An indirect ELISA kit for distinguishing between wild-type actinobacillus pleuropneumoniae infection and vaccine immunization of pigs and application thereof

Abstract

The application discloses an indirect ELISA kit for distinguishing between wild-type Actinobacillus pleuropneumoniae (APP) infection and vaccine immunization and application thereof, and belongs to the technical field of biology.The indirect ELISA kit is characterized in that a screened ApxIVA truncated protein is used as a coating antigen, the amino acid sequence of the ApxIVA truncated protein is shown as SEQ ID NO.3, the ApxIVA truncated protein can be expressed in a soluble manner in Escherichia coli, only carries one His tag and S tag, and is close to natural ApxIVA in structure and function.The indirect ELISA kit is characterized in that the ApxIVA truncated protein is used as an antigen, an indirect ELISA method and kit for distinguishing between wild-type APP infection and gene deletion vaccine immunization are established, the kit has the advantages of high specificity, low background value, low cost and short detection time, and has important significance in improving the diagnostic accuracy of livestock and poultry APP, optimizing prevention and control measures, guaranteeing animal health and production efficiency.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to an indirect ELISA kit for distinguishing between wild toxin infection and vaccine immunization of porcine pleuropneumonia actinobacillus and application. BACKGROUND

[0002] Porcine pleuropneumonia (PCP) is a serious respiratory bacterial disease caused by Actinobacillus pleuropneumoniae (APP) infection, which is widespread in the world and mainly transmitted between pig populations through direct contact, aerosol and contamination. The disease is mainly characterized by acute hemorrhagic fibrocavitary pneumonia and chronic fibrocavitary necrotizing pleuropneumonia. Acute infected pigs often have nasal and oral bleeding and sudden death. Chronic infected pigs often become a latent source of infection, leading to sub-health state of pig population, which is prone to secondary infection with other pathogens. The disease has caused great economic losses to the global pig industry. At present, many diagnostic methods have been developed for APP, mainly including routine microbiological diagnosis, serological identification and molecular biological diagnosis.

[0003] According to the difference of capsule antigens, APP can be divided into 19 serotypes, and ApxIV is considered as a target protein for diagnosing all serotype strains of APP. Obtaining the natural structure of apxIV protein is helpful for in-depth understanding and diagnosis of APP. However, the natural apxIV is often secreted in a small amount after the strain infects a pig, and it is difficult to isolate and obtain. The recombinant soluble apxIV protein on the market often has a large tag or uses a vector carrying a large tag, and its structure and function are quite different from those of the natural apxIV. In addition, the applicant has successfully developed an APP gene deletion vaccine (APP-HB-04M), and has obtained a new veterinary drug certificate (2022, new veterinary drug certificate No. 73). The gene deletion vaccine has been widely used in pig farms of several leading pig breeding enterprises in China and has achieved good protection effect. However, there is currently no serological diagnostic method to distinguish APP wild strain infection from gene deletion vaccine (APP-HB-04M) immunization in the clinic. When the ordinary serological detection method is used to detect the pig population inoculated with the APP gene deletion vaccine APP-HB-04M, APP wild strain infection and gene deletion vaccine immunization will be confused. The serological differential diagnosis method for distinguishing wild strain infection from gene deletion vaccine APP-HB-04M immunization is urgently needed to be established. SUMMARY

[0004] The application aims to solve the problems of the prior art APP apxIVA expression, and provides an APP apxIVA truncated protein and a preparation method thereof, the ApxIVA truncated protein can be expressed in E. coli, only carries 1 His tag (about 0.84 kDa) and S tag (about 2 kDa), and the structure and function are close to the natural ApxIVA.

[0005] The application also aims to provide the application of the APP apxIVA truncated protein as an antigen in an indirect ELISA method or in the preparation of an indirect ELISA kit, and the APP wild strain infection and the gene deletion vaccine immunization can be distinguished by using the indirect ELISA method or the indirect ELISA kit.

[0006] The application aims to solve the problems of the prior art APP apxIVA expression, and provides an APP apxIVA truncated protein and a preparation method thereof, the ApxIVA truncated protein can be expressed in E. coli, only carries 1 His tag (about 0.84 kDa) and S tag (about 2 kDa), and the structure and function are close to the natural ApxIVA.

[0007] An apxIVA truncated protein of Actinobacillus pleuropneumoniae, the amino acid sequence of which is shown as SEQ ID NO. 3.

