Cell strain secreting anti-swine erysipelothrix spaA protein and application thereof

Abstract

The invention provides a cell strain capable of secreting anti-swine erysipelothrix spaA protein and application of the cell strain, and belongs to the technical field of antibodies. According to the invention, swine erysipelothrix truncation SpaA protein (SpaA-N) is used as an immunizing antigen for screening hybridoma cell strains secreting monoclonal antibodies for the first time to obtain hybridoma cells, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.45348. The hybridoma cell not only can generate a monoclonal antibody with strong specific binding capacity with an immune antigen or swine erysipelothrix, but also has higher hereditary stability in the performance of secreting the monoclonal antibody. Meanwhile, the monoclonal antibody secreted by the hybridoma cell strain SpaA-21D can realize immunohistochemical detection and indirect ELISA detection of swine erysipelothrix, and has good detection specificity. Therefore, the hybridoma cell strain and the monoclonal antibody secreted by the hybridoma cell strain provide a new means for detection of swine erysipelothrix.

Description

Technical Field

[0001] The invention belongs to the technical field of antibodies, and specifically relates to a cell strain secreting anti-Erysipelothrix rhizoctonia solium SpaA protein and application thereof. Background Art

[0002] Swine erysipelas is a zoonosis caused by Erysipelothrix rhusiopathiae (ER) that seriously endangers the healthy development of the pig industry. Clinically, it can be divided into four types: peracute, acute septic, subacute and chronic. The diagnostic features are mainly high fever, acute septicemia, skin rash, endocarditis and arthritis. The mortality rate of acute septicemia is as high as 80%, which is a great threat to the pig farming industry. With the in-depth implementation of the Ministry of Agriculture and Rural Affairs' action to reduce the use of veterinary antibiotics, mixed infections of multiple bacteria such as swine erysipelas or secondary viral infections have become an important part of disease control in farms. Therefore, the research on the detection method of swine erysipelas is of great technical significance to the prevention and control of epidemics in the pig industry.

[0003] At present, the PCR method is mainly used for differential diagnosis of swine erysipelas in diagnostic work. Makino established a PCR method based on the 16S RNA sequence of Erysipelothrix rhizogenes to identify Erysipelothrix rhizogenes and Tonsilrothrix rhizogenes, laying the foundation for molecular biological detection methods of Erysipelothrix rhizogenes. Takeshi used 4 sets of oligonucleotide primers to amplify 4 16S rRNA sequences of Erysipelothrix rhizogenes and established a PCR method for rapid screening of Erysipelothrix rhizogenes in slaughterhouses. Compared with traditional sampling detection and isolation culture, the specificity and time consumption have been improved. Pal et al. created a multiplex RT-PCR method to identify two strains of tonsils and Erysipelothrix rhizogenes of swine, and then multiplex RT-PCR was used to identify mixed infections of multiple diseases. The PCR method has the characteristics of high sensitivity, rapidity and high efficiency, but it has the following shortcomings: first, it is impossible to localize the antigen in the tissue; second, it is necessary to diagnose samples mixed with other highly infectious diseases in high-level biosafety facilities. The immunohistochemical detection method can not only achieve tissue localization, but also the test samples are inactivated by formaldehyde, which can be carried out in a conventional testing laboratory, minimizing the biosafety risk. At the same time, there is currently a lack of effective antibodies to detect Erysipelothrix rhusiopathiae, which hinders the specific detection of animals infected with Erysipelothrix rhusiopathiae. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a hybridoma cell capable of secreting anti-Erysipelothrix rhusiopathiae monoclonal antibodies, wherein the anti-Erysipelothrix rhusiopathiae monoclonal antibodies can specifically bind to Erysipelothrix rhusiopathiae antigens, thereby providing a guarantee for the prevention and control of swine erysipelas.

[0005] The invention provides a mouse hybridoma cell SpaA-21D, whose deposit number is CGMCC No.45348.

[0006] The invention provides an anti-Erysipelothrix rhizoctoniae monoclonal antibody, which is obtained by secretion of the mouse hybridoma cell SpaA-21D.

