CN113461815B - Monoclonal antibody for recognizing botrytis cinerea and hybridoma cell strain BcA4 thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody for identifying botrytis cinerea and a hybridoma cell strain thereof, wherein the monoclonal antibody for identifying botrytis cinerea is prepared from the following materials in percentage by mass: cctccc No: the hybridoma cell strain of C2021115 is secreted; the hybridoma cell strain is named BcA4 and is preserved in China Center for Type Culture Collection (CCTCC) at the year 2021, 5 and 13, and the preservation number is: cctccc No: C2021115. the monoclonal antibody is used for identification and dynamic monitoring of animal and plant diseases caused by botrytis cinerea infection and biological research of botrytis cinerea, and a large amount of monoclonal antibodies can be obtained by intraperitoneal injection of the cell strain into BaL b/c mice.

Description

Monoclonal antibody that recognizes Botrytis cinerea and its hybridoma cell strain BcA4

Technical field

The invention belongs to the field of animals, and in particular relates to a monoclonal antibody that recognizes Botrytis cinerea and its hybridoma cell strain BcA4.

Background technique

The Ascomycota fungus Botrytis cinerea is one of the pathogenic fungi of Fritillary gray mold. The fungus overwinters as hyphae left in the soil along with the diseased tissue, and infects the caladium again the following year. Botrytis cinerea is also the causative agent of gray mold disease in many crops, causing serious losses to agricultural production every year. There are many types of fungi in the genus Sporosporium, and the colonies, hyphae and spores of closely related species are similar in shape, making it difficult to distinguish them based on the above characteristics. The monoclonal antibody (monoclonal antibody) combined with enzyme-linked immunosorbent assay (ELISA) against Botrytis cinerea disclosed in the present invention has the characteristics of high sensitivity, strong specificity, and is suitable for detecting a large number of Botrytis cinerea in the field. It is suitable for the application of this method. Antibodies are used for the identification and biological research of Botrytis cinerea, which lays the foundation for dynamic monitoring of the occurrence of diseases infected by Botrytis cinerea, such as Fritillaria cinerea.

Contents of the invention

The invention provides a monoclonal antibody that recognizes Botrytis cinerea, which is secreted by a hybridoma cell strain deposited with the deposit number: CCTCC No: C2021115.

Further, the present invention also provides a hybridoma cell strain, which is classified and named BcA4, and was deposited in the China Type Culture Collection Center (CCTCC), Wuhan University, Wuhan, China, on May 13, 2021. The deposit number is: CCTCC No: C2021115.

The invention has the following beneficial effects:

(1) The specificity of the monoclonal antibody is strong: the monoclonal antibody reacts strongly with the antigen of Botrytis cinerea and does not react with Alternaria alternata, Alternaria tenuifolia, Fusarium oxysporum, Fusarium solani, and Fusarium equiseti Bacteria, Fusarium seminude, Phytophthora phyllophthora and Phomopsis pseudophyllum react with antigens, and can be used for the detection of Botrytis cinerea and the Botrytis cinerea caused by Fritillaria cinerea.

(2) The detection sensitivity of the monoclonal antibody is high: The indirect ELISA test results show that the detection sensitivity of the monoclonal antibody against the antigen prepared from Botrytis cinerea hyphae and spores is 39.06ng/mL (i.e. 3.906ng per well), which is good. development and application prospects.

(3) The type of monoclonal antibody is Igλ.

(4) The monoclonal antibody binds to a single target protein: the monoclonal antibody only binds to the approximately 25 kDa antigen protein of Botrytis cinerea.

Description of the drawings

Figure 1 BcA4 titer detection chart;

Figure 2 Reaction chart of BcA4 with different fungal antigens;

Figure 3 BcA4 detection sensitivity measurement chart;

Figure 4 Identification diagram of types and subclasses of BcA4 antibodies;

Figure 5BcA4 antibody bound Botrytis cinerea target protein map.

Detailed ways

The present invention is further explained below in conjunction with the drawings and examples.

Example 1: Preparation of hybridoma cell lines

(1) Antigen preparation: Pick single colonies of Botrytis cinerea and inoculate them into potato glucose liquid culture medium. After shaking culture at 25°C for 4 to 5 days, use a 50mL centrifuge tube to collect spores and hyphae, 6000r/min Centrifuge for 20 minutes, wash twice with PBS, and then ultrasonic disrupt (power 200W, disrupt for 2 seconds, 2 seconds interval), centrifuge the disrupted liquid at 6000 r/min for 20 minutes, collect the supernatant, and measure the protein content of the supernatant using the Coomassie Brilliant Blue method, and adjust The protein concentration is 1000 μg/mL. As an immune antigen and an initial antigen for later detection, the antigen solution is aliquoted in small quantities and stored frozen at -80°C. Take a small amount and store at -20°C before use.

(2) Select 3 healthy BaL b/c mice aged 6 to 8 weeks and use intraperitoneal injection. Each mouse is injected with 200 μL of the antigen emulsified with the same amount of Freund's complete adjuvant. After 3 weeks of immunization, the same amount of 200 μL of Freund's incomplete adjuvant emulsified antigen was used. Immunization with unadjuvanted antigen was performed another 3 weeks later. Three days after the last immunization, antiserum (polyclonal antibodies) were collected, and mouse spleen lymphocytes were collected under sterile conditions for cell fusion.

