Indirect ELISA kit for distinguishing porcine actinobacillus pleuropneumoniae wild virus infection and vaccine immunity and application

Abstract

The invention discloses an indirect ELISA kit for distinguishing porcine actinobacillus pleuropneumoniae wild virus infection and vaccine immunity and application, and belongs to the technical field of biology. According to the indirect ELISA kit, screened ApxIVA truncated protein serves as a coating antigen, the amino acid sequence of the ApxIVA truncated protein is shown in SEQ ID NO.3, the ApxIVA truncated protein can be subjected to soluble expression in escherichia coli, only one His tag and one S tag are carried, and the structure and the function of the ApxIVA truncated protein are close to those of natural ApxIVA. The pxIVA truncated protein is used as an antigen, an indirect ELISA method and kit for distinguishing APP wild strain infection and gene deletion vaccine immunity are established, the kit has the advantages of high specificity, low background value, low cost and short detection time, and the kit has the advantages of improving animal husbandry APP diagnosis accuracy and improving animal husbandry APP diagnosis accuracy. And the method has important significance in the aspects of optimizing prevention and control measures and guaranteeing animal health and production benefits.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an indirect ELISA kit for distinguishing wild-type infection of porcine Actinobacillus pleuropneumoniae from vaccine immunity and application thereof. Background Art

[0002] Porcine contagious pleuropneumonia (PCP) is a serious respiratory bacterial disease caused by infection with Actinobacillus pleuropneumoniae (APP). It is widely prevalent throughout the world and is mainly transmitted between pigs through direct contact, aerosols and contaminants. The disease is characterized by acute hemorrhagic fibrinous pneumonia and chronic fibrinous necrotizing pleuropneumonia. Acutely infected pigs often bleed from the mouth and nose and die suddenly. Chronically infected pigs often become hidden sources of infection, causing the pig herd to be in a sub-healthy state and are extremely susceptible to secondary infections with other pathogens. The disease causes great economic losses to the global pig industry. At present, many diagnostic methods have been developed for APP, mainly including conventional microbiological diagnostic methods, serological identification and molecular biological diagnosis.

[0003] According to the difference in capsule antigens, APP can be divided into 19 serotypes, among which ApxIV is considered to be the target protein for diagnosing all serotypes of APP strains. Obtaining the natural structure of apxIV protein is helpful for the in-depth understanding and diagnosis of APP. However, natural apxIV is often secreted in small quantities after the strain infects pigs, and it is difficult to separate and obtain. At present, the recombinantly expressed soluble apxIV protein on the market often carries a large tag or uses a vector carrying a large tag, and its structure and function are very different from natural apxIV. In addition, the applicant has successfully developed an APP gene-deficient vaccine (APP-HB-04M) and has obtained a new veterinary drug certificate (2022, New Veterinary Drug Certificate No. 73). This gene-deficient vaccine has been widely used in pig farms of several leading pig farming companies in my country and has achieved good protection effects. However, there is currently no serological diagnostic method for distinguishing APP wild strain infection from gene-deficient vaccine (APP-HB-04M) immunity in clinical practice. When ordinary serological detection methods are used to detect pigs vaccinated with the APP gene-deficient vaccine APP-HB-04M, they will confuse APP wild strain infection with gene-deficient vaccine immunity. A serological differential diagnostic method for distinguishing wild-type virus infection from immunity with the gene-deficient vaccine APP-HB-04M needs to be established urgently. Summary of the invention

[0004] The purpose of the present invention is to solve the problems existing in the prior art of expressing APP apxIVA, and to provide an APPapxIVA truncated protein and a preparation method thereof. The ApxIVA truncated protein can be expressed soluble in Escherichia coli, carries only one His tag (about 0.84 kDa) and an S tag (about 2 kDa), and its structure and function are close to natural ApxIVA.

[0005] The present invention also aims to provide the use of the APP apxIVA truncated protein as an antigen in an indirect ELISA method or in the preparation of an indirect ELISA kit. By using the indirect ELISA method or the indirect ELISA kit, APP wild strain infection and gene-deficient vaccine immunity can be distinguished.