[0008] A nucleotide for encoding the apxIVA truncated protein, the nucleotide sequence of which is shown as SEQ ID NO. 4, which is based on a part of the apxIVA gene deleted in the APP gene deletion vaccine APP-HB-04M, and is obtained by 2 times of 6 truncation screening.

[0009] An expression vector comprising the nucleotide for encoding the apxIVA truncated protein, which is introduced into a host cell to express the apxIVA truncated protein. The expression vector is preferably pET-30a, and can express the soluble apxIVA truncated protein carrying only 1 His (about 0.84 kDa) and S tag (about 2 kDa), and the structure and function are closer to the natural state of apxIVA.

[0010] A host cell comprising the nucleotide for encoding the apxIVA truncated protein or the above-mentioned expression vector, which can express the apxIVA truncated protein. The host cell is preferably E. coli.

[0011] The preparation method of the apxIVA truncated protein of Actinobacillus pleuropneumoniae comprises the following steps:

[0012] (1) cloning the nucleotide for encoding the apxIVA truncated protein into pET-30a to obtain a recombinant expression plasmid;

[0013] (2) transforming the recombinant expression plasmid into E. coli BL21 to obtain a recombinant expression strain;

[0014] (3) The recombinant expression strain is cultured, IPTG induced expression, and the expressed bacteria are broken, centrifuged, and the supernatant is purified to obtain the apxIVA truncated protein.

[0015] Step (1) is specifically as follows: taking the genomic DNA of APP 4074T strain (serum type 1 standard strain) as a template, and performing PCR amplification by using the following primers.

[0016] ApxIVA-N2 forward primer: 5'-CCGGAATTCATGGAGAACCTGTACTTCCAAGGGCCTAAGGCGGATCCTAAGCGG-3',

[0017] ApxIVA-N2 reverse primer: 5'-CCGCTCGAGTTACCAGCCCGTTGCGGTACGAAT-3'.

[0018] The amplification product and pET-30a are subjected to EcoRI / Xhol double enzyme digestion, enzyme ligation, and the enzyme ligation product is transformed into DH5α competent cells, and a recombinant expression plasmid is obtained through identification.

[0019] The apxIVA truncated protein of Actinobacillus pleuropneumoniae can be used as an antigen in the establishment of an indirect ELISA method for Actinobacillus pleuropneumoniae or in the preparation of an indirect ELISA kit for Actinobacillus pleuropneumoniae.

[0020] An indirect ELISA method is an indirect ELISA method for detecting Actinobacillus pleuropneumoniae ApxIV antibody or an indirect ELISA method for distinguishing between Actinobacillus pleuropneumoniae wild strain infection and immunization of gene deletion vaccine APP-HB-04M, which uses the above-mentioned apxIVA truncated protein as a coating antigen and comprises the following steps:

[0021] (1) Coating: dilute the ApxIVA truncated protein with coating buffer, add it to the reaction wells of an enzyme-labeled plate, and coat at 4°C for more than 10 hours;

[0022] (2) Discard the solution in the wells, block with 2% BSA blocking solution at 4°C, and discard the solution in the wells after blocking;

[0023] (3) Sample addition: after dilution, add the serum to be tested to the above-mentioned coated reaction wells, incubate at 37°C, and then wash; meanwhile, prepare blank wells, negative control wells, and positive control wells;

[0024] (4) Add enzyme-labeled antibody: add freshly diluted horseradish peroxidase-labeled goat anti-pig IgG to each reaction well, incubate at 37°C, and wash;

[0025] (5) Substrate solution color development: add TMB color developing solution to each reaction well, and react at room temperature in the dark;

[0026] (6) Reaction termination: add termination solution to each reaction well;

[0027] (7) Result determination: measure the OD630nm value of each well on an ELISA detector.

[0028] The condition corresponding to the highest value of the positive OD value / negative OD value (P / N value) is the optimal condition of ELISA. The optimal condition is as follows: the coating concentration of the ApxIVA truncated protein is 0.625 μg / mL; the dilution of the serum sample to be detected is 1:20, and incubation is performed at 37℃ for 30 minutes; the dilution of the horseradish peroxidase-labeled goat anti-swine IgG is 1:5000, and incubation is performed at 37℃ for 30 minutes; and the TMB color developing condition is reaction at room temperature (20-25℃) in the dark for 10 minutes.