[0007] The invention provides an Erysipelothrix rhizome detection kit based on immunological detection technology, comprising the anti-Erysipelothrix rhizome monoclonal antibody and other detection reagents.

[0008] Preferably, the immunological detection technique comprises at least one of the following techniques: immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting and immunochromatography.

[0009] Preferably, it is an immunohistochemical detection kit for Erysipelothrix rhizogenes;

[0010] Other detection reagents in the Erysipelothrix rhizogenes immunohistochemical detection kit include antibody diluent, secondary antibody, washing solution and color development kit.

[0011] Preferably, the working dilution of the anti-Erysipelothrix rhizoctonia solium monoclonal antibody is 1:100.

[0012] Preferably, the working dilution of the secondary antibody is 1:400.

[0013] Preferably, the washing solution is a PBS solution; and the antibody diluent is a PBS solution.

[0014] Preferably, the secondary antibody is goat anti-mouse IgG.

[0015] Preferably, an indirect ELISA detection kit is also included; other detection reagents in the indirect ELISA detection kit include at least one of the following components: a detection plate coated with a modified Erysipelothrix rhizogenes antigen, a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a color developing solution, and a stop solution;

[0016] The amino acid sequence of the modified Erysipelothrix rhizoctonia solium antigen is as shown in SEQ ID NO: 2.

[0017] The present invention provides a mouse hybridoma cell SpaA-21D, with a deposit number of CGMCCNo.45348. The present invention uses an Escherichia coli expression system for the first time to obtain a truncated SpaA protein (SpaA-N) of Erysipelothrix rhusiopathiae (deposit number CVCC43008) expressed in a soluble form as an immune antigen, and after immunizing mice, screening of hybridoma cell strains secreting monoclonal antibodies is performed. The mouse hybridoma cells obtained by screening can not only produce monoclonal antibodies with strong specific binding ability to immune antigens or Erysipelothrix rhusiopathiae, but also have high genetic stability in the performance of secreting monoclonal antibodies. At the same time, the present invention verifies that the mouse hybridoma cell SpaA-21D can be used to achieve immunohistochemical detection and indirect ELISA detection of Erysipelothrix rhusiopathiae after preparation and purification through ascites, and has good detection specificity. It can be seen that the mouse hybridoma cell provided by the present invention provides a detection basis for the detection of Erysipelothrix rhusiopathiae.

[0018] The present invention provides an Erysipelothrix rhizome detection kit based on immunological detection technology, comprising the anti-Erysipelothrix rhizome monoclonal antibody and other detection reagents. Based on the fact that the anti-Erysipelothrix rhizome monoclonal antibody can have good affinity with the immune antigen (SpaA-N) or Erysipelothrix rhizome, the present invention prepares an Erysipelothrix rhizome detection kit based on immunological detection technology, which can achieve the purpose of rapid and efficient detection of Erysipelothrix rhizome, thereby realizing rapid diagnosis of swine erysipelas disease.

[0019] Furthermore, the present invention specifically defines the types of kits to include Erysipelothrix rhizogenes immunohistochemical detection kits. The present invention is the first to establish an Erysipelothrix rhizogenes immunohistochemical detection method using anti-Erysipelothrix rhizogenes SpaA protein monoclonal antibodies, which also has the characteristics of simple sample operation, low cost, sensitive reaction, strong specificity, etc., thereby providing a basis and technical support for differential diagnosis of swine erysipelas and research on pathogenic mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The SDS-PAGE identification results of the purified SpaA-N protein; M: Protein marker; 1: Purified SpaA-N;

[0021] Figure 2 The results of the purification of SpaA-21D monoclonal antibody are shown in Figure 1. M: Protein marker; 1: Purified SpaA-21D;

[0022] Figure 3 The results of SpaA-21D monoclonal antibody cardiac immunohistochemistry. Note: The arrows point to positive staining.

