A colloidal gold test strip for distinguishing between actinobacillus pleuropneumoniae wild virus infection and vaccine immunization and application thereof

Abstract

The application discloses a colloidal gold test strip for distinguishing between wild toxin infection and vaccine immunization of porcine pleuropneumonia actinobacillus and application. The test strip of the application takes ApxIVA truncated protein with an amino acid sequence shown as SEQ ID NO. 3 as an antigen, the truncated protein can be soluble expressed in E. coli, and the structure and function of the truncated protein are close to those of natural apxIVA. The test strip of the application comprises a sample pad, a colloidal gold pad, an NC membrane, a water absorption filter paper and a bottom plate, the colloidal gold pad is coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody, and the C line and the T line of the NC membrane are fixed with goat anti-chicken IgY antibody and apxIVA truncated protein. The test strip only needs 25 muL of pig serum to make detection within 5-10 min, and can distinguish between APP wild strain infection and gene deletion vaccine APP-HB-04M immunized serum samples.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology and relates to a colloidal gold test strip for distinguishing between wild-type Actinobacillus pleuropneumoniae infection and vaccine immunization, and its application. Background Technology

[0002] Porcine contagious pleuropneumonia (PCP) is a serious bacterial respiratory disease in pigs caused by infection with Actinobacillus pleuropneumoniae (APP). It is characterized by acute hemorrhagic fibrinous pneumonia and chronic fibrinous necrotizing pleuropneumonia. Acute infection often causes bleeding from the mouth and nose, leading to sudden death; chronic infection often results in pigs becoming asymptomatic sources of infection. Pigs infected with APP are in a prolonged sub-healthy state, highly susceptible to secondary infections by other pathogens, causing stunted growth and even death, resulting in significant economic losses to my country's pig industry.

[0003] Based on differences in capsular antigens, APP can be classified into 19 serotypes, and ApxIVA is considered the target protein for diagnosing all serotypes of APP. Currently, various APP serological and nucleic acid diagnostic products based on apxIVA have been developed. These products all suffer from drawbacks such as requiring cumbersome operating procedures or demanding specialized equipment and experimental skills. While designated veterinary stations can provide testing, the time-consuming sample transportation process leads to lower detection rates and higher infection rates. Products that provide veterinarians and farmers (especially in underdeveloped and remote areas) with on-site, immediate testing that is simple to operate, saves sample delivery time, and eliminates the need for specialized equipment are urgently needed. APP colloidal gold antibody test strips are an ideal detection method; however, to date, no related products have been successfully marketed globally. Furthermore, the applicant has successfully developed an APP gene-deleted vaccine (APP-HB-04M), which has obtained a new veterinary drug certificate (2022, New Veterinary Drug Certificate No. 73) and has been widely used in several large-scale pig farms in my country, achieving excellent protective effects. However, a differential diagnostic method for distinguishing between wild-type virus infection and immunization with the gene-deleted vaccine APP-HB-04M has not yet been established. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide an application of soluble truncated apxIVA protein from *Actinobacillus pleuropneumoniae* as an antigen in the preparation of colloidal gold antibody test strips for *Actinobacillus pleuropneumoniae* (APP). The truncated apxIVA protein can be solublely expressed in *Escherichia coli*, carrying only one His tag (approximately 0.84 kDa) and an S tag (approximately 2 kDa), and its structure and function are close to those of natural apxIVA.

[0005] Another objective of this invention is to provide a colloidal gold antibody test strip that can distinguish between APP wild-type virus infection and immunization with the gene-deleted vaccine APP-HB-04M. The test strip has a simple structure, low cost, and the detection process is simple, time-saving and labor-saving. Only 25 μL of swine serum is needed to make a rapid diagnosis in 5-10 minutes, without the need for instruments and equipment such as multi-channel pipettes, circulating heating instruments, and electrophoresis.

[0006] The present invention also aims to provide a method for preparing and using the test strip.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The application of a soluble truncated apxIVA protein from *Actinomyces pleuropneumoniae* as an antigen in the preparation of APP colloidal gold antibody test strips. The amino acid sequence of the truncated apxIVA protein is shown in SEQ ID NO.3.

[0009] Furthermore, the truncated apxIVA protein is prepared by a method comprising the following steps:

[0010] (1) The nucleotide encoding the truncated apxIVA protein is cloned into pET-30a to obtain a recombinant expression plasmid; wherein the sequence of the nucleotide encoding the truncated apxIVA protein is preferably as shown in SEQ ID NO.4;

[0011] (2) The recombinant expression plasmid was transformed into Escherichia coli BL21 to obtain the recombinant expression strain;

[0012] (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.