[0029] Determination of the critical value of indirect ELISA: critical value = average value + 2 x standard deviation; the average value is the average value of the indirect ELISA results of the negative serum, and the standard deviation is the standard deviation of the indirect ELISA results of the negative serum; the indirect ELISA result determination standard is that the OD value of the serum sample is greater than or equal to the critical value, which is judged as wild strain infection positive, and the OD value of the serum sample is less than the critical value, which is judged as wild strain infection negative; the determination is not affected by the inoculation of the gene deletion vaccine (APP-HB-04M). In the result determination, the critical value is 0.3500.

[0030] An indirect ELISA kit for detecting Actinobacillus pleuropneumoniae ApxIV antibody, or an indirect ELISA kit for distinguishing between wild strain infection of Actinobacillus pleuropneumoniae and immunization of the gene deletion vaccine APP-HB-04M, which comprises an enzyme-labeled plate coated with the ApxIVA truncated protein; and can further comprise APP wild strain positive and negative serum, sample diluent, washing buffer, horseradish peroxidase (HRP)-labeled goat anti-swine secondary antibody, TMB (tetramethylbenzidine), and enzyme reaction termination solution.

[0031] The present application has the following advantages and effects relative to the prior art:

[0032] The present application screens an ApxIVA truncated protein to obtain an N2 protein sequence that can be expressed in a soluble form. The recombinant protein only carries a His tag (about 0.84 kDa) and an S tag (about 2 kDa) at the N terminus, and the structure and function are closer to the natural state of ApxIVA. The N2 protein expression procedure is simple, the cost is low, and the protein purity is high, and is suitable for large-scale production.

[0033] The application establishes an indirect ELISA method and kit for distinguishing APP wild strain infection and gene deletion vaccine immunization by using purified N2 protein as an antigen, which has the advantages of high specificity, low background value, low cost and short detection time, and the incubation time of the primary antibody and the secondary antibody is only 30 minutes each. The indirect ELISA kit of the application is the first product that can distinguish APP wild strain infection and gene deletion vaccine APP-HB-04M immunization. The ELISA method and kit can accurately distinguish APP wild strain infection and gene deletion vaccine immunization, and have important significance in improving the accuracy of APP diagnosis in animal husbandry, optimizing prevention and control measures, protecting animal health and production efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 A schematic diagram for screening and expression of ApxIVA truncated protein, establishment of ELISA method and application of the application.

[0035] Figure 2 A bioinformatics analysis result of APP apxIVA.

[0036] Figure 3 A schematic diagram of APP apxIVAN terminal truncation.

[0037] Figure 4 An amplification result of APP apxIVAN1, N2 and N3 target genes.

[0038] Figure 5 A bacterial liquid PCR identification result of plasmid pET-30a-APP apxIVAN1, N2 and N3.

[0039] Figure 6 An SDS-PAGE result of expression of recombinant N1, N2 and N3 truncated proteins.

[0040] Figure 7 A western blot result of recombinant N1, N2 and N3 truncated proteins.

[0041] Figure 8 A specificity analysis result of N2 ELISA method.

[0042] Figure 9 A sensitivity analysis result of N2 ELISA method.

[0043] Figure 10 A detection (part) result of N2 ELISA kit for APP gene deletion vaccine APP-HB-04M immunization serum sample.

[0044] Figure 11 A clinical serum detection (part) result of N2 ELISA kit. DETAILED DESCRIPTION

[0045] The application provides an APP apxIVA truncated protein and a preparation method thereof, and application of the APP apxIVA truncated protein as an antigen in an indirect ELISA method or in preparation of an indirect ELISA kit, and the indirect ELISA method or the indirect ELISA kit can be used to distinguish APP wild strain infection and gene deletion vaccine immunization.

[0046] The application will be further described in detail below in combination with examples, but the embodiments of the application are not limited thereto.

[0047] Figure 1 A schematic diagram for screening expression of an ApxIVA truncated protein, establishment of an ELISA method and application thereof in the following examples.

[0048] Example 1 Screening of soluble expression of an ApxIVA protein

[0049] (1) Primary screening of the ApxIVA protein

[0050] ① Construction and identification of a recombinant expression plasmid

[0051] First, the hydrophilicity / hydrophobicity and transmembrane domain of APP apxIVA are analyzed by bioinformatics, and then the antigen epitope prediction and three-dimensional structure prediction are performed, so as to preliminarily truncate APP apxIVA into three segments N1, N2 and N3 with sizes of 918 bp, 756 bp and 1035 bp respectively. Bioinformatics analysis results are shown in Figure 2 , and a truncation schematic diagram is shown in Figure 3 , wherein the amino acid sequence and the nucleotide sequence of N1 are shown as SEQ ID NO. 1 and 2 respectively, the amino acid sequence and the nucleotide sequence of N2 are shown as SEQ ID NO. 3 and 4 respectively, and the amino acid sequence and the nucleotide sequence of N3 are shown as SEQ ID NO. 5 and 6 respectively.