[0023] Figure 4The Western blot results of the monoclonal antibody SpaA-21D, where M: protein standard mass; 1: whole bacterial protein of pGEX-4T-1 / SpaA-N recombinant bacteria; 2: empty vector protein of GST tag; 3: wall protein of C43008 strain; 4: whole bacterial protein of bovine Pasteurella A; 5: whole bacterial protein of bovine Pasteurella B; 6: whole bacterial protein of bovine Pasteurella E.

[0024] Biomaterial Deposit Information

[0025] The mouse hybridoma cell SpaA-21D was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, abbreviated as CGMCC, and the deposit date is January 9, 2023. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No. 45348. DETAILED DESCRIPTION

[0026] The invention provides a mouse hybridoma cell SpaA-21D, whose deposit number is CGMCC No.45348.

[0027] In the present invention, the murine hybridoma cell SpaA-21D is obtained by immunizing BALB / c mice with the modified Erysipelothrix suis SpaA protein (Span-N, amino acid sequence see SEQ ID NO: 2) as an immune antigen, expressing it in Escherichia coli, obtaining a recombinant protein, and fusion of myeloma cells and spleen cells isolated from the immune mice. The screening method preferably includes the following: screening positive hybridoma cells using indirect ELISA with SpaA-N coated antigen, performing 3 to 5 subclone screenings by limiting dilution method to obtain three hybridoma cells, namely SpaA-21D, SpaA-25C and SpaA-42G, wherein SpaA-25C and SpaA-42G cannot stably secrete antibodies during passage and the hybridoma cell state is poor, and finally screening a hybridoma cell that can stably secrete anti-Erysipelothrix suis monoclonal antibody, named as murine hybridoma cell SpaA-21D.

[0028] The invention provides an anti-Erysipelothrix rhizoctoniae monoclonal antibody, which is obtained by secretion of the mouse hybridoma cell SpaA-21D.

[0029] In the present invention, the method for preparing the monoclonal antibody against Erysipelothrix rhizogenes preferably comprises obtaining the antibody by separation and purification from the culture medium of the mouse hybridoma cell SpaA-21D, or obtaining the antibody by separation and purification after inoculating the mouse hybridoma cell SpaA-21D into the peritoneal cavity to produce ascites. In an embodiment of the present invention, the method of inoculating the mouse peritoneal cavity with a hybridoma cell line to produce ascites is preferably to use BALB / c mice as the subjects, sensitize them with Freund's incomplete adjuvant intraperitoneally, inject the hybridoma cell line SpaA-21D 7 days later, and collect the ascites 7 to 14 days after the injection. The cell density of the hybridoma cell line SpaA-21D is 10 6 / mL, 0.5mL was injected into the peritoneal cavity of each mouse. The method for collecting ascites is preferably to puncture the bulging part of the mouse abdomen, gently knead and press to make the ascites flow out. The purification method is to purify using Protein A (GE Healthcare 17-5079-01) affinity column.

[0030] In the present invention, immunohistochemical detection and blocking ELISA detection of Erysipelothrix rhusiopathiae are realized based on the anti-Erysipelothrix rhusiopathiae monoclonal antibody. The results show that the detection method provided by the present invention has good detection specificity and high detection sensitivity.

[0031] The invention provides an Erysipelothrix rhizome detection kit based on immunological detection technology, comprising the anti-Erysipelothrix rhizome monoclonal antibody and other detection reagents.

[0032] In the present invention, the immunological detection technology preferably includes at least one of the following technologies: immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting and immunochromatography. The kit is preferably an immunohistochemical detection kit for Erysipelothrix rhusiopathiae. Other detection reagents in the immunohistochemical detection kit for Erysipelothrix rhusiopathiae include antibody diluent, secondary antibody, washing solution and color development kit. The working dilution of the anti-Erysipelothrix rhusiopathiae monoclonal antibody is preferably 1:100; the working dilution of the secondary antibody is preferably 1:400. The secondary antibody is goat anti-mouse IgG. The washing solution is PBS solution. The color development kit is preferably a VectorVIP HRP color development kit. The method for using the immunohistochemical detection kit for Erysipelothrix rhusiopathiae preferably includes the following steps:

