CN109884314B

CN109884314B - Indirect ELISA antibody detection method for porcine epidemic diarrhea virus S2 protein and kit thereof

Info

Publication number: CN109884314B
Application number: CN201910140122.3A
Authority: CN
Inventors: 秦毅斌; 赵武; 刘磊; 卢冰霞; 何颖; 陈忠伟; 周英宁; 李斌; 梁家幸; 闭炳芬; 段群棚; 蒋冬福; 卢敬专; 苏乾莲
Original assignee: Guangxi Veterinary Research Institute
Current assignee: Guangxi Veterinary Research Institute
Priority date: 2019-02-26
Filing date: 2019-02-26
Publication date: 2022-06-03
Anticipated expiration: 2039-02-26
Also published as: CN109884314A

Abstract

The invention discloses an indirect ELISA antibody detection kit for porcine epidemic diarrhea virus S2 protein, which comprises a coated ELISA plate, wherein the coated ELISA plate takes recombinant S2 protein as a coating antigen. Therefore, a corresponding ELISA detection method is established and is clinically applied, and a diagnostic tool with practical value is provided for detecting the PEDV antibody. The method for identifying the antibody generated by the porcine epidemic diarrhea virus infection can be established, has higher specificity and sensitivity, is simple to operate, short in time consumption and low in cost, can be used for large-scale detection, and has important significance for prevention, control and purification of the porcine epidemic diarrhea virus infection.

Description

Indirect ELISA antibody detection method for porcine epidemic diarrhea virus S2 protein and kit thereof

Technical Field

The invention belongs to the technical field of porcine epidemic diarrhea virus, and particularly relates to an indirect ELISA antibody detection method for porcine epidemic diarrhea virus S2 protein and a kit thereof.

Background

Porcine Epidemic Diarrhea (PED) is a highly-contact acute infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV), and mainly transmitted by a fecal oral route, infected pigs have symptoms of vomiting, watery diarrhea, dehydration and the like, piglets die from dehydration and acidosis, and the death rate is higher when the pigs are younger. Porcine epidemic diarrhea was first reported in the uk and belgium in the 70 s of the 20 th century, and then the occurrence of this disease was also reported in succession in a number of asian countries including china, philippines, korea, etc. Since 2010, large-scale outbreaks of PED are continuously reported in a plurality of provinces or regions in China, and a large-scale pig farm immunized with epidemic diarrhea vaccines also outbreaks of PED, so that newborn piglets die in a large amount and are disastrous. PED was also first outbreak in 2013 in America, and later it was confirmed that the virus variation caused the pandemic of PEDV in countries and regions in America, causing enormous economic loss to the pig industry.

Porcine Epidemic Diarrhea Virus (PEDV) is a single-stranded positive strand RNA virus and belongs to the genus coronavirus of the family coronaviridae, the order capsuloviridae, on a genetic classification. The PEDV genome encodes 4 major structural proteins in total, namely nucleocapsid protein (N), envelope protein (E), membrane protein (M) and spike protein (S). The S protein is located on the surface of the virion, consists of 1383 amino acids and is divided into two functional regions, namely S1 (1-789 aa) and S2 (790-1383 aa), and plays a key role in mediating the virion to enter cells and inducing an organism to generate neutralizing antibodies. After 2010 in China, PEDV epidemic strains separated from diseased pig intestines or excrement are mainly variant strains, and compared with a classical vaccine strain CV777, the PEDV epidemic strains have a lot of variations on an S gene, so that virus toxicity is enhanced or an immune escape mechanism of the virus is changed, and the PED is promoted to be greatly outbreak in China. Sun et al report that the protein 636-789 aa of S1 is a highly conserved region capable of inducing the body to produce neutralizing antibodies. Sundonbo identified the S1 region of the S protein as having 5 linear epitope regions and 1 neutralizing epitope using a filamentous phage display system. The pronucleus such as guirui expresses PEDV partial S1 protein (60-845bp), and the recombinant protein is expressed and has good reactogenicity, and can be identified by rabbit PEDV polyclonal antiserum. The studies described above mainly address the function of the PEDV S protein S1 domain, while there are few studies on the function of the PEDV S protein S2 domain, such as antigenicity.