[0006] The purpose of the present invention is achieved through the following technical solutions:

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

[0008] A nucleotide encoding the apxIVA truncated protein, whose nucleotide sequence is shown in SEQ ID NO.4, is based on a part of the apxIVA gene deleted in the APP gene deletion vaccine APP-HB-04M, and is obtained through two 6 truncation screenings.

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

[0010] A host cell, comprising a nucleotide encoding the apxIVA truncated protein or the above expression vector, capable of expressing the apxIVA truncated protein. The host cell is preferably Escherichia coli.

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

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

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

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

[0015] Step (1) is specifically as follows: using APP 4074T strain (serotype 1 standard strain) genomic DNA as a template, PCR amplification is performed using the following primers.

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

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

[0018] The amplified product and pET-30a were double-digested with EcoRI / Xhol and ligated with enzymes. The ligated product was transformed into DH5α competent cells and the recombinant expression plasmid was obtained after identification.

[0019] The truncated protein of porcine Actinobacillus pleuropneumoniae apxIVA is used as an antigen in establishing an indirect ELISA method for porcine Actinobacillus pleuropneumoniae or in preparing an indirect ELISA kit for porcine Actinobacillus pleuropneumoniae.

[0020] An indirect ELISA method, which is an indirect ELISA method for detecting porcine Actinobacillus pleuropneumoniae ApxIV antibodies, or an indirect ELISA method for distinguishing porcine Actinobacillus pleuropneumoniae wild strain infection and gene-deficient vaccine APP-HB-04M immunization, 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 the ELISA plate, and coat it at 4° C. for more than 10 h;

[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: dilute the serum to be tested and add it to the coated reaction wells, incubate at 37°C, and then wash; prepare blank wells, negative control wells, and positive control wells at the same time;

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

[0025] (5) Add substrate solution for color development: Add TMB color development solution to each reaction well and react at room temperature in the dark;

[0026] (6) Stop reaction: Add stop solution to each reaction well;

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

[0028] The condition corresponding to the highest value of positive OD value / negative OD value (P / N value) is the optimal ELISA condition. The optimal conditions are as follows: the coating concentration of the ApxIVA truncated protein is 0.625 μg / mL; the dilution of the serum sample to be tested is 1:20, and it is incubated at 37°C for 30 minutes; the dilution of the goat anti-swine IgG labeled with horseradish peroxidase is 1:5000, and it is incubated at 37°C for 30 minutes; the TMB color development condition is room temperature (20-25°C) and light-proof reaction for 10 minutes.

[0029] Determine the critical value of indirect ELISA: critical value = mean value + 2 × standard deviation; the mean value is the mean value of the negative serum indirect ELISA results, and the standard deviation is the standard deviation of the negative serum indirect ELISA results; indirect ELISA result judgment criteria: serum sample OD value greater than or equal to the critical value is judged as positive for wild strain infection, and serum sample OD value less than the critical value is judged as negative for wild strain infection; this judgment is not affected by gene deletion vaccine (APP-HB-04M) vaccination. In the result judgment, the critical value is 0.3500.

[0030] An indirect ELISA kit is an indirect ELISA kit for detecting porcine Actinobacillus pleuropneumoniae ApxIV antibodies, or an indirect ELISA kit for distinguishing porcine Actinobacillus pleuropneumoniae wild strain infection and gene-deficient vaccine APP-HB-04M immunization, comprising an ELISA plate coated with the ApxIVA truncated protein; and may further comprise: APP wild strain positive and negative sera, sample diluent, washing buffer, horseradish peroxidase (HRP)-labeled goat anti-swine secondary antibody, TMB (tetramethylbenzidine), and enzyme reaction stop solution.

[0031] Compared with the prior art, the present invention has the following advantages and effects:

[0032] The present invention obtains a soluble expressed N2 protein sequence by screening ApxIVA truncated protein. The recombinant protein only carries a His tag (about 0.84kDa) and an S tag (about 2kDa) at the N-terminus, and its structure and function are closer to the natural state of ApxIVA. The N2 protein expression steps are simple, the cost is low, and the protein purity is high, which is suitable for large-scale production.