[0013] Step (1) specifically involves using the genomic DNA of APP 4074T strain (serum type 1 standard strain) as a template and performing PCR amplification with the following primers.

[0014] ApxIVA-N2 forward primer: 5'-CCGGAATTCATGGAGAACCTGTACTTCCAAGGGCCTAAGGCGGATCCTAAGCGG-3'.

[0015] ApxIVA-N2 reverse primer: 5'-CCGCTCGAGTTACCAGCCCGTTGCGGTACGAAT-3'.

[0016] 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.

[0017] An APP colloidal gold antibody test strip is disclosed, which is used to distinguish between APP wild-type strain infection and immunization with the gene-deleted vaccine APP-HB-04M. The strip includes a sample pad, a colloidal gold pad, a nitrocellulose (NC) membrane, absorbent filter paper, and a base plate. The sample pad, colloidal gold pad, NC membrane, and absorbent filter paper are sequentially overlapped and adhered to the base plate. The colloidal gold pad is coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody. The NC membrane has C-lines and T-lines spaced at intervals. Goat anti-chicken IgY antibody is immobilized on the C-lines, and apxIVA truncated protein is immobilized on the T-lines. The amino acid sequence of the apxIVA truncated protein is shown in SEQ ID NO.3.

[0018] The preparation method of the above-mentioned APP colloidal gold antibody test strip includes the following steps:

[0019] (1) Soak the sample pad in the sample pad pretreatment solution for 4-7 minutes and then dry it for later use. The sample pad pretreatment solution is preferably a 0.02M PBS buffer with pH=7.4 containing 10g / L NaCl and 0.5g / L ProClin 300 antibacterial agent.

[0020] (2) Immerse the colloidal gold pad in the colloidal gold pad pretreatment solution for 4-7 minutes and then dry it for later use. The colloidal gold pad pretreatment solution is preferably a 0.02M PBS buffer with pH=7.4 containing 5g / L casein, 10g / L BSA, 50g / L trehalose and 0.5g / L ProClin 300 antibacterial agent.

[0021] (3) The colloidal gold-labeled apxIVA truncated protein and the colloidal gold-labeled chicken IgY were immobilized on the colloidal gold pad to obtain a colloidal gold pad coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody.

[0022] (4) The goat anti-chicken IgY solution and the apxIVA truncated protein solution were streaked onto the NC membrane and dried to obtain an NC membrane containing C-lines and T-lines. The concentrations of the goat anti-chicken IgY solution and the apxIVA truncated protein solution were preferably 0.5 mg / mL and 1.0 mg / mL, respectively.

[0023] (5) Assemble the processed sample pad, the colloidal gold pad coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody, the nitrocellulose membrane containing C and T lines, the absorbent filter paper and the base plate in sequence to obtain the colloidal gold antibody test strip.

[0024] The method of using the above-mentioned APP colloidal gold antibody test strip includes the following steps: dilute the serum to be tested with the sample diluent at a volume ratio of 1:2, drop the solution into the sample pad, and observe the results after 5-10 minutes. If the C line and T line show color, the serum to be tested is positive for porcine infectious pleuropneumonia wild-type virus. If the C line shows color but the T line does not, the serum to be tested is APP-HB-04M gene-deleted vaccine immune serum or negative serum. Other color development results are invalid. The sample diluent is preferably 0.01M PBS buffer with pH=7.4 containing 1% BSA.

[0025] The present invention has the following advantages and effects compared with the prior art:

[0026] 1. This invention obtains a soluble N2 protein segment by screening truncated apxIVA proteins. This recombinant protein carries only one His tag (approximately 0.84 kDa) and one S tag (approximately 2 kDa) at its N-terminus, and its structure and function are closer to the native state of apxIVA. The N2 protein expression procedure is simple, low-cost, and produces high protein purity, making it suitable for large-scale production. The results of this invention suggest that inclusion body expression of apxIVA may be one of the reasons why APP colloidal gold test strip products have been delayed in their market launch.

[0027] 2. The test strip of this invention has a simple structure, and the detection process is convenient, time-saving, and labor-saving. Only 25 μL of swine serum is needed for detection within 5-10 minutes. It can distinguish between APP wild-type strain infection and gene-deleted vaccine immune serum samples, and eliminates the need for instruments such as multi-channel pipettes, circulating heating devices, and electrophoresis. It is very suitable for on-site and remote area testing, has a low barrier to entry, and can be mass-produced and used. Furthermore, this product is the world's first colloidal gold diagnostic test strip for APP, which is not only of great significance in vaccine strain identification and diagnosis and swine disease prevention and control breeding, but also has broad application prospects and practical value in on-site APP diagnosis, especially in remote areas lacking experimental equipment and skills. Attached Figure Description

[0028] Figure 1 This is a schematic diagram (A) showing the structure of the colloidal gold antibody test strip of the present invention and the determination of positive and negative results (B).

