An ELISA antibody detection kit for Pasteurella multocida capsular type A in cattle, its preparation method and application

Abstract

The present invention belongs to the technical field of pathogen detection, and specifically relates to an ELISA antibody detection kit for bovine Pasteurella multocida capsule type A, and its preparation method and application. The gene with the sequence shown in SEQ ID NO.1 is integrated into an expression vector and transferred into a host bacterium; the host bacterium is induced to express and purified to obtain a PmA153 recombinant antigen protein with the sequence shown in SEQ ID NO.2; PmA153 is coated on a solid phase carrier, and the detection method of bovine Pasteurella multocida capsule type A antibodies in the sample can be used to detect the bovine Pasteurella multocida capsule type A antibodies, thereby achieving the diagnostic purpose. The kit provided by the present invention does not use Pasteurella multocida as an antigen source, has good biosafety and low production costs, has good specificity for the detection of bovine Pasteurella multocida capsule type A antibodies, can accurately diagnose bovine Pasteurella multocida capsule type A pneumonia, and has important clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen detection, and particularly relates to a bovine Pasteurella multocida capsule type A ELISA antibody detection kit and a preparation method and application thereof. Background Art

[0002] Pasteurella multocida (Pm) capsular type B is the pathogen that causes hemorrhagic septicemia in cattle in my country. Hemorrhagic septicemia is an infectious disease characterized by high fever, pneumonia, acute gastroenteritis, and extensive bleeding in internal organs. It has been prevalent in my country for decades and continues to occur sporadically or endemically in many areas. In 2008, a bovine respiratory infection characterized by fibrinous pneumonia emerged on Chinese cattle farms and became prevalent in several provinces and municipalities, including Heilongjiang, Gansu, Guizhou, Chongqing, and Sichuan. Clinical manifestations of the disease are characterized by lethargy, loss of appetite, cough, runny nose, elevated temperature, and weight loss. The main pathological anatomical changes are in the lungs, showing widespread congestion and scattered abscesses of varying sizes, acute, subacute, or chronic purulent fibrinous exudates and necrosis, and edema and suppuration of interlobular septa. In severe cases, adhesions between the pericardium and pleura, and fibrinous or purulent pleurisy may occur. The Harbin Veterinary Research Institute and other institutions isolated and identified the pathogen in diseased samples from several provinces and cities, and conducted animal regression tests, and determined that the pathogen was bovine Pasteurella multocida capsular type A.

[0003] Pasteurella multocida capsular types A and B differ significantly. For example, type A bacteria grow in the lungs of cattle and are virtually undetectable in the blood of sick cattle. Therefore, PCR testing of serum samples for pathogen detection is not feasible, and ELISA antibody testing is the preferred method. However, because many genes of type A capsular type B of Pasteurella multocida share high homology with those of type B, cross-reactivity between antigens makes it difficult to identify a specific protein antigen for ELISA testing. Traditional capsular antigens use type A-specific capsular antigens, but these cannot be expressed in large quantities in genetically engineered bacteria such as Escherichia coli. They must be extracted from pathogenic bacteria cultures, resulting in low yields and potential biosafety concerns. Furthermore, quantification and coating of polysaccharides are significantly more difficult than with genetically engineered proteins. Therefore, using genetically engineered proteins makes it easier to prepare and manufacture ELISA antibody test kits than using capsular antigens. Summary of the Invention

[0004] The present invention aims to provide an ELISA antibody detection kit for bovine Pasteurella multocida capsule type A, so as to solve the technical problem in the prior art of lacking a specific antigen for bovine Pasteurella multocida capsule type A that can be prepared in large quantities.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A bovine Pasteurella multocida capsule type A ELISA antibody detection kit comprises a solid phase carrier coated with PmA153; the amino acid sequence of PmA153 is shown in SEQ ID NO.2.

[0007] This solution also provides an application of an antigen protein in the preparation of a kit for specifically detecting bovine Pasteurella multocida capsule type A, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] This solution also provides a method for preparing a bovine Pasteurella multocida capsular type A ELISA antibody detection kit, comprising the steps of preparing a PmA153-coated solid phase carrier:

[0009] Obtaining the S1 antigen protein: Integrate the gene with the sequence shown in SEQ ID NO.1 into the expression vector to obtain a recombinant plasmid; transfer the recombinant plasmid into the host bacteria to induce the host bacteria to express the antigen protein; then disrupt the bacteria, centrifuge and obtain the supernatant and purify to obtain PmA153.

[0010] S2 coating: Prepare a coating solution containing PmA153, allow the coating solution to contact and incubate with the solid phase carrier, and then perform a blocking treatment to obtain a PmA153-coated solid phase carrier.

