Hybridoma cell strain Mhr0461-8E3, anti-mycoplasma hyorhinis monoclonal antibody secreted by hybridoma cell strain Mhr0461-8E3 and application of monoclonal antibody

Abstract

The invention belongs to the field of immunology, and discloses a hybridoma cell strain Mhr0461-8E3, an anti-mycoplasma hyorhinis monoclonal antibody secreted by the hybridoma cell strain Mhr0461-8E3 and application of the monoclonal antibody. The preservation number of the hybridoma cell strain is CCTCC (China Center For Type Culture Collection) NO: C2024162, the hybridoma cell strain can generate a monoclonal antibody aiming at the mycoplasma hyorhinis outer membrane protein Mhr0461, and the Mhr0461 is the outer membrane protein which is specific to the mycoplasma hyorhinis and has unknown functions. The ELISA method established by using the Mhr0461-8E3 monoclonal antibody only can specifically recognize the mycoplasma hyorhinis, and has no cross reaction with other mycoplasmas, bacteria and viral proteins; the established indirect immunofluorescence method can specifically mark the mycoplasma hyorhinis and shows specific fluorescence under a microscope; the monoclonal antibody has good reaction activity, high titer and strong specificity, and provides a technical means for diagnosis, prevention and treatment of mycoplasma hyorhinis.

Description

Technical Field

[0001] The invention belongs to the field of immunology, and specifically relates to a hybridoma cell line Mhr_0461-8E3, and anti-hyorhinis mycoplasma monoclonal antibodies secreted by the hybridoma cell line and applications. Background Art

[0002] Mycoplasma hyorhinis (Mhr) is a cell wall-less, self-replicating prokaryotic microorganism. The infection rate of Mhr is extremely high and is commonly found in the nasal, tracheal and bronchial secretions of sick pigs. It is mainly transmitted through droplets and direct contact. Infection with Mhr can cause symptoms such as polyserositis, arthritis, and pneumonia. It is often mixed with other pathogens, which increases the difficulty of disease treatment and causes huge economic losses to the breeding industry.

[0003] Mycoplasma is a recognized source of contamination in the field of laboratory cells and biological products. Nature magazine published a special article emphasizing the prevalence and severity of mycoplasma contamination. Researchers at the University of Pennsylvania found that mycoplasma contamination is more common than we thought. The U.S. Food and Drug Administration tested more than 20,000 cell cultures and found that more than 15% of cell cultures were contaminated with mycoplasma. A study by the German DSMZ (National Culture Collection Center) showed that 28% of 440 cell lines (mostly leukemia and lymphoma cells) were contaminated with mycoplasma. Among the eight common sources of mycoplasma contamination, Mycoplasma hyorhinis is an extremely important source of contamination for cells and biological products. It not only seriously affects the orderly progress of scientific research, but also threatens public health safety. It has become a global problem.

[0004] Studies in recent years have found that Mycoplasma hyorhinis can be detected and directly isolated from human gastric cancer tissue. Further studies have shown that there is a significant correlation between infection with Mycoplasma hyorhinis and a variety of tumors such as gastric cancer, lung cancer, pancreatic cancer, colorectal cancer, and prostate cancer, posing a threat to human health. Although Mycoplasma hyorhinis is highly correlated with tumor occurrence, little is known about its infection, pathogenic mechanism, and immunological status. At present, there is no diagnostic kit for Mycoplasma hyorhinis in China, so there is an urgent need to establish a method that can directly detect Mycoplasma hyorhinis to meet the growing demand for Mycoplasma hyorhinis prevention and control.

[0005] Mycoplasma hyorhinis has no cell wall structure, and its outer membrane protein undertakes multiple important functions such as colonization, infection, pathogenicity, and interaction with host cells. The immunogen Mhr_0461 (CN113444157B) used in the present invention for preparing monoclonal antibodies is a Mycoplasma hyorhinis outer membrane protein unique to Mycoplasma hyorhinis discovered by the applicant in the early stage. It is an immunogenic protein with extremely high specificity and can be used as a molecular target for the detection of Mycoplasma hyorhinis or its antibodies. At present, there is no monoclonal antibody prepared against the immune protein.