[0029] Figure 2 The results are the bioinformatics analysis results of APP apxIVA in Example 1.

[0030] Figure 3 This is a schematic diagram of the N-end truncation of ApxIVA in Example 1 (including initial screening and secondary screening).

[0031] Figure 4 The results of APP apxIVA N1, N2, and N3 target gene amplification in Example 1 are shown.

[0032] Figure 5 The results of bacterial culture PCR identification of plasmids pET-30a-APP apxIVA N1, N2, and N3 in Example 1 are shown.

[0033] Figure 6 The SDS-PAGE results are for the expression of recombinant N1, N2, and N3 truncated proteins in Example 1.

[0034] Figure 7 The results are Western blots of the recombinant N1, N2, and N3 truncated proteins from Example 1.

[0035] Figure 8 This is the detection result of the test strip of the present invention on APP positive and negative serum samples.

[0036] Figure 9 This is the detection result of the test strip of the present invention on the immune serum sample of gene-deleted vaccine APP-HB-04M.

[0037] Figure 10 This is the sensitivity test result of the test strip of the present invention.

[0038] Figure 11 This is the specificity test result of the test strip of the present invention.

[0039] Figure 12 This is the result of the shelf life test of the test strip of the present invention.

[0040] Figure 13 This is the repeatability test result of the test strip of the present invention.

[0041] Figure 14 These are partial clinical serum test results for the test strip of this invention. Detailed Implementation

[0042] To more clearly illustrate the technical content of the present invention, it is described in detail here with reference to specific embodiments and accompanying drawings. Obviously, the listed embodiments are only preferred embodiments of the present technical solution, and other technical solutions that can be obviously derived by those skilled in the art based on the disclosed technical content still fall within the protection scope of the present invention.

[0043] like Figure 1As shown, the APP colloidal gold antibody test strip of the present invention specifically includes a sample pad, a colloidal gold pad, an NC membrane, absorbent filter paper, and a PCV substrate. The NC membrane has control lines (C lines) and test lines (T lines) spaced apart sequentially. Goat anti-chicken IgY is immobilized on the C line, and apxIVA truncated N2 protein is immobilized on the T line. The colloidal gold pad is labeled with N2 protein and chicken IgY antibody. After the serum sample to be tested is diluted 1:2 and added to the sample pad at one end of the test strip, it moves forward through chromatography, reacting with the colloidal gold-labeled reagent on the colloidal gold pad. When it moves to the area where the antigen is immobilized, the colorimetric result can be observed with the naked eye. If both the C and T lines react, it indicates that the sample is serum infected with the APP wild-type strain; if only the C line reacts, it indicates that the sample is serum immunized with the gene-deleted vaccine APP-HB-04M or APP-negative serum; if the C line does not react, the test strip is invalid.

[0044] Example 1: Screening for soluble expression of ApxIVA protein

[0045] (1) Initial screening of ApxIVA protein

[0046] ① Construction and identification of recombinant expression plasmids

[0047] First, bioinformatics analysis was used to determine the hydrophilicity / hydrophobicity and transmembrane domain of APP apxIVA. Then, through antigenic epitope prediction and its three-dimensional structure prediction, the N-terminus of APP apxIVA was preliminarily truncated into three segments: N1, N2, and N3, with sizes of 918 bp, 756 bp, and 1035 bp, respectively. The bioinformatics analysis results are shown below. Figure 2 See the truncated diagram. Figure 3 The amino acid and nucleotide sequences of N1 are shown in SEQ ID NO.1 and 2, respectively; the amino acid and nucleotide sequences of N2 are shown in SEQ ID NO.3 and 4, respectively; and the amino acid and nucleotide sequences of N3 are shown in SEQ ID NO.5 and 6, respectively.

[0048] Based on the nucleotide sequence of the ApxIVA gene (GenBank: AF021919), the following primers were designed:

[0049] ApxIVA-N1 forward primer: 5'-CCGGAATTCATGCGCGCCTATATCTGGAAT-3'.

[0050] ApxIVA-N1 reverse primer: 5'-CCGCTCGAGTTTTATTTCTTCTTTCGTTATGTACTCGCT-3'.