[0011] The principles and advantages of this technical solution are:

[0012] By screening numerous databases, including VISTA and NCBI, and combining experimental analysis, the inventors obtained a recombinant protein, PmA153, that specifically detects antibodies against bovine Pasteurella multocida capsular type A. The pET30a-PmA153 plasmid was constructed. The PmA153 recombinant protein was successfully expressed in Escherichia coli and purified using a nickel column to achieve a high protein purity. Detection of the PmA153 protein can specifically detect bovine Pasteurella multocida capsular type A, allowing for more targeted treatments based on the test results. Using the PmA153 antigen protein, a series of immune-reaction-based detection methods and kits for the specific detection of bovine Pasteurella multocida capsular type A strains can be established, including but not limited to various types of ELISA and immunochromatography. For example, based on the PmA153 antigen protein, antibodies can also be determined using the colloidal gold method, the latex agglutination method, and the countercurrent electrophoresis and agar diffusion methods. These methods differ only in their specific implementation methods, but are all based on the PmA153 antigen protein.

[0013] As an example, the inventors developed an ELISA kit for detecting bovine Pasteurella multocida capsular type A antibodies using the PmA153 recombinant protein. The kit demonstrated ideal detection specificity, stability, and sensitivity. The inventors further applied the kit developed in this protocol to clinical testing, achieving excellent results.

[0014] Furthermore, a bovine Pasteurella multocida capsule type A ELISA antibody detection kit also includes a standard positive control serum and a standard negative control serum; the standard positive control serum is obtained by immunizing cattle with a bovine Pasteurella multocida capsule type A strain; the standard negative control serum is the serum of cattle that are not immunized and not infected with Pasteurella multocida capsule type A.

[0015] Furthermore, a bovine Pasteurella multocida capsule type A ELISA antibody detection kit also includes an enzyme-labeled secondary antibody, a color developing solution, a stop solution, a concentrated washing solution and a sample diluent.

[0016] Furthermore, the enzyme-labeled secondary antibody is rabbit anti-bovine IgG labeled with horseradish peroxidase; the color developing solution is a two-component TMB color developing solution; the stop solution is a sulfuric acid solution; the concentrated washing solution is a PBST washing solution; and the sample diluent is a PBS solution containing bovine serum albumin, Tween-20, EDTA and sodium chloride.

[0017] Furthermore, a bovine Pasteurella multocida capsule type A antibody detection kit is provided, wherein the judgment criteria are: when the S / P value of the sample to be tested is ≥0.161, it is judged as positive; when the S / P value of the sample to be tested is <0.161, it is judged as negative; the calculation method of the S / P value is: (sample OD 450 Value - Standard negative control serum OD 450 mean) / (standard positive control serum OD 450 Mean - standard negative control serum OD 450 mean); OD 450 Refers to the optical density at 450nm wavelength; and the standard positive control serum OD 450 The values were all ≥2.0, and the standard negative control serum OD 450 The values are all <0.25.

[0018] Furthermore, in obtaining the S1 antigen protein, the expression vector was pET30a; the reagent for inducing the host bacteria to express the antigen protein was isopropyl-β-D-thiogalactoside; the host bacteria was Rosseta DE3; and the purification method was purification through Ni-NTA Superflow Cartridges His affinity chromatography column.

[0019] Furthermore, in S2 coating, the content of PmA153 in the coating solution was 10 μg / mL, and the incubation conditions were 4° C. for 8-12 h; the blocking treatment was performed using a protein-free blocking buffer at 37° C. for 2 h.

[0020] Furthermore, a method for preparing a bovine Pasteurella multocida capsular type A ELISA antibody detection kit also includes a step of preparing a standard positive control serum: healthy cattle are immunized with an inactivated vaccine of a bovine Pasteurella multocida type A strain, a booster immunization is performed 14 days later, and serum is collected one month after the booster immunization to obtain a standard positive control serum.

[0021] In summary, the beneficial effects of this technical solution are:

[0022] 1) The present invention provides a preparation and application method of an ELISA antibody detection kit for bovine Pasteurella multocida capsule type A. The coating antigen is a Pm153 recombinant protein antigen expressed by genetic engineering in Escherichia coli. Bovine Pasteurella multocida is not used as the source of antigen preparation, and the kit has good biosafety.

[0023] 2) The present invention provides a preparation and application method of an ELISA antibody detection kit for bovine Pasteurella multocida capsule type A. The coating antigen is a Pm153 recombinant protein antigen expressed by genetic engineering in Escherichia coli. Bovine Pasteurella multocida capsule antigen is not used for coating. The quantification is convenient and the coating method is simple. The coating stability is reliable and the repeatability is good.

[0024] 3) The present invention provides a preparation and application method of an ELISA antibody detection kit for bovine Pasteurella multocida capsular type A. The coating antigen is a Pm153 recombinant protein antigen expressed by genetically engineered Escherichia coli. Bovine Pasteurella multocida capsular antigen is not used for coating, so a higher antigen yield can be obtained, which is conducive to mass production.

