:400, the P/N value is maximum.

TABLE 2

(2) Selection of working concentration of enzyme-labeled antibody

On the basis of the determined optimal antigen coating concentration and serum dilution factor, diluting the enzyme-labeled secondary antibody by 1:1000, 1:2000, 1:5000 and 1:10000 respectively, performing ELISA detection, and selecting the dilution factor of the enzyme-labeled antibody with the maximum P/N value as the optimal working concentration. Through analysis, when the dilution multiple of the enzyme-labeled antibody is 1:10000, the P/N is the maximum, and the detection function can be realized.

TABLE 3

(3) Selection of coating liquid

Separately distilling with single distilled water (H)₂O, pH5.0), 0.9% NaCl physiological saline (NS, pH7.0), 0.01M phosphate buffer (PBS, pH7.4), 0.05M carbonate buffer (CB, pH9.6) as coating solution, ELISA was performed, and the coating solution corresponding to the maximum P/N value was selected. The P/N value is maximum when 0.05M carbonate buffer solution with pH9.6 is used as the coating solution.

TABLE 4

(4) Selection of confining liquids

And (3) respectively using 2% of skimmed milk powder, 5% of skimmed milk powder, 7.5% of skimmed milk powder, 0.1% of BSA, 0.5% of BSA and 1% of BSA as blocking solutions, performing ELISA detection, and selecting the blocking solution corresponding to the maximum P/N value. Through analysis, when the blocking solution is 5 percent and 7.5 percent of skim milk or 0.2 percent of BSA, the P/N is larger, and the detection function can be realized. Optimally, the P/N value is maximal when 5% skim milk is the confining liquid.

TABLE 5

(5) Selection of primary anti-dilution solution

The ELISA was performed using PBS, 0.05% Tween-20 PBS (PBST1), 0.1% Tween-20 PBS (PBST2), O.1% BSA PBS, 1% BSA PBS, 5% skim milk PBS as primary dilutions, and the primary dilution corresponding to the highest P/N value was selected. Through analysis, when PBS of 1% BSA, PBS of 1% BSA and PBS of 5% skimmed milk are used as diluents, the P/N value is large, and the detection function can be realized. Optimally, the P/N value is maximal when 5% skim milk is included as the sample diluent.

TABLE 6

(6) Selection of the Primary antibody reaction time

Adding the diluted primary antibody, respectively incubating at 37 deg.C for 10min, 30min, 45min, 60min, and 90min, performing ELISA detection, and selecting the primary antibody reaction time corresponding to the maximum P/N value. Through analysis, when the reaction time of the serum to be detected is 60-90 min, the P/N value is larger, and the detection function can be realized. Most preferably, the P/N value is maximal when the reaction time of the serum to be tested is 60 min.

TABLE 7

(7) Selection of enzyme-labeled Secondary antibody reaction time

Adding diluted enzyme-labeled antibody, incubating at 37 deg.C for 10min, 30min, 45min, 60min and 90min, performing ELISA detection, and selecting the enzyme-labeled antibody reaction time corresponding to the maximum P/N value. Through analysis, when the reaction time of the enzyme-labeled secondary antibody is 45min and 90min, the P/N value is larger, and the detection function can be realized. Optimally, when the reaction time of the enzyme-labeled secondary antibody is 45min (45min is the time after optimization), the P/N value is maximum.

TABLE 8

(8) Selection of reaction time for TMB substrate

Adding color developing solution, incubating at 37 deg.C for 3min, 5min, 8min, 10min, 15min, 20min, and 25min, respectively, and adding 100 μ l/well of 2M H₂SO₄And (4) stopping, and selecting the TMB substrate reaction time corresponding to the maximum P/N value. Through analysis, when the reaction time of the substrate is 8-15 min, the P/N value is large, and the detection can be realizedAnd (4) performing functions. Optimally, the P/N value is maximal when the substrate reaction time is 10 min.

TABLE 9

The optimal reaction conditions to be finally determined are therefore: and (3) optimizing the reaction conditions to determine the optimal reaction conditions: the antigen coating concentration is 5 mug/ml, the dilution ratio of the serum to be detected is 1:400, and the dilution ratio of the enzyme-labeled antibody is 1: 10000; carbonate buffer solution with 0.05MpH9.6 is used as coating solution; 5% skimmed milk powder as sealing liquid; 5% skimmed milk as sample diluent; the reaction time of the serum to be detected and the enzyme-labeled secondary antibody is 60min and 45min respectively, and the reaction time of the substrate is 10 min.

Example 5 determination of the criteria

30 parts of negative serum is selected, and an optimized ELISA reaction system is used for detecting the OD450 value of the serum antibody. The results were statistically analyzed to obtain the mean (X) and standard deviation (S) of OD450 values as the threshold criteria for positive and negative determinations in ELISA.

TABLE 10 ELISA assay results

The mean X of the above 30 negative sera was calculated to be 0.194, standard deviation S to be 0.0475, and ELISA yin-yang cut-off value X +3S to be 0.336, so the OD450 value was calculated to be 0.336 negative-positive serum cut-off value.

Example 6 specificity test

Detecting Newcastle Disease Virus (NDV) positive serum, Avian Influenza Virus (AIV) positive serum and avian leukemia virus (ALV-K) positive serum by using the established ELISA method optimal conditions, establishing IBV positive and negative serum controls, and identifying the specificity of the method. The detection result shows that the serum results are negative, which indicates that the recombinant antigen has no cross reaction with the serum and has better specificity.