[0006] Monoclonal antibodies have the characteristics of high purity, good repeatability, and strong specificity in binding to antigens. However, the preparation of monoclonal antibodies, especially the screening of hybridoma cells, is accidental. Using the same immunogen, the same immunization method, the same screening method, and the same person for testing, the hybridoma cell lines finally screened out are often different. The specificity, sensitivity, stability, titer, function, application range, etc. of the antibodies produced are also uncertain. It takes multiple rounds of screening to obtain good monoclonal antibodies.

[0007] The present invention lays a foundation for preparing high-quality hyorhinitis mycoplasma detection reagents or kits, and provides technical support for the prevention of hyorhinitis mycoplasma. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the applicant provides a hybridoma cell line Mhr_0461-8E3, and the deposit number of the hybridoma cell line is CCTCC NO: C2024162.

[0009] Another object of the present invention is to provide a monoclonal antibody secreted by hybridoma cell line Mhr_0461-8E3.

[0010] The last object of the present invention is to provide the use of the hybridoma cell line Mhr_0461-8E3 or the monoclonal antibody secreted by it. In order to achieve the above object, the present invention adopts the following technical measures:

[0011] Based on the previous research results, the applicant used Mhr_0461 protein as an antigen, and then obtained a hybridoma cell line by immunizing experimental animals and fusing the spleen cells of immunized animals with myeloma cells. The hybridoma cell line was screened through multiple rounds of stability, titer, etc., and finally a hybridoma cell line Mhr_0461-8E3 was obtained. The hybridoma cell line was sent to the China Center for Type Culture Collection for preservation on May 27, 2024, with the preservation number: CCTCC NO: C2024162, and the classification name is: Hybridoma cell line (Hybridoma cell line) Mhr_0461-8E3, address: Wuhan University, Wuhan, China.

[0012] The hybridoma cell line Mhr_0461-8E3 described above is stable in passage, has an average chromosome number of 102, and secretes monoclonal antibodies of which the heavy chain subtype is IgM and the light chain subtype is Kappa.

[0013] The protection scope of the present invention includes:

[0014] Monoclonal antibody secreted by hybridoma cell line Mhr_0461-8E3.

[0015] Application of hybridoma cell line Mhr_0461-8E3 or the monoclonal antibody secreted by it in the preparation of a kit for detecting Mycoplasma hyorhinis.

[0016] Application of hybridoma cell line Mhr_0461-8E3 or the monoclonal antibody secreted by it in the preparation of a kit for detecting Mycoplasma hyorhinis antigen.

[0017] In the above application, preferably, the kit is an ELISA detection kit.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a monoclonal antibody capable of specifically identifying a Mycoplasma hyorhinis antigen. The Mycoplasma hyorhinis ELISA, Western blot detection method and immunofluorescence detection method established using the monoclonal antibody can specifically identify the Mycoplasma hyorhinis and have strong reactivity and specificity.

[0020] The ELISA detection method for Mycoplasma hyorhinis established using this monoclonal antibody has a minimum detection amount of 1.5625 ng / mL for the whole bacterial antigen of Mycoplasma hyorhinis.

[0021] The immunofluorescence detection method established using this monoclonal antibody can directly bind specifically to hyorhinis Mycoplasma and label it with fluorescence, thereby achieving visual labeling and tracing of hyorhinis Mycoplasma under a microscope.

[0022] Monoclonal antibodies have good immunological activity, and the titer of antibodies secreted in the supernatant by hybridoma cells is as high as 1×10 6 The purified ascites fluid has a titer of up to 10 8 .

[0023] The hybridoma cell line Mhr_0461-8E3 is stable in culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is the chromosome map of hybridoma cell line Mhr_0461-8E3.

[0025] Figure 2 The Western blot test results of Mhr_0461-8E3 monoclonal antibody;

[0026] In the figure, M: protein marker; 1: BSA protein; 2: Mhr_0461 recombinant protein.

[0027] Figure 3 This is an ELISA diagram of the ELISA method based on the Mhr_0461-8E3 monoclonal antibody reacting with other common pathogen antigens.