[0025] 4) The present invention provides a method for preparing and using an ELISA antibody detection kit for bovine Pasteurella multocida capsular type A. The detection method has good specificity, repeatability, and stability. It can detect bovine Pasteurella multocida capsular type A serum, but not types B, D, E, or F.

[0026] 5) The present invention provides a preparation and application method of an ELISA antibody detection kit for bovine Pasteurella multocida capsule type A, which can accurately provide diagnosis and treatment for pneumonia caused by bovine Pasteurella multocida type A. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the pET30a-PmA153 plasmid structure in Example 1.

[0028] Figure 2This is a graph showing the agarose electrophoresis results of the pET30a-PmA153 plasmid PCR product in Example 1.

[0029] Figure 3 This is a diagram showing the results of SDS-PAGE electrophoresis analysis of the PmA153 recombinant protein before and after expression in Example 1.

[0030] Figure 4 This is a diagram showing the results of SDS-PAGE electrophoresis analysis of the purified Pm153 recombinant protein in Example 1.

[0031] Figure 5 This is a diagram showing the Western Blot identification results of the PmA153 recombinant protein antigen in Example 1. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0033] Example 1: Construction and expression of pET30a-PmA153 expression vector

[0034] (1) Gene design

[0035] A nucleotide fragment of PmA153 from the bovine Pasteurella multocida type A protein (GenBank: QGV31027.1) was extracted and optimized based on codon degeneracy and codon preferences in various organisms. The optimized nucleic acid sequence is shown in SEQ ID NO. 1, and the encoded amino acid is shown in SEQ ID NO. 2.

[0036] PmA153 nucleotide sequence (SEQ ID NO. 1, 5'→3')

[0037] atgaccgaggagaacaagggcaagcgctacttcctgtggtttatcctgttcatcctgtctatctatctgttcattactattcaggaacgtcgtggttactgcttcgataaacgt gcttacatccacgaactgtacaccgaacaggaactgatcgatcgtggcattgaatacgtcgtgagcactatgccgtccggtgtaatcaaaccggatggcacgatcaaggaagtaa aacgttatacttctgttgaagaatttaaacagatgaacccggcatgctgcactctgactacctttatcgatgaaggtggtgatggttacccggatgatgacggttatggctacgt gcgcatcgagtacctgcgtcactacgttgaaaacctgaaaccgtatcaccgtgttatctacctggaatacactccgtgcggtgaactgcgtgaagaggctgcattctccaaaaac

[0038] PmA153 amino acid sequence (SEQ ID NO. 2, N→C)

[0039] MTEENKGKRYFLWFILFILSIYLFITIQERRGYCFDKRAYIHELYTEQELIDRGIEYVVSTMPSGVIKPDGTIKEVKRYTSVEEFKQMNPACCTLTTFIDEGGDGYPDDDGYGYVRIEYLRHYVENLKPYHRVIYLEYTPCGELREEAAFSKN

[0040] (2) Construction of recombinant plasmid

[0041] The PmA153 gene was synthesized by Shanghai Bioengineering using conventional methods and connected to the pET30a vector, and then transformed into the TOP10 strain. The pET30a-PmA153 recombinant plasmid was constructed (see the attached plasmid structure). Figure 1 ).

[0042] (3) Transformation of recombinant plasmid

[0043] The TOP10 strain was inoculated into 100 mL of LB medium containing AMP (100 μg / mL) and cultured in a shaker at 37°C for 10 hours. The plasmid was extracted and transferred into the host strain Rosseta (DE3). 200 μl of the transformation product was spread on an LB agar plate containing AMP (100 μg / mL), and the plate was cultured in a 37°C incubator for 18 hours.

[0044] (4) Identification and screening of expressing bacteria

[0045] Pick a single colony from the plate and inoculate it into 10 mL of LB medium containing AMP (100 μg / mL). Place it in a shaker at 37°C and culture overnight to serve as the original bacterial solution. PCR amplify the bacteria using pET30a universal primers, and compare the target fragments of nucleic acid electrophoresis with the theoretical value. The results are shown in the attached figure. Figure 2 The PCR results showed that the target band was 798 bp, which was consistent with the theoretical value. The PCR product was sent to Shanghai Bioengineering for sequence determination and comparison.

[0046] Take 1 mL of the original bacterial solution and transfer it to 100 mL of LB liquid medium containing AMP (100 μg / mL) at a 1% inoculum volume, place it in a shaking incubator at 37°C and shake at 150 r / min. When the bacterial solution OD 600 When the value reaches about 0.6, take 1 mL and place it in an EP tube. Centrifuge at 3000 r / min for 1 minute to obtain the pre-induced bacteria.

