Colloidal gold test strip for distinguishing porcine actinobacillus pleuropneumoniae wild virus infection and vaccine immunity and application of colloidal gold test strip

Abstract

The invention discloses a colloidal gold test strip for distinguishing porcine actinobacillus pleuropneumoniae wild virus infection and vaccine immunity and application. According to the test strip, ApxIVA truncated protein with the amino acid sequence shown as SEQ ID NO.3 serves as an antigen, the truncated protein can be subjected to soluble expression in escherichia coli, and the structure and the function of the truncated protein are close to those of natural apxIVA. The test strip comprises a sample pad, a colloidal gold pad, an NC membrane, water absorption filter paper and a bottom plate, the colloidal gold pad is coated with gold-labeled apxIVA truncated protein and a gold-labeled chicken IgY antibody, and a goat anti-chicken IgY antibody and apxIVA truncated protein are fixed on a C line and a T line of the NC membrane. The method can be used for distinguishing APP wild strain infection and gene deletion seedling APP-HB-04M immune serum samples.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and relates to a colloidal gold test strip for distinguishing wild-type infection of porcine Actinobacillus pleuropneumoniae from vaccine immunity and an application thereof. Background Art

[0002] Porcine Pleuropneumonia (PCP) is a serious bacterial respiratory disease of pigs caused by Actinobacillus pleuropneumoniae (APP), characterized by acute hemorrhagic fibrinous pneumonia and chronic fibrinous necrotizing pleuropneumonia. Acute infection often causes bleeding from the mouth and nose and sudden death of pigs; chronic infection of pigs often becomes a hidden source of infection. Pigs infected with APP are in a sub-healthy state for a long time and are very likely to be infected by other pathogens, causing stunted development or even death of pigs, which has brought huge economic losses to my country's pig industry.

[0003] According to the differences in capsular antigens, APP can be divided into 19 serotypes, and ApxIV is considered to be the target protein for diagnosing all serotype strains of APP. At present, a variety of APP serological and nucleic acid diagnostic products based on apxIVA have been developed. These products all have the disadvantages of requiring cumbersome operating steps or rigidly requiring instruments and equipment, experimental skills, etc. Designated veterinary workstations can provide inspections but require long sample transportation, resulting in lower detection rates and more infections. Products that can provide veterinarians and farmers (especially in underdeveloped and remote areas) with on-site, instant detection, simple operation, saving sample delivery time, and exempting instruments and equipment are urgently needed to be developed. APP colloidal gold antibody test strips are an ideal detection method. However, to date, no related products have been successfully launched worldwide. In addition, the applicant has successfully developed an APP gene-deficient 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 good protection effects. However, the differential diagnostic method for distinguishing wild-type virus infection from immunity with the gene-deficient vaccine APP-HB-04M has not yet been established. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a colloidal gold antibody test strip for porcine Actinobacillus pleuropneumoniae (APP) using a soluble apxIVA truncated protein as an antigen. The apxIVA truncated protein can be expressed soluble in Escherichia coli and only carries a His tag (about 0.84 kDa) and an S tag (about 2 kDa). Its structure and function are close to those of natural apxIVA.

[0005] Another object of the present invention is to provide a colloidal gold antibody test strip for distinguishing APP wild-type virus infection from APP-HB-04M gene-deficient vaccine immunity. The test strip has a simple structure, low cost, and a simple and time-saving and labor-saving detection process. Only 25 μL of pig serum is required to make a rapid diagnosis in 5-10 minutes, without the need for a volley gun, a circulating heater, an electrophoresis instrument, or other instruments and equipment.

[0006] The present invention also aims to provide a preparation method and a use method of the test strip.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The invention discloses an application of a soluble apxIVA truncated protein of Actinobacillus pleuropneumoniae as an antigen in the preparation of an APP colloidal gold antibody detection test strip. The amino acid sequence of the apxIVA truncated protein is shown in SEQ ID NO.3.

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

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

[0011] (2) transforming the recombinant expression plasmid into Escherichia coli BL21 to obtain a recombinant expression strain;

[0012] (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.

[0013] 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.

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

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

[0016] 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.