[0051] ApxIVA-N2 forward primer: 5'-CCGGAATTCATGGAGAACCTGTACTTCCAAGGGCCTAAGGCGGATCCTAAGCGG-3', ApxIVA-N2 reverse primer: 5'-CCGCTCGAGTTACCAGCCCGTTGCGGTACGAAT-3'.

[0052] ApxIVA-N3 forward primer: 5'-CCGGAATTCATGGGGCGACAAGGCGCGTTATTC-3'.

[0053] ApxIVA-N3 reverse primer: 5'-CCGCTCGAGTTTCCCTTCGAATTGTTTCGCATTAACGC-3'.

[0054] Using APP 4074T strain (serum type 1 standard strain) as a template, the N1, N2, and N3 fragments were amplified using the primers designed above. Amplification conditions were as follows: denaturation at 95℃ for 5 min, followed by cycling parameters: 95℃ for 1 min, annealing for 1 min (annealing temperatures for N1, N2, and N3 were 55℃, 55℃, and 50℃, respectively), extension at 72℃ (extension times for N1, N2, and N3 were all 1 min 10 s), 35 cycles, and a final extension at 72℃ for 10 min. After PCR, the products were analyzed by 1% agarose gel electrophoresis. Amplification results are shown below. Figure 4 The sizes of each fragment were the same as expected, indicating that the amplification was successful.

[0055] After gel recovery, the PCR product was double-digested with the pET-30a vector (EcoRI, Xhol), ligated, and then transformed into DH5α competent cells. Single colonies were picked for culture PCR and sent to Qingke Company for sequencing, successfully constructing the recombinant expression plasmid. The results of the culture identification are shown below. Figure 5 The size is the same as expected, and the sequencing results are correct.

[0056] ② Expression and purification of the target gene

[0057] The recombinant expression plasmid was transformed into competent *E. coli* BL21(DE3) cells, plated on LA plates containing kanamycin, and incubated upside down in an incubator. Single colonies were then picked and transferred to LB medium containing kanamycin, incubated overnight at 37°C and 180 rpm. Single colonies were then transferred to fresh LB medium containing kanamycin and cultured to OD0.05. 600When the value was 0.6, 1 mmol / L IPTG was added, and the cells were induced at 16℃ and 120 rpm for 16 h. After cell disruption by high-pressure cell disruption and centrifugation, the supernatant of the soluble expressed protein was used for nickel column purification. First, the column and packing material were washed and equilibrated with ddH2O and Binding Buffer (20 mM imidazole, 0.5 M NaCl, 20 mM Na3PO4·12H2O, pH=7.4). The supernatant and precipitate were collected after disruption and centrifugation. The supernatant and packing material were incubated at 4℃ for 0.5 h, and the effluent was used as a flow-through sample after standing. The column was cleared with Binding Buffer, and the effluent was used as a washing sample. The column was eluted with Elution Buffer (500 mM imidazole, 0.5 M NaCl, 20 mM Na3PO4·12H2O, pH=7.4), and the effluent was the elution sample, i.e., the liquid containing the target protein. The sample was analyzed by 12% SDS-PAGE electrophoresis.

[0058] Purification of inclusion body expressed proteins by bacterial cell precipitation: First, collect undisturbed bacterial culture as whole bacterial sample, and collect the supernatant and precipitate from the lysed and centrifuged bacterial culture. Add 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 sarcosinate stock solution, and 20 μL of 0.5 M DTT to every 200 mL of the precipitate after lysing the bacterial culture. Stir vigorously to allow it to dissolve slowly, incubate at room temperature for 30 min to 2 h, centrifuge, and collect the supernatant as a denaturation sample. Add 210 μL of 20% glycerol... 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) were incubated at room temperature for 0.5–2 h, and samples were taken for renaturation. After dialysis with TE buffer (10 mM Tris-Base, 1 mM EDTA) for 72 h, sucrose was added to the surface of the dialysis bag to absorb excess moisture, and samples were taken for concentration. 30a served as the pET-30a empty vector control. 12% SDS-PAGE electrophoresis analysis was performed.

[0059] SDS-PAGE results ( Figure 6 The results showed that N2 protein was soluble and expressed, with a protein band of 42 kDa, consistent with the expected size, indicating successful purification of protein N2. N1 and N3 proteins were expressed as inclusion bodies, with values ​​of 43 and 45 kDa, respectively. Using the Beyotime Bradford protein assay kit, the expression levels of N1, N2, and N3 proteins per liter of bacterial culture were 13, 42, and 55 mg, respectively.