[0047] Add an appropriate amount of IPTG to the remaining culture to induce protein expression. Incubate the culture in a shaker at 37°C, 150 rpm, for 6 hours. Take 1 mL of the bacterial solution and centrifuge it as above to obtain the induced bacteria. Perform SDS-PAGE electrophoresis on the bacteria before and after induction to identify protein expression. Then, select single colonies with good expression and preserve the strain. During the induction process, the final concentration of IPTG is between 0.25 and 0.5 mmol / L. In this specific example, the amount added is 0.5 mmol / L. (The final concentration of IPTG is 0.5 mmol / L).

[0048] (5) Protein expression and purification

[0049] Take the preserved strain, transfer 100mL of LB liquid culture medium containing AMP (100μg / mL) at 1% inoculation volume, place in a 37℃ shaker, shake at 150r / min for 8h, transfer 3000mL of LB liquid culture medium containing AMP (100μg / mL) at 1% inoculation volume. When the bacterial solution OD 600 When the value reaches about 0.6, add IPTG at an appropriate final concentration to induce protein expression, and incubate at 37°C in a shaker at 150 rpm for 6 hours. Centrifuge the culture at 6000 rpm at 4°C for 15 minutes, and collect the precipitate.

[0050] The precipitate was washed once with Buffer A, centrifuged, and the supernatant removed. The precipitate was then ultrasonically disrupted with 10 volumes of Buffer A (weight-to-volume ratio) at 300W for 3 seconds on, 3 seconds off, for 120 minutes. The supernatant was collected after centrifugation at 12,000 rpm for 15 minutes at 4°C. The Buffer A formula is as follows: 29.22 g NaCl, 20 mL 1 M Tris-HCl, pH 8.0, and 1000 mL with deionized water. Protein expression was assessed by SDS-PAGE electrophoresis. The results are shown in the attached figure. Figure 3 The results showed that the expressed protein was soluble.

[0051] The supernatant protein solution was purified using a Ni-NTA Superflow Cartridges His affinity chromatography column (referred to as the Ni column). The nickel column was equilibrated with 10 column volumes of Buffer A. The supernatant was loaded at a flow rate of 1 mL / min. Impurities were washed away with 5 column volumes of Buffer B at a flow rate of 1.5 mL / min. The target protein was eluted with Buffer C at a flow rate of 1.5 mL / min. The formula for Buffer B is as follows: 29.22 g NaCl, 20 mL 1 M Tris-HCl, pH 8.0, and 1.7 g imidazole, and the volume was adjusted to 1000 mL with deionized water. The formula for Buffer C is as follows: 29.22 g NaCl, 20 mL 1 M Tris-HCl, pH 8.0, and 34 g imidazole, and the volume was adjusted to 1000 mL with deionized water.

[0052] Take an appropriate amount of purified sample and determine the total protein content by Bradford method. SDS-PAGE electrophoresis was used to identify the protein purification status. The results are shown in the attached Figure 4 The results showed that the protein could be effectively purified by Ni column to obtain the target protein of 25KD.

[0053] (6) Western Blot

[0054] The purified protein was subjected to SDS-PAGE gel electrophoresis and electrophoresis at 60V for 1 hour to transfer the protein to a nitrocellulose membrane. 5% skim milk powder was used to block for 2 hours. A confirmed Pasteurella multocida type A positive serum (PmCQ2 strain secondary immune serum) was used as the primary antibody at a dilution of 1:500 and incubated at room temperature for 2 hours. Rabbit anti-bovine IgG was used as the secondary antibody at a dilution of 1:500 and incubated at room temperature for 2 hours. DAB colorimetry was used for color development. The results are shown in the attached figure. Figure 5 The results showed that the protein had good immunogenicity.

[0055] Example 2: Establishment of kit detection method

[0056] (1) Coating and blocking of antibody detection plates

[0057] The solid phase carrier was coated with purified PmA153 recombinant protein antigen at a concentration of 10 μg / mL at 4°C overnight, and then the solid phase carrier was coated with the Thermo Scientific TM The cells were blocked with Pierce protein-free blocking buffer at 37° C. for 2 h. In this embodiment, the solid phase carrier was a 96-well polystyrene ELISA plate, and 100 μL of purified PmA153 recombinant protein antigen solution or protein-free blocking buffer was added to each well.

[0058] (2) Preparation of control serum

[0059] Inactivated vaccines were prepared using the bacterial culture of bovine Pasteurella multocida capsular type A, B, D, E, and F strains (using conventional formaldehyde inactivation methods in the prior art). Each vaccine was administered to a group of healthy susceptible cattle weighing about 100 kg, aged 3 to 6 months (4 cattle / group). 4 mL of the vaccine (bacterial concentration 5 × 10 9 CFU / mL, 4 mL / head immunized). Sera from the vaccine group were collected one month later as positive sera. Cattle immunized with PmCQ2 received a booster immunization 14 days later, and serum was collected one month after the booster immunization as a positive control for the test kit. Sera from unimmunized Pasteurella multocida type A cattle were also collected as a negative control. Strain sources and numbers are shown in Table 1.

