CN113461815B - Monoclonal antibody for recognizing botrytis cinerea and hybridoma cell strain BcA4 thereof - Google Patents
Monoclonal antibody for recognizing botrytis cinerea and hybridoma cell strain BcA4 thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/56961—Plant cells or fungi
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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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
Technical Field
The invention belongs to the field of animals, and particularly relates to a monoclonal antibody for recognizing botrytis cinerea and a hybridoma cell strain BcA4 thereof.
Background
The ascomycete phylum fungus Botrytis cinerea is one of pathogenic fungi of Botrytis cinerea. The germ is in the soil with the hypha and the residual tissue is lost, the winter is over, and the fritillary bulb is infected again in the next year. Botrytis cinerea is also pathogenic bacteria of gray mold disease of numerous crops, causing serious losses to agricultural production every year. While the fungus of the genus sporophyte is various, the colony, hypha and spore of the related species are similar in morphology and are difficult to distinguish by virtue of the characteristics. The monoclonal antibody (monoclonal antibody) combined enzyme-linked immunosorbent assay (ELISA) for the botrytis cinerea disclosed by the invention has the characteristics of high sensitivity and strong specificity, is suitable for detecting a large amount of botrytis cinerea in the field, and lays a foundation for carrying out the identification and biological research of the botrytis cinerea by using the antibody and dynamically monitoring the occurrence of diseases infected by the botrytis cinerea, such as Bolbostemma thunbergii gray mold and the like.
Disclosure of Invention
The invention provides a monoclonal antibody for identifying botrytis cinerea, which is prepared from the following materials in percentage by mass: cctccc No: the hybridoma cell line of C2021115 was produced by secretion.
Further, the invention also provides a hybridoma cell strain which is classified and named as BcA4 and is preserved in China Center for Type Culture Collection (CCTCC) for type 13 (5 months of 2021), and has the preservation number of university of Wuhan in Wuhan, china: cctccc No: C2021115.
the invention has the following beneficial effects:
(1) The specificity of the monoclonal antibody is strong: the monoclonal antibody has strong reaction with the botrytis cinerea antigen, does not react with antigens of Alternaria alternata, alternaria minutissima, fusarium oxysporum, fusarium solani, fusarium equisetosum, fusarium seminude, phoma, phomopsis, and can be used for detecting Botrytis cinerea and the Botrytis cinerea induced by the Botrytis cinerea.
(2) The detection sensitivity of the monoclonal antibody is high: the indirect ELISA detection result shows that the detection sensitivity of the monoclonal antibody to the antigen prepared by the botrytis cinerea mycelium and spore is 39.06ng/mL (namely 3.906ng per hole), and the monoclonal antibody has good development and application prospects.
(3) The type of mab is igλ.
(4) Target protein single of monoclonal antibody combination: the monoclonal antibody binds only to an antigen protein of about 25kDa from Botrytis cinerea.
Drawings
FIG. 1BcA4 potency detection graph;
FIG. 2 shows a reaction diagram of BcA4 with different fungal antigens;
FIG. 3BcA4 detection sensitivity measurement chart;
FIG. 4 is a diagram of identification of BcA4 antibody types and subclasses;
FIG. 5BcA4 antibody binding to Botrytis cinerea target protein.
Detailed Description
The invention is further explained below with reference to the drawings and the examples.
Example 1: hybridoma cell line preparation
(1) Antigen preparation: single bacterial colonies of Botrytis cinerea are selected and respectively inoculated into potato glucose liquid culture medium, after shaking culture is carried out for 4-5 d at 25 ℃, spores and hyphae are collected by a 50mL centrifuge tube, after centrifugation is carried out for 20min at 6000r/min, PBS is used for washing for 2 times, ultrasonic crushing (power is 200W, crushing is carried out for 2s, intermittent is carried out for 2 s), crushing liquid is centrifuged for 20min at 6000r/min, supernatant is collected, the content of protein in the supernatant is measured by a Coomassie brilliant blue method, the protein concentration is adjusted to 1000 mug/mL, the protein is used as an initial antigen for immune antigen and later detection, and the antigen liquid is frozen and stored at-80 ℃ after a small amount of subpackage. Taking a small amount of the extract and preserving at-20deg.C.
(2) 3 healthy BaL b/c mice with the age of 6-8 weeks are selected, and 200 mu L of antigen emulsified by equivalent Freund's complete adjuvant is injected by an intraperitoneal injection method. Immunization was performed after 3 weeks using 200 μl of antigen emulsified with equal amount of incomplete Freund's adjuvant. After a further 3 weeks immunization with antigen without adjuvant. 3 days after the last immunization, antisera (polyclonal antibodies) were collected and mouse spleen lymphocytes were taken under sterile conditions for cell fusion.