[0028] Figure 4 This is a fluorescence micrograph of cells infected with Mycoplasma hyorhinis detected by indirect immunofluorescence method based on the Mhr_0461-8E3 monoclonal antibody;

[0029] A: porcine alveolar macrophages artificially infected with Mycoplasma hyorhinis; B: porcine alveolar macrophages not infected with Mycoplasma hyorhinis. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Embodiment 1:

[0032] Prokaryotic expression and purification of Mycoplasma hyorhinis Mhr_0461 protein:

[0033] 1. Synthesis of Mycoplasma hyorhinis Mhr_0461 gene

[0034] The Mhr_0461 (GenBank accession number ADM21918.1) in the genome of Mycoplasma hyorhinis HUB-1 strain (Liu W, Fang LR, Li S, et al. Complete genome sequence of Mycoplasma hyorhinis Strain HUB-1. Journal of Bacteriology. Nov. 2010, p. 5844-5845) was used as the research object, its transmembrane region was deleted, and one UGA codon was adjusted to UGG according to the codon preference of Escherichia coli. The optimized Mycoplasma hyorhinis outer membrane gene Mhr_0461 (nucleotide sequence is shown in SEQ ID NO.1 in the sequence table) was artificially synthesized.

[0035] 2. Construction of pET30a-Mhr_0461 recombinant plasmid

[0036] The artificially synthesized Mhr_0461 gene was cloned into the corresponding sites (BamH I and Xho I) of the prokaryotic expression vector pET-30a(+) (Novagen, Germany) through BamH I and Xho I to obtain the recombinant plasmid pET30a-Mhr_0461 expressing the Mhr_0461 gene.

[0037] 3. Prokaryotic expression and purification of recombinant plasmid pET30a-Mhr_0461

[0038] The expression plasmid pET30a-Mhr_0461 (kanamycin resistance) containing the gene of Mycoplasma hyorhinis Mhr_0461 was transformed into Escherichia coli BL21 competent cells. Induction was carried out under the conditions of a final concentration of 1.0 mM IPTG, 37°C and 180 r / min. The Mhr_0461 fusion protein expressed in a soluble form was purified by AKTA FPLC (Amersham Biosciences UPC-900), the concentration of the purified protein was determined by a spectrophotometer, and its purity was detected by SDS-PAGE, and stored at -80°C for use. The prokaryotic expressed Mhr_0461 protein here carries a 6×His tag and is named as the recombinant protein rMhr_0461 (the sequence is shown in SEQ ID NO.2).

[0039] Embodiment 2:

[0040] Preparation of monoclonal antibody against Mycoplasma hyorhinis Mhr_0461

[0041] 1. Animal immunization

[0042] Purified recombinant protein rMhr_0461 was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant in equal amounts, and used as an immunogen to immunize Balb / c mice after complete emulsification. The immunization scheme is as follows: multiple subcutaneous injections (rMhr_0461 protein + Freund's complete adjuvant) were given at the back of the neck of the mice for the first immunization; the second immunization was given subcutaneously (rMhr_0461 protein + Freund's incomplete adjuvant), two weeks apart from the first immunization. The third immunization was given subcutaneously (rMhr_0461 protein + Freund's incomplete adjuvant), three weeks apart from the second immunization. Blood was collected from the retro-orbital venous plexus 7 days after the second immunization and 7 days after the third immunization to measure the antibody titer. One day before fusion, rMhr_0461 protein was injected intraperitoneally to boost immunization.

[0043] 2. Cell fusion

[0044] SP2 / 0 myeloma cells were revived and injected subcutaneously into the back of Balb / c mice. After the tumors grew, they were separated to obtain rejuvenated SP2 / 0 myeloma cells. The rMhr_0461 protein was used as the coating antigen to establish an ELISA method, and the mice with the highest serum antibody titer were screened. The spleens of the mice were removed aseptically to prepare spleen cells. 1×10 8 splenocytes and 2×10 7 The suspension of myeloma cells was fused under the action of fusion agent PEG4000. The fused cells were resuspended in HAT selection medium and added to a 96-well cell plate with feeder cells, 100 μL per well, and placed in a 5% CO 2 Culture in an incubator and observe and record cell growth every day.

[0045] 3. Screening and cloning of hybridoma cells

[0046] After the fused cell clones grow to cover 1 / 4 to 1 / 3 of the bottom of the cell well, the indirect ELISA method is used for detection, and the hybridoma cell wells that are positive in the ELISA test are cloned by limiting dilution. The specific steps are as follows: prepare feeder cells, plate them on the night before cloning, one drop per well; blow the cells evenly with a broken 200μL gun tip, and count them according to the amount of cells; in a 96-well cell culture plate, follow 10 / well, 5 / well, 1 / well, 4 columns for each concentration; dilute to an appropriate concentration with complete medium containing HT and inoculate on a culture plate. Subsequent cloning uses complete medium without HT. Place in an incubator for culture, observe the growth of hybridoma cells, and when the cell culture supernatant turns yellow or the clones are distributed to more than 1 / 10 of the bottom area of ​​the well, aspirate 100μL of cell supernatant for antibody detection.