[0060] In addition, several clinically confirmed positive sera for bovine hemolytic Mannheimia, bovine heterologous Mannheimia, bovine mycoplasma, bovine viral diarrhea, and bovine foot-and-mouth disease were obtained for use in specific research.

[0061] Table 2: Strain sources and numbers

[0062] strain number Host Capsular serotype source PmCQ1 ox A Separation of Southwest University PmCQ2 ox A Separation of Southwest University PmCQ3 ox A Separation of Southwest University PmCQ4 ox A Separation of Southwest University PmCQ5 ox A Separation of Southwest University PmCQ6 ox A Separation of Southwest University PmCQ7 ox B Separation of Southwest University CVCC390 ox B China Veterinary Drug Administration PmAL1 ox B Chongqing Aolong Biological Products Co., Ltd. PmAL2 ox B Chongqing Aolong Biological Products Co., Ltd. PmAL3 ox B Chongqing Aolong Biological Products Co., Ltd. PmAL4 ox B Chongqing Aolong Biological Products Co., Ltd. CVCC391 ox B China Veterinary Drug Administration CVCC44502 ox B China Veterinary Drug Administration CVCC44702 ox B China Veterinary Drug Administration NCTC10323 ox B NCTC Culture Collection Center CVCC392 ox D China Veterinary Drug Administration CVCC393 ox E China Veterinary Drug Administration CVCC395 ox F China Veterinary Drug Administration

[0063] (3) Composition of the PmA153 kit

[0064] The components of the kit include a solid phase carrier coated with purified bovine Pasteurella multocida type A PmA153 recombinant protein antigen, standard positive control serum, standard negative control serum, enzyme-labeled secondary antibody, color development solution, stop solution, concentrated washing solution, sample diluent and serum dilution plate.

[0065] The specific details of each component of the kit are described as follows:

[0066] The solid phase carrier is a 96-well polystyrene ELISA plate.

[0067] The bovine Pasteurella multocida PmA153 recombinant protein antigen was purified after recombinant expression in Escherichia coli using the method of Example 1. The positive control serum was prepared using bovine Pasteurella multocida capsular type A serum. Specifically, healthy susceptible cattle were immunized with the PmCQ2 strain, followed by a booster immunization 14 days later. Serum was collected one month after the booster immunization to serve as the standard positive control serum. Information on the PmCQ2 strain can be found in the literature "Comparative Genomics Analysis of Two Different Virulent Bovine Pasteurella multocida Isolates, Huihui Du et al., International Journal of Genomics, 2016," and is a bovine Pasteurella multocida capsular type A strain known in the art. The PmCQ2 strain is used as an example for the purpose of illustrating the composition of the kit. In addition to this strain, other bovine Pasteurella multocida capsular type A strains can also be used to immunize cattle and obtain standard positive control serum. Using inactivated strains as vaccines to immunize animals to obtain antibody-containing serum is a conventional operating method in the existing technology. The kit of this scheme can be used to detect all Pasteurella multocida capsular type A strains (as demonstrated by the experimental data below), rather than detecting the PmCQ2 strain alone. Therefore, it is feasible to use bovine Pasteurella multocida capsular type A strains, including the PmCQ2 strain, to immunize cattle and obtain standard positive control serum.

[0068] The negative control serum was the serum of non-immunized and non-infected bovine Pasteurella multocida capsular type A.

[0069] The colorimetric solution is TMB colorimetric solution (two-component), a conventional reagent in the prior art. TMB is a substrate for horseradish peroxidase (HRP). Under the action of horseradish peroxidase, it reacts with the oxidant hydrogen peroxide to develop color. The intensity of the color is proportional to the activity of HRP, making it suitable for detecting HRP-labeled substances. The TMB colorimetric solution (two-component) consists of two parts: Solution A and Solution B. Solution A contains hydrogen peroxide and is packaged in a white bottle; Solution B contains 3,3',5,5'-tetramethylbenzidine (TMB) and is packaged in a brown bottle. More specifically, Solution A consists of 13.6g of sodium acetate, 1.6g of citric acid, and 0.3mL of 30% hydrogen peroxide, brought to 500mL with distilled water. Solution B consists of 0.2g of disodium EDTA, 0.95g of citric acid, 50mL of glycerol, and 0.15g of TMB powder (dissolved in 3mL of DMSO), brought to 500mL with distilled water. After adding the distilled water to 500mL and filtering and sterilizing, the mixture is dispensed into 30mL plastic bottles, 10mL per bottle.

[0070] The reaction termination solution is 2 mol / L H2SO4.