[0017] A colloidal gold antibody test strip for APP, which is a colloidal gold antibody test strip for distinguishing APP wild strain infection from gene-deficient vaccine APP-HB-04M immunization, comprising a sample pad, a colloidal gold pad, a nitrocellulose (NC) membrane, a water-absorbing filter paper, and a bottom plate, wherein the sample pad, the colloidal gold pad, the NC membrane, and the water-absorbing filter paper are overlapped and adhered to the bottom plate in sequence. The colloidal gold pad is coated with a gold-labeled apxIVA truncated protein and a gold-labeled chicken IgY antibody; the NC membrane is sequentially arranged with C lines and T lines, the C line is fixed with sheep anti-chicken IgY antibody, and the T line is fixed with apxIVA truncated protein; 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 detection test strip comprises the following steps:

[0019] (1) Soak the sample pad in a 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 solution with a pH of 7.4 and 10 g / L NaCl and 0.5 g / L ProClin 300 antibacterial agent added thereto.

[0020] (2) Soak the colloidal gold pad in a 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 solution with a pH of 7.4 to which 5g / L casein, 10g / L BSA, 50g / L trehalose and 0.5g / L ProClin 300 antibacterial agent are added.

[0021] (3) Immobilizing colloidal gold-labeled apxIVA truncated protein and colloidal gold-labeled chicken IgY on a 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 are streaked onto the NC membrane and dried to obtain an NC membrane containing lines C and T. The concentrations of the goat anti-chicken IgY solution and the apxIVA truncated protein solution are preferably 0.5 mg / mL and 1.0 mg / mL, respectively.

[0023] (5) The treated sample pad, the colloidal gold pad coated with the gold-labeled apxIVA truncated protein and the gold-labeled chicken IgY antibody, the nitrocellulose membrane containing the C line and the T line, the absorbent filter paper and the bottom plate are assembled in sequence to obtain a colloidal gold antibody detection test strip.

[0024] The method for using the APP colloidal gold antibody test strip comprises the following steps: diluting the serum to be tested with the sample diluent in a volume ratio of 1:2, dripping into the sample pad, observing the results after 5-10 minutes, if the C line and the T line are colored, the serum to be tested is a wild-type positive serum of porcine contagious pleuropneumonia, if the C line is colored and the T line is not colored, the serum to be tested is a gene-deficient vaccine APP-HB-04M immune serum or a negative serum, and the other color development conditions are invalid. The sample diluent is preferably a 0.01M PBS buffer with a pH of 7.4 containing 1% BSA.

[0025] Compared with the prior art, the present invention has the following advantages and effects:

[0026] 1. The present invention obtains a soluble expressed N2 protein by screening apxIVA truncated protein. The recombinant protein only carries one His tag (about 0.84 kDa) and one S tag (about 2 kDa) at the N-terminus, and its 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, which is suitable for large-scale production. The results of the present invention show that the inclusion body expression of apxIVA may be one of the reasons why the APP colloidal gold test strip product has not been launched yet.

[0027] 2. The test strip of the present invention has a simple structure, and the detection process is easy to operate, time-saving and labor-saving. Only 25 μL of pig serum is needed to make a test within 5-10 minutes, and it can distinguish between APP wild strain infection and gene-deficient vaccine immune serum samples, and no need for equipment such as volley guns, circulating heaters, and electrophoresis. It is very suitable for on-site and remote area detection, has a low threshold for use, and can be mass-produced and used. At the same time, this product is the first APP colloidal gold diagnostic test strip product in the world. It is not only of great significance in vaccine strain differential diagnosis and pig prevention and control breeding, but also has broad application prospects and practical value in on-site diagnosis of APP, especially in remote areas lacking experimental equipment and experimental skills. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The structure of the APP colloidal gold antibody test strip of the present invention (A) and the schematic diagram of positive and negative determination (B).

[0029] Figure 2 This is the bioinformatics analysis result of APP apxIVA in Example 1.

[0030] Figure 3 Schematic diagram of the N-terminal truncation of ApxIVA in Example 1 (including primary screening and secondary screening).

[0031] Figure 4 These are the amplification results of the APP apxIVA N1, N2, and N3 target genes in Example 1.

