CN119220501A - A cell line secreting SpaA protein against Erysipelothrix rhizogenes and its application - Google Patents

Abstract

The invention provides a cell strain secreting anti-erysipelas spaA protein and application thereof, and belongs to the technical field of antibodies. The invention firstly uses the erysipelas pig truncated SpaA protein (SpaA-N) as an immune antigen to screen hybridoma cell strains secreting monoclonal antibodies, and the hybridoma cell is obtained with the preservation number of CGMCC No.45348. The hybridoma cell not only can generate the monoclonal antibody with strong specific binding capacity with immune antigen or erysipelothrix rhusiopathiae, but also has higher genetic 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 erysipelothrix rhusiopathiae, 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 detecting erysipelothrix rhusiopathiae.

Description

Cell strain secreting anti-erysipelas SpaA protein and application thereof

Technical Field

The invention belongs to the technical field of antibodies, and particularly relates to a cell strain secreting anti-erysipelothrix rhusiopathiae SpaA protein and application thereof.

Background

Erysipelas are a zoonotic disease caused by erysipelas of swine (Erysipelothrix rhusiopathiae, ER) that seriously jeopardizes the healthy development of the pig industry. Clinically, the method can be divided into four types of severe acute, acute sepsis, subacute and chronic, and the diagnosis is mainly characterized by symptoms such as hyperpyrexia, acute septicemia, skin eruption, endocarditis and arthritis, wherein the death rate of the acute septicemia is about 80%, and the method has great harm to pig farming. With the deep implementation of the use reduction action of veterinary antibacterial agents in rural areas, the mixing of various bacteria such as swine erysipelas and the like or secondary virus infection become important contents for controlling diseases of farms. Therefore, the research of developing the swine erysipelas detection method has important technical significance for prevention and control of swine fever.

At present, a PCR method is mainly used for differential diagnosis of swine erysipelas in diagnostic work. Makino establishes a PCR method for identifying erysipelas and amygdalina based on erysipelas 16S RNA sequence, and lays the foundation of the erysipelas molecular biology detection method. Takeshi adopts 4 groups of oligonucleotide primers to amplify 4 erysipelas bacillus 16S rRNA sequences, and establishes a PCR method for rapidly screening erysipelas bacillus in slaughterhouses, and compared with the traditional sampling detection and isolated culture, the specificity and the time consumption are improved. Pal et al created a multiplex RT-PCR method for identifying 2 strains of tonsils and erysipelothrix suis, followed by multiplex RT-PCR for identifying mixed infections for a variety of diseases. The PCR method has the characteristics of high sensitivity, rapidness and high efficiency, but has the defects that firstly, the positioning of antigens in tissues cannot be realized, and secondly, the diagnosis of samples mixed with other infections of the virulent infectious diseases needs to be carried out in a high-grade biosafety facility. The immunohistochemical detection method not only can realize tissue positioning, but also can be used for inactivating detection samples by formaldehyde and can be developed in a conventional detection laboratory, so that the biosafety risk is reduced to the greatest extent. Meanwhile, at present, the lack of effective antibodies for detecting erysipelas on pigs brings barriers to specific detection of animals infected with erysipelas on pigs.

Disclosure of Invention

Therefore, the invention aims to provide a hybridoma cell capable of secreting the anti-erysipelas monoclonal antibody, wherein the anti-erysipelas monoclonal antibody can specifically bind to erysipelas antigen, and a guarantee is provided for prevention and control of erysipelas.

The invention provides a murine hybridoma cell SpaA-21D with a preservation number of CGMCC No.45348.

The invention provides an anti-erysipelas monoclonal antibody which is secreted by the murine hybridoma cell SpaA-21D.

The invention provides a erysipelothrix rhusiopathiae detection kit based on an immunological detection technology, which comprises the erysipelothrix rhusiopathiae monoclonal antibody and other detection reagents.

Preferably, the immunological detection technique includes at least one of immunohistochemistry, enzyme linked immunosorbent assay, immunoblotting and immunochromatography techniques.