The detection method of the porcine epidemic diarrhea antibody mainly comprises an indirect immunofluorescence assay (IFA), a virus neutralization assay (VN), an immunoperoxidase monolayer assay (IPMA) and an enzyme-linked immunosorbent assay (ELISA). Compared with other detection methods, the ELISA method is simple, convenient and sensitive, and is suitable for detection and evaluation of batch samples. Scholars at home and abroad establish an ELISA antibody detection method aiming at the PEDV holovirus and based on the recombinant N protein and the S1 protein. The detection method of the PEDV indirect ELISA antibody is established by taking prokaryotic expression recombinant N protein as a detection antigen, optimizing ELISA reaction conditions and the like. Gerber and the like establish an indirect ELISA detection method of an S1 protein antibody based on eukaryotic expression PEDV S1 recombinant protein, and have good reaction on IgG and IgA; the recombinant PEDV partial spike protein is used as a detection antigen, an indirect ELISA antibody detection method based on PEDV S1 protein is established, and the method is used for detecting PEDV antibodies in sow serum, colostrum and serum of piglets of different ages on the day of delivery of sows immunized with PED inactivated vaccines 4 weeks before delivery. However, no indirect ELISA antibody detection method established based on the PEDV S2 recombinant protein is reported at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing a swine epidemic diarrhea virus S2 protein indirect ELISA antibody detection method and a kit thereof.

In order to solve the technical problem, the invention adopts the following technical scheme:

the indirect ELISA antibody detection kit for the porcine epidemic diarrhea virus S2 protein comprises a coated ELISA plate, wherein the coated ELISA plate takes recombinant S2 protein as a coating antigen.

The recombinant S2 protein is encoded by a gene base sequence of a sequence table SEQ ID No.1 or has an amino acid sequence of SEQ ID No. 2.

The ELISA detection kit also comprises negativesSerum, positive serum, goat anti-pig IgG antibody marked by HRP, washing liquid, diluent, coating liquid, confining liquid, substrate liquid and stop solution; the washing solution is PBS + 0.05% Tween-20; the diluent is PBS + 2% skimmed milk powder + 0.05% Tween-20; the coating liquid is 0.05M PH9.6Na₂CO₃-NaHCO₃A buffer solution; the substrate solution is a single-component TMB substrate color development solution; the confining liquid is PBS + 5% skimmed milk powder; the stop solution is 2M H₂SO₄And (3) solution.

The indirect ELISA antibody detection method for the porcine epidemic diarrhea virus S2 protein is carried out according to the following operations:

coating antigen: diluting the recombinant S2 protein with a coating solution, adding the diluted protein into an ELISA plate for coating, and obtaining a coated ELISA plate with 100 mu L/hole;

washing: discarding the coating solution and washing with a washing solution;

and (3) sealing: adding 250 μ L of sealing solution into each well, sealing at 37 deg.C, discarding the sealing solution, and washing;

incubating the primary antibody: mixing pig serum with the diluent, incubating at 37 deg.C with a volume of 100 μ L/hole, discarding and washing;

incubation of secondary antibody: uniformly mixing the goat anti-pig IgG antibody marked by the HRP with a diluent, incubating at the temperature of 37 ℃ and discarding and washing;

color reading: adding substrate solution into 100 μ L/hole, performing color reaction at room temperature, adding stop solution, and reading OD with enzyme-labeling instrument₄₅₀The value is obtained.

The recombinant S2 protein was diluted to 8. mu.g/mL with the coating solution, and the dilution of the goat anti-pig IgG antibody labeled with HRP was 1: 4000.

Coating was 4 ℃ overnight plus 37 ℃ for 2 h.

The blocking time was 120 minutes.

The dilution of pig serum was 1:40, and the incubation time was 120 min.

The reaction time for incubating the secondary antibody was 45 minutes.

The color reaction time was 15 minutes.