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

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

[0034] Figure 7 The western blot results of the recombinant N1, N2, and N3 truncated proteins in Example 1 are shown.

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

[0036] Fig. 9 The figure is the test result of the test strip of the present invention on the immune serum sample of the gene-deficient vaccine APP-HB-04M.

[0037] Fig.10 It is the sensitivity test result of the test strip of the present invention.

[0038] Fig.11 It is the specific detection result of the test strip of the present invention.

[0039] Fig.12 It is the shelf life test result of the test strip of the present invention.

[0040] Fig.13 It is the repeatability test result of the test strip of the present invention.

[0041] Fig.14 These are some clinical serum test results of the test strips of the present invention. DETAILED DESCRIPTION

[0042] In order to more clearly explain the technical content of the present invention, it is described in detail in conjunction with specific embodiments and drawings. Obviously, the enumerated embodiments are only preferred implementation schemes of the present technical solution, and other technical solutions that can be obviously derived by technicians in this field based on the disclosed technical content still belong to the protection scope of the present invention.

[0043] like Figure 1As shown, the APP colloidal gold antibody detection test strip of the present invention specifically includes a sample pad, a colloidal gold pad, an NC membrane, a water-absorbing filter paper and a PCV bottom plate, and the control line (C line) and the test line (T line) are arranged in sequence on the NC membrane, and the C line is fixed with sheep anti-chicken IgY, and the T line is fixed with apxIVA truncated N2 protein; 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, reacts with the colloidal gold labeling reagent on the colloidal gold pad, and then moves to the area where the antigen is fixed, the color development result can be observed by the naked eye. If the C line and the T line react, it means that this sample is serum infected with APP wild strain; if only the C line reacts, it means that the sample is serum immunized with the gene-deficient seedling APP-HB-04M or APP negative serum; if the C line does not react, it means that this test strip is invalid.

[0044] Example 1 Screening of soluble expressed ApxIVA protein

[0045] (1) Initial screening of ApxIVA protein

[0046] ① Construction and identification of recombinant expression plasmid

[0047] 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.

[0048] According to the nucleotide sequence of 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 (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.

[0055] 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.

[0056] ②Expression and purification of target gene

[0057] 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.

[0058] 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.

[0059] 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.

[0060] ③Western blot analysis

[0061] 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.

[0062] Western blot results are shown in Figure 7 , indicating 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 plasmid

[0065] 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.

[0066] According to the nucleotide sequence of 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 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.

[0074] (3) Immunogenicity verification

[0075] Indirect ELISA was performed using N1, N2, N3 and N2c2 as coating antigens to test their effectiveness in distinguishing APP wild strains from the gene-deficient vaccine APP-HB-04M. Ten sera of pigs immunized with the gene-deficient vaccine APP-HB-04M were randomly selected (sera collected from pigs in the farm 70 days after secondary immunization). The test results are shown in Table 1. 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 327 bp, was expressed in a soluble form, but the immunogenicity did not increase significantly, and the background value of the gene-deficient vaccine serum samples increased.

[0076] Table 1 Results of ELISA for distinguishing gene-deficient seedling samples from N1, N2, N3, and N2c2

[0077]

[0078]

[0079] Example 2 Preparation of APP Colloidal Gold Antibody Detection Test Strips

[0080] (1) Sample pad pretreatment

[0081] The sample pad is placed in a sample pad pretreatment solution and soaked for 4-7 minutes, and then dried for later use. The sample pad pretreatment solution is a 0.02M PBS buffer solution with a pH of 7.4 and 10 g / L NaCl and 0.5 g / L ProClin 300 antibacterial agent added thereto.

[0082] (2) Preparation of colloidal gold pad

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

[0084] Preparation of colloidal gold: Add 1L ultrapure water to a round-bottom flask and heat to boiling, add 10mL 1% chloroauric acid solution, and 2 minutes later, add 10mL 1% trisodium citrate solution. At this time, the color of the solution gradually changes from black to wine red. Continue to boil for 15 minutes, then stop heating and cool to room temperature to obtain a colloidal gold solution.