Preferably, the kit is used for detecting erysipelas Sus domestica immunohistochemical detection;

Other detection reagents in the erysipelas immune histochemical detection kit comprise antibody diluent, secondary antibody, washing liquid and color development kit.

Preferably, the working dilution of the anti-erysipelothrix rhusiopathiae monoclonal antibody is 1:100.

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

Preferably, the washing solution is PBS solution, and the antibody diluent is PBS solution.

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

Preferably, the kit also comprises an indirect ELISA detection kit, wherein other detection reagents in the indirect ELISA detection kit comprise at least one component selected from a detection plate coated with modified erysipelas antigen, a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a chromogenic solution and a stop solution;

The amino acid sequence of the modified erysipelas Sus Domestica antigen is shown as SEQ ID NO. 2.

The invention provides a murine hybridoma cell SpaA-21D with a preservation number of CGMCC No.45348. According to the invention, a colibacillus expression system is used for the first time to obtain a SpaA protein (SpaA-N) truncated by erysipelas suis (preservation number CVCC 43008) expressed in a soluble form as an immune antigen, and after mice are immunized, screening of hybridoma cell strains secreting monoclonal antibodies is performed. The screened mouse hybridoma cells not only can generate monoclonal antibodies with strong specific binding capacity with immune antigens or erysipelothrix rhusiopathiae, but also have higher genetic stability in the performance of secreting monoclonal antibodies. Meanwhile, the invention verifies that the immunohistochemical detection and indirect ELISA detection of erysipelas of pigs can be realized by utilizing the mouse hybridoma cell SpaA-21D after ascites preparation and purification, and the detection specificity is good. Therefore, the mouse hybridoma cell provided by the invention provides a detection basis for detecting erysipelas Sus domestica.

The invention provides a erysipelothrix rhusiopathiae detection kit based on an immunological detection technology, which comprises the erysipelothrix rhusiopathiae monoclonal antibody and other detection reagents. Based on the fact that the monoclonal antibody against erysipelas is capable of having good affinity with immune antigen (SpaA-N) or erysipelas, the erysipelas detection kit is prepared based on an immunological detection technology, and the aim of rapid and efficient detection of erysipelas can be achieved, so that rapid diagnosis of erysipelas diseases of pigs is achieved.

Furthermore, the invention specifically defines the types of the kit including the erysipelas suis immunohistochemical detection kit. The invention establishes the swine erysipelas immunohistochemical detection method by utilizing the swine erysipelas-resistant SpaA protein monoclonal antibody for the first time, and the method has the characteristics of simple and convenient sample operation, low cost, sensitive response, strong specificity and the like, thereby providing basis and technical support for swine erysipelas differential diagnosis and pathogenic mechanism research.

Drawings

FIG. 1 shows the SDS-PAGE identification of purified SpaA-N protein, M: proteinmarker, 1: purified SpaA-N;

FIG. 2 shows the results of the identification of the purification effect of the SpaA-21D monoclonal antibody, M: proteinmarker, 1: purified SpaA-21D;

FIG. 3 shows the results of cardiac immunohistochemistry with SpaA-21D monoclonal antibody, with the arrow indicated as positive color development;

FIG. 4 shows the Westernblot results of monoclonal antibodies SpaA-21D, wherein M is the standard mass of protein, 1 is pGEX-4T-1/SpaA-N recombinant whole-cell protein, 2 is GST-tagged empty carrier protein, 3 is C43008 strain wall protein, 4 is Pasteurella bovis A whole-cell protein, 5 is Pasteurella bovis B whole-cell protein, and 6 is Pasteurella bovis E whole-cell protein.

Biological material preservation information

The murine hybridoma cells SpaA-21D are preserved in China general microbiological culture Collection center (CGMCC), and the preservation time is 2023, 01 and 09. The address is the institute of microbiology of the national academy of sciences of China, the Korean area North Star, the West way 1, the 3 rd. The preservation number is CGMCC No.45348.

Detailed Description

The invention provides a murine hybridoma cell SpaA-21D with a preservation number of CGMCC No.45348.