Aiming at the problem that the detection technology of the Porcine Epidemic Diarrhea Virus (PEDV) antibody is relatively deficient, the inventor combines the S protein to play a key role in the process of mediating virus particles to enter cells and inducing an organism to generate a neutralizing antibody, and designs an indirect ELISA antibody detection kit of the porcine epidemic diarrhea virus S2 protein on the basis of obtaining S2 recombinant protein by expression and purification, wherein the kit comprises a coated enzyme label plate, and the coated enzyme label plate takes the recombinant S2 protein as a coating antigen. Therefore, a corresponding ELISA detection method is established and clinically applied, and a diagnostic tool with practical value is provided for detecting the PEDV antibody. The method for identifying the antibody generated by the porcine epidemic diarrhea virus infection can be established, has higher specificity and sensitivity, is simple to operate, short in time consumption and low in cost, can be used for large-scale detection, and has important significance for prevention, control and purification of the porcine epidemic diarrhea virus infection.

Detailed Description

Establishment of indirect ELISA antibody detection method for porcine epidemic diarrhea virus S2 protein

1. Materials, reagents and apparatus

1.1 Primary reagents

PBS powder: products of the company doctor de;

and (3) skim milk powder: products of the Solarbio corporation;

HRP-labeled goat anti-pig IgG antibody: product of the easthox corporation, usa;

the single-component TMB substrate color developing solution comprises the following components: tiangen products;

96-well enzyme label plate: products of the firm Constar.

Recombinant S2 protein: self-made (coded by a gene base sequence of a sequence table SEQ.ID.No.1 and provided with an amino acid sequence of SEQ.ID.No. 2).

1.2 Main instruments

A water-proof electric heating constant temperature incubator (Shanghai leap forward), an ultraviolet spectrophotometer (Tianpu), an enzyme labeling instrument (Berle) and the like.

1.3 preparation of Indirect ELISA related reagent

1) Coating liquid: 0.05M PH9.6Na was used₂CO₃-NaHCO₃Buffer, weighing Na₂CO₃ 0.159g，NaHCO₃0.293g, sterile ddH₂O to 100mL, and storing at 4 ℃;

2) serum and enzyme-labeled antibody diluent: PBS + 2% skimmed milk powder + 0.05% Tween-20;

3) washing solution: PBS + 0.05% Tween-20;

4) sealing liquid: PBS + 5% skim milk powder;

5) substrate solution: the single-component TMB substrate color developing solution is purchased from Tiangen company;

6) stopping liquid: 2M H₂SO₄And (3) solution. 11.1mL of concentrated sulfuric acid was slowly added to 80mL of water to make a volume of 100 mL.

2. Methods, procedures and results

2.1 optimization of Indirect ELISA

2.1.1 optimization of optimal coating concentration of recombinant protein and optimal working concentration of Secondary antibody

The recombinant S2 protein was diluted with the coating solution to 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL and 0.25. mu.g/mL of 8 dilutions in order, each dilution coating 1 vertical row of ELISA plate, two. Goat anti-pig IgG HRP-labeled antibody was diluted sequentially at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000 fold ratios with antibody dilutions in the same horizontal row at 100. mu.L/well to form a matrix. Selection of Positive serum OD₄₅₀Value and negative serum OD₄₅₀The antigen concentration and the secondary antibody dilution with the maximum value ratio (P/N value) are used as the optimal antigen coating concentration and the optimal secondary antibody reaction concentration. The results showed that the P/N value was maximal at an antigen coating concentration of 8. mu.g/mL and an enzyme-labeled secondary antibody concentration of 1:4000 (Table 1).

TABLE 1 optimization of optimal antigen coating concentration and optimal dilution factor of secondary antibody

2.1.2 determination of antigen coating conditions

Coating the ELISA plate with the optimal antigen coating concentration obtained in 2.1.1 at a concentration of 100 muL/hole; the experiments were divided into three groups, the first group was coated overnight at 4 ℃; a second group of 4 ℃ coating overnight and 37 ℃ wet box coating for 2 h; the third group was coated for 2h at 37 ℃ wet box. Each group is provided with 3 repeats, and ELISA and enzyme-linked immunosorbent assay are respectively carried out to determine the positive and negativeOD of serum₄₅₀And calculating the P/N value. Selecting the P/N value as the best antigen coating condition. The results show that 4 ℃ coating overnight plus 37 ℃ coating for 2h is the optimal coating condition, when the P/N value is maximal (Table 2).