[0033] (1) Fixing the pig heart tissue to be tested;

[0034] (2) trimming the tissue sections, dehydrating, transparentizing and waxing the trimmed tissue blocks;

[0035] (3) embedding the wax-impregnated tissue sample with embedding wax;

[0036] (4) Slice the wax-embedded tissue sample;

[0037] (5) Antigen retrieval of tissue sections after dewaxing;

[0038] (6) After antigen retrieval, tissue sections are washed and blocked;

[0039] (7) After discarding the blocking solution, add anti-Erysipelothrix rhizoctonia solium monoclonal antibody SpaA-21D for incubation, wash, add secondary antibody, incubate at room temperature, and wash;

[0040] (8) Use VectorVIP HRP color development kit for color development. Stop color development after completion, depending on the microscopic examination results. Counterstain with hematoxylin solution for 10 seconds, wash off the solution with distilled water, bluing with tap water for 10 minutes, and dehydrate and seal the slides.

[0041] In the present invention, the fixation method of the heart tissue is preferably to place the heart tissue in a 4% paraformaldehyde solution for 48 hours. The dehydration, transparency and waxing of the repaired tissue block are preferably completed in an automatic dehydrator. The thickness of the slice during the slicing process is preferably 5 μm. The dewaxing reagent is preferably treated with a xylene solution. The antigen repair method is preferably to hydrate the tissue slices to distilled water through gradient alcohol, then soak them in 0.01M PBS for 5 minutes, replace PBS, wash 3 times, and place the tissue slices in the repair solution at 90°C to 95°C for 20 minutes. The repair solution is preferably a sodium citrate solution. The solution used for dilution is preferably 0.01M PBS. The number of washes preferably includes 3 times, each time for 5 minutes. The blocking method preferably includes first using a 3% methanol-hydrogen peroxide solution for blocking for 30 minutes, then washing, and then dripping 5% goat serum for blocking for 30 minutes. The incubation condition is preferably overnight at 4°C. The color development time is preferably 50 to 70 seconds. The solution for stopping color development is preferably distilled water.

[0042] The present invention establishes an immunohistochemical detection method for Erysipelothrix rhusiopathiae using monoclonal antibodies against SpaA protein of Erysipelothrix rhusiopathiae. The method also has the characteristics of simple sample operation, low cost, sensitive reaction, strong specificity, etc., thereby providing a basis and technical support for differential diagnosis and pathogenic mechanism research of swine erysipelas.

[0043] In the present invention, the kit preferably also includes an indirect ELISA detection kit. Other detection reagents in the indirect ELISA detection kit include at least one of the following components: a detection plate coated with a modified Erysipelothrix rhusiopathiae antigen, a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a color developing solution, and a stop solution; the amino acid sequence of the modified Erysipelothrix rhusiopathiae antigen is such as SEQ ID NO: 2. The coating concentration of the modified Erysipelothrix rhusiopathiae antigen in the detection plate coated with the modified Erysipelothrix rhusiopathiae antigen is preferably 0.05-0.625 μg / ml, more preferably 0.2 μg / ml. The washing solution is preferably a PBS solution. The enzyme-labeled secondary antibody is preferably an enzyme-labeled goat anti-mouse IgG antibody. The color developing solution is determined according to the type of the marker enzyme. The present invention has no special restrictions on the type of marker enzyme, and the marker enzyme commonly used in enzyme-linked immunosorbent assay and immunoblotting known in the art can be used, such as horseradish peroxide. The stop solution is preferably a strong acid or a strong base, such as a sulfuric acid solution or a sodium hydroxide solution. The present invention has no particular limitation on the preparation and use of the indirect ELISA detection kit, and the preparation and use methods known in the art can be used.