In the invention, the murine hybridoma cell SpaA-21D is obtained by taking modified erysipelas Sus domestica SpaA protein (Span-N, amino acid sequence is shown in SEQ ID NO: 2) as an immune antigen, performing escherichia coli soluble expression to obtain a recombinant protein immune BALB/c mouse, fusing myeloma cells with spleen cells separated from the immune mouse, and screening. The screening method preferably comprises the following steps of screening positive hybridoma cells by indirect ELISA of a SpaA-N coated antigen, performing subcloning screening for 3-5 times by a limiting dilution method to obtain three hybridoma cells, namely SpaA-21D, spaA-25C and SpaA-42G, wherein the SpaA-25C and the SpaA-42G can not stably secrete antibodies in the passage process and the hybridoma cells are poor in state, and finally screening out a hybridoma cell capable of stably secreting anti-erysipelothrix rhusiopathiae monoclonal antibody, and the hybridoma cell is named as murine hybridoma cell SpaA-21D.

The invention provides an anti-erysipelas monoclonal antibody which is secreted by the murine hybridoma cell SpaA-21D.

In the invention, the preparation method of the anti-erysipelas monoclonal antibody preferably comprises the steps of separating and purifying from a culture solution of murine hybridoma cells SpaA-21D, or inoculating the murine hybridoma cells SpaA-21D into abdominal cavities of mice to generate ascites, and separating and purifying. In the embodiment of the invention, the method for generating ascites by inoculating the hybridoma cell strain into the abdominal cavity of the mouse preferably takes a BALB/c mouse as a subject, and is sensitized by injecting Freund's incomplete adjuvant into the abdominal cavity, injecting the hybridoma cell strain SpaA-21D after 7D, and collecting the ascites after 7-14D injection. The cell density of the hybridoma cell strain SpaA-21D is 10 ⁶/mL, and each mouse is injected with 0.5mL intraperitoneally. The method for collecting the ascites is preferably to puncture the swelling part of the abdomen of the mouse, and the ascites can flow out by gentle kneading and pressing. The purification method adopts Protein A (GE HEALTHCARE-5079-01) affinity column for purification.

In the invention, immunohistochemical detection and blocking ELISA detection of erysipelothrix rhusiopathiae are realized based on the anti-erysipelothrix rhusiopathiae monoclonal antibody. The result shows that the detection method provided by the invention has good detection specificity and higher detection sensitivity.

The invention provides a erysipelothrix rhusiopathiae detection kit based on an immunological detection technology, which comprises the erysipelothrix rhusiopathiae monoclonal antibody and other detection reagents.

In the present invention, the immunological detection technique preferably includes at least one of immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting and immunochromatography techniques. The kit is preferably an immunohistochemical detection kit for erysipelas Sus Domestica. Other detection reagents in the erysipelas immune histochemical detection kit comprise antibody diluent, secondary antibody, washing liquid and color development kit. The working dilution of the monoclonal antibody against erysipelas Sus is preferably 1:100, and the working dilution of the secondary antibody is preferably 1:400. The secondary antibody is goat anti-mouse IgG. The washing liquid is PBS solution. The color development kit is preferably a Vector VIP HRP color development kit. The application method of the erysipelas immune histochemical detection kit preferably comprises the following steps:

(1) Fixing heart tissue of a pig to be tested;

(2) Trimming the section of the tissue, and dehydrating, transparentizing and waxing the trimmed tissue block;

(3) Embedding the waxed tissue sample with an embedding wax;

(4) Slicing the tissue sample embedded with the wax;

(5) Tissue sections were subjected to antigen retrieval after deparaffinization;

(6) Washing the tissue slice subjected to antigen repair and then sealing;

(7) Dropping the blocking solution, then dropwise adding a monoclonal antibody SpaA-21D for resisting erysipelas, incubating, washing, adding a secondary antibody, incubating at room temperature, and washing;

(8) And (3) performing color development by using a VectorVIP HRP color development kit, stopping the color development after the color development is finished according to the microscopic examination condition, counterstaining the hematoxylin dye solution for 10s, washing the dye solution with distilled water, bluing for 10min by using tap water, and dehydrating and sealing the wafer.