[0047] The purified Mhr_0461 protein was used as the coating antigen, and the antibody detection was performed on 240 cell wells by indirect ELISA method. The cell wells with strong ELISA positive were further cloned by limiting dilution method. The negative wells were tested again after 3 days. If they were still negative, they were discarded. The cloning steps were repeated 4 times. After 4 rounds of screening and cloning culture, 6 positive clones with high antibody titers were obtained, namely 8B3, 8G3, 8C3, 8C4, 8E3, and 8B4. The screened cell lines were further expanded and cultured. Combined with the subtype of secreted antibodies, the titer of produced antibodies, passage and growth characteristics, a hybridoma cell line 8E3 with good growth, stable passage, and efficient secretion of anti-swine rhinoplasma Mhr_0461 monoclonal antibodies was finally screened. The hybridoma cell was still stable at the D60th generation, and the one sent for preservation was the D20th generation, named Mhr_0461-8E3. The indirect ELISA method was used to detect the titer of the antibody secreted in the supernatant of hybridoma cells. The results showed that the antibody titer was 1×106 The hybridoma cell line was sent to China Center for Type Culture Collection on May 27, 2024 for preservation, with the preservation number: CCTCC NO: C2024162, the classification name: Hybridoma cell line Mhr_0461-8E3, address: Wuhan University, Wuhan, China.

[0048] 4. Chromosome counting of hybridoma cells

[0049] When the hybridoma cells are in the logarithmic growth phase 36-48 hours after subculturing, add colchicine to a final concentration of 0.3μg / mL and continue to culture for 6 hours; gently blow the cells and centrifuge at 1000r / min for 10 minutes, discard the supernatant; add 3mL of 0.075mol / L KCL hypotonic solution preheated at 37℃, blow the cells to suspend and mix, and bathe at 37℃ for 15-20 minutes; add 1mL of fixative and centrifuge at 1000r / min for 10 minutes , discard the supernatant; add 5mL of fixative dropwise, let stand for 30min, centrifuge at 1000r / min for 10min, and discard the supernatant; fix again, same as above; add 5mL of fixative, mix well, seal the tube, and keep at 4℃ overnight; 1000r / min, 10min, discard the supernatant, leave 0.2mL of supernatant, mix well, use a dropper to absorb the cell suspension, drop it on a 4℃ precooled slide from a height of 10-20cm, blow it away immediately, spread the cells on it, and dry naturally; stain with 10% Giemsa for 10min, wash off the stain with tap water, and dry naturally; select cells with well-dispersed chromosomes, no overlap, and no loss for observation and counting. Count the number of chromosomes in 10 cells and calculate the average value. The average value is 102 / cell, see Figure 1 .

[0050] 5. Preparation and purification of ascites

[0051] Sterile liquid paraffin was intraperitoneally injected into BALB / c mice at a dose of 0.5 mL / mouse. One week later, hybridoma cells 8E3 (in logarithmic growth phase) diluted with 1640 medium were intraperitoneally injected at a dose of 2×10 6 When the abdomen of the mouse was obviously bulging, the ascites was collected and centrifuged at 8000 rpm for 10 min to remove the foam and grease on the surface, and the supernatant was aspirated and stored at -80°C for later use.