[0052] According to the nucleotide sequence of the ApxIVA gene (GenBank: AF021919), the following primers are designed:

[0053] ApxIVA-N1 forward primer: 5'-CCGGAATTCATGCGCGCCTATATCTGGAAT-3',

[0054] ApxIVA-N1 reverse primer: 5'-CCGCTCGAGTTTTATTTCTTCTTTCGTTATGTACTCGCT-3'.

[0055] ApxIVA-N2 forward primer: 5'-CCGGAATTCATGGAGAACCTGTACTTCCAAGGGCCTAAGGCGGATCCTAAGCGG-3',

[0056] ApxIVA-N2 reverse primer: 5'-CCGCTCGAGTTACCAGCCCGTTGCGGTACGAAT-3'.

[0057] ApxIVA-N3 forward primer: 5'-CCGGAATTCATGGGGCGACAAGGCGCGTTATTC-3',

[0058] ApxIVA-N3 reverse primer: 5'-CCGCTCGAGTTTCCCTTCGAATTGTTTCGCATTAACGC-3'.

[0059] The N1, N2 and N3 fragments were amplified using the above-mentioned primers and the APP 4074T strain (serum type 1 standard strain) as a template. The amplification conditions were as follows: denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, annealing at 55°C for N1, 55°C for N2 and 50°C for N3 for 1 min, extension at 72°C for 1 min 10 s, and extension at 72°C for 10 min. After PCR, the products were subjected to 1% agarose gel electrophoresis detection. The amplification results are shown in Figure 4 The sizes of the fragments were the same as expected, indicating successful amplification.

[0060] After the PCR products were recovered, they were double-digested with EcoRI and Xhol, ligated with the pET-30a vector, and then transformed into DH5a competent cells. After single colony bacteria were picked and subjected to bacterial liquid PCR, they were sent to GenScript for sequencing, and the recombinant expression plasmid was successfully constructed. The bacterial liquid identification results are shown in Figure 5 The size was the same as expected, and the sequencing results were correct.

[0061] 2. Expression and purification of the target gene

[0062] The recombinant expression plasmid was transformed into E. coli BL21 (DE3) competent cells, spread on LA plates containing kanamycin, and then cultured in an incubator. After single colonies were picked and transferred to fresh LB medium containing kanamycin, the bacterial liquid was cultured to an OD 600The value is 0.6, 1 mmol / L IPTG is added, 16 ℃, 120 rpm induction for 16 h. After high-pressure cell disruptor crushing and centrifugation, the soluble expression protein is taken from the supernatant of the bacteria and purified by a nickel column. First, the column and the filler are washed and balanced with ddH2O and Binding Buffer (20 mM imidazole, 0.5 M NaCl, 20 mM Na3PO4·12H2O, pH = 7.4), and the supernatant and the precipitate of the bacteria after crushing and centrifugation are taken as samples. The supernatant is incubated with the filler at 4 ℃ for 0.5 h, and the effluent is taken as a flow-through sample after standing. The column is washed with Binding Buffer, and the effluent is taken as a washing sample. Elution Buffer (500 mM imidazole, 0.5 M NaCl, 20 mM Na3PO4·12H2O, pH = 7.4) is used to elute the column, and the effluent is taken as an elution sample, i.e., the final liquid containing the target protein. 12% SDS-PAGE electrophoresis analysis is performed.