In the present invention, the method of fixing heart tissue is preferably to treat heart tissue in a 4% paraformaldehyde solution for 48 hours. The desiccation, transparency and waxing of the trimmed tissue mass is preferably accomplished in an automatic desiccator. The thickness of the slice at the time of the slicing process is preferably 5 μm. The dewaxing reagent is preferably a xylene solution treatment. In the antigen retrieval method, tissue slices are preferably hydrated to distilled water through gradient alcohol, then soaked in 0.01M PBS for 5min, PBS is replaced, washed for 3 times, and the tissue slices are placed in a retrieval liquid and treated for 20min at 90-95 ℃. The repair liquid is preferably a sodium citrate solution. The solution used for the dilution is preferably 0.01M PBS. The number of washes preferably includes 3 times, each for 5 minutes. The blocking method preferably comprises the steps of blocking for 30min by using a 3% methanol-hydrogen peroxide solution, and then dropwise adding 5% goat serum for blocking for 30min after washing. The incubation conditions are preferably overnight at 4 ℃. The color development time is preferably 50 to 70 seconds. The solution for stopping the color development is preferably distilled water.

The invention establishes the swine erysipelas immune histochemical detection method by utilizing the swine erysipelas resisting SpaA protein monoclonal antibody, and the method has the characteristics of simple and convenient sample operation, low cost, sensitive response, strong specificity and the like, thereby providing basis and technical support for swine erysipelas differential diagnosis and pathogenic mechanism research.

In the present invention, the kit preferably further comprises an indirect ELISA detection kit. The other detection reagents in the indirect ELISA detection kit comprise at least one component of a detection plate coated with the modified erysipelas, a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a chromogenic solution and a stop solution, wherein the amino acid sequence of the modified erysipelas is shown as SEQ ID NO. 2. The coating concentration of the modified erysipelas antigen in the detection plate coated with the modified erysipelas antigen is preferably 0.05-0.625 mug/ml, more preferably 0.2 mug/ml. The washing solution is preferably 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 marking enzyme. The invention is not particularly limited in the type of the labeling enzyme, and the labeling enzyme commonly used in enzyme-linked immunosorbent assay and immunoblotting, such as peroxidase, are well known in the art. The stop solution is preferably a strong acid or base, such as sulfuric acid solution or sodium hydroxide solution. The method for preparing and using the indirect ELISA kit is not particularly limited, and can be prepared and used by methods well known in the art.

The following examples are provided to illustrate a cell strain secreting anti-erysipelothrix rhusiopathiae SpaA protein and its use in detail, but they should not be construed as limiting the scope of the invention.

Example 1

Preparation method of erysipelas suis antigen SpaA-N

Construction method of SpaA-N expression vector

(1) Gene synthesis

The N-terminal 342 amino acids of the SpaA protein of erysipelas Sus Domestica (Genbank: AB 259654.1) are cut off the C-terminal 20 repeated amino acid sequence according to the preferred codon of Escherichia coli, bamHI and XhoI enzyme cutting sites are added at the two ends of the sequence, GST tag is introduced at the C-terminal, and the gene fragment SpaA-N is artificially synthesized by chemical synthesis method and contains 1053 nucleotides. The two ends of the sequence contain enzyme cutting sites and do not contain GST sequences, and the specific nucleic acid sequence is shown as SEQ ID NO. 1:

ggatcccctaaaggctatcagagctttgaagccgtgaatgaagaaattaatagcattgtgagtgagctgaaaaatgaaggtatgagcctgcagaatattcatcatatgtttaaacagagcatccagaatctggcaacccgcattggctatcgcagttttatgcaggatgccatgtatctggaaaattttgaacgtctgaccattccggaactggatgaagcctatgtggatctgctggttaattatgaagttaaacatcgcattctggtgaaatatgaaggtaaagttaagggccgcgccccgctggaagcatttattgtgccgctgcgtgatcgtattcgtagtatgaatgaaattgcagcagaagtgaattatctgccggaagcccatgaagattttctggttagtgatagcagtgaatataatgataagctgaataacatcaacttcgccctgggtctgggcgttagtgaatttattgattataatcgcctggaaaacatgatggaaaaagaaattcatccgctgtatctggaactgtatgcaatgcgccgtaatcgccagattcaggttgttcgcgatgtgtatccgaatctggaacgtgcaaatgccgttgttgaaagcctgaaaaccattaaagatattaagcagcgtggcaaaaaactgcaggaactgctggaaatttatattcagcgcagcggtgatgtgcgtaaaccggatgttctgcagcgctttattggtaaatatcagagtgtggtggatgaagaaaaaaataaactgcaggattacctggaaagtgatatttttgatagttacagcgttgatggtgaaaaaattcgcaataaagaaatcaccctgattaatcgcgatgcatatctgagcatgatttatcgtgcacagagcattagcgaaattaaaaccattcgtgccgatctggaaagtctggttaaaagctttcagaatgaagaaagcgatagcaaagtggaaccggaaagcccggttaaagttgaaaaaccggttgatgaagaaaagccgaaagatcagtaactcgag.

the amino acid sequence is shown in SEQ ID NO. 2:

GSPKGYQSFEAVNEEINSIVSELKNEGMSLQNIHHMFKQSIQNLATRIGYRSFMQDAMYLENFERLTIPELDEAYVDLLVNYEVKHRILVKYEGKVKGRAPLEAFIVPLRDRIRSMNEIAAEVNYLPEAHEDFLVSDSSEYNDKLNNINFALGLGVSEFIDYNRLENMMEKEIHPLYLELYAMRRNRQIQVVRDVYPNLERANAVVESLKTIKDIKQRGKKLQELLEIYIQRSGDVRKPDVLQRFIGKYQSVVDEEKNKLQDYLESDIFDSYSVDGEKIRNKEITLINRDAYLSMIYRAQSISEIKTIRADLESLVKSFQNEESDSKVEPESPVKVEKPVDEEKPKDQLE.

(2) Construction of fusion expression vectors

PGEX-4T-1 and the synthetic plasmid containing the SpaA-N gene were digested simultaneously with BamHI and XhoI. The reaction system was 20. Mu.L of 3. Mu.L of plasmid, 2. Mu.L of 10 XBuffer, 1. Mu. LBamHI, 1. Mu. LXhoI, 13. Mu.L of ddH ₂ O. The target gene fragment was purified and recovered, 3. Mu.L of the vector (pGEX-4T-1), 1. Mu.L of the recovered product was digested, 1. Mu.L of the ligase, and 1. Mu.L of the 10 XT 4 DNAbuffer mixture were mixed and ligated overnight at 4 ℃. Transferring the recombinant plasmid into cloning competent cells DH5 alpha, selecting ampicillin-resistant LB coated plates, airing, inverting a 37 ℃ incubator overnight, picking single colonies, shaking to a logarithmic phase, and extracting the recombinant plasmid for later use.

Expression and purification of SpaA-N

(1) Construction of genetically engineered Strain of recombinant SpaA-N Gene

The extracted recombinant plasmid is transformed into competent cells of escherichia coli BL21 (DE 3), monoclonal is selected and cultured in LB liquid medium containing ampicillin at 37 ℃ for overnight in a shaking way, after PCR identification, the recombinant plasmid contains target DNA fragments, the recombinant plasmid is named as escherichia coli (ESCHERICHIA COLI) BL/SpaA-N strain, and 50% glycerol LB with the same volume is added for freezing at-70 ℃.

(2) Expression and purification of SpaA-N

Recombinant escherichia coli (E.coli) BL/SpaA-N strain is inoculated into 1L of LB liquid medium containing ampicillin for culture, when OD ₆₀₀ is 0.6-0.8 in shaking culture at 37 ℃, IPTG solution with the final concentration of 0.5mM is added for low-temperature induction culture for 16h. After the bacterial liquid culture is completed, the bacterial bodies are collected by centrifugation, the bacterial bodies are resuspended according to the proportion of adding 10mL PBS (phosphate buffered saline) into each gram of bacterial body weight, and the bacterial bodies are crushed by ultrasound in an ice-water bath for 30min under the conditions of 9s working, 9s intermittence and 400W ultrasonic power. The crushed bacterial liquid is centrifuged at 12000r/min for 10min at 4 ℃ and the supernatant is collected.