(3) Polyethylene glycol (PEG-4000) was used as the fusion agent, and the mouse myeloma cell line was SP 2/0. The entire process is performed under sterile conditions. Put the mouse splenocytes and myeloma cells into a 50 mL centrifuge tube, mix and centrifuge (1200 r/min, 2 min), absorb the supernatant, and smooth the cells with your fingers. Place the centrifuge tube in a water beaker pre-warmed in a 37°C water bath, and slowly add 0.7 mL of cell fusion agent (50% PEG, pH adjusted to 9.0) within 1 min. After resting for 1 minute, gradually add 40 mL of RMPI-1640 culture medium that has been prewarmed to 37°C to dilute the PEG and lose its effect. After centrifugation (1000r/min, 2min), discard the supernatant. The precipitated cells were suspended in 40 mL of HAT medium, distributed into 96-well cell culture plates with feeder cells added in advance, and cultured at 37°C in a 5% (V/V) _CO2 incubator. After 3 days, half of the HAT medium is replaced, after 5 days, half of the HAT medium is replaced, and after another 5 days, the HT medium is used to continue culturing. At this time, the parents are dead. If there are cells growing, they are hybridoma cells.

(4) When the cells in the well grow to cover a quarter of the bottom area of the well, the supernatant can be drawn and the antibodies detected using indirect ELISA. Select the antigens that are strongly positive and do not react with Fusarium equiseti, Fusarium seminata, Alternaria alternata, Alternaria tenuifolia, Fusarium oxysporum, Fusarium solani, Pseudomonas solani, and Phomopsis pseudopathogens The cell line was cloned and cultured multiple times using the limiting dilution method until all wells were positive, and the monoclonal antibody-secreting cell line BcA4 was obtained. The cell line BcA4 was further expanded and cultured for the preparation of monoclonal antibody ascites and liquid nitrogen cryopreservation. . The hybridoma cells were deposited at the China Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on May 13, 2021, with the deposit number: CCTCC No: C2021115.

Example 2: Production of monoclonal antibodies

Take BaLb/c mice about 8 weeks old, intraperitoneally inject 0.2 mL of phytane, and 7 days later, inject 0.2 mL of cell suspension containing 5-10×10 ⁵ hybridoma cells intraperitoneally. The mice can be seen 7-10 days after injection. If the abdominal cavity is obviously distended, take the ascites, centrifuge it at 2000r/min for 3 minutes, and collect the supernatant, which is the ascites-type monoclonal antibody. Purify monoclonal antibodies by Protein A column chromatography and store at -80°C. The monoclonal antibody prepared from the BcA4 cell line is a monoclonal antibody that can specifically recognize Botrytis cinerea.

Example 3: Monoclonal antibody titer detection experiment

An indirect ELISA method was used to determine the antibody titer. Dilute 1000 μg/mL Botrytis cinerea antigen 500 times with coating solution and coat the entire enzyme plate (i.e. 2 μg/mL). Leave it overnight at 4°C to allow it to adsorb to the polystyrene plate wells. After washing three times with PBST Block with skim milk for 60 min. Dilute the monoclonal antibody BcA 4 times and add it to the coated wells. Add 100 μL to each well and incubate at 37°C for 1 hour. After washing three times with PBST, add 100 μL of horseradish peroxidase-labeled goat anti-mouse (Sigma) diluted 5000 times according to the instructions into the wells. 37 °C for 1 hour, add OPD substrate chromogenic solution after washing with PBST to develop color, use 50 μL 2M H ₂ SO ₄ to terminate the reaction, read OD _490nm with a microplate reader, and determine the monoclonal antibody ascites efficacy by taking the ratio of negative to negative greater than 2 as positive. price.

After testing, it was determined that the potency of BcA4 was diluted 1.28×10 ⁴ times, and BcA4 was determined to be diluted 1000 times for other detection experiments (Figure 1).

Example 4: Specific detection experiment of monoclonal antibodies

The detection objects are Botrytis cinerea, Fusarium equiseti, Fusarium seminude, Alternaria alternata, Alternaria tenuifolia, Fusarium oxysporum, Fusarium solani, Phytophthora solani and Phomophomonas. The above antigen (1000 μg/mL) was diluted to 2 μg/mL with coating solution and then coated on the enzyme plate. Using the coating solution as a blank control, indirect ELISA was used to detect the specificity of the monoclonal antibody, in which the monoclonal antibody was diluted 1000 times and the enzyme-labeled secondary antibody was diluted 5000 times.

After the above detection, the antibody did not cross-react with the antigens of the above-mentioned fungi, but the antibody reacted strongly with the Botrytis cinerea antigen, and the presence of the Botrytis cinerea antigen could be clearly distinguished (Figure 2).