[0063] Inclusion body expressed protein is purified from the precipitate of the bacteria. First, the whole bacteria sample is collected from the uncrushed bacteria liquid, and the supernatant and the precipitate of the bacteria after crushing and centrifugation are taken as samples. In the precipitate of 200 mL of bacteria liquid after crushing, 19.7 mL of buffer A (50 mM Tris-Base, 0.5 mM EDTA, 100 mM NaCl, 0.05% glycerol), 0.3 mL of 20% sodium dodecyl sulfate stock solution, and 20 μL of 0.5 M DTT are added, and the mixture is stirred vigorously to allow it to dissolve slowly. After standing at room temperature for 30 min to 2 h, the supernatant is taken as a denaturation sample after centrifugation. The supernatant is added with 210 μL of 20% PEG4000 (0.2 g / mL, final concentration 0.2%), 420 μL of 50 mM (0.03 g / mL) oxidized glutathione (final concentration 1 mM), and 420 μL of 100 mM (0.03 g / mL) reduced glutathione (final concentration 2 mM), and the mixture is allowed to stand at room temperature for 0.5-2 h. The sample is taken as a renaturation sample. After 72 h of dialysis using TE Buffer (10 mM Tris-Base, 1 mM EDTA), the surface of the dialysis bag is dried by sucking off the excess water, and the sample is taken as a concentration sample. 30a is a pET-30a empty vector control. 12% SDS-PAGE electrophoresis analysis is performed.

[0064] SDS-PAGE results Figure 6 show that N2 protein is soluble expression, the protein band is 42 kDa, which is consistent with the expected size, and the purified protein N2 is successfully obtained. N1 and N3 proteins are inclusion body expression, and the molecular weights are 43 kDa and 45 kDa, respectively. According to the detection of Biyun Bradford protein concentration determination kit, the expression amounts of N1, N2, and N3 proteins in 1 L of bacteria liquid are 13 mg, 42 mg, and 55 mg, respectively.

[0065] ③ Western blot analysis

[0066] After SDS-PAGE electrophoresis, the gel was directly transferred to a membrane without staining. The gel was cut to an appropriate size, and 6 pieces of 3 mm filter paper and 1 piece of polyvinylidene fluoride membrane (PVDF) were cut. The 6 pieces of filter paper were soaked in the transfer buffer. The transfer device was installed, 3 pieces of soaked filter paper were placed on the graphite plate, aligned accurately, and bubbles were removed. The gel was placed on the 3 layers of filter paper, then the PVDF membrane was placed on the gel, and finally the 3 layers of filter paper were placed on the membrane to ensure alignment and remove bubbles. The upper electrode (anode) was pressed on the gel, and the power supply was connected. The transfer was performed at 200 mA and 4°C for 32 minutes. After the transfer was completed, the filter membrane was placed in a dish and sealed with blocking solution (TBST containing 2% BSA) at room temperature for 2 hours (also more than 6 hours at 4°C). Then the membrane was placed in a new dish, and His-tag primary antibody diluted with blocking solution was added. After mixing, it was incubated at 4°C overnight. The membrane was washed with TBST 5 times, each for 3 minutes, and horseradish peroxidase-labeled secondary antibody diluted with blocking solution was added. It was incubated at room temperature on a shaker for 1 hour. The membrane was washed with TBST 5 times, each for 3 minutes, and the ECL developing kit was used according to the instructions. The chemiluminescence developing instrument was used for detection.

[0067] The results of Western blot are shown in Figure 7 , indicating that the N1, N2 and N3 proteins are expressed correctly.

[0068] (2) Secondary screening based on soluble N2 protein sequence

[0069] ① Construction and identification of recombinant expression plasmid

[0070] APP apxIVAN2 protein is a soluble protein with a sequence size of 756 bp. To maximize immunogenicity, the N2 sequence was extended and subjected to secondary screening under the premise of maintaining soluble expression. The extension schematic is shown in Figure 3 , wherein the amino acid sequence and nucleotide sequence of N2a2 are shown in SEQ ID NO. 7 and 8, respectively, the amino acid sequence and nucleotide sequence of N2b2 are shown in SEQ ID NO. 9 and 10, respectively, and the amino acid sequence and nucleotide sequence of N2c2 are shown in SEQ ID NO. 11 and 12, respectively.

[0071] According to the nucleotide sequence of ApxIVA gene (GenBank: AF021919), the following primers were designed:

[0072] ApxIVA-N2a2 forward primer: 5'-CCGGAATTCATGGACCCATCCGGTATCGGTGGAACGGTAA A-3',

[0073] ApxIVA-N2a2 reverse primer: 5'-CCGCTCGAGTTACGTTGCCGCCCATTTATCTAAAATGGC AG-3'.

[0074] ApxIVA-N2b2 forward primer: 5'-CCGGAATTCATGGGCACTAAAATCACCCGTAGGATTGCG G-3',

[0075] ApxIVA-N2b2 reverse primer: 5'-CCGCTCGAGTTAGCCCATTTGTGCAAAAGTACCGTCCG-3'.