[0033] The present invention uses purified N2 protein as an antigen to establish an indirect ELISA method and kit for distinguishing APP wild strain infection and gene-deficient vaccine immunization, which exhibits 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 only takes 30 minutes each. The indirect ELISA kit of the present invention is the first product that can distinguish APP wild strain infection from gene-deficient vaccine APP-HB-04M immunization. The ELISA method and kit can accurately distinguish APP wild strain infection from gene-deficient vaccine immunization, which is of great significance in improving the accuracy of APP diagnosis in animal husbandry, optimizing prevention and control measures, and ensuring animal health and production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the screening expression of the ApxIVA truncated protein of the present invention, the establishment of the ELISA method and its application.

[0035] Figure 2 Bioinformatics analysis results of APP apxIVA.

[0036] Figure 3 Schematic diagram of APP apxIVA N-terminal truncation.

[0037] Figure 4 These are the amplification results of the APP apxIVA N1, N2, and N3 target genes.

[0038] Figure 5 These are the PCR identification results of the bacterial solution of plasmids pET-30a-APP apxIVA N1, N2, and N3.

[0039] Figure 6 The figure shows the SDS-PAGE results of the expression of recombinant N1, N2, and N3 truncated proteins.

[0040] Figure 7 Shown are the western blot results of recombinant N1, N2, and N3 truncated proteins.

[0041] Figure 8 The results are the specificity analysis of the N2 ELISA method.

[0042] Fig. 9 The results of sensitivity analysis of N2 ELISA method are shown in Figure 2.

[0043] Fig.10 These are the (partial) results of the N2 ELISA kit's test of the APP gene-deficient vaccine APP-HB-04M immune serum sample.

[0044] Fig.11 These are the (partial) results of clinical serum testing using the N2 ELISA kit. DETAILED DESCRIPTION

[0045] The invention provides an APP apxIVA truncated protein and a preparation method thereof, as well as an application of the APP apxIVA truncated protein as an antigen in an indirect ELISA method or in the preparation of an indirect ELISA kit. By using the indirect ELISA method or the indirect ELISA kit, APP wild-type strain infection and gene-deficient vaccine immunity can be distinguished.

[0046] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0047] Figure 1 Schematic diagram of the screening expression of ApxIVA truncated proteins, the establishment of ELISA method and its application in the following examples.

[0048] Example 1 Screening of soluble expressed ApxIVA protein

[0049] (1) Initial screening of ApxIVA protein

[0050] ① Construction and identification of recombinant expression plasmid

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

[0052] According to the nucleotide sequence of ApxIVA gene (GenBank: AF021919), the following primers were 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] Using APP 4074T strain (serotype 1 standard strain) as template, the primers designed above were used to amplify N1, N2 and N3 fragments. Amplification conditions: 95°C denaturation for 5 minutes before entering the cycle, cycle parameters: 95°C for 1 minute, annealing for 1 minute (N1, N2, N3 annealing temperatures are 55°C, 55°C, 50°C, respectively), 72°C extension (N1, N2, N3 extension time is 1min 10s), 72°C extension for 10 minutes after 35 cycles. After the PCR was completed, the product was taken out and tested by 1% agarose gel electrophoresis. The amplification results are shown in Figure 4 The sizes of each fragment were the same as expected, indicating that the amplification was successful.

[0060] After the PCR product was recovered by gel, it was double-digested with pET-30a vector (EcoRI, Xhol), enzyme-ligated, and then transformed into DH5α competent cells. The single clone colony was picked for bacterial liquid PCR and sent to Qingke Company for sequencing. The recombinant expression plasmid was successfully constructed. The bacterial liquid identification results are shown in Figure 5 , the size is the same as expected, and the sequencing result is correct.