(3) Polyethylene glycol (PEG-4000) was used as a fusion agent, and the mouse myeloma cell line was SP 2/0. The whole process is operated under aseptic conditions. The spleen cells and myeloma cells of the mice are placed into a 50mL centrifuge tube, mixed and centrifuged (1200 r/min,2 min), and the supernatant is sucked out and the cells are flicked by fingers. The centrifuge tube was placed in a water-containing beaker pre-warmed in a 37 ℃ water bath, and 0.7mL of cell fusion agent (50% peg, ph modulation 9.0) was slowly added over 1 min. After 1min of rest, 40mL of RMPI-1640 medium, which had been pre-warmed to 37℃was added gradually, and PEG was diluted to lose effect. After centrifugation (1000 r/min,2 min) the supernatant was discarded. Suspending the precipitated cells in 40mL HAT medium, sub-packaging in 96-well cell culture plate with feeder cells, and placing in 5% (V/V) CO 2 In a temperature boxCulturing at 37 ℃. After 3 days, half of the HAT medium is changed, after 5 days, half of the HAT medium is changed, and after 5 days, HT medium is changed to continue culturing, at the moment, the parents are dead, and if cells grow, the hybridoma cells are obtained.
(4) When cells in the wells grew to cover one quarter of the area of the bottom of the wells, the supernatant was aspirated and the antibodies were detected by indirect ELISA. Selecting cell strains which are strong positive and do not react with antigens of fusarium equiseti, fusarium seminude, alternaria alternata, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma and phomopsis, performing cloning culture for a plurality of times by using a limiting dilution method until all holes are positive, obtaining cell strain BcA4 secreting monoclonal antibody, and further expanding and culturing the cell strain BcA4 for preparing monoclonal antibody ascites and freezing liquid nitrogen. The hybridoma is preserved in China Center for Type Culture Collection (CCTCC) at 5 and 13 days of 2021, and the preservation number is as follows: cctccc No: C2021115.
example 2: production of monoclonal antibodies
Taking about 8 weeks old BaLb/c mice, injecting 0.2mL pristane into the abdominal cavity, and injecting 0.2mL containing 5-10×10 into the abdominal cavity after 7 days 5 The cell suspension of the hybridoma cells can be obviously swelled in the abdominal cavity of the mouse 7-10 days after injection, the ascites is taken, the centrifugation is carried out for 3min at 2000r/min, and the supernatant is collected, thus obtaining the ascites type monoclonal antibody. Purifying monoclonal antibody by Protein A column chromatography, and preserving at-80 ℃. BcA4 cell strain is prepared into monoclonal antibody which can specifically identify Botrytis cinerea.
Example 3: potency detection experiment of monoclonal antibody
The titers of the antibodies were determined by indirect ELISA. The 1000. Mu.g/mL of Botrytis cinerea antigen was diluted 500 times with the coating solution, and then the whole ELISA plate (i.e., 2. Mu.g/mL) was coated overnight at 4℃to adsorb onto the polystyrene plate wells, and after three times of PBST washing, the ELISA plate was blocked with skimmed milk for 60min. Adding monoclonal antibody BcA 4-fold dilution into coated wells, adding 100 μl of the mixture into each well, washing with PBST for three times at 37deg.C for 1H, adding 100 μl of horseradish peroxidase-labeled goat anti-mouse (Sigma Co.) diluted 5000-fold according to instructions into the wells, washing with PBST for 1H at 37deg.C, adding OPD substrate color development solution, and developing with 50 μl of 2M H 2 SO 4 After the termination of the reaction, the reaction mixture,reading OD with an ELISA reader 490nm The positive determination of the primary ascites titer is carried out by taking the ratio of the primary ascites titer to the negative value of the primary ascites titer to be more than 2.
The potency of BcA4 was determined to be diluted 1.28X10 by detection 4 The BcA4 dilution by a factor of 1000 was determined for other detection experiments (see fig. 1).
Example 4: specificity detection experiment of monoclonal antibody
The detection objects are Botrytis cinerea, fusarium equisetum, fusarium seminude, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma and Phomopsis respectively, and the antigen (1000 mug/mL) is diluted to 2 mug/mL by a coating liquid and then an enzyme-labeled plate is coated. And (3) taking the coating liquid as a blank control, and detecting the specificity of the monoclonal antibody by an indirect ELISA method, wherein the monoclonal antibody is diluted 1000 times, and the enzyme-labeled secondary antibody is diluted 5000 times.
The detection of the antibody did not cross-react with the antigen of the fungus, but the antibody reacted strongly with the botrytis cinerea antigen, so that it was possible to clearly determine whether or not the botrytis cinerea antigen was present (FIG. 2).
Example 5: sensitivity detection experiment of monoclonal antibody
The sensitivity of the monoclonal antibodies was determined by indirect ELISA. The botrytis cinerea antigen of 1000 mug/mL is diluted to 1:100 multiplied by 2 by the ratio of carbonate coating liquid 0 ～1:80×2 14 Coating the ELISA plate, and the concentration is from top to bottom. The procedure was repeated 8 times, with column 12 being a blank, coated only with coating solution. Monoclonal antibody dilution 1000 times, enzyme-labeled secondary antibody dilution 5000 times.
The maximum dilution factor of the botrytis cinerea antigen detected by 1000 mug/mL is 25600 times, namely the detection sensitivity is: 1000 μg/mL/25600=39.06 ng/mL, i.e. 3.906ng per well (fig. 3).