[0076] ApxIVA-N2c2 forward primer: 5'-CCGGAATTCATGCCCTTAGCCCCTTACACTAAAAATGGC GTGG-3',

[0077] ApxIVA-N2c2 reverse primer: 5'-CCGCTCGAGTTAGGCTAATGTCGCAAAACCGTGTGCAG-3'.

[0078] The remaining steps were consistent with the above (1), and the annealing temperature of the three genes was 57°C, the extension time of N2a2 was 2min10s, and the extension time of N2b2 and N2c2 was 1min30s. The results showed that only N2c2 (1083bp) was expressed in soluble state; N2a2 (2046bp) and N2b2 (1545bp) were expressed in inclusion body state.

[0079] Example 2 Establishment of indirect ELISA method and evaluation of its potential for distinguishing gene deletion vaccine samples

[0080] Take the clinical APP positive pig serum (purchased from Wuhan Keqian Biological Co., Ltd., same below) and SPF pig negative serum (purchased from Guangzhou Hongquan Biological Technology Co., Ltd., same below), and four proteins of purified ApxIVAN1, N2, N3 and N2c2 as antigens to establish indirect ELISA method respectively, and detect serum antibodies. In this process, first of all, the chessboard method is used to determine the optimal antigen coating concentration and serum dilution of the four ELISA methods. Next, the type of blocking solution, blocking condition, serum incubation time, secondary antibody dilution, incubation condition and color development time will be determined. The OD value of the sample is determined, and the optimal reaction condition is the condition when the positive OD value / negative OD value (P / N value) is the highest. The optimal coating concentration of N1, N2, N3 and N2c2 antigens and serum dilution are shown in Tables 1, 2, 3 and 4. 630

[0081] ​Table 1 Determination of optimal coating concentration of recombinant protein Nl antigen and serum dilution

[0082]

[0083]

[0084] Table 2 Determination of optimal coating concentration of recombinant protein N2 antigen and serum dilution

[0085]

[0086] Table 3 Determination of optimal coating concentration of recombinant protein N3 antigen and serum dilution

[0087]

[0088] Table 4 Determination of optimal coating concentration of recombinant protein N2c2 antigen and serum dilution

[0089]

[0090]

[0091] Next, the type of blocking solution was determined as shown in Table 5, the type of serum dilution was determined as shown in Table 6, the serum incubation time was determined as shown in Table 7, the enzyme-labeled secondary antibody incubation time was determined as shown in Table 8, and the optimal substrate incubation time was determined as shown in Table 9. The OD values of the samples were measured 630 The optimal reaction conditions were conditions in which the highest value of positive OD value / negative OD value (P / N value) was obtained.

[0092] Table 5 Determination of optimal blocking solution type for Nl, N2, N3, and N2c2 ELISA

[0093]

[0094] Table 6 Determination of optimal sample dilution for Nl, N2, N3, and N2c2 ELISA

[0095]

[0096] Table 7 Determination of optimal sample incubation time for Nl, N2, N3, and N2c2 ELISA

[0097]

[0098] Table 8 Determination of optimal enzyme-labeled secondary antibody incubation time for Nl, N2, N3, and N2c2 ELISA

[0099]

[0100]

[0101] Table 9 N1, N2, N3, N2c2 ELISA optimal substrate incubation time determination

[0102]

[0103] The ELISA method steps are as follows:

[0104] (1) Coating: After the purified protein is diluted to the optimal concentration with coating buffer (1.59 g Na2CO3, 2.93 g NaHCO3 dissolved in 1 L ddH2O, pH = 9.6), 100 μL is added to each reaction well of the enzyme-labeled plate, and the plate is coated at 4°C overnight (10-14 h);

[0105] (2) Discard the solution in the well, add 100 μL of 2% BSA blocking solution (137 mM NaCl, 2.68 mM KCL, 10 mM Na2HPO4, 2 mM KH2PO4, 0.05% Tween 20, 2% BSA, pH = 7.4) to the well, and incubate at 4°C overnight. The next day, discard the solution in the well;

[0106] (3) Sample addition: After the serum to be tested is diluted 20 times with the blocking solution, 100 μL is added to the above-mentioned blocked reaction well, and the well is incubated at 37°C for 30 minutes. Then, 300 μL of washing solution (137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.05% Tween 20, pH = 7.4) is used to wash the well 5 times without standing each time. At the same time, blank wells, negative control wells, and positive control wells are prepared;

[0107] (4) Addition of enzyme-labeled antibody: Freshly diluted horseradish peroxidase-labeled goat anti-pig IgG is added to each reaction well at a dilution of 1:5000 with the blocking solution. The well is incubated at 37°C for 30 minutes, and then washed with 300 μL of washing solution 5 times without standing each time;

[0108] (5) Color development with substrate solution: 100 μL of TMB color developing solution is added to each reaction well, and the well is reacted at room temperature (20-25°C) for 10 minutes in the dark;

[0109] (6) Reaction termination: 50 μL of termination solution is added to each reaction well;

[0110] (7) Result determination: The OD value of each well is measured at 630 nm on an ELISA detector.