[0061] ②Expression and purification of target gene

[0062] The recombinant expression plasmid was transferred into competent E. coli BL21 (DE3), coated with LA plates containing kanamycin, incubated in an incubator, and single colonies were picked and transferred to LB medium containing kanamycin at 37°C and 180 rpm overnight. Single clones were picked and transferred to fresh LB medium containing kanamycin, and the culture was cultured until OD 600When the value was 0.6, 1mmol / L IPTG was added, and the cells were induced at 16℃ and 120rpm for 16h. The cells were broken by high-pressure cell disruptor and centrifuged. The soluble expressed protein was purified by nickel column using bacterial supernatant. First, the column and filler were washed and balanced with ddH2O and Binding Buffer (20mM imidazole, 0.5M NaCl, 20mM Na3PO4·12H2O, pH=7.4). The bacterial liquid after crushing and centrifugation was taken as supernatant and precipitate sample; the supernatant and filler were incubated at 4℃ for 0.5h, and the effluent was taken as flow-through sample after standing; the column was cleared by Binding Buffer, and the effluent was taken as washing sample; the column was eluted with Elution Buffer (500mM imidazole, 0.5M NaCl, 20mM Na3PO4·12H2O, pH=7.4), and the effluent was the elution sample, i.e., the liquid containing the target protein in the end; 12% SDS-PAGE electrophoresis analysis.

[0063] Purification of protein expressed in inclusion bodies by bacterial precipitation: First, collect the unbroken bacterial solution as the whole bacterial sample, and take the broken and centrifuged bacterial solution as the supernatant and precipitate samples; add 19.7mL buffer A (50mM Tris-Base, 0.5mM EDTA, 100mM NaCl, 0.05% glycerol), 0.3mL 20% sodium lauryl sarcosyl sarcosinate storage solution and 20μL 0.5M DTT to the precipitate after each 200mL of bacterial solution, stir vigorously to dissolve it slowly, let it stand at room temperature for 30min to 2h, and take the supernatant after centrifugation as the denatured sample; add 210μL 20% PEG4000 (0.2 g / mL final concentration of 0.2%), 420 μL of 50 mM (0.03 g / mL) oxidized glutathione (final concentration of 1 mM), and 420 μL of 100 mM (0.03 g / mL) reduced glutathione (final concentration of 2 mM), let stand at room temperature for 0.5-2 h, and take samples for renaturation; after dialysis with TE Buffer (10 mM Tris-Base, 1 mM EDTA) for 72 h, add sucrose to the surface of the dialysis bag to absorb excess water, take samples for concentration, 30a is the pET-30a empty vector control; 12% SDS-PAGE electrophoresis analysis.

[0064] SDS-PAGE results ( Figure 6 ) showed that N2 protein was expressed in a soluble form, with a protein band of 42 kDa, which was consistent with the expected size, and the purified protein N2 was successfully obtained. N1 and N3 proteins were expressed in inclusion bodies, with a size of 43 and 45 kDa, respectively. The expression levels of N1, N2, and N3 proteins were 13, 42, and 55 mg per liter of bacterial solution as detected by the Biyuntian Bradford protein concentration assay kit.

[0065] ③Western blot analysis

[0066] After SDS-PAGE electrophoresis, the gel is directly transferred to the membrane using a transfer instrument without staining. Cut the gel of appropriate size, cut 6 3mm filter papers and 1 polyvinylidene fluoride membrane (PVDF). Soak the 6 filter papers in electrotransfer buffer. Install the transfer device, place 3 soaked filter papers on the graphite plate, align them accurately, drive out bubbles, first place the gel on the 3 layers of filter paper, then place the PVDF membrane on the gel, and finally place the 3 layers of filter paper on the membrane, ensure alignment, and scrape off bubbles. Press the upper electrode (anode) on the gel, connect the power supply, and transfer the membrane at 200mA and 4℃ for 32 minutes. After the transfer, place the filter membrane in a plate and block it with blocking solution (TBST containing 2% BSA) at room temperature for 2h (or 4℃ for more than 6h). Then put the membrane in a new plate, add the His tag primary antibody diluted in the blocking solution at an appropriate ratio, mix well, and incubate at 4℃ overnight. The membrane was washed 5 times with TBST for 3 min each time, and the secondary antibody labeled with horseradish peroxidase diluted in blocking solution at an appropriate ratio was added, and incubated on a shaker at room temperature for 1 h. The membrane was washed 5 times with TBST for 3 min each time, and the ECL color development kit was followed according to the operating instructions, and the detection was performed in a chemiluminescence colorimeter.