The target protein expressed in a soluble form in the cell lysate supernatant was purified according to the instructions of GST fusion protein purification kit (Nanjing gold Style Co.), and filtered through a 0.22 μm pore size filter membrane to obtain the target protein for preliminary purification.

SDS-PAGE identification is carried out on the purified target protein, and the electrophoresis result is shown in figure 1. As can be seen from FIG. 1, the purified protein size is consistent with the expected size of the target protein. The invention shows that the SpaA-N protein antigen is obtained by successful recombinant expression.

Example 2

Establishment of indirect ELISA detection method

Using a square matrix titration method, the SpaA-N protein was diluted in a horizontal double ratio with ELISA coating solution at a mass concentration of 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.3. Mu.g/mL, 0.2. Mu.g/mL, 0.1. Mu.g/mL, 0.05. Mu.g/mL, 100. Mu.L/well coated 96-well ELISA plate, and washed three times with PBST overnight at 4 ℃.

The wells were blocked with 5% skim milk for 2h, 100. Mu.L/well, and washed 3 times with PBST. Mouse positive and negative sera were diluted in longitudinal gradients at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 100 μl/well, 37 ℃ for 2h, and pbst washed 3 times. Goat anti-mouse secondary antibody was added at 1:10000 dilution of HRP, incubated at 37℃for 1h at 100. Mu.L per well, and washed 3 times with PBST.

50. Mu.L of each of the color-developing solution and the stop solution was added, the D _450nm value was measured, and the P/N was calculated.

The detection results are shown in Table 1.

The results are shown in Table 1. As can be seen from the data in Table 1, the P/N value was greater than 2.0 and the N value was minimal at a protein coating concentration of 0.2. Mu.g/ml at a serum dilution of 1:3200. Thus, an indirect ELISA assay kit was prepared at a serum dilution of 1:3200 at a selectin coating concentration of 0.2 μg/ml.

TABLE 1 protein coating concentration and serum dilution assay results

Example 3

Screening of SpaA protein monoclonal antibody hybridoma cell strain secreting erysipelas and preparation of monoclonal antibody ascites

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

4 BALB/c mice with 6-8 weeks of age are immunized by using the erysipelas Sus Domestica strain (preservation number CVCC 43008) wall protein as an immunization antigen, wherein the immunization is performed for 4 times at intervals of 2 weeks. Adding equal volume Freund complete adjuvant into the protein for primary immunization, emulsifying, immunizing a mouse by a neck and back multipoint subcutaneous injection way, changing the adjuvant into Freund incomplete adjuvant after immunization, and taking blood from the tail of the mouse after the immunization for 7-10 days, coating an ELISA plate with purified SpaA-N protein by 0.2 mug/mL, measuring serum titer by an indirect ELISA method, selecting the mouse with titer higher than 1:1600, enhancing immunity three days before fusion, directly injecting the mouse into the protein abdominal cavity, and obtaining the dose of 120 mug/mouse. Higher titers of mouse spleen cells were taken 3-5 days after the last immunization for cell fusion.

2. Cell fusion and selection of hybridoma cell lines

The method comprises the steps of taking myeloma cells SP2/0 with good growth state, detecting titers by using indirect ELISA of SpaA-N coated antigen, and selecting mouse spleen cells with highest titers to carry out chemical fusion by using PEG 1450. Positive hybridoma cells are screened by indirect ELISA of the SpaA-N coated antigen prepared in example 2, and three strains of hybridoma cells, namely SpaA-21D, spaA-25C and SpaA-42G, are obtained by screening after 3-5 subcloning by a limiting dilution method. The SpaA-21D, spaA-25C and SpaA-42G were passaged multiple times and the stability of antibody secretion by the three hybridoma cell lines was assessed by indirect ELISA. The results show that SpaA-25C and SpaA-42G do not stably secrete antibodies during passage and hybridoma cells are not in good condition, while SpaA-21D stably secrete antibodies during passage and hybridoma cells are in good condition. Identification was performed using the AMb subtype kit, and the hybridoma cell line SpaA-21D secreted a monoclonal antibody subtype that was G2b.