[0111] After comparing ELISA methods using N1, N2, N3, and N2c2 as coating antigens, the potential of these methods to distinguish gene-deleted vaccine samples was evaluated. Ten swine serum samples immunized with the gene-deleted vaccine APP-HB-04M (serum collected 70 days after secondary immunization in pig farms, the same below) were randomly selected. Table 10 shows that N1 and N3 proteins expressed in inclusion body form did not significantly distinguish between APP wild-type strain and gene-deleted vaccine serum samples; while the 327bp extended N2c2 protein, although expressed in a soluble form, did not show a significant increase in immunogenicity and even increased the background value for gene-deleted vaccine serum samples. Therefore, the ELISA method using soluble N2 protein as the coating antigen was ultimately selected.

[0112] Table 10. Potential assessment of N1, N2, N3, and N2c2 ELISA for distinguishing gene-deleted seedling samples.

[0113]

[0114] In summary, after screening and comparison, the optimal conditions for the N2 ELISA method are as follows: N2 protein coating concentration of 0.625 μg / mL; serum sample dilution of 1:20, incubation at 37℃ for 30 minutes; horseradish peroxidase-labeled goat anti-pig IgG dilution of 1:5000, incubation at 37℃ for 30 minutes; TMB color development conditions of room temperature (20-25℃) in the dark for 10 minutes.

[0115] Example 3: Determination of the indirect ELISA cutoff value

[0116] Fifteen swine serum samples immunized with the gene-deleted vaccine APP-HB-04M (serum collected 70 days after secondary immunization of pigs in the farm, the same below) were randomly selected to determine the cutoff value for the indirect ELISA method. The results are shown in Table 11. The negative serum was tested under the optimal conditions of the indirect ELISA method in Example 2, and the OD was measured. 630 The values ​​are then calculated, along with the mean and standard deviation. The formula for calculating the critical value is: Critical value = Mean + 2 × Standard deviation.

[0117] Based on the measured OD 630 The mean and standard deviation of 15 serum samples were calculated. The mean was 0.234 and the standard deviation was 0.057, meaning the critical value was 0.3500. This indicates that the indirect ELISA method using ApxIVAN2 protein as the antigen established in this invention has an OD value of [missing value]. 630 A value greater than or equal to 0.3500 indicates infection with a wild-type virus strain, while a value less than 0.3500 indicates a negative result.

[0118] Table 1. Results of immune serum testing on 115 gene-deleted vaccine samples

[0119]

[0120] Example 4 Indirect ELISA specificity test

[0121] The positive swine sera of each type, including Streptococcus suis (SS), Rotavirus (RV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine pseudorabies virus (PRV), Porcine circovirus type 3 (PCV3), Classical swine fever virus (CSFV), Japanese encephalitis virus (JEV), and APP-HB-04M immunized swine sera were detected by the optimal conditions of the indirect ELISA method established in Example 2 (N2 ELISA method), respectively.

[0122] Figure 8 The results showed that the N2 ELISA method could specifically detect APP positive sera, and could not detect the positive swine sera of various microorganism infections, indicating that the method had good specificity. APP-HB-04M immunized swine sera were also not detected, indicating that the method could be used to distinguish APP wild strain infection and gene deletion vaccine immunization.

[0123] Example 5 Indirect ELISA sensitivity test

[0124] The clinical APP positive serum was diluted by 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and detected according to the conditions of the indirect ELISA method in Example 2 to determine the sensitivity of the method.

[0125] Figure 9 The results of Table 12 showed that the positive serum was still positive after being diluted by the highest dilution of 1:160, indicating that the method had high sensitivity.

[0126] Table 12 N2 ELISA sensitivity test

[0127]

[0128] Example 6 Indirect ELISA coincidence rate test

[0129] The optimal conditions of the ELISA in Example 2 were used to test the coincidence rate of 60 clinical swine sera (from a pig farm in Mianyang, Sichuan Province). At the same time, the Porcine Actinobacillus pleuropneumoniae ApxIV-ELISA antibody detection kit (Wuhan Keygen Biotech Co., Ltd.) was used as a control.