TABLE 2 optimization of optimal coating conditions

2.1.3 determination of optimal seal time

The enzyme label plate is coated with the optimal recombinant protein antigen coating concentration and the optimal coating condition, and each well is 100 mu L. The microplate was divided into four groups. Sealing the first group at 37 deg.C for 30min, the second group at 37 deg.C for 60min, the third group at 37 deg.C for 90min, and the fourth group at 37 deg.C for 120 min. After washing, ELISA reaction was performed. Each group was assayed for positive and negative serum OD₄₅₀And calculating the P/N value to determine the optimal closing time. The results show that blocking at 37 ℃ for 120min is the optimal blocking condition, at which the P/N value is maximal (Table 3).

TABLE 3 determination of optimal seal time

2.1.4 determination of optimal serum dilution and serum incubation time

The enzyme label plate is coated with the optimal recombinant protein antigen coating concentration and the optimal coating condition, and each well is 100 mu L. After blocking, the mixture was divided into three groups. Adding 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640 times diluted negative and positive sera into the first group from top to bottom respectively, repeating the steps, and reacting at 37 ℃ for 30 min; adding 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640 times diluted negative and positive sera into the second group from top to bottom respectively, repeating the two groups, and reacting at 37 ℃ for 60 min; the third group is added with 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640 times diluted negative and positive serum respectively from top to bottom, and the reaction is repeated for 120min at 37 ℃. Respectively carrying out ELISA reaction, and respectively measuring the average O of negative and positive serum of each dilution in each group after the reaction is finishedD₄₅₀And calculating the P/N value to determine the optimal serum dilution and the optimal serum reaction time. The results show that the P/N values were maximal at 120min incubation with 1:40 fold dilutions of serum (Table 4).

TABLE 4 optimization of optimal serum dilution and action time

2.1.5 determination of optimal Secondary antibody reaction time

The enzyme label plate is coated with the optimal recombinant protein antigen coating concentration and the optimal coating condition, and each well is 100 mu L. After washing, blocking at 37 ℃ for 2h, adding 1:40 diluted negative and positive serum into the reaction vessel, 100. mu.L/well, and reacting at 37 ℃ for 2 h. After washing, HRP-goat anti-porcine IgG antibody diluted 1:4000 was added at 100. mu.L/well. And dividing into four groups, the first group reacting at 37 deg.C for 15min, the second group reacting at 37 deg.C for 30min, the third group reacting at 37 deg.C for 45min, the fourth group reacting at 37 deg.C for 60min, and each group repeating for 3 times. After washing, the color is developed, and the OD of each group of positive and negative serum is determined on an enzyme-linked immunosorbent assay₄₅₀And calculating the P/N value to determine the optimal reaction time of the secondary antibody. As a result, the maximum P/N value was observed when the secondary antibody reaction time was 45min (Table 5).

TABLE 5 optimization of optimal Secondary antibody reaction time

2.1.6 determination of the color development time of the substrate

The enzyme label plate is coated with the optimal recombinant protein antigen coating concentration and the optimal coating condition, and each well is 100 mu L. After washing, blocking at 37 ℃ for 2h, adding 1:40 diluted negative and positive serum at 100. mu.L/well, and reacting at 37 ℃ for 90 min. After washing, HRP-goat anti-pig IgG antibody diluted 1:4000 times was added thereto at 100. mu.L/well, and the reaction was carried out at 37 ℃ for 45 min. After washing, the mixture was divided into four groups. Adding 100 μ L/well substrate solution into the first group, developing at room temperature for 5min, adding 100 μ L/well substrate solution into the second group, and developing at room temperature for 10 min; adding 100 μ L/well substrate solution into the third group, and developing at room temperature for 15 min; first, theThe four groups were added with 100. mu.L/well substrate solution and developed for 20min at room temperature. Repeating each group for 3 times, adding 50 μ L/well stop solution to stop color development, and measuring OD of positive and negative serum of each group₄₅₀And calculating the P/N value to determine the optimal substrate color development time. As a result, the P/N value was the largest at 15min of color development (Table 6).