[0085] Take 1mL of the above colloidal gold solution, add 6μL 0.2mol / L K2CO3 buffer, add 40μg apxIVA truncated antigen N2, the amount of antigen is adjusted according to the actual results, in this embodiment, N2 antigen is labeled with 6μL 0.2mol / L K2CO3, and the amount of antigen is 40μg / mL; add a final concentration of 1% BSA solution for blocking, react for 10min, centrifuge at 10000r / min for 10min, discard the supernatant, suspend with 100μL 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 0.5μL / mm gold spraying amount after resuspension, and dry at 37°C for 2h. Take another 1mL of colloidal gold solution, add 4μL of 0.2mol / L K2CO3 solution, add chicken IgY labeled at 20μg / mL, block with final concentration of 1% BSA, centrifuge at 10000r / min for 10min, remove the supernatant, resuspend with 100μL phosphate suspension, resuspend and fix on the colloidal gold pad at a gold spraying amount of 0.5μL / mm, dry at 37°C for 2h, and obtain a colloidal gold pad coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody.

[0086] (3) Film cutting:

[0087] Line C: Take out the NC membrane, equilibrate it at room temperature and normal humidity for 1 hour, clean 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 sheep anti-chicken IgY antibody to 0.5 mg / mL, and spray as line C.

[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 as the T line.

[0089] (4) Assemble the test strips and add desiccant for later use. Figure 1 shown.

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

[0091] According to the method in Example 2, test strips with colloidal gold labeled different antigens and NC membrane T lines coated with different antigen combinations (Table 2) were prepared, and the serum to be tested and the sample diluent (0.01M PBS buffer with pH=7.4 containing 1% BSA) were diluted in a volume ratio of 1:2, dropped into the sample pad and reacted for 5 minutes, and the color development was observed. The results are shown in Table 3. When the colloidal gold pad was coated with gold-labeled N1 antigen, after the positive serum sample was added, only the A4 group showed slight bands, and the test strips of the other groups did not show bands. When the colloidal gold pad was coated with gold-labeled N3 antigen, positive serum samples and negative serum samples were added, and the C1-C6 groups of test strips all showed bands, all of which were invalid test strips. When the colloidal gold pad was coated with gold-labeled N2 antigen, bands appeared in groups B2, B3, and B4, among which group B4 was the most obvious. 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 subsequent research and development.

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

[0093]

[0094] Table 3 Test results of the combination of T-line coated antigen and colloidal gold labeled antigen

[0095]

[0096] Example 4 Detection of APP wild virus infected serum, gene-deficient seedling APP-HB-04M immune serum and negative serum samples

[0097] Clinical APP positive pig serum (purchased from Wuhan Keqian Biological Co., Ltd., the same below), SPF antibody negative pig serum (purchased from Guangzhou Hongquan Biotechnology Co., Ltd., the same below) and gene-deficient vaccine APP-HB-04M immunized pig serum (serum collected 70 days after secondary immunization of pigs in the farm, the same below) were diluted 1:2 with sample diluent (0.01M PBS buffer with pH = 7.4 containing 1% BSA), and dropped into the sample pad of the test strip prepared in Example 2 (or the test strip was loaded into the test paper card). The results after 5-10 minutes of reaction are as follows: Figure 8 and Fig. 9 The results show that the test strip can well distinguish APP positive serum from negative pig serum, and can distinguish the serum samples of pigs vaccinated with APP gene deletion vaccine, and the detection compliance rate is 100%.

[0098] Example 5 Performance determination of APP colloidal gold antibody test strips

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

[0100] APP positive serum was selected and diluted with PBS at multiple ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. The titer of each gradient dilution serum was tested using a porcine actinobacillus pleuropneumoniae ApxIV-ELISA antibody detection kit (Wuhan Keqian Biological Co., Ltd., the same below). At the same time, the test strip prepared in Example 2 (the test strip was loaded into a test strip card) was used for detection to evaluate the sensitivity of the test strip.

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

[0102] Table 4 Sensitivity test results

[0103]

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

[0105] In order to evaluate the specificity of the test strip prepared in Example 2, 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) positive serum were detected simultaneously, 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 Fig.11 As shown, except for the APP positive serum, the other test results were all negative, indicating that the test strip had good specificity and had no cross reaction with the above disease antibodies.