3. Preparation of SpaA protein monoclonal antibody ascites against erysipelas Sus domestica

6-8 Month old and healthy warp-produced BALB/c mice are selected, freund's incomplete adjuvant is injected for sensitization, 0.5 mL/mouse is used, and hybridoma cells are injected after 7 d. Collecting the three hybridoma cells in logarithmic growth phase and SP2/0 cells, centrifuging for 10min at 800r/min, washing the cells once with DMEM culture solution, re-suspending, adjusting the cell density to 10 ⁶/mL, and injecting 0.5mL into the abdominal cavity of each mouse. After 7-14 days, when the abdomen of the mouse is obviously swelled, a needle of a 20ml syringe is used for aseptically puncturing the lower peripheral swelled part of the abdomen of the mouse, gentle kneading is carried out to enable ascites to flow out or drip out, a 15ml centrifuge tube is used for collecting the ascites, 3000r/min is used for centrifugation for 10min, the ascites is marked as SpaA-21D and ascites control (S) of myeloma cells SP2/0, and the ascites is kept at-20 ℃ for standby after sub-packaging.

Example 4

Purification and detection of monoclonal ascites

The ascites prepared in example 3 was purified by the ProteinA (GE HEALTHCARE-5079-01) affinity column purification method as follows:

Sample pretreatment, namely, diluting ascites with a coupling buffer (20 mM sodium phosphate buffer, pH 7.0) at a ratio of 1:3, centrifuging at 12000rpm and 4 ℃ for 10min, and filtering with a 0.22 μm filter membrane to remove fat, cell residues and small particulate matters.

Balancing, namely balancing the column by using coupling buffer solution with the volume of 5-10 times of the column, and keeping the flow rate at 2 s/drop.

Loading the sample into the upper end of the column with a syringe, collecting the effluent in a 50ml centrifuge tube, and maintaining the flow rate at 4 s/drop.

Washing impurities, namely passing the coupling buffer solution through a column with the volume of 5 times of the column, and keeping the flow rate at 2 s/drop.

Elution the antibody was eluted with 5 column volumes of elution buffer (0.1M sodium citrate buffer, pH 9.0) and collected in the EP tube described above, maintaining a flow rate of 4 s/drop.

Sample detection, namely performing SDS-PAGE identification (see figure 3) on the monoclonal antibody obtained by purification, obtaining the monoclonal antibody with high purity by a method of purifying by a ProteinA affinity column, and measuring the concentration of the monoclonal antibody by adopting a BCA method, wherein the concentration can reach 4.2mg/mL.

Example 5

Establishment of immunohistochemical detection method of erysipelas suis antibody based on monoclonal antibody SpaA-21D

1. Paraffin section preparation:

(1) Placing pig heart tissue infected with swine erysipelas in 4% paraformaldehyde solution, and fixing for 48h;

(2) Trimming the section of the tissue, and placing the trimmed tissue block into an automatic dehydrator for dehydration, transparency and wax dipping;

(3) Embedding the waxed tissue sample with an embedding wax;

(4) Slicing with paraffin slicer with thickness of 5 μm, and storing;

2. Determination of immunohistochemical detection method conditions of erysipelas suis antibody

(1) Tissue sections were dewaxed in xylene solution, hydrated with gradient alcohol to distilled water, then soaked in 0.01M PBS for 5min, PBS replaced, and washed 3 times. Placing the tissue slice into the prepared sodium citrate repairing solution, and performing antigen repairing by using a microwave oven, wherein the temperature is kept at 90-95 ℃ for 20min;

(2) The repair box was taken out of the microwave oven, after it was returned to room temperature, washed 3 times with 0.01MPBS for 5min each with 3% methanol-hydrogen peroxide solution and blocked for 30min. Washing with 0.01MPBS for 3 times for 5min each time, dripping 5% goat serum, and sealing for 30min;