TABLE 6 optimization of optimal substrate development time

2.1.7 optimized Indirect ELISA detection method

In conclusion, the indirect ELISA antibody detection method for the porcine epidemic diarrhea virus S2 protein is carried out according to the following steps:

coating antigen: diluting the recombinant S2 protein to 8 mu g/mL by using a coating solution, adding the diluted recombinant S2 protein into an ELISA plate, coating the ELISA plate at 4 ℃ overnight, coating the ELISA plate at 37 ℃ for 2h, and coating the ELISA plate at 100 mu L/hole to obtain a coated ELISA plate;

washing: discarding the coating solution and washing with a washing solution;

and (3) sealing: adding 250 mu L of sealing liquid into each hole, sealing for 120 minutes at 37 ℃, and removing the sealing liquid for washing;

incubating the primary antibody: mixing pig serum with the diluent at a dilution ratio of 1:40, incubating at 37 deg.C for 120min at 100 μ L/hole, discarding and washing;

incubation of secondary antibody: uniformly mixing the goat anti-pig IgG antibody marked by HRP with the diluent according to the dilution ratio of 1:4000, incubating at the temperature of 37 ℃ for 45 minutes at a concentration of 100 mu L/hole, discarding and washing;

color reading: adding substrate solution into 100 μ L/hole, performing color reaction at room temperature for 15min, adding stop solution, and reading OD with microplate reader₄₅₀The value is obtained.

2.2 Indirect ELISA Positive and negative cut-off values

Randomly selecting a plurality of fresh pig sera, obtaining 30 pig sera which can not be specifically combined with the recombinant S2 protein through Western-blot analysis, detecting by using established and optimized indirect ELISA, and reading OD of each serum₄₅₀And calculating the S/P value. S/P valueBetula (sample serum OD)₄₅₀Value-negative serum OD₄₅₀Value)/(positive serum OD₄₅₀Value-negative serum OD₄₅₀Value). The mean and Standard Deviation (SD) of the S/P values of 30 sera were calculated. When the S/P value of the sample is>^-Judging the X +3SD to be positive at any time; when the S/P value of the sample is<The result is judged to be negative when the product is X +2 SD; and when the S/P value of the sample is less than or equal to X +2SD and less than or equal to X +3SD, determining the sample as suspicious.

The mean value X of S/P values of 30 negative sera was calculated to be 0.140 and the standard deviation SD was calculated to be 0.024, thus X +3SD was calculated to be 0.212 and X +2SD was calculated to be 0.188. Therefore, when the S/P value of the serum sample to be detected is greater than 0.212, the serum sample is judged to be positive; judging the sample to be detected as negative when the S/P value of the sample is less than 0.188; and when the S/P value of the serum sample to be detected is less than or equal to 0.188 and less than or equal to 0.212, the serum sample is judged to be suspicious.

2.3 specificity test

The enzyme-labeled hole is coated with the purified recombinant protein PEDV S2 protein, and then classical swine fever virus standard positive serum, porcine reproductive and respiratory syndrome virus standard positive serum, porcine circovirus standard positive serum, porcine foot-and-mouth disease virus standard positive serum, porcine pseudorabies virus standard positive serum and porcine delta coronavirus standard positive serum are detected according to the ELSIA method established and optimized in the foregoing, and meanwhile, a PEDV negative positive serum control is set to judge whether the PEDV S2 protein indirect ELISA antibody detection method established in the experiment has cross reaction with antibodies of other pathogens. The results show that the S/P values of the classical swine fever virus standard positive serum, the porcine reproductive and respiratory syndrome virus standard positive serum, the porcine circovirus standard positive serum, the foot-and-mouth disease virus standard positive serum, the porcine pseudorabies virus standard positive serum and the porcine delta coronavirus standard positive serum are all less than 0.188 (Table 7), so that the PEDV S2 protein indirect ELISA antibody detection method established in the research has good specificity.

TABLE 7 Indirect ELISA antibody specificity test results for PEDV S2 protein

2.4 repeatability test

Three batches of PEDV S2 protein were coated on days 1, 3, and 5, respectively, and negative and positive sera were tested by ELISA on day 6, with 5 replicates in a batch, for OD determination₄₅₀And calculating the average value, the standard deviation and the coefficient of variation.