[0071] The concentrated washing solution is 25 times PBST washing solution, that is, phosphate buffered saline containing Tween-20.

[0072] The sample diluent was PBS containing 0.5% BSA, 0.05% Tween-20, 5 mmol / L EDTA, 0.5 mol / L NaCl, and pH 7.2.

[0073] The enzyme-labeled secondary antibody is a rabbit anti-bovine IgG labeled with horseradish peroxidase (HRP), which can be obtained through immunoreaction, screening, purification, and HRP conjugation using conventional methods. To achieve excellent production simplicity and reproducibility, this patent uses the commercial HRP Anti-Bovine Serum Albumin antibody (abcam, product number: ab7637).

[0074] Specifically, this embodiment designs the following specifications of the kit product (Table 2):

[0075] Table 2: Kit Composition

[0076] Kit components Specification quantity Coated board 96 wells / block 1 piece / box Serum dilution plate 96 wells / block 1 piece / box Negative control serum 30μL / vial 1 tube / box Positive control serum 30μL / vial 1 tube / box Sample diluent 50mL / bottle 1 bottle / box 25× PBST concentrated washing buffer 30mL / bottle 1 bottle / box Rabbit anti-bovine IgG enzyme-linked antibody HRP 50μL / vial 1 bottle / box Substrate color development solution 10mL / bottle 1 bottle / box Stop solution 10mL / bottle 1 bottle / box manual Double-sided 1 serving / box

[0077] (4) Detection method

[0078] The specific detection steps are as follows:

[0079] 1) Before the experiment begins, all reagents should be equilibrated to room temperature. Serum samples to be tested should be diluted with sample diluent at a volume ratio of 1:50 so that the absorbance of the diluted samples is within the detection range of the kit. Positive and negative control serum should also be diluted with sample diluent at a volume ratio of 1:50.

[0080] 2) Sample Addition: Set up blank wells, control wells, and test sample wells in a 96-well plate. Add 100 μL of sample diluent to the blank wells, 100 μL of diluted positive control serum to the positive control wells, 100 μL of diluted negative control serum to the negative control wells, and 100 μL of diluted test sample to the test sample wells. Be careful not to allow air bubbles to form in the wells when adding samples. Add each solution to the bottom of the plate wells, minimizing contact with the well walls. Gently shake to mix. Cover or film the plate and incubate at 37°C for 30 minutes. To ensure the validity of the results, use a fresh standard solution for each experiment.

[0081] 3) Washing: After step 2) incubation, discard and spin dry the liquid in the wells. Add 350 μL of washing solution to each well and soak for 4-5 minutes. Spin dry (or tap the wells to dry the liquid) and then wash a second time. Wash a total of 5 times.

[0082] 4) Add 100 μL of diluted enzyme-labeled secondary antibody (enzyme-labeled secondary antibody and sample diluent are diluted at a volume ratio of 1:100, diluted within ten minutes before use) to each well after washing in step 3) and incubate at 37° C. for 30 minutes.

[0083] 5) Washing: After incubation in step 4), discard and spin dry the liquid in the wells, and wash the wells 5 times using the same method as in step 3).

[0084] 6) Color development: Add 100 μL of color development solution to each well and incubate at 37°C in the dark for 10 minutes.

[0085] 7) Stop the reaction: Add 50 μL of stop solution to each well to stop the reaction. The blue color will turn yellow immediately.

[0086] 8) Determination: After adding the stop solution, the optical density (OD) of each well was measured in sequence using a microplate reader at a wavelength of 450 nm. 450 The determination should be carried out within 15 minutes after adding the stop solution.

[0087] 9) Result determination: S / P value calculation The S / P value was calculated according to the following formula.

[0088]

[0089] Positive control serum OD 450 The values were all ≥2.0, and the negative control serum OD 450 If the values are all less than 0.25, the test is considered to be successful.

[0090] When the S / P value of the sample to be tested is ≥0.161, it is judged as positive; when the S / P value of the sample to be tested is <0.161, it is judged as negative.

[0091] The specificity, stability and sensitivity of the kit and detection method of the present invention were tested and verified:

[0092] Specificity tests showed that the coated Pm153 recombinant protein antigen reacted positively with bovine Pasteurella multocida type A sera, but negatively with sera positive for bovine Pasteurella multocida types B, D, E, and F. The coated Pm153 recombinant protein antigen also reacted negatively with sera positive for Mannheimia hemolytica, Mannheimia heterologous to bovine, Mycoplasma bovis, bovine viral diarrhea, and foot-and-mouth disease. The indirect ELISA method constructed with the Pm153 recombinant protein antigen exhibited good specificity (Table 3).