[0060] ③Western blot analysis

[0061] After SDS-PAGE electrophoresis, the gel is transferred directly to a membrane using a transfer apparatus without staining. Cut a gel of appropriate size, and prepare six 3mm filter papers and one polyvinylidene fluoride (PVDF) membrane. Soak the six filter papers in electrotransfer buffer. Assemble the transfer apparatus, place three soaked filter papers on a graphite plate, aligning them precisely and removing any air bubbles. First, place the gel on the three layers of filter paper, then place the PVDF membrane on the gel, and finally place the three layers of filter paper on the membrane, ensuring alignment and removing any air bubbles. Press the upper electrode (anode) onto the gel, connect the power supply, and perform the transfer at 200mA and 4℃ for 32 minutes. After the transfer, place the membrane in a petri dish and block it with blocking buffer (TBST containing 2% BSA) at room temperature for 2 hours (or at 4℃ for 6 hours or more). Then, place the membrane in a new petri dish, add His-tagged primary antibody diluted with blocking buffer at an appropriate ratio, mix well, and incubate overnight at 4℃. Wash the membrane 5 times with TBST for 3 min each time, add horseradish peroxidase-labeled secondary antibody diluted with blocking buffer at an appropriate ratio, and incubate on a shaker at room temperature for 1 h. Wash the membrane 5 times with TBST for 3 min each time, follow the operating instructions of the ECL colorimetric kit, and detect the membrane using a chemiluminescence colorimeter.

[0062] The results of the Western blot are shown below. Figure 7 This indicates that N1, N2, and N3 proteins are expressed correctly.

[0063] (2) Secondary screening based on soluble N2 protein sequence

[0064] ① Construction and identification of recombinant expression plasmids

[0065] The APP apxIVA N2 protein is a soluble protein with a sequence size of 756 bp. To maximize immunogenicity while maintaining soluble expression, the N2 sequence was extended and subjected to secondary screening. See the diagram for the extension illustration. Figure 3 The amino acid and nucleotide sequences of N2a2 are shown in SEQ ID NO.7 and 8, respectively; the amino acid and nucleotide sequences of N2b2 are shown in SEQ ID NO.9 and 10, respectively; and the amino acid and nucleotide sequences of N2c2 are shown in SEQ ID NO.11 and 12, respectively.

[0066] Based on the nucleotide sequence of the ApxIVA gene (GenBank: AF021919), the following primers were designed:

[0067] ApxIVA-N2a2 forward primer: 5'-CCGGAATTCATGGACCCATCCGGTATCGGTGGAACGGTA AA-3',

[0068] ApxIVA-N2a2 reverse primer: 5'-CCGCTCGAGTTACGTTGCCGCCCATTTATCTAAAATGGC AG-3'.

[0069] ApxIVA-N2b2 forward primer: 5'-CCGGAATTCATGGGCACTAAAATCACCCGTAGGATTGCG G-3'.

[0070] ApxIVA-N2b2 reverse primer: 5'-CCGCTCGAGTTAGCCCATTTGTGCAAAAGTACCGTCCG-3'.

[0071] ApxIVA-N2c2 forward primer: 5'-CCGGAATTCATGCCCTTAGCCCCTTACACTAAAAATGGC GTGG-3'.

[0072] ApxIVA-N2c2 reverse primer: 5'-CCGCTCGAGTTAGGCTAATGTCGCAAAACCGTGTGCAG-3'.

[0073] The remaining steps were performed in the same manner as described in (1) above. The annealing temperature for all three genes was 57°C, the extension time for N2a2 was 2 min 10 s, and the extension times for N2b2 and N2c2 were 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 an inclusion body state.

[0074] (3) Immunogenicity verification

[0075] Indirect ELISA was performed using N1, N2, N3, and N2c2 as coating antigens to detect their effectiveness in distinguishing between wild-type APP virus and the gene-deleted vaccine APP-HB-04M. Ten swine serum samples immunized with the gene-deleted vaccine APP-HB-04M were randomly selected (serum collected 70 days after secondary immunization of pigs in a farm). The results are shown in Table 1. The N1 and N3 proteins expressed in inclusion body form did not significantly distinguish between APP wild-type virus and gene-deleted vaccine serum samples. Although the N2c2 protein, which was extended by 327 bp, was expressed in a soluble manner, its immunogenicity did not increase significantly, and the background value of the gene-deleted vaccine serum sample was also higher.

[0076] Table 1. Results of N1, N2, N3, and N2c2 ELISA in differentiating gene-deleted seedling samples.