2.4.1 in-batch repeatability: and calculating the variation coefficient among 5 repeats in each batch, and determining the repeatability in the batch according to the magnitude of the variation coefficient. OD of in-batch Positive sera₄₅₀The coefficient of variation of the value is between 1.4 and 3.0 percent, and the OD of the negative serum₄₅₀The coefficient of variation of the values was between 3.6% and 4.9% (table 8).

2.4.2 batch to batch repeatability: and calculating the variation coefficient among the three batches, and determining the repeatability among the batches according to the variation coefficient. OD of batch Positive sera₄₅₀The value variation coefficient was 1.1%, OD of negative serum₄₅₀The value variation coefficient was 2.6% (Table 8).

TABLE 8 result of repetitive test of indirect ELISA antibody with PEDV S2 protein

The results of the in-batch and inter-batch tests show that the ELISA detection method established by the research has better repeatability.

2.5 sensitivity test

3 parts of PEDV positive serum are diluted in a ratio of 1:40, and are detected by an established ELISA kit, and a negative positive control is set. Determination of OD₄₅₀And calculating S/P value, and analyzing the sensitivity. As a result: OD of negative and positive controls in this experiment₄₅₀The values were 0.086 and 1.085, respectively, and the S/P values at each dilution were calculated for 3 positive sera, respectively. The results showed that 2 positive sera were negative at 1:640 (S/P values)<0.188), 1 part of positive serum becomes negative when the ratio of the positive serum to the negative serum is 1:1280, which indicates that the kit has better sensitivity, and the results are shown in a table 9.

TABLE 9 Indirect ELISA antibody sensitivity test results for PEDV S2 protein

2.6 preliminary application of Indirect ELISA antibody detection method of PEDV S2 protein

The indirect ELISA antibody of PEDV S2 protein established in the research is used for detecting 385 total swine herd serum samples in different stages in Guangxi area, the positive rate is calculated, and the PEDV antibody level of the swine herd in each stage in the Guangxi area is analyzed, and the result is shown in Table 10. From the detection results, the total sample positive rate is 76.36%, the positive rates at different stages are greatly different, the highest positive rate of the sows is 86.36%, and the lowest positive rate of the nursery pigs is 53.57%.

TABLE 10 results of clinical serum PEDV antibody detection

Sequence listing

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Thr Gln Tyr Thr Ala Ala Cys Lys Thr Ile Glu Ser Ala Leu Gln Leu

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Ser Ala Arg Leu Glu Ser Val Glu Val Asn Ser Met Leu Thr Ile Ser

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Glu Glu Ala Leu Gln Leu Ala Thr Ile Ser Ser Phe Asn Gly Asp Gly

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Tyr Asn Phe Thr Asn Val Leu Gly Val Ser Val Tyr Asp Pro Ala Ser

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Gly Arg Val Val Gln Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn

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Lys Val Val Thr Asn Gly Leu Gly Thr Val Asp Glu Asp Tyr Lys Arg

100 105 110

Cys Ser Asn Gly Arg Ser Val Ala Asp Leu Val Cys Ala Gln Tyr Tyr

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Ser Gly Val Met Val Leu Pro Gly Val Val Asp Ala Glu Lys Leu His

130 135 140

Met Tyr Ser Ala Ser Leu Ile Gly Gly Met Val Leu Gly Gly Phe Thr

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Ser Ala Ala Ala Leu Pro Phe Ser Tyr Ala Val Gln Ala Arg Leu Asn

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Tyr Leu Ala Leu Gln Thr Asp Val Leu Gln Arg Asn Gln Gln Leu Leu

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Ala Glu Ser Phe Asn Ser Ala Ile Gly Asn Ile Thr Ser Ala Phe Glu

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Ser Val Lys Glu Ala Ile Ser Gln Thr Ser Lys Gly Leu Asn Thr Val

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Ala His Ala Leu Thr Lys Val Gln Glu Val Val Asn Ser Gln Gly Ala

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Ala Leu Thr Gln Leu Thr Val Gln Leu Gln His Asn Phe Gln Ala Ile

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Ser Ser Ser Ile Asp Asp Ile Tyr Ser Arg Leu Asp Ile Leu Ser Ala

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Asp Val Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ala Leu Asn

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Ala Phe Val Ala Gln Thr Leu Thr Lys Tyr Thr Glu Val Gln Ala Ser