[0093] Table 3: Specificity test results

[0094]

[0095]

[0096] The stability test showed that the OD of 10 serum samples was detected by the same batch of coated plates. 450 The coefficient of variation of the values was between 2.03% and 5.22%. The OD values of 10 serum samples were tested on different batches of coated plates. 450 The coefficient of variation of the values was between 5.03% and 9.15%, indicating that the method had good repeatability.

[0097] Positive samples were randomly selected and diluted in multiples before testing using the established ELISA method. The results showed that when the serum was diluted at a volume ratio of 1:2560, positive results could still be detected, indicating that this method has good sensitivity (Table 4).

[0098] Table 4: Sensitivity test results

[0099] Dilution multiple 20 40 80 160 320 640 1280 2560 5120 <![CDATA[Sample OD 450 > 1.689 1.486 1.278 1.091 0.879 0.764 0.682 0.463 0.32 Positive control 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 Negative control 0.185 0.185 0.185 0.185 0.185 0.185 0.185 0.185 0.185 S / P 0.931 0.806 0.677 0.561 0.430 0.359 0.308 0.172 0.084 Test results + + + + + + + + -

[0100] Example 3: Clinical application of kit detection

[0101] In addition, 250 clinically confirmed bovine type A positive sera and 240 negative sera were collected, and the negative and positive coincidence rates were calculated.

[0102] Among the 250 positive samples, 238 were positive, with a compliance rate of 95.2%. Among the 240 negative samples, 220 were negative, with a compliance rate of 91.7%, which has a good compliance rate.

[0103] Example 4: Screening process for the bovine Pasteurella multocida capsule type A specific gene PmA153

[0104] In the NCBI database, the whole genome data of PmCQ2 strain (type A) (GenBank: NZ_CP033599.1) and NCTC 10323 strain (type B) (GenBank: LR134532.1) were used as typical representatives for comparison. Because the genome is relatively large and difficult to complete using ordinary programs, a powerful tool was used: mVISTA (https: / / genome.lbl.gov / vista / mvista / submit.shtml). Through the comparison of mVISTA results, it was found that the PmCQ2 strain genome has a region around 1040k, which is completely missing in the NCTC 10323 strain. The gene in the missing segment was named PmA153 gene:

[0105] ATGACAGAAGAAAATAAAGGAAAGAGATATTTTTTATGGTTCATATTGTTTATCCTTTCAATCTATTTATTTATTACCATACAAGAAAGACGAGGTTATTGTTTTGACAAACGTGCATATATTCATGAGCTTTATACTGAGCAAGAGTTAATTGATCGGGGGATTGAATATGTGGTATCCACCATGCCGTCAGGTGTTATTAAACCAGATGGCACAATAAAAGAAGTAAAG CGTTACACGAGTGTCGAGGAGTTTAAACAGATGAACCCAGCTTGTTGTACATTAACCACCTTTATTGATGAAGGAGGCGATGGCTATCCAGATGATGATGGATATGGTTATGTCAGAATTGAATATTTAAGACATTATGTTGAGAATCTAAAACCTTATCATAGAGTGATTTATCTTGAATATACGCCCTGTGGAGAGTTAAGGGAAGAGGCGGCTTTTTCAAAAAATTAA.

[0106] Blast analysis using the PmA153 gene revealed the presence of the PmA153 gene in all type A strains, not just the PmCQ2 strain. (A database of type A strains, such as PmCQ2, Pm1BS168, EB168, TB168, and Pm8-1, was used to compare the specific gene fragment (HyaD gene) of Bacillus multocida type A.) Further analysis of type B, D, E, and F strains revealed the absence of the PmA153 gene in strains other than type A, indicating that PmA153 is a gene fragment unique to type A. This technical solution identifies gene fragments specific to type A strains and can be used to detect pathogenic microorganisms.

[0107] However, despite the existence of this gene fragment, which is specific to type A, PCR cannot be used to detect infected cattle. Pasteurella multocida type A grows in the lungs of cattle, but this strain cannot be isolated from blood, and the pathogen cannot be collected through nasal or throat swabs. Therefore, without sacrificing the cattle to obtain lung tissue, it is impossible to detect this pathogen. Therefore, despite the existence of this gene, the sample size is limited, and the PCR method is ineffective on live cattle. Collecting peripheral blood for ELISA is more convenient and overcomes this problem. If an animal's infection is to be determined based on antibodies produced in serum by antigenic substances, the antigen must be capable of producing large quantities of antibodies. Specifically, not only must the PmA153 gene be specific to other types, but it must also be able to produce large quantities of antigenic proteins, with good immunogenicity, capable of generating large quantities of antibodies circulating in the blood. Only when these conditions are met can this gene be used in the development of ELISA antibody detection kits.

[0108] Through sequence comparison, the inventors discovered four type A-specific genes (for example, PmA181, PmA153, etc.). Furthermore, through experimental analysis, they found that the proteins (antigens) expressed by these specific genes had poor immunogenicity and were difficult to produce detectable antibodies in organisms, making them unusable for diagnosing whether an organism was infected with type A. Therefore, they finally chose to use the PmA153 antigen protein for the production of an ELISA detection kit.