Example 6: type and subclass detection experiments of monoclonal antibodies
Monoclonal antibodies were identified using a mouse monoclonal antibody subclass identification kit (Baioton laboratory materials center, lot number 20200809). Diluting 1000 μg/mL of Botrytis cinerea antigen with coating solution for 1000 times, coating ELISA plate, 100 μl/well, repeating 16 wells, incubating at 37deg.C for 2 hr or placing at 4deg.C for 12 hr, removing coating solution, and washing with PBST1 time. Adding 100 μL of 1000-fold single antibody diluted by PBST into each hole of an ELISA plate, incubating at 37deg.C for 30min, washing with PBST for 5 times, adding 100 μL of 8 ELISA antibodies of Ig type and subclass in the kit, respectively, adding 2 repeats of each ELISA antibody, incubating at 37deg.C for 30min, washing with PBST for 5 times, adding TMB color development liquid, developing in dark for 15min, and adding 50 μL of 2M H 2 SO 4 The reaction was terminated. Detection OD of enzyme label instrument 450nm The antibody Ig type and subclass corresponding to the positive hole are the antibody type and subclass of the monoclonal antibody.
The antibody type of BCA4 was determined to be igλ (fig. 4) by detection.
Example 7: detection experiment of monoclonal antibody binding protein
The antibody binding protein is detected by adopting SDS-PAGE and Western blot technology, and the steps are as follows:
(1) A5% (M/V) concentrated gel and a 10% (M/V) separate gel were prepared.
(2) 80. Mu.L of Botrytis cinerea antigen and 20. Mu.L of 5 Xsample buffer are mixed in a 1.5mL centrifuge tube, and immediately after 10min of metal bath at 99 ℃, the mixture is put in an ice bath for 3min, and then centrifuged at 10000r/min for 5min.
(3) Samples were added to the wells with a microscale applicator, 2 wells per sample, and protein markers.
(4) Electrophoresis: the constant voltage of 80V is firstly kept, after the bromophenol blue indicator enters the separation gel, the constant voltage is changed into 120V, and when the bromophenol blue indicator moves to the lower opening of the gel plate for about 1cm, the electrophoresis is stopped.
(5) Dividing the gel into two parts, immersing half of the gel into the staining solution, staining on a shaker for 45min, and decolorizing with the decolorizing solution until the background is clear.
(6) The other half of the gel was transferred to a nitrocellulose membrane by semi-dry transfer at 25V constant pressure for 30 min.
(7) The transferred film was washed with PBST 4 times for 5min each.
(8) The membrane was immersed in 3% (M/V) skimmed milk and blocked at room temperature for 1h.
(9) The membrane was immersed in a solution of mab diluted 2000-fold with 3% (M/V) skimmed milk, and after shaking at 75r/min for 1h at room temperature, incubated overnight at 4 ℃.
(10) Washing in the same way as (7).
(11) The membrane was immersed in a 8000-fold dilution of horseradish peroxidase-labeled antibody with 3% (M/V) BSA, and shaken at room temperature for 1h at 75 r/min.
(12) Washing in the same way as (7).
(13) The membrane was immersed in 10mL of freshly prepared DAB chromogenic solution and slowly shaken in the dark until developed, and the chromogenic reaction was stopped with distilled water. The chromogenic band on the membrane is the specific protein bound by the antibody, and the relative molecular weight of the bound protein is calculated according to the protein Marker.
The antibody was detected to bind specifically to a protein of about 25kDa in relation to Botrytis cinerea (FIG. 5).
Finally, it should also be noted that the above list is merely a specific example of the invention. Obviously, the invention is not limited to the above examples of facts, but many variants are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the 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.
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| WO2013063613A2 (en) * | 2011-10-28 | 2013-05-02 | University Of Maryland | Methods and compositions related to intracellular neutralization by igg |
| CN110804094A (en) * | 2019-12-03 | 2020-02-18 | 浙江中医药大学 | Monoclonal antibody for identifying alternaria tenuis and hybridoma cell strain AtD2 thereof |
| CN110894234A (en) * | 2019-11-29 | 2020-03-20 | 浙江中医药大学 | Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaC5 thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013063613A2 (en) * | 2011-10-28 | 2013-05-02 | University Of Maryland | Methods and compositions related to intracellular neutralization by igg |
| CN110894234A (en) * | 2019-11-29 | 2020-03-20 | 浙江中医药大学 | Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaC5 thereof |
| CN110804094A (en) * | 2019-12-03 | 2020-02-18 | 浙江中医药大学 | Monoclonal antibody for identifying alternaria tenuis and hybridoma cell strain AtD2 thereof |
Non-Patent Citations (2)
| Title |
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| Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea;R. BOSSI等;Plant Pathology;第41卷(第4期);摘要，第473页右栏最后1段到第474页左栏第3段，第480页左栏第3段 * |
| 灰霉菌多克隆抗体的制备与鉴定;张银志等;食品与生物技术学报(第6期);第82-85页 * |
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