[0130] The results of Table 13 showed that the positive coincidence rate of N2 ELISA was 100%, the negative coincidence rate was 92.8%, and the total coincidence rate was 54 / 60 (90%), indicating that the method had high coincidence rate.

[0131] Table 13 Comparison of N2 ELISA and commercial ELISA

[0132]

[0133] Example 7 Preparation of N2 ELISA kit

[0134] The N2 ELISA kit mainly contains: ApxIV AN2 antigen coated ELISA plate, APP wild strain positive and negative serum, sample diluent, washing buffer, HRP labeled goat anti-pig secondary antibody, TMB, enzyme reaction termination solution.

[0135] Example 8 Shelf life detection of indirect ELISA

[0136] The N2 ELISA kit prepared in Example 7 (the detection conditions are the same as the optimal conditions in Example 2) was used to detect APP clinical positive serum at 0, 1-6 months, and there was no obvious change in OD value, indicating that the shelf life of the kit can be more than 6 months.

[0137] Example 9 Clinical application of indirect ELISA method

[0138] 574 clinical pig sera (from a pig farm in Guangxi Zhuang Autonomous Region and a pig farm in Hubei Province) were detected using the N2 ELISA kit prepared in Example 7 (the detection conditions are the same as the optimal conditions in Example 2), which contains the genetically deleted vaccine APP-HB-04M immunized farm and the non-immunized farm. Figure 10 、 11 The results of Table 14 show that the N2 ELISA kit can well distinguish APP wild strain infection and genetically deleted vaccine APP-HB-04M immunization in clinic.

[0139] Table 14 Clinical detection results of N2 ELISA kit

[0140]

[0141] The above examples are only used to help illustrate the present application, and the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A truncated protein of Actinobacillus pleuropneumoniae apxIVA, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. A biomaterial expressing the truncated apxIVA protein of claim 1, characterized in that: The biomaterial is an expression vector or a host cell; a) The expression vector comprises a polynucleotide encoding the truncated apxIVA protein of claim 1; b) The host cell contains a polynucleotide encoding the truncated apxIVA protein of claim 1 or the expression vector of a).

3. The method for preparing the truncated apxIVA protein according to claim 1, characterized in that, Includes the following steps: (1) The nucleotide encoding the truncated apxIVA protein was cloned into pET-30a to obtain a recombinant expression plasmid; (2) The recombinant expression plasmid was transformed into Escherichia coli BL21 to obtain the recombinant expression strain; (3) The recombinant expression strain was cultured and induced to express by IPTG. After expression, the bacterial cells were broken and centrifuged. The supernatant was purified to obtain the truncated apxIVA protein.

4. The preparation method according to claim 3, characterized in that, Step (1) is as follows: Using genomic DNA of Actinobacillus pleuropneumoniae as a template, PCR amplification was performed using the following primers; ApxIVA-N2 forward primer: 5'-CCGGAATTCATGGAGAACCTGTACTTCCAAGGGCCTAAGGCGGATCCTAAGCGG-3'. ApxIVA-N2 reverse primer: 5'-CCGCTCGAGTTACCAGCCCGTTGCGGTACGAAT-3'; The amplification product and pET-30a were double-digested and ligated with EcoRI / Xhol. The ligation product was transformed into DH5α competent cells, and the recombinant expression plasmid was obtained after identification.

5. The use of the truncated apxIVA protein of claim 1 as an antigen in the preparation of an indirect ELISA kit for Actinobacillus pleuropneumoniae.

6. An indirect ELISA kit, characterized in that: The indirect ELISA kit is an indirect ELISA kit for detecting Actinobacillus pleuropneumoniae ApxIV antibodies, or an indirect ELISA kit for distinguishing between infection with wild-type Actinobacillus pleuropneumoniae and immunization with the gene-deleted vaccine APP-HB-04M, comprising an enzyme-labeled plate coated with the truncated ApxIVA protein as described in claim 1.

7. The indirect ELISA kit according to claim 6, characterized in that, It also includes: positive and negative sera of wild-type Actinobacillus pleuropneumoniae, sample diluent, washing buffer, horseradish peroxidase-labeled goat anti-pig secondary antibody, TMB, and enzyme reaction termination solution.

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