[0077]

[0078]

[0079] Example 2: Preparation of APP colloidal gold antibody test strip

[0080] (1) Sample pad pretreatment

[0081] Immerse the sample pads in the sample pad pretreatment solution for 4-7 minutes, then dry them for later use. The sample pad pretreatment solution is a 0.02M PBS buffer with pH=7.4 containing 10g / L NaCl and 0.5g / L ProClin 300 antibacterial agent.

[0082] (2) Preparation of colloidal gold pads

[0083] Colloidal gold pad pretreatment: Immerse the colloidal gold pad in the colloidal gold pad pretreatment solution for 4-7 minutes, then dry for later use. The colloidal gold pad pretreatment solution is a 0.02M PBS buffer at pH 7.4 with 5 g / L casein, 10 g / L BSA, 50 g / L trehalose, and 0.5 g / L ProClin300 antibacterial agent added.

[0084] Preparation of colloidal gold: 1 L of ultrapure water was added to a round-bottom flask and heated to boiling. 10 mL of 1% chloroauric acid solution was added, and after 2 min, 10 mL of 1% trisodium citrate solution was added. At this point, the solution color gradually changed from black to wine red. Boiling was continued for 15 min, then heating was stopped, and the solution was cooled to room temperature to obtain the colloidal gold solution.

[0085] Take 1 mL of the above colloidal gold solution, add 6 μL of 0.2 mol / L K2CO3 buffer, add 40 μg apxIVA to truncate antigen N2. The amount of antigen used should be adjusted according to the actual results. In this example, N2 antigen is labeled with 6 μL of 0.2 mol / L K2CO3, and the amount of antigen used is 40 μg / mL. Add 1% BSA solution for blocking, react for 10 min, centrifuge at 10000 r / min for 10 min and discard the supernatant. Resuspend in 100 μL of phosphate suspension (phosphate suspension is 0.01M PBS buffer with pH=7.4 containing 1% BSA), resuspend, and fix on the colloidal gold pad at a gold spraying rate of 0.5 μL / mm. Dry at 37℃ for 2 h. Take another 1 mL of colloidal gold solution, add 4 μL of 0.2 mol / L K2CO3 solution, add chicken IgY labeled at a concentration of 20 μg / mL, block with 1% BSA, centrifuge at 10000 r / min for 10 min, remove the supernatant, suspend in 100 μL of phosphate suspension, resuspend, fix on the colloidal gold pad at a gold spraying rate of 0.5 μL / mm, and dry at 37℃ for 2 h to obtain a colloidal gold pad coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody.

[0086] (3) Membrane scratching:

[0087] C-line: Remove the NC membrane and equilibrate it for 1 hour at room temperature and normal humidity. After cleaning the instrument with ultrapure water, set the spray point parameters as follows: SHAPE:LINE, LENGTH (mm):300.0, SPEED (mm / s):50.0, ACC (mm / s2):1000, Y START (mm):16.5. Dilute the goat anti-chicken IgY antibody to 0.5 mg / mL and spray it as C-line.

[0088] T-line: After cleaning the instrument again with ultrapure water, set the spray point parameters as follows: SHAPE:LINE, LENGTH (mm):300.0, SPEED (mm / s):50.0, ACC (mm / s2):1000, Y START (mm):12.5. Dilute the N2 antigen to 1 mg / mL and spray it as a T-line.

[0089] (4) Assemble the test strip and add the desiccant. A schematic diagram of the APP colloidal gold antibody test strip structure and result interpretation is shown below. Figure 1 As shown.

[0090] Example 3: The effect of the combination of coated antigen and labeled antigen on the detection effect of the test strip

[0091] Test strips with different combinations of colloidal gold-labeled antigens and NC membrane T-line coated with different antigens (Table 2) were prepared according to the method in Example 2. The serum to be tested was diluted with sample diluent (0.01M PBS buffer containing 1% BSA at pH 7.4) at a volume ratio of 1:2. The solutions were added to the sample pad and reacted for 5 minutes. The color development was observed, and the results are shown in Table 3. When the colloidal gold pad was coated with gold-labeled N1 antigen, only group A4 showed a slight band after adding a positive serum sample; the other groups of test strips did not show any bands. When the colloidal gold pad was coated with gold-labeled N3 antigen, bands appeared in groups C1-C6 after adding both positive and negative serum samples; these were all invalid test strips. When the colloidal gold pad was coated with gold-labeled N2 antigen, bands appeared in groups B2, B3, and B4, with group B4 showing the most prominent band. Therefore, group B4 (colloidal gold pad coated with gold-labeled N2 antigen, T-line coated with 1.0 mg / mL N2 antigen) was selected for further research and development.

[0092] Table 2. Combinations of colloidal gold-labeled antigen and T-line coated antigen on test strips.