[0052] The ascites was purified using a subclass mouse monoclonal antibody purification kit (purchased from Beijing Biolong Immunotechnology Co., Ltd.). The purification steps are as follows: rewarm at room temperature for 30 minutes; add the purification solution A in the kit to the supernatant and mix well, and stand at room temperature in the dark for 30 minutes; centrifuge at 8000rpm for 10 minutes, discard the supernatant, dissolve the precipitate with purification solution B with half the volume of the original ascites, add purification solution C and stand at room temperature in the dark for 10 minutes; centrifuge at 8000rpm for 10 minutes, discard the precipitate; add purification solution D to the supernatant, stand at room temperature in the dark for 20 minutes; centrifuge at 8000rpm for 10 minutes, discard the supernatant, dissolve the precipitate with purification solution B to 1 / 3 of the volume of the original ascites, put it into a dialysis bag, and dialyze at 4°C overnight; after dialysis, mix the antibody and purification solution E at a volume ratio of 9:1, stand for 5 minutes; centrifuge at 8000rpm for 10 minutes, collect the supernatant and dialyze again at 4°C, change the solution once every 4 hours, collect the antibody after three times, centrifuge at 8000rpm for 10 minutes, collect the supernatant and measure the concentration, and the concentration reaches 1.47 mg / mL. The purification effect was detected by SDS-PAGE, and the results showed that the impurities were basically removed after purification. The ELISA method was used to detect the titer of the purified antibody, and the titer was 10 8 , used in the following examples.

[0053] 6. Monoclonal antibody subtype identification

[0054] The subtype identification of monoclonal antibodies was performed using a mouse monoclonal antibody subtype identification kit (purchased from Proteintech). The specific steps are as follows: equilibrate the monoclonal antibody subtype identification kit at room temperature for 30 minutes; take the hybridoma cell supernatant, dilute it with PBST at 1:100, and add it to the sample wells in turn, 50μL / well; add HRP-labeled goat anti-mouse secondary antibody (IgA+IgM+IgG) to the sample wells, 50μL / well; mix thoroughly, incubate at room temperature for 1h, discard the liquid in the well, wash 3 times with PBST, 5min each time, and discard the liquid in the well; add color development solution, 100μL / well; color development at room temperature in the dark for 10-20min; add stop solution, 100μL / well; determine the OD by naked eye according to the instructions or measure it with an enzyme reader 450nm The monoclonal antibody subtype was determined, the antibody heavy chain subtype was IgM, and the light chain subtype was Kappa.

[0055] Embodiment 3:

[0056] Reactivity verification of monoclonal antibody against Mycoplasma hyorhinis Mhr_0461

[0057] Purified rMhr_0461 protein and control protein BSA were added to 5× Loading Buffer, boiled in boiling water at 100°C for 10 minutes, and then ice-bathed for 10 minutes before 12% SDS-PAGE electrophoresis. After electrophoresis, the protein was transferred to a nitrocellulose membrane. After transfer, the membrane was blocked with 5% skim milk at 4°C overnight, washed with TBST three times for 5 minutes each time, and then incubated with Mhr_0461 monoclonal antibody for 1 hour (monoclonal antibody was diluted with TBST at a volume ratio of 1:5000). The membrane was washed with TBST three times for 5 minutes each time, and then incubated with HRP-labeled goat anti-mouse secondary antibody for 1 hour (volume ratio 1:5000). After incubation, the membrane was washed with TBST five times for 5 minutes each time, and chemiluminescence was used for color development and image acquisition.

[0058] The results of Western Blot test showed that the Mhr_0461 monoclonal antibody could specifically bind to the rMhr_0461 protein, but did not react with the control protein BSA, indicating that the Mhr_0461 monoclonal antibody had good reactivity ( Figure 2 ).

[0059] Embodiment 4:

[0060] Specificity verification of monoclonal antibody against Mycoplasma hyorhinis Mhr_0461

[0061] In this embodiment, the applicant used the Mhr_0461 monoclonal antibody and used the ELISA method to detect antigenic proteins of common pig pathogens in clinical practice, including proteins encoded by Mycoplasma hyopneumoniae, Streptococcus suis, porcine rotavirus, and Actinobacillus pleuropneumoniae.

[0062] The specific operations are as follows:

[0063] (1) The codon-optimized Mycoplasma hyopneumoniae LppB gene (WP_014579843.1), Streptococcus suis codY gene (WP_011921926.1), porcine rotavirus VP1 gene (MZ165445.1), and Actinobacillus pleuropneumoniae afuC gene (AAB05030.1) were cloned into prokaryotic expression plasmids, transformed into Escherichia coli BL21 competent cells, and induced by IPTG for expression. The proteins were purified using AKTA FPLC (Amersham Biosciences UPC-900) to obtain the purified products of the above proteins.