[0044] The cell strain secreting the anti-Erysipelothrix SpaA protein and its application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] A preparation method of Erysipelothrix rhizogenes antigen SpaA-N

[0047] 1. Construction of the expression vector of SpaA-N

[0048] (1) Gene synthesis

[0049] The N-terminal 342 amino acids of the SpaA protein of Erysipelothrix rhizogenes (Genbank: AB259654.1) were optimized by truncating the C-terminal 20 repeated amino acid sequence according to the preferred codons of Escherichia coli, and BamHI and XhoI restriction sites were added at both ends of the sequence, and a GST tag was introduced at the C-terminus. The gene fragment SpaA-N was artificially synthesized by chemical synthesis, which contained a total of 1053 nucleotides. The sequence contained restriction sites at both ends and did not contain a GST sequence. The specific nucleic acid sequence is shown in SEQ ID NO: 1:

[0050] ggatcc ctcgag .

[0051] The amino acid sequence is shown in SEQ ID NO: 2:

[0052] GSPKGYQSFEAVNEEINSIVSELKNEGMSLQNIHHMFKQSIQNLATRIGYRSFMQDAMYLENFERLTIPELDEAYVDLLVNYEVKHRILVKYEGKVKGRAPLEAFIVPLRDRIRSMNEIAAEVNYLPEAHEDFLVSDSSEYNDKLNNINFALGLGVSEFIDYNRLENMMEKEIHP LYLELYAMRRNRQIQVVRDVYPNLERANAVVESLKTIKDIKQRGKKLQELLEIYIQRSGDVRKPDVLQRFIGKYQSVVDEEKNKLQDYLESDIFDSYSVDGEKIRNKEITLINRDAYLSMIYRAQSISEIKTIRADLESLVKSFQNEESDSKVEPESPVKVEKPVDEEKPKDQLE.

[0053] (2) Construction of fusion expression vector

[0054] pGEX-4T-1 and the synthetic plasmid containing the SpaA-N gene were double-digested with BamHI and XhoⅠ at the same time. The reaction system was 20μL: 3μL plasmid, 2μL 10×Buffer, 1μL BamHI, 1μL XhoⅠ, 13μL ddH2O. Purify and recover the target gene fragment, mix 3μL vector (pGEX-4T-1), 1μL enzyme recovery product, 1μL ligase, and 1μL 10×T4 DNA buffer mixture, and connect at 4℃ overnight. The recombinant plasmid was transferred into the cloning competent cell DH5α, and the LB plate with ampicillin resistance was selected, dried, inverted in a 37℃ incubator overnight, and a single colony was picked and shaken to the logarithmic phase, and the recombinant plasmid was extracted for use.

[0055] 2. Expression and Purification of SpaA-N

[0056] (1) Construction of genetically engineered strains carrying the recombinant SpaA-N gene

[0057] The extracted recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and a single clone was picked out and placed in LB liquid medium containing ampicillin, and cultured at 37°C overnight with shaking. After PCR identification, the strain containing the target DNA fragment was named Escherichia coli BL / SpaA-N strain, and an equal volume of 50% glycerol LB was added and frozen at -70°C.

[0058] (2) Expression and purification of SpaA-N

[0059] The recombinant Escherichia coli (E. coli) BL / SpaA-N strain was inoculated into 1 L LB liquid medium containing ampicillin and cultured at 37°C with shaking until OD 600 When the pH value is 0.6-0.8, add IPTG solution with a final concentration of 0.5mM to induce culture at low temperature for 16h. After the bacterial culture is completed, centrifuge and collect the cells. Resuspend the cells by adding 10mL PBS per gram of cell weight. Ultrasonicate the cells in an ice water bath for 30min. The conditions for disruption are: 9s working, 9s rest, and 400W ultrasonic power. Centrifuge the disrupted bacterial solution at 4℃ and 12000r / min for 10min, and collect the supernatant.

[0060] The soluble target protein expressed in the supernatant of bacterial lysis was purified according to the instruction manual of GST fusion protein purification kit (Nanjing GenScript Co., Ltd.) and filtered through a 0.22 μm pore size filter membrane to obtain the initially purified target protein.

[0061] The purified target protein was identified by SDS-PAGE. The electrophoresis results are shown in Figure 1 .Depend on Figure 1 It can be seen that the size of the purified protein is consistent with the expected size of the target protein, indicating that the SpaA-N protein antigen is successfully obtained by recombinant expression in the present invention.