[0093]

[0094] Table 3. Detection results of the combination of T-line coated antigen and colloidal gold-labeled antigen on the test strips.

[0095]

[0096] Example 4: Detection of APP wild-type virus infected serum, gene-deleted vaccine APP-HB-04M immune serum and negative serum samples.

[0097] Clinical APP-positive swine serum (purchased from Wuhan Keqian Biotechnology Co., Ltd., hereinafter the same), SPF antibody-negative swine serum (purchased from Guangzhou Hongquan Biotechnology Co., Ltd., hereinafter the same), and swine serum immunized with the gene-deleted vaccine APP-HB-04M (serum collected 70 days after secondary immunization of pigs in a farm, hereinafter the same) were diluted 1:2 with sample dilution buffer (0.01M PBS buffer containing 1% BSA at pH=7.4). The diluted solution was then applied to the sample pad of the test strip prepared in Example 2 (or the test strip was inserted into the test strip card). The results after 5-10 minutes of reaction are as follows: Figure 8 and Figure 9 As shown in the figure. The results indicate that this test strip can effectively distinguish between APP-positive and APP-negative swine sera, and can also differentiate between swine serum samples vaccinated with the APP gene deletion vaccine, with a detection concordance rate of 100%.

[0098] Example 5: Performance Determination of APP Colloidal Gold Antibody Test Strip

[0099] (1) Sensitivity detection of the colloidal gold antibody test strip method of the present invention

[0100] APP-positive serum was serially diluted with PBS at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. The titer of each serially diluted serum was tested using the Actinobacillus pleuropneumoniae ApxIV-ELISA antibody detection kit (Wuhan Keqian Biotechnology Co., Ltd., hereinafter the same). At the same time, the test strips prepared in Example 2 (test strips inserted into test strip cards) were used to evaluate the sensitivity of the test strips.

[0101] The results are shown in Table 4 and Figure 10 As shown, when APP-positive serum is diluted serially, the test strip shows a color gradient change. When the serum is diluted to 1:64, a weak positive result can still be detected, which indicates that the test strip has good sensitivity.

[0102] Table 4 Sensitivity Detection Results

[0103]

[0104] (2) Specificity detection of the APP colloidal gold antibody test strip of the present invention

[0105] To evaluate the specificity of the test strip prepared in Example 2, positive sera for APP, Streptococcus suis (SS), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), African swine fever virus (ASFV), Mycoplasma hyopneumoniae (Mhp), and porcine circovirus type 3 (PCV3) were simultaneously detected, and the color development of the test strip was observed to determine the specificity of the test strip of the present invention.

[0106] The results are as follows Figure 11 As shown, except for APP positive serum, all other test results were negative, indicating that the test strip has good specificity and no cross-reaction with the above-mentioned disease antibodies.

[0107] (3) Shelf life detection of the APP colloidal gold antibody test strip of the present invention

[0108] To evaluate the shelf life of the test strips prepared in Example 2, they were stored for 0, 2, 4, and 6 months before being tested for APP clinical positive serum.

[0109] The results are as follows Figure 12 As shown, test strips stored for 0, 2, 4, and 6 months can all detect APP-positive serum, and their shelf life can reach more than 6 months.

[0110] (4) Repeatability test of the APP colloidal gold antibody test strip of the present invention

[0111] SPF swine serum, APP weakly positive and strongly positive serum were collected and tested 8-10 times using the same batch of test strips prepared according to the method described in Example 2. The test results were compared. The repeatability experiment of the test strips can verify the stability, accuracy and reliability of the test strips.

[0112] Repeatability test results as follows Figure 13 As shown, the results were: the T line of SPF porcine serum was not colored, the T line of APP weakly positive serum was light pink, and the T line of APP positive serum was red. The color development was basically consistent in 8-10 replicates with the same serum, indicating that the test strip of this invention has good repeatability.

[0113] Example 6: Clinical application and concordance rate detection of APP colloidal gold antibody test strips

[0114] One hundred clinical swine serum samples (from a pig farm in Guangxi Zhuang Autonomous Region) were diluted 1:2 with sample diluent and then dropped into the sample pad of the test strip prepared in Example 2. At the same time, the serum titer was tested using the ApxIV-ELISA antibody detection kit (Keqian) to observe the accuracy of the test results.

[0115] The test results are shown in Table 5 and Figure 14As shown, the positive concordance rate of the test strip was 100%, the negative concordance rate was 87.0%, and the total concordance rate was 85 / 100 (85%). The results show that the test strip of the present invention has a high concordance rate and has clinical application value.