[0067] Western blot results are shown in Figure 7 , indicating that 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 apxIVA N2 protein is a soluble protein with a sequence size of 756 bp. In order to maximize immunogenicity, the N2 sequence was extended and screened again while maintaining soluble expression. See the schematic diagram of the extension. Figure 3 Among them, the amino acid sequence and nucleotide sequence of N2a2 are shown in SEQ ID NOs.7 and 8 respectively, the amino acid sequence and nucleotide sequence of N2b2 are shown in SEQ ID NOs.9 and 10 respectively, and the amino acid sequence and nucleotide sequence of N2c2 are shown in SEQ ID NOs.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'-CCGGAATTCATGGACCCATCCGGTATCGGTGGAACGGTA AA-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), with the annealing temperature of the three genes being 57°C, the extension time of N2a2 being 2 min 10 s, and the extension time of N2b2 and N2c2 being 1 min 30 s. The results showed that only N2c2 (1083 bp) was expressed in a soluble state; N2a2 (2046 bp) and N2b2 (1545 bp) were both expressed in inclusion bodies.

[0079] Example 2 Establishment of the indirect ELISA method and evaluation of its potential to distinguish gene-deficient seedling samples

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

[0081] Table 1 Determination of the optimal coating concentration and serum dilution of recombinant protein N1 antigen

[0082]

[0083]

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

[0085]

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

[0087]

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

[0089]

[0090]

[0091] Next, the type of blocking solution is determined in Table 5, the type of serum diluent is determined in Table 6, the serum incubation time is determined in Table 7, the enzyme-labeled secondary antibody incubation time is determined in Table 8, and the optimal substrate incubation time is determined in Table 9. Determine the sample OD 630 The best reaction condition is the condition when the positive OD value / negative OD value (P / N value) is the highest.

[0092] Table 5 Determination of the best blocking solution for N1, N2, N3, and N2c2 ELISA

[0093]

[0094] Table 6 Determination of optimal sample dilutions for N1, N2, N3, and N2c2 ELISA

[0095]

[0096] Table 7 Determination of optimal sample incubation time for N1, N2, N3, and N2c2 ELISA

[0097]

[0098] Table 8 Determination of the optimal enzyme-labeled secondary antibody incubation time for N1, N2, N3, and N2c2 ELISA

[0099]

[0100]

[0101] Table 9 Determination of optimal substrate incubation time for N1, N2, N3, and N2c2 ELISA

[0102]

[0103] The ELISA method steps are as follows:

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

[0105] (2) Discard the solution in the wells, 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), incubate at 4°C overnight, and discard the solution the next day;

[0106] (3) Sample addition: dilute the serum to be tested 20 times with blocking solution and add 100 μL to the blocked reaction wells. Incubate at 37°C for 30 minutes. Then wash five times with 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). Do not let stand each time. At the same time, make blank wells, negative control wells and positive control wells.

[0107] (4) Add enzyme-labeled antibody: Add freshly diluted horseradish peroxidase-labeled goat anti-swine IgG to each reaction well, diluted with blocking solution at a ratio of 1:5000. Incubate at 37°C for 30 minutes, wash 5 times with 300 μL washing solution, without standing each time;

[0108] (5) Add substrate solution for color development: Add 100 μL of TMB color development solution to each reaction well and react at room temperature (20-25°C) in the dark for 10 minutes;

[0109] (6) Stop reaction: Add 50 μL of stop solution to each reaction well;

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

[0111] After comparing the ELISA methods using N1, N2, N3 and N2c2 as coating antigens, the potential of these methods to distinguish gene-deficient vaccine samples was evaluated. Ten sera of pigs immunized with the gene-deficient vaccine APP-HB-04M were randomly selected (sera collected 70 days after the second immunization of pigs in the farm, the same below). The results in Table 10 showed that the N1 and N3 proteins expressed in the form of inclusion bodies did not significantly distinguish between APP wild strains and gene-deficient vaccine serum samples; and the N2c2 protein, which was extended by 327bp, was expressed in a soluble manner, but the immunogenicity did not increase significantly, and the background value of the gene-deficient vaccine serum samples also increased. Therefore, the ELISA method using N2 soluble protein as the coating antigen was finally selected.