TABLE 11

Example 7 repeatability test

And (3) selecting 3 positive sera and 1 negative sera to perform a repeated experiment by using an optimized ELISA reaction system, simultaneously setting a blank control hole, performing statistical analysis on the result, and respectively calculating the average (X), the standard deviation (S) and the coefficient of variation (C.V) of each group. The detection results show that the coefficient of variation is less than 5%, which indicates that the same sample has small degree of variation and good repeatability in different batches of tests.

TABLE 12

EXAMPLE 85 b preliminary application of ELISA method

SPF chickens infected with LX4 strain and SPF chickens infected with QXIBV2 strain as well as SPF chickens of the immune inactivated vaccine and the control group are subjected to saphenous vein blood collection at 3d, 5d, 7d, 10d, 13d and 21d respectively and serum is separated and stored at-80 ℃. The method for detecting SPF chicken serum of 3d, 5d, 7d, 10d, 13d and 21d by adopting the established 5b-ELISA method comprises the following specific steps: diluting the antigen to 5 mu g/ml by using an ELISA coating solution, coating an ELISA plate according to 200 mu l/hole, coating overnight at 4 ℃, and washing the plate for 5 times by using PBST; and (3) sealing: adding 200 mul/well of 5% skimmed milk powder sealing solution, sealing at 37 deg.C for 2h, and washing the plate with PBST for 5 times; incubation of primary antibody: adding serum to be detected diluted by 5% skimmed milk at a ratio of 1:400 into 100 μ l/well, incubating in a thermostat at 37 deg.C for 1h, and washing the plate with PBST for 5 times; and (3) incubation of the secondary antibody: adding HRP-labeled goat anti-chicken IgG diluted to 1:10000 into the sample at the amount of 100 mu l/hole, incubating the mixture for 1h in a 37 ℃ incubator, and washing the plate for 5 times by PBST; color development: adding TMB substrate in an amount of 100 μ l/hole, and developing in a 37 deg.C incubator for 10min in dark;

and (4) terminating: adding 2M H2SO4 stop solution in an amount of 100 mu l/hole to terminate the reaction; reading: the absorbance at OD450nm was determined for each well using a microplate reader. The judgment results are shown in FIGS. 3 and 4.

The result shows that when the 5b-ELISA method is used for detecting the serum of the challenge experimental animal, the control group and the

inactivated vaccine groups

3d, 5d, 7d, 10d, 13d and 21d are all negative, and the OD450 value is less than 0.336.

Challenge groups

5d and 7d were shown negative, 10d, 13d and 21d were shown positive. The detection result of the commercial kit IDEXX IBV detection kit is consistent with the detection result of 5b ELSIA, 3d, 5d, 7d, 10d, 13d and 21d of the control group are all negative, and the successful virus attacking group and the inactivated vaccine group are positive. The established 5b-ELISA detection method has the potential of distinguishing inactivated vaccine immunity from live virus infection.

Sequence listing

<110> southern China university of agriculture

<120> avian infectious bronchitis virus 5b ELISA antibody detection kit and application thereof

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cccgcgtgca catcactagt attccagggg aaaacttgtg aagagcacat acgtaataat 180

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Claims

1. An ELISA antibody detection kit for chicken infectious bronchitis, which is characterized in that the kit contains an ELISA reaction plate coated by IBV non-structural protein 5 b;

further comprising:

enzyme-labeled reagent;

sealing liquid;

the coating solution used for preparing the ELISA reaction plate is 0.05M carbonate buffer solution with pH of 9.6;

the enzyme labeling reagent is a goat anti-chicken IgG antibody labeled by horseradish peroxidase;

the confining liquid is PBS containing 5% skimmed milk powder;

the nucleotide sequence of the IBV non-structural protein 5b is shown as SEQ ID NO. 1; the amino acid sequence of the IBV non-structural protein 5b is shown as SEQ ID NO. 2.

2. The kit of claim 1, wherein the IBV nonstructural protein 5b is produced by recombinant expression.

3. The kit of claim 2, wherein the method for recombinant expression of the IBV non-structural protein 5b comprises:

s1, amplifying an IBV 5b gene by RT-PCR;

s2, integrating the gene obtained in the step S1 into a plasmid vector to obtain a recombinant plasmid;

s3, transforming the recombinant plasmid constructed in the step S2 into host bacteria and carrying out induced expression to obtain a bacterial liquid;

4. The kit according to claim 3,

in step S1, the primer pair for amplifying the IBV 5b gene is shown as SEQ ID NO.3 and SEQ ID NO. 4;

in step S2, the plasmid vector is pET-32 a;

in step S3, the host bacterium is Escherichia coli.

5. The kit of claim 1, further comprising:

washing liquid;

diluting the solution;

a positive control sample;

a negative control sample;

a color developing solution;

and (4) stopping the solution.

6. The kit according to claim 5,

the washing solution is PBS containing 0.05 percent of Tween-20;

the diluent is PBST containing 5% of skimmed milk powder;

the positive control sample is anti-IBV 5b chicken serum;

the negative control sample is negative SPF chicken serum;

the color developing liquid is TMB single-component color developing liquid;

the stop solution is 2M H₂SO₄。