[0062] Example 2

[0063] Establishment of indirect ELISA detection method

[0064] The SpaA-N protein was diluted horizontally in two-fold ratios according to the mass concentration of 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.3 μg / mL, 0.2 μg / mL, 0.1 μg / mL, and 0.05 μg / mL using ELISA coating solution. 100 μL / well was coated on a 96-well ELISA plate, incubated at 4°C overnight, and washed three times with PBST.

[0065] Block with 5% skim milk for 2 hours, 100 μL / well, and wash 3 times with PBST. Dilute the mouse positive and negative sera at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400, 100 μL / well, incubate at 37°C for 2 hours, and wash 3 times with PBST. Add 1:10000 diluted HRP-labeled goat anti-mouse secondary antibody, 100 μL per well, incubate at 37°C for 1 hour, and wash 3 times with PBST.

[0066] Add 50 μL of colorimetric solution and stop solution, and measure D 450nm value, calculate P / N.

[0067] The test results are shown in Table 1.

[0068] The results are shown in Table 1. From the data in Table 1, it can be seen that when the protein coating concentration is 0.2 μg / ml and the serum dilution is 1:3200, the P / N value is greater than 2.0, and the N value is the smallest. Therefore, the protein coating concentration is 0.2 μg / ml and the serum dilution is 1:3200 to prepare the indirect ELISA detection kit.

[0069] Table 1 Protein coating concentration and serum dilution determination results

[0070]

[0071] Example 3

[0072] Screening of hybridoma cell lines secreting monoclonal antibodies against SpaA protein of Erysipelothrix rhusiopathiae and preparation of monoclonal antibody ascites

[0073] 1. Immunization of BALB / c mice and determination of antibody titer

[0074] Four BALB / c mice aged 6 to 8 weeks were immunized with the wall protein of Erysipelothrix rhizogenes strain (deposit number CVCC 43008) as the immune antigen, for a total of four immunizations, with an interval of 2 weeks between each immunization. For the first immunization, the protein was added with an equal volume of Freund's complete adjuvant, emulsified, and immunized with mice via multiple subcutaneous injections at the back of the neck, 120 μg / mouse; for subsequent immunizations, the adjuvant was replaced with Freund's incomplete adjuvant, and the rest was the same as the first immunization; 7 to 10 days after the third immunization, a small amount of blood was collected from the tail, and the purified SpaA-N protein was coated on an enzyme-labeled plate at 0.2 μg / mL and the serum titer was determined by indirect ELISA. Mice with a titer higher than 1:1600 were selected for booster immunization three days before fusion, and the protein was directly injected intraperitoneally at a dose of 120 μg / mouse. 3-5 days after the last immunization, the spleen cells of mice with higher titers were taken for cell fusion.

[0075] 2. Cell fusion and screening of hybridoma cell lines

[0076] Take the myeloma cells SP2 / 0 with good growth status, use the indirect ELISA of SpaA-N coated antigen to detect the titer, select the mouse spleen cells with the highest titer and use PEG1450 for chemical fusion. Use the indirect ELISA of SpaA-N coated antigen prepared in Example 2 to screen positive hybridoma cells, and after 3 to 5 subclones by limiting dilution method, three hybridoma cells were screened, namely SpaA-21D, SpaA-25C and SpaA-42G. SpaA-21D, SpaA-25C and SpaA-42G were passaged for many times, and the indirect ELISA method was used to evaluate the stability of antibody secretion of the three hybridoma cell lines. The results showed that SpaA-25C and SpaA-42G could not stably secrete antibodies during passage and the hybridoma cells were in poor condition, while SpaA-21D could stably secrete antibodies during passage and the hybridoma cells were in good condition. The AMB subtype kit was used for identification, and the monoclonal antibody subtype secreted by the hybridoma cell line SpaA-21D was G2b.