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

[0108] In order to evaluate the shelf life of the test strip prepared in Example 2, the test strip was stored for 0, 2, 4, and 6 months and then tested for APP clinical positive serum.

[0109] The results are as follows Fig.12 As shown, test strips stored for 0, 2, 4, and 6 months can 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 pig serum, APP weak positive and strong positive serum were taken and repeated tests were performed 8-10 times using the test strips of the same batch prepared according to the method described in Example 2, and 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 such as Fig.13 As shown, the results are: SPF pig serum T line does not show color, APP weak positive serum T line is light pink, APP positive serum T line is red. The same serum, 8-10 times repeated color development is basically the same, indicating that the test strip of the present invention has good repeatability experimental results.

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

[0114] 100 portions of clinical pig sera (from a pig farm in Guangxi) were diluted with sample diluent at 1:2 and then dropped onto 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 Fig.14As shown, the positive compliance rate of the test strip is 100%, the negative compliance rate is 87.0%, and the total compliance rate is 85 / 100 (85%). The results show that the test strip of the present invention has a high compliance rate and has clinical application value.

[0116] Table 5 Clinical test results of test strips and comparison with commercial ELISA kit test results

[0117]

[0118] 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. Use of a soluble apxIVA truncated protein of porcine Actinobacillus pleuropneumoniae as an antigen in the preparation of a colloidal gold antibody detection test strip for porcine Actinobacillus pleuropneumoniae, characterized in that: The amino acid sequence of the apxIVA truncated protein is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The apxIVA truncated protein is prepared by a method comprising the following steps: (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.

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

4.

4. The use according to claim 2, characterized in that: Step (1) is: using the genomic DNA of Actinobacillus pleuropneumoniae as a template, PCR amplification is performed using 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. A colloidal gold antibody test strip for porcine Actinobacillus pleuropneumoniae, characterized in that: It includes a sample pad, a colloidal gold pad, an NC membrane, a water-absorbing filter paper, and a bottom plate, wherein the sample pad, the colloidal gold pad, the NC membrane, and the water-absorbing filter paper are overlapped and adhered to the bottom plate in sequence; The colloidal gold pad is coated with gold-labeled apxIVA truncated protein and gold-labeled chicken IgY antibody; the NC membrane is provided with C line and T line in sequence, the C line is fixed with sheep anti-chicken IgY antibody, and the T line is fixed with apxIVA truncated protein; the amino acid sequence of the apxIVA truncated protein is shown in SEQ ID NO.

3.

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

7. The method for preparing the colloidal gold antibody detection test strip for Actinobacillus pleuropneumoniae according to claim 5 or 6, characterized in that: The following steps are involved: (1) Soak the sample pad in the sample pad pretreatment solution evenly and then dry it for later use; (2) soaking the colloidal gold pad in the colloidal gold pad pretreatment solution evenly and then drying it for later use; (3) fixing the colloidal gold-labeled apxIVA truncated protein and the colloidal gold-labeled chicken IgY on the colloidal gold pad to obtain a colloidal gold pad coated with the gold-labeled apxIVA truncated protein and the gold-labeled chicken IgY antibody; (4) streaking and coating the sheep anti-chicken IgY solution and the apxIVA truncated protein solution on the NC membrane and drying them to obtain a NC membrane containing C lines and T lines; (5) The treated sample pad, the colloidal gold pad coated with the gold-labeled apxIVA truncated protein and the gold-labeled chicken IgY antibody, the nitrocellulose membrane containing the C line and the T line, the absorbent filter paper and the bottom plate are assembled in sequence to obtain a colloidal gold antibody detection test strip.

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

9. The method for using the colloidal gold antibody detection test strip for Actinobacillus pleuropneumoniae according to claim 5 or 6, characterized in that: The following steps are involved: Dilute the test serum and sample diluent in a volume ratio of 1:2, drop into the sample pad, and observe the results after 5-10 minutes. If the C line and T line are colored, the test serum is a positive serum for porcine contagious pleuropneumonia wild virus. If the C line is colored and the T line is not colored, the test serum is the gene-deficient vaccine APP-HB-04M immune serum or negative serum. The other color development conditions are invalid.

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

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