[0064] (2) Antigen coating: Use coating solution (Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, add ddH 20 to 1000 mL) dilute the purified proteins of the above pathogens and the rMhr_0461 protein of Mycoplasma hyorhinis to 1 μg / mL, add 100 μL of each diluted protein to each well of a 96-well ELISA plate, and coat overnight at 4°C. Discard the liquid in the wells, add 200 μL of PBST (PBS: NaCl 8.0 g, KCl 0.2 g, Na 2 HPO 4 12H 2 O 2.9 g, NaH 2 PO 4 0.2g, add ddH 2 0 to 1000mL; PBST: add 0.05% Tween-20 to PBS), wash 5 times, and discard the liquid in the wells after 2 minutes each time. In the last wash, fully discard the liquid in the wells.

[0065] (3) Blocking: Add 100 μL of 5% skim milk (5 g skim milk plus PBST to 100 mL) to each well, place in a 37°C incubator for 1 h, discard the liquid in the wells, add 200 μL of PBST, wash 5 times, and shake off the liquid in the wells after 2 minutes each time. During the last wash, fully discard the liquid in the wells.

[0066] (4) Sample addition: Dilute the Mhr_0461 monoclonal antibody with PBST at a ratio of 1:5000 (V / V), add 100 μL of the diluted sample to each well, incubate at 37°C for 1 h, discard the liquid in the wells, add 200 μL PBST, wash 5 times, 2 minutes each time, and then discard the liquid in the wells. During the last wash, fully discard the liquid in the wells.

[0067] (5) Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse secondary antibody (purchased from ProteinTech) with PBST at a ratio of 1:5000 (V / V), add 100 μL of diluted secondary antibody to each well, and incubate at 37°C for 60 minutes. Discard the liquid in the well, add 200 μL PBST, wash 5 times, 2 minutes each time, and discard the liquid in the well. During the last wash, fully discard the liquid in the well.

[0068] (6) Add substrate for color development: 50 μL each of TMB color development solution A and TMB color development solution B (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) were added in sequence, and the color was developed for 10 min in the dark. Subsequently, 50 μL of stop solution (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) was added to each well to terminate the reaction.

[0069] (7) Read the absorbance (OD) at 450 nm using an ELISA reader. 450nm value).

[0070] Specific test results such as Figure 3 The results showed that the Mhr_0461 monoclonal antibody was highly specific only to the antigen of Mycoplasma hyorhinis, and had no cross-reaction with other common pathogen antigens such as Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Streptococcus suis, indicating that the screened Mhr_0461 monoclonal antibody had good specificity.

[0071] Embodiment 5:

[0072] Sensitivity test of monoclonal antibody against Mycoplasma hyorhinis Mhr_0461

[0073] In this example, the applicant used the Mhr_0461 monoclonal antibody and used the ELISA method to detect whole bacterial proteins of Mycoplasma hyorhinis at different concentrations to study the sensitivity of the Mhr_0461 monoclonal antibody.

[0074] The specific operations are as follows:

[0075] (1) Use coating solution (Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, add ddH 2 The whole bacterial protein of Mycoplasma hyorhinis after ultrasonic lysis was diluted to 10000ng / mL, 1000ng / mL, 100ng / mL, 50ng / mL, 12.5ng / mL, 6.25ng / mL, 3.125ng / mL, 1.5625ng / mL and 0.7813ng / mL respectively. The diluted proteins and negative control BSA protein were added to 96-well ELISA plates and coated overnight at 4°C. The liquid in the wells was discarded, 200μL PBST was added to each well, and the wells were washed 5 times, and the liquid in the wells was discarded after 2 minutes each time. During the last wash, the liquid in the wells was fully discarded.

[0076] (2) Blocking: Add 100 μL of 5% skim milk to each well, place in a 37°C incubator for 1 hour, discard the liquid in the wells, add 200 μL of PBST, wash 5 times, and shake off the liquid in the wells after 2 minutes each time. During the last wash, fully discard the liquid in the wells.

[0077] (3) Sample addition: Dilute the Mhr_0461 monoclonal antibody with PBST at a ratio of 1:100 (V / V) (the monoclonal antibody solution used for dilution is the purified monoclonal antibody in Example 2), add 100 μL of the diluted sample to each well, incubate at 37°C for 1 h, discard the liquid in the wells, add 200 μL of PBST, wash 5 times, 2 minutes each time, and then discard the liquid in the wells. During the last wash, fully discard the liquid in the wells.