[0077] 3. Preparation of monoclonal antibodies against SpaA protein of Erysipelothrix rhizogenes in ascites

[0078] BALB / c mice aged 6 to 8 months were selected for sensitization by intraperitoneal injection of Freund's incomplete adjuvant (0.5 mL / mouse). Hybridoma cells were injected 7 days later. The above three hybridoma cells in logarithmic growth phase and SP2 / 0 cells were collected and centrifuged at 800 r / min for 10 min. The cells were washed once with DMEM culture medium and resuspended to adjust the cell density to 10 6 / mL, 0.5mL was injected into the peritoneal cavity of each mouse. The mouse abdomen was gently rubbed with alcohol cotton to evenly disperse the cells in the abdominal cavity; 7 to 14 days later, when the mouse abdomen was obviously swollen, the needle of a 20ml syringe was aseptically pierced into the swollen part of the mouse abdomen, and gently rubbed and pressed to make the ascites flow out or drip out; the ascites was collected in a 15ml centrifuge tube, centrifuged at 3000r / min for 10min, and marked as SpaA-21D and the ascites control (S) of myeloma cells SP2 / 0, and stored at -20℃ for later use.

[0079] Example 4

[0080] Purification and detection of monoclonal ascites

[0081] The ascites prepared in Example 3 was purified using a Protein A (GE Healthcare 17-5079-01) affinity column purification method, and the purification steps were as follows:

[0082] Sample pretreatment: dilute the ascites with coupling buffer (20 mM sodium phosphate buffer, pH 7.0) at a ratio of 1:3, centrifuge at 12,000 rpm and 4°C for 10 min, and filter with a 0.22 μm filter membrane to remove fat, cell debris and small particles.

[0083] Equilibration: Equilibrate the column with 5 to 10 column volumes of coupling buffer, maintaining a flow rate of 2 s / drop.

[0084] Loading: Use a syringe to inject the sample into the upper interface of the column, collect the effluent in a 50ml centrifuge tube, and maintain a flow rate of 4s / drop.

[0085] Washing: Pass 5 column volumes of coupling buffer through the column, maintaining a flow rate of 2s / drop.

[0086] Elution: Elute the antibody with 5 column volumes of elution buffer (0.1 M sodium citrate buffer, pH 9.0), collect in the above EP tube, and maintain the flow rate at 4 s / drop.

[0087] Sample detection: The purified monoclonal antibody was identified by SDS-PAGE (see Figure 3 ), a monoclonal antibody with high purity was obtained by purification using a Protein A affinity column; the concentration of the monoclonal antibody was determined using the BCA method, and its concentration could reach 4.2 mg / mL.

[0088] Example 5

[0089] Establishment of an immunohistochemical detection method for Erysipelothrix rhizogenes antibodies based on monoclonal antibody SpaA-21D

[0090] 1. Preparation of paraffin sections:

[0091] (1) Place the heart tissue of pigs infected with swine erysipelas in 4% paraformaldehyde solution and fix it for 48 hours;

[0092] (2) trimming the tissue section and placing the trimmed tissue block in an automatic dehydrator for dehydration, transparency, and wax impregnation;

[0093] (3) embedding the wax-impregnated tissue sample with embedding wax;

[0094] (4) Slice with a paraffin slicer to a thickness of 5 μm and store for later use;

[0095] 2. Determination of the conditions for an immunohistochemical detection method for Erysipelothrix rhizogenes antibodies

[0096] (1) After dewaxing in xylene solution, the tissue sections were hydrated with gradient alcohol to distilled water, then soaked in 0.01M PBS for 5 min, replaced with PBS, and washed three times. The tissue sections were placed in the prepared sodium citrate repair solution and antigen repair was performed in a microwave oven at 90°C to 95°C for 20 min;

[0097] (2) Take out the repair box from the microwave oven, wait for it to return to room temperature, wash it three times with 0.01M PBS, 5 minutes each time, and block it with 3% methanol-hydrogen peroxide solution for 30 minutes. Then wash it three times with 0.01M PBS, 5 minutes each time, add 5% goat serum, and block it for 30 minutes;

[0098] (3) Discard the serum and add the primary antibody (monoclonal antibody SpaA-21D against Erysipelothrix rhizogenes) at a dilution concentration of 1:100. Place in a 4°C refrigerator overnight and take out the next day to warm up for 30 minutes. Wash three times with 0.01M PBS, 5 minutes each time. Dilute the secondary antibody (goat anti-mouse IgG) with PBS to 1:400, add the diluted secondary antibody and incubate at room temperature for 2 minutes. Wash three times with PBS, 5 minutes each time.