[0109] In addition, the existing technology primarily uses type A and type B capsular antigens for detecting type A and type B strains. Compared to type A capsular antigens, the PmA153 antigen has better specificity for type A serotypes and weaker cross-reactivity. The results of ELISA tests using the PmA153 antigen were compared with those using type A and type B capsular antigens, as shown in Table 5.

[0110] Table 5: Comparison of PmA153 antigen and capsular antigen coating

[0111]

[0112] Therefore, capsular antigens extracted from type A and type B bacteria cannot be directly used in ELISA to distinguish between type A and type B sera. Cross-reactivity is observed, and the values are large. Protein antigens, however, do not have this problem and are therefore superior to using capsular antigens.

[0113] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A bovine Pasteurella multocida capsule type A antibody ELISA kit, characterized in that: The invention comprises a solid phase carrier coated with PmA153; the amino acid sequence of PmA153 is shown in SEQ ID NO.

2.

2. A bovine Pasteurella multocida capsule type A ELISA antibody detection kit according to claim 1, characterized in that, It also includes a standard positive control serum and a standard negative control serum; the standard positive control serum is obtained by immunizing cattle with a bovine Pasteurella multocida capsule type A strain; the standard negative control serum is the serum of cattle that are not immunized and not infected with Pasteurella multocida capsule type A.

3. A bovine Pasteurella multocida capsule type A ELISA antibody detection kit according to claim 2, characterized in that, It also includes enzyme-labeled secondary antibody, color development solution, stop solution, concentrated washing solution and sample diluent.

4. A bovine Pasteurella multocida capsule type A ELISA antibody detection kit according to claim 3, characterized in that, The enzyme-labeled secondary antibody is rabbit anti-bovine IgG labeled with horseradish peroxidase; the color developing solution is a two-component TMB color developing solution; the stop solution is a sulfuric acid solution; the concentrated washing solution is a PBST washing solution; and the sample diluent is a PBS solution containing bovine serum albumin, Tween-20, EDTA and sodium chloride.

5. A bovine Pasteurella multocida capsule type A ELISA antibody detection kit according to claim 4, characterized in that, The judgment criteria are: when the S / P value of the sample to be tested is ≥0.161, it is judged as positive; when the S / P value of the sample to be tested is <0.161, it is judged as negative; the calculation method of the S / P value is: (sample OD 450 Value - Standard negative control serum OD 450 mean) / (standard positive control serum OD 450 Mean - standard negative control serum OD 450 mean); OD 450 Refers to the optical density at 450nm wavelength; and the standard positive control serum OD 450 The values were all ≥2.0, and the standard negative control serum OD 450 The values are all <0.

25.

6. Use of an antigen protein in preparing a kit for specifically detecting bovine Pasteurella multocida capsule type A, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

7. A method for preparing a bovine Pasteurella multocida capsule type A ELISA antibody detection kit, characterized in that: The preparation steps of the PmA153-coated solid phase carrier include: Acquisition of S1 antigen protein: The gene sequence as shown in SEQ ID NO.1 is integrated into the expression vector to obtain a recombinant plasmid; the recombinant plasmid is transferred into the host bacteria to induce the host bacteria to express the antigen protein; the bacteria are then broken, the supernatant is collected by centrifugation and purified to obtain PmA153, S2 coating: Prepare a coating solution containing PmA153, allow the coating solution to contact and incubate with the solid phase carrier, and then perform a blocking treatment to obtain a PmA153-coated solid phase carrier.

8. The method for preparing a bovine Pasteurella multocida capsule type A ELISA antibody detection kit according to claim 7, characterized in that: In obtaining the S1 antigen protein, the expression vector is pET30a; the reagent for inducing the host bacteria to express the antigen protein is isopropyl-β-D-thiogalactoside; the host bacteria is Rosseta DE3; and the purification method is purification through Ni-NTA Superflow Cartridges His affinity chromatography column.

9. The method for preparing a bovine Pasteurella multocida capsule type A ELISA antibody detection kit according to claim 8, characterized in that: In S2 coating, the content of PmA153 in the coating solution was 10 μg / mL, and the incubation conditions were 4°C for 8-12 hours; the blocking treatment was performed using a protein-free blocking buffer at 37°C for 2 hours.

10. The method for preparing a bovine Pasteurella multocida capsule type A ELISA antibody detection kit according to claim 8, characterized in that: The method also includes the steps of preparing a standard positive control serum: immunizing healthy cattle with an inactivated vaccine of bovine Pasteurella multocida type A strain, performing a booster immunization 14 days later, and collecting serum one month after the booster immunization to obtain the standard positive control serum.

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