[0112] Table 10 Evaluation of the potential of N1, N2, N3, and N2c2 ELISA to distinguish gene-deficient seedling samples

[0113]

[0114] In summary, the N2 ELISA method was established after screening and comparison, and its optimal conditions were as follows: the coating concentration of N2 protein was 0.625 μg / mL; the dilution of the serum sample to be tested was 1:20, and incubated at 37°C for 30 minutes; the dilution of horseradish peroxidase-labeled goat anti-pig IgG was 1:5000, and incubated at 37°C for 30 minutes; the TMB color development conditions were room temperature (20-25°C) and dark reaction for 10 minutes.

[0115] Example 3 Determination of indirect ELISA critical value

[0116] 15 sera from pigs immunized with the gene-deficient vaccine APP-HB-04M (sera collected from pigs in the farm 70 days after secondary immunization, the same below) were randomly selected to determine the critical value of the indirect ELISA method. The results are shown in Table 11. The negative sera were tested under the optimal conditions of the indirect ELISA method in Example 2, and the OD was determined. 630 The critical value is calculated as follows: critical value = average value + 2 × standard deviation.

[0117] According to the measured OD 630 The average and standard deviation of the 15 serum samples were calculated, and the average was 0.234, the standard deviation was 0.057, and the critical value was 0.3500; that is, the indirect ELISA method using ApxIVAN2 protein as antigen established by the present invention, OD 630 A value greater than or equal to 0.3500 can be considered as wild strain infection, and a value less than 0.3500 is negative.

[0118] Table 11 Test results of 15 gene-deficient vaccine immune sera

[0119]

[0120] Example 4 Indirect ELISA specificity experiment

[0121] The optimal conditions of the indirect ELISA method (N2 ELISA method) established in Example 2 were used to detect various types of positive pig sera, including Streptococcus suis (SS), rotavirus (RV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine circovirus type 3 (PCV3), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV), and pig sera immunized with the gene-deficient vaccine APP-HB-04M.

[0122] Figure 8 The results showed that the N2 ELISA method could specifically detect APP-positive serum, but could not detect positive sera of pigs infected with the above-mentioned microorganisms, indicating that the method had good specificity. The gene-deficient vaccine APP-HB-04M immunized pig serum was also not detected, indicating that the method can be used to distinguish between APP wild-type virus infection and gene-deficient vaccine immunity.

[0123] Example 5 Indirect ELISA sensitivity test

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

[0125] Fig. 9 The results in Table 12 show that the positive serum was still positive after being diluted to a maximum dilution of 1:160, indicating that the method has a high sensitivity.

[0126] Table 12N2 ELISA sensitivity test

[0127]

[0128] Example 6 Indirect ELISA compliance test

[0129] The compliance rate of 60 clinical pig sera (from a pig farm in Mianyang City, Sichuan Province) was tested using the optimal conditions of ELISA in Example 2. At the same time, a porcine Actinobacillus pleuropneumoniae ApxIV-ELISA antibody detection kit (Wuhan Keqian Biological Co., Ltd.) was used as a control.

[0130] The results in Table 13 show that the positive coincidence rate of N2 ELISA is 100%, the negative coincidence rate is 92.8%, and the total coincidence rate is 54 / 60 (90%). This method has a high coincidence rate.

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

[0132]

[0133] Example 7 Preparation of N2 ELISA Kit

[0134] The N2 ELISA kit mainly includes: ApxIVAN2 antigen-coated ELISA plate, APP wild-type positive and negative sera, sample diluent, washing buffer, HRP-labeled goat anti-pig secondary antibody, TMB, and enzyme reaction termination solution.

[0135] Example 8 Indirect ELISA Shelf Life Detection

[0136] The N2 ELISA kit prepared in Example 7 (the detection conditions were the same as the optimal conditions in Example 2) was used to detect APP clinical positive serum at 0 and 1-6 months, and the OD value did not change significantly, indicating that the shelf life of the kit can reach more than 6 months.