[0078] (4) Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse secondary antibody (purchased from ProteinTech) with PBST at a ratio of 1:5000 (V / V), add 100 μL of diluted secondary antibody to each well, and incubate at 37°C for 60 minutes. Discard the liquid in the well, add 200 μL PBST, wash 5 times, 2 minutes each time, and discard the liquid in the well. During the last wash, fully discard the liquid in the well.

[0079] (5) Add substrate for color development: 50 μL each of TMB color development solution A and TMB color development solution B (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) were added in sequence, and the color was developed for 10 min in the dark. Subsequently, 50 μL of stop solution (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) was added to each well to terminate the reaction.

[0080] (6) Read the absorbance (OD) at 450 nm using an ELISA reader. 450nm value).

[0081] The results are as follows:

[0082]

[0083] The results were analyzed using the formula S / N=(sample A-blank A) / (negative control A-blank A) and the judgment criteria, S / N≥2.1 was positive, and S / N<2.1 was negative. The results showed that the detection limit of the monoclonal antibody of the present invention for the whole bacterial protein antigen of Mycoplasma hyorhinis was 1.5625 ng / mL.

[0084] Embodiment 6:

[0085] Application of monoclonal antibody Mhr_0461 in the preparation of indirect immunofluorescence detection kit for Mycoplasma hyorhinis

[0086] 1. Assembly of the indirect immunofluorescence detection kit for Mycoplasma hyorhinis based on Mhr_0461 monoclonal antibody The kit includes Mycoplasma hyorhinis Mhr_0461 monoclonal antibody, reaction slide, fixative (high-purity 4% formalin PBS solution, pH 7.3), permeabilization solution (0.5% Triton X-100), blocking solution (phosphate buffer containing 0.5% skim milk powder), washing solution (PBS, pH 7.4), Tween 20, goat anti-mouse secondary antibody (Cy3 labeled, purchased from Biolong Biotechnology Co., Ltd.), DAPI staining solution (purchased from Biosharp), sealing solution, negative control, positive control, and cover glass.

[0087] Operation steps: Fix the cell sample to be tested (cell slides are placed in advance in a 24-well cell plate) with pre-cooled fixative at room temperature for 15 minutes, wash with PBS three times, 5 minutes each time, take out and dry; add blocking solution, block at room temperature for 1 hour, wash with PBS three times, 5 minutes each time; use 1:5000 (V / V) diluted Mycoplasma hyorhinis Mhr_0461 monoclonal antibody as the primary antibody, incubate at 4°C overnight; wash with PBS three times, 5 minutes each time; use 1:1000 (V / V) diluted goat anti-mouse IgG (CY3 labeled) as the secondary antibody, incubate at room temperature for 1 hour in the dark; wash with PBS three times, 5 minutes each time; incubate with DAPI staining solution for 15 minutes; take out the slide, add sealing solution on the slide, and fix the cover slip with nail polish; set up negative and positive controls at the same time; observe and take pictures under a fluorescence microscope.

[0088] 2. Application of Mhr_0461 monoclonal antibody in indirect immunofluorescence detection kit for Mycoplasma hyorhinis

[0089] Using the monoclonal antibody Mhr_0461-8E3 of Mycoplasma hyorhinis, indirect immunofluorescence detection was performed on cells infected with Mycoplasma hyorhinis and cells not infected with Mycoplasma hyorhinis. Under a fluorescence microscope, Mycoplasma hyorhinis was distributed on the outside of the cell membrane and appeared as red fluorescent bright spots ( Figure 4 ), while no obvious red fluorescent bright spot was seen in the negative control. In summary, the monoclonal antibody against Mycoplasma hyorhinis Mhr_0461-8E3 can be used for the rapid diagnosis of Mycoplasma hyorhinis infection.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A hybridoma cell line Mhr_0461-8E3, characterized in that: Its accession number is CCTCC NO:C2024162.

2. The monoclonal antibody secreted by the hybridoma cell according to claim 1.

3. Use of the hybridoma cell line Mhr_0461-8E3 or the monoclonal antibody secreted by it according to claim 1 in the preparation of a kit for detecting Mycoplasma hyorhinis.

4. Use of the hybridoma cell line Mhr_0461-8E3 or the monoclonal antibody secreted by it according to claim 1 in the preparation of a kit for detecting Mycoplasma hyorhinis antigen.

5. According to the use of claim 3 or 4, the kit is an ELISA detection kit.

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