[0099] (4) Use VectorVIP HRP colorimetric kit for color development. The color development time is controlled at about 1 min, depending on the microscopic examination. After color development is completed, use distilled water to terminate the color development. Re-stain with hematoxylin solution for 10 s, wash off the solution with distilled water, bluing with tap water for 10 min, dehydrate and seal the slide.

[0100] (5) Determination of the criteria for judging the results

[0101] The established immunohistochemical detection method for Erysipelothrix rhusiopathiae was used to detect the heart tissue infected with Erysipelothrix rhusiopathiae, and a negative control was set up. Using the VIP HRP colorimetric system, the positive signal was colored purple (Erysipelothrix rhusiopathiae). If purple color is visible in the infected heart tissue, Figure 3 If the purple color is not found in the tissue, it is judged as negative.

[0102] Example 6

[0103] Specificity detection method of monoclonal antibody SpaA-21D

[0104] The whole bacterial protein of SpaA-N recombinant bacteria, empty vector protein of GST tag, wall protein of C43008 strain, whole bacterial protein of Pasteurella bovis A, whole bacterial protein of Pasteurella bovis B and whole bacterial protein of Pasteurella bovis E were used as materials to prepare protein solutions for immunoblotting detection.

[0105] Results Figure 4 .Depend on Figure 4It can be seen that the monoclonal antibody SpaA-21D of the present invention has binding properties only to the whole bacterial protein of the pGEX-4T-1 / SpaA-N recombinant bacteria, but cannot bind to other types of proteins.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mouse hybridoma cell SpaA-21D, characterized in that: The deposit number is CGMCC No.45348.

2. An anti-Erysipelothrix rhizoctonia solium monoclonal antibody, characterized in that: It is secreted by the mouse hybridoma cell SpaA-21D as claimed in claim 1.

3. A swine Erysipelothrix detection kit based on immunological detection technology, characterized in that: The invention comprises the anti-Erysipelothrix rhizoctoniae monoclonal antibody as described in claim 2 and other detection reagents.

4. The swine Erysipelothrix detection kit based on immune technology according to claim 3, characterized in that: The immunological detection technique includes at least one of the following techniques: immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting and immunochromatography.

5. The swine Erysipelothrix detection kit based on immune technology according to claim 4, characterized in that: It is an immunohistochemical detection kit for Erysipelothrix rhizogenes; Other detection reagents in the Erysipelothrix rhizogenes immunohistochemical detection kit include antibody diluent, secondary antibody, washing solution and color development substrate.

6. The swine Erysipelothrix detection kit based on immune technology according to claim 5, characterized in that: The working dilution of the anti-Erysipelothrix rhizoctonia solium monoclonal antibody is 1:

100.

7. The Erysipelothrix rhizoctoniae detection kit based on immune technology according to claim 5, characterized in that: The working dilution of the secondary antibody was 1:

400.

8. The swine Erysipelothrix detection kit based on immune technology according to claim 5, characterized in that: The washing solution is PBS solution; The antibody diluent is PBS solution.

9. The swine Erysipelothrix detection kit based on immune technology according to claim 5, characterized in that: The secondary antibody was goat anti-mouse IgG.

10. The Erysipelothrix rhizoctoniae detection kit based on immune technology according to claim 3 or 4, characterized in that: Also included are indirect ELISA test kits; The other detection reagents in the indirect ELISA detection kit include at least one of the following components: a detection plate coated with a modified Erysipelothrix rhizogenes antigen, a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a color developing solution, and a stop solution; The amino acid sequence of the modified Erysipelothrix rhizoctonia solium antigen is as shown in SEQ ID NO: 2.

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