[0137] Example 9 Clinical application of indirect ELISA method

[0138] The N2 ELISA kit prepared in Example 7 (the detection conditions were the same as the optimal conditions in Example 2) was used to detect 574 clinical pig sera (from a pig farm in Guangxi and a pig farm in Hubei Province), including those from the immunized and non-immunized fields with the gene-deficient vaccine APP-HB-04M. Fig.10 , 11 The results in Table 14 show that the N2 ELISA kit can well distinguish APP wild-type virus infection and gene-deficient vaccine APP-HB-04M immunity in clinical practice.

[0139] Table 14N2 ELISA kit clinical test results

[0140]

[0141] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

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 apxIVA truncated protein of claim 1, characterized in that: The biological material is an expression vector or a host cell; The expression vector comprises a nucleotide encoding the apxIVA truncated protein of claim 1; The host cell contains the nucleotide encoding the apxIVA truncated protein of claim 1 or the above-mentioned expression vector.

3. The method for preparing the apxIVA truncated protein according to claim 1, characterized in that: The following steps are involved: (1) cloning the nucleotide encoding the apxIVA truncated protein into pET-30a to obtain a recombinant expression plasmid; (2) transforming the recombinant expression plasmid into Escherichia coli BL21 to obtain a recombinant expression strain; (3) The recombinant expression strain is cultured and induced to express by IPTG. After expression, the bacteria are broken and centrifuged, and the supernatant is purified to obtain the apxIVA truncated protein.

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

5. Use of the apxIVA truncated protein according to claim 1 as an antigen in establishing an indirect ELISA method for porcine Actinobacillus pleuropneumoniae or in preparing an indirect ELISA kit for porcine Actinobacillus pleuropneumoniae.

6. An indirect ELISA method, characterized in that: The indirect ELISA method is an indirect ELISA method for detecting porcine Actinobacillus pleuropneumoniae ApxIV antibodies, or an indirect ELISA method for distinguishing porcine Actinobacillus pleuropneumoniae wild strain infection and gene-deficient vaccine APP-HB-04M immunity, comprising the following steps: (1) Coating: dilute the ApxIVA truncated protein of claim 1 with coating buffer, add it to the reaction wells of the ELISA plate, and coat at 4° C. for more than 10 hours; (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; (3) Sample addition: dilute the serum to be tested and add it to the coated reaction wells, incubate at 37°C, and then wash; prepare blank wells, negative control wells, and positive control wells at the same time; (4) Adding enzyme-labeled antibodies: Add freshly diluted horseradish peroxidase-labeled goat anti-swine IgG to each reaction well, incubate at 37°C, and wash; (5) Add substrate solution for color development: Add TMB color development solution to each reaction well and react at room temperature in the dark; (6) Stop reaction: Add stop solution to each reaction well; (7) Result determination: Measure the OD630nm value of each well on the ELISA detector.

7. The indirect ELISA method according to claim 6, characterized in that: The coating concentration of the ApxIVA truncated protein is 0.625 μg / mL; the dilution of the serum sample to be tested is 1:20, and it is incubated at 37°C for 30 minutes; the dilution of the goat anti-swine IgG labeled with horseradish peroxidase is 1:5000, and it is incubated at 37°C for 30 minutes; the TMB color development condition is 20-25°C in the dark for 10 minutes.

8. An indirect ELISA kit, characterized in that: The indirect ELISA kit is an indirect ELISA kit for detecting ApxIV antibodies of porcine Actinobacillus pleuropneumoniae, or an indirect ELISA kit for distinguishing porcine Actinobacillus pleuropneumoniae wild strain infection and gene-deficient vaccine APP-HB-04M immunization, comprising an ELISA plate coated with the ApxIVA truncated protein of claim 1.

9. The indirect ELISA kit according to claim 8, characterized in that Also contains: positive and negative sera of wild strain of Actinobacillus pleuropneumoniae, sample diluent, washing buffer, goat anti-swine secondary antibody labeled with horseradish peroxidase, TMB, enzyme reaction stop solution.

10. Use of the indirect ELISA method according to claim 6 or 7 or the indirect ELISA kit according to claim 8 or 9 in distinguishing infection with wild strain of Actinobacillus pleuropneumoniae from immunity with gene-deficient vaccine APP-HB-04M.

Images