290 295 300

Arg Lys Leu Ala Gln Gln Lys Val Asn Glu Cys Val Lys Ser Gln Ser

305 310 315 320

Gln Arg Tyr Gly Phe Cys Gly Gly Asp Gly Glu His Ile Phe Ser Leu

325 330 335

Val Gln Ala Ala Pro Gln Gly Leu Leu Phe Leu His Thr Val Leu Val

340 345 350

Pro Gly Asp Phe Val Asp Val Ile Ala Ile Ala Gly Leu Cys Val Asn

355 360 365

Asp Glu Ile Ala Leu Thr Leu Arg Glu Pro Gly Leu Val Leu Phe Thr

370 375 380

His Glu Leu Gln Asn His Thr Ala Thr Glu Tyr Phe Val Ser Ser Arg

385 390 395 400

Arg Met Phe Glu Pro Arg Lys Pro Thr Val Ser Asp Phe Val Gln Ile

405 410 415

Glu Ser Cys Val Val Thr Tyr Val Asn Leu Thr Arg Asp Gln Leu Pro

420 425 430

Asp Val Ile Pro Asp Tyr Ile Asp Val Asn Lys Thr Leu Asp Glu Ile

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Leu Ala Ser Leu Pro Asn Arg Thr Gly Pro Ser Leu Pro Leu Asp Val

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Phe Asn Ala Thr Tyr Leu Asn Leu Thr Gly Glu Ile Ala Asp Leu Glu

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Gln Arg Ser Glu Ser Leu Arg Asn Thr Thr Glu Glu Leu Gln Ser Leu

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Ile Tyr Asn Ile Asn Asn Thr Leu Val Asp Leu Glu

500 505

Claims

1. The indirect ELISA antibody detection kit for the porcine epidemic diarrhea virus S2 protein comprises a coated ELISA plate, and is characterized in that: the coated enzyme label plate takes recombinant S2 protein as a coating antigen; the recombinant S2 protein is encoded by a gene base sequence of a sequence table SEQ ID No.1 or is an amino acid sequence of SEQ ID No. 2.

2. The ELISA antibody detection kit of claim 1, characterized by further comprising a negative serum, a positive serum, a goat anti-pig IgG antibody labeled with HRP, a washing solution, a diluent, a coating solution, a blocking solution, a substrate solution, a stop solution; the washing solution is PBS + 0.05% Tween-20; the diluent is PBS + 2% skimmed milk powder + 0.05% Tween-20; the coating solution is 0.05M pH9.6Na₂CO₃-NaHCO₃A buffer solution; the substrate solution is a single-component TMB substrate color development solution; the confining liquid is PBS + 5% skimmed milk powder; the stop solution is 2M H₂SO₄And (3) solution.

3. An indirect ELISA antibody detection method for porcine epidemic diarrhea virus S2 protein is characterized by comprising the following steps: coating antigen: diluting the recombinant S2 protein with a coating solution, adding the diluted protein into an ELISA plate for coating, and obtaining a coated ELISA plate with 100 mu L/hole; the recombinant S2 protein is encoded by a gene base sequence of a sequence table SEQ ID No.1 or is an amino acid sequence of SEQ ID No. 2;

washing: discarding the coating solution and washing with a washing solution;

incubating the primary antibody: mixing pig serum with the diluent, incubating at 37 deg.C with 100 μ L/hole, discarding and washing;

4. The ELISA antibody detection method of claim 3, wherein: the recombinant S2 protein was diluted to 8. mu.g/mL with coating solution, and the dilution of the HRP-labeled goat anti-pig IgG antibody was 1: 4000.

5. The ELISA antibody detection method of claim 4, wherein: the coating was 4 ℃ overnight plus 37 ℃ for 2 h.

6. The ELISA antibody detection method of claim 5, wherein: the blocking time was 120 minutes.

7. The ELISA antibody detection method of claim 6, wherein: the dilution of the pig serum is 1:40, and the incubation time is 120 min.

8. The ELISA antibody detection method of claim 7, wherein: the reaction time for the incubation of the secondary antibody was 45 minutes.

9. The ELISA antibody detection method of claim 8, wherein: the color reaction time was 15 minutes.