Example 5: Sensitivity detection experiment of monoclonal antibodies

The indirect ELISA method was used to determine the sensitivity of monoclonal antibodies. Dilute 1000 μg/mL Botrytis cinerea antigen with carbonate coating solution to a ratio of 1:100×2 ⁰ to 1:80×2 ¹⁴ , and coat it on an enzyme plate, with the concentration increasing from top to bottom. to low. Repeat 8 times, and the 12th column is a blank control, which is only coated with coating solution. The monoclonal antibody was diluted 1000 times, and the enzyme-labeled secondary antibody was diluted 5000 times.

The maximum dilution factor of 1000 μg/mL Botrytis cinerea antigen is 25600 times, that is, the detection sensitivity is: 1000 μg/mL÷25600=39.06ng/mL, that is, 3.906ng per well (Figure 3).

Example 6: Detection experiment of types and subtypes of monoclonal antibodies

The monoclonal antibodies were identified using a mouse monoclonal antibody subclass identification kit (Biaotong Experimental Materials Center, batch number: 20200809). Dilute 1000 μg/mL Botrytis cinerea antigen 1000 times with coating solution and coat the enzyme plate, 100 μL/well, repeat 16 wells, incubate at 37°C for 2 hours or leave at 4°C for 12 hours, shake off the coating solution Then wash once with PBST. Add 100 μL of monoclonal antibody diluted 1000 times with PBST to each well of the enzyme plate, incubate at 37°C for 30 minutes, wash 5 times with PBST, and take 100 μL of each of the eight enzyme-labeled antibodies for detecting Ig types and subclasses in the kit. Add each enzyme-labeled antibody to each well separately, set up 2 replicates, incubate at 37°C for 30 minutes, wash with PBST 5 times, add TMB chromogenic solution to develop color in the dark for 15 minutes, and terminate the reaction with 50 μL of 2M H ₂ SO ₄ . The microplate reader detects OD _450nm . The antibody Ig type and subclass corresponding to the positive well is the antibody type and subclass of the monoclonal antibody.

After testing, it was determined that the BCA4 antibody type was Igλ (Figure 4).

Example 7: Detection experiment of monoclonal antibody binding proteins

SDS-PAGE and Western blot techniques are used to detect antibody-binding proteins. The steps are as follows:

(1) Prepare a 5% (M/V) stacking gel and a 10% (M/V) separating gel.

(2) Mix 80 μL of Botrytis cinerea antigen and 20 μL of 5× sample buffer in a 1.5 mL centrifuge tube, place in a metal bath at 99°C for 10 minutes, immediately put it into an ice bath for 3 minutes, and then centrifuge at 10000 r/min for 5 minutes.

(3) Use a micropipette to add the sample to the sample well, add 2 wells for each sample, and add a protein marker at the same time.

(4) Electrophoresis: First use a constant voltage of 80V. After the bromophenol blue indicator enters the separation gel, then change to a constant voltage of 120V. Stop the electrophoresis when the bromophenol blue indicator moves about 1cm below the gel plate.

(5) Divide the gel into two parts, immerse half in the staining solution, stain on a shaking table for 45 minutes, and then change to the destaining solution to destain until the background is clear.

(6) Use the semi-dry transfer method to transfer the other half of the gel at a constant voltage of 25V for 30 minutes, and transfer the protein to the nitrocellulose membrane.

(7) The transferred membrane was washed 4 times with PBST, 5 minutes each time.

(8) Immerse the membrane in 3% (M/V) skim milk and block at room temperature for 1 hour.

(9) Immerse the membrane in a monoclonal antibody solution diluted 2000 times with 3% (M/V) skim milk, shake at 75 r/min for 1 hour at room temperature, and incubate at 4°C overnight.

(10) Same as (7) for washing.

(11) Immerse the membrane in horseradish peroxidase-labeled antibody diluted 8000 times with 3% (M/V) BSA, and shake at room temperature at 75 r/min for 1 hour.

(12) Same as (7) for washing.

(13) Immerse the membrane in 10 mL of newly prepared DAB chromogenic solution, avoid light and shake slowly until color develops, then use distilled water to terminate the color reaction. The colored band on the membrane is the specific protein bound by the antibody. The relative molecular weight of the bound protein is calculated according to the protein marker.

After testing, the antibody specifically binds to a protein of Botrytis cinerea with a relative molecular weight of approximately 25 kDa (Figure 5).

Finally, it should be noted that the above examples are only specific implementation examples of the present invention. Obviously, the present invention is not limited to the above factual examples, and many modifications are possible. All modifications that a person of ordinary skill in the art can directly derive or associate from the disclosure of the present invention should be considered to be within the protection scope of the present invention.

Claims (2)

1. A monoclonal antibody for identifying Botrytis cinerea is prepared from the polypeptide with the preservation number of CCTCC No: the hybridoma cell strain of C2021115 is secreted; the monoclonal antibody for recognizing Botrytis cinerea only specifically binds to a protein of about 25kDa of Botrytis cinerea, and does not bind to antigens of Fusarium equisetum, fusarium seminude, alternaria alternata, alternaria tenuifolia, fusarium oxysporum, fusarium solani, phoma and Phomopsis.

2. A hybridoma cell line designated BcA4, deposited with the chinese collection center (cctccc) at 2021, 5 and 13 days, with deposit numbers: cctccc No: C2021115.