(3) The serum is discarded, primary antibody (monoclonal antibody SpaA-21D against erysipelas Sus) is added dropwise, the dilution concentration of the antibody is 1:100, the mixture is placed in a refrigerator at 4 ℃ overnight, the mixture is taken out and rewarmed for 30min in the next day, and the mixture is washed with 0.01M PBS for 3 times for 5min each time. Diluting the secondary antibody (goat anti-mouse IgG) to 1:400 by using PBS, dropwise adding the diluted secondary antibody, incubating for 2min at room temperature, and washing with PBS for 3 times and 5min each time;

(4) And (3) performing color development by using VectorVIP HRP color development kit, wherein the color development time is controlled to be about 1min, and stopping the color development by using distilled water after the color development is completed according to the microscopic examination condition. The hematoxylin dye liquor is counterstained for 10s, distilled water is used for washing out the dye liquor, tap water is used for bluing for 10min, and the wafer is dehydrated and sealed.

(5) Determination of result decision criteria

The established swine erysipelas immunohistochemical detection method is used for detecting heart tissues infected with swine erysipelas, and negative controls are set. Using the VIP HRP chromogenic system, the positive signal developed purple (erysipelothrix rhusiopathiae). A positive test was performed if purple coloration was observed in the infected heart tissue, as indicated by the arrow in FIG. 3, and a negative test was performed if purple coloration was not observed in the tissue.

Example 6

Specific detection method of monoclonal antibody SpaA-21D

The SpaA-N recombinant bacteria whole-cell protein, GST-tagged empty carrier protein, C43008 strain wall protein, bulbumin bovis Seu Bubali bacterium A whole-cell protein, bulbumin bovis Seu Bulbumin B whole-cell protein and Bulbumin bovis Seu Bulbumin E whole-cell protein are respectively used as materials to prepare protein solutions, and immunoblotting detection is carried out.

The results are shown in FIG. 4. As shown in FIG. 4, the monoclonal antibody SpaA-21D of the invention only has binding property to pGEX-4T-1/SpaA-N recombinant bacteria whole protein, but other kinds of proteins can not be bound.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A murine hybridoma cell SpaA-21D is characterized by having a preservation number of CGMCC No.45348.

2. An anti-erysipelas monoclonal antibody, which is secreted by the murine hybridoma cell SpaA-21D of claim 1.

3. An immunological detection technology-based erysipelas detection kit is characterized by comprising the erysipelas-resistant monoclonal antibody and other detection reagents.

4. The swine erysipelothrix rhusiopathiae detection kit based on the immunological technique according to claim 3, wherein the immunological detection technique comprises at least one of the following techniques of immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting and immunochromatography.

5. The swine erysipelothrix rhusiopathiae detection kit based on the immune technology of claim 4, wherein the swine erysipelothrix rhusiopathiae detection kit is used for immunohistochemical detection of swine erysipelothrix rhusiopathiae;

other detection reagents in the erysipelas immune histochemical detection kit comprise antibody diluent, secondary antibody, washing liquid and chromogenic substrate.

6. The kit for detecting erysipelothrix rhusiopathiae according to claim 5, wherein the working dilution of the monoclonal antibody against erysipelothrix rhusiopathiae is 1:100.

7. The swine erysipelothrix rhusiopathiae detection kit based on the immunological technique of claim 5, wherein the working dilution of the secondary antibody is 1:400.

8. The immunoassay technology-based erysipelothrix rhusiopathiae detection kit of claim 5, wherein the washing solution is a PBS solution;

the antibody diluent is PBS solution.

9. The swine erysipelothrix rhusiopathiae detection kit based on an immune technique of claim 5, wherein the secondary antibody is goat anti-mouse IgG.

10. The immunoassay-based erysipelothrix rhusiopathiae detection kit of claim 3 or 4, further comprising an indirect ELISA detection kit;

the other detection reagents in the indirect ELISA detection kit comprise at least one component of a detection plate coated with the modified erysipelas treponema hyopneum antigen, a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a color development solution and a termination solution;

The amino acid sequence of the modified erysipelas Sus Domestica antigen is shown as SEQ ID NO. 2.