[0116] Table 5. Clinical test results of the test strips and comparison with those of commercial ELISA kits.

[0117]

[0118] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A soluble apxIVA truncated protein of Actinobacillus pleuropneumoniae as an antigen in the preparation of a colloidal gold antibody test strip for Actinobacillus pleuropneumoniae, characterized in that: The amino acid sequence of the apxIVA truncated protein is shown as SEQ ID NO.

3. ​ 2. Use according to claim 1, characterized in that: The apxIVA truncated protein is prepared by a method comprising the following steps: (1) cloning a nucleotide encoding the apxIVA truncated protein into pET-30a to obtain a recombinant expression plasmid; (2) transforming the recombinant expression plasmid into E. coli BL21 to obtain a recombinant expression strain; (3) culturing and IPTG inducing expression of the recombinant expression strain, breaking the bacterial cells after expression and centrifuging, and purifying the supernatant to obtain the apxIVA truncated protein.

3. Use according to claim 2, characterized in that: In step (1), the sequence of the nucleotide encoding the apxIVA truncated protein is shown as SEQ ID NO.

4.

4. Use according to claim 2, characterized in that: Step (1) is: using porcine pleuropneumonia actinobacillus genomic DNA as a template, and performing PCR amplification with the following primers: ApxIVA-N2 forward primer: 5'-CCGGAATTCATGGAGAACCTGTACTTCCAAGGGCCTAAGGCGGATCCTAAGCGG-3', ApxIVA-N2 reverse primer: 5'-CCGCTCGAGTTACCAGCCCGTTGCGGTACGAAT-3'; The amplification product and pET-30a are subjected to EcoRI / Xhol double enzyme digestion and ligation, the ligation product is transformed into DH5α competent cells, and the recombinant expression plasmid is obtained after identification.

5. A colloidal gold antibody test strip for detecting Actinobacillus pleuropneumoniae, characterized in that: The sample pad, the colloidal gold pad, the NC membrane, and the water absorption filter paper are sequentially and adjacently adhered to the bottom plate. The colloidal gold pad is coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody.

6. The colloidal gold antibody test strip for detecting Actinobacillus pleuropneumoniae according to claim 5, characterized in that: The colloidal gold antibody test strip is used for distinguishing APP wild-type infection from gene deletion vaccine APP-HB-04M immunization.

7. The method of producing the colloidal gold antibody test strip for Actinobacillus pleuropneumoniae of claim 5 or 6, characterized in that, The following steps are included: (1) Soak the sample pad in the sample pad pretreatment solution until it is evenly soaked, and then dry it for standby use; (2) Soak the colloidal gold pad in the colloidal gold pad pretreatment solution until it is evenly soaked, and then dry it for standby use; (3) Fix the gold-labeled apxIVA truncated protein and the gold-labeled chicken IgY on the colloidal gold pad to obtain a colloidal gold pad coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody; (4) Line-coat the goat anti-chicken IgY solution and the apxIVA truncated protein solution on the NC membrane respectively and dry it to obtain an NC membrane containing C line and T line; (5) Assemble the treated sample pad, the colloidal gold pad coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody, the NC membrane containing C line and T line, the water absorption filter paper, and the bottom plate in order to obtain the colloidal gold antibody test strip.

8. The method for preparing the colloidal gold antibody test strip of Actinobacillus pleuropneumoniae according to claim 7, characterized in that: The sample pad pre-treatment liquid in step (1) is 0.02M PBS buffer solution with pH=7.4, to which 10g / L NaCl and 0.5g / L ProClin 300 bacteriostatic agent are added; the sample pad pre-treatment liquid in step (2) is 0.02M PBS buffer solution with pH=7.4, to which 5g / L casein, 10g / L BSA, 50g / L trehalose and 0.5g / L ProClin 300 bacteriostatic agent are added.

9. The method of using the colloidal gold antibody test strip for Actinobacillus pleuropneumoniae of claim 5 or 6, characterized in that, The method comprises the following steps: The serum to be tested is diluted with the sample diluent at a volume ratio of 1:2, and then dropped into the sample pad; the result is observed after 5-10 minutes; if the C line and the T line develop color, the serum to be tested is positive serum of the wild porcine contagious pleuropneumonia virus; if the C line develops color and the T line does not develop color, the serum to be tested is the immune serum or negative serum of the gene deletion vaccine APP-HB-04M; and the remaining color development conditions are invalid.

10. The method of using the colloidal gold antibody test strip for Actinobacillus pleuropneumoniae according to claim 9, characterized in that: The sample diluent is 0.01M PBS buffer solution with pH=7.4, containing 1% BSA.

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