CN110862452B - Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaA1 thereof - Google Patents
Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaA1 thereof Download PDFInfo
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- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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Abstract
The invention discloses a monoclonal antibody for identifying alternaria and a hybridoma cell strain thereof, wherein the monoclonal antibody for identifying alternaria is prepared from the following components in parts by weight: CCTCC No: c2019218 hybridoma cell strain secretion; the hybridoma cell strain is named as AaA1 and is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 17 months, and the preservation number is as follows: CCTCC No: C2019218. the monoclonal antibody is used for the identification and dynamic monitoring of animal and plant diseases caused by Alternaria alternata infection and the biological research of Alternaria alternata, and a large amount of monoclonal antibody can be obtained by injecting the cell strain into the abdominal cavity of a Balb/c mouse.
Description
Technical Field
The invention belongs to the field of animals, and particularly relates to a monoclonal antibody for identifying alternaria alternata and a hybridoma cell strain thereof.
Background
Alternaria alternata, a fungus of Alternaria of the subdivision Deuteromycotina, is one of the pathogenic fungi of fritillaria melasma. The pathogenic bacteria can be lost in soil with the hypha to live through the winter, and the fritillaria is infected again in the next year. Alternaria is also the pathogenic bacterium of alternaria leaf spot of many crops, causing serious losses to agricultural production every year. The alternaria fungus is of various types, and the colony, hypha and spore forms of related species are similar, so that the alternaria fungus is difficult to distinguish by means of the characteristics. The monoclonal antibody (monoclonal antibody) combined with enzyme-linked immunosorbent assay (ELISA) for Alternaria alternata disclosed by the invention has the characteristics of high sensitivity and strong specificity, and is suitable for detecting Alternaria alternata in fields in large quantities, so that a foundation is laid for the identification and biological research of Alternaria alternata by applying the antibody and dynamically monitoring the occurrence of diseases infected by Alternaria alternata, such as Fritillaria thunbergii black spot and the like.
Disclosure of Invention
The invention provides a monoclonal antibody for identifying alternaria alternate, which is prepared from the following components in parts by weight: CCTCC No: c2019218 hybridoma cell strain secretion.
The invention also provides a hybridoma cell strain which is named as AaA1 and is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 17 days, and the preservation number is as follows: CCTCC No: C2019218.
the invention has the following beneficial effects:
(1) the monoclonal antibody has strong specificity: the monoclonal antibody has strong reaction with Alternaria alternata antigen, does not react with Alternaria alternata, Fusarium oxysporum, Fusarium solani, Fusarium equiseti, Fusarium semitectum, Botrytis cinerea, Phoma niponicum and phomopsis longipes antigen, and can be used for detecting Alternaria alternata and thunberg fritillary black spot caused by the Alternaria alternata.
(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 from the alternaria alternate hypha and spore is 12.21ng/mL (namely 1.221ng per hole), and the monoclonal antibody has good development and application prospects.
(3) The monoclonal antibody is IgG2 a.
(4) The monoclonal antibody-bound target protein is single: the monoclonal antibody binds only to an antigen protein of about 37kDa of Alternaria alternata.
(5) The detection sensitivity of the thunberg fritillary bulb to the monoclonal antibody has no influence: the monoclonal antibody has no cross reaction to the thunberg fritillary bulb protein extracting solution; after the thunberg fritillary bulb protein extracting solution is added into the antigen prepared from the alternaria alternate hypha and the spores, the detection sensitivity of the thunberg fritillary bulb extracting solution on the monoclonal antibody is not influenced, the content of the thunberg fritillary bulb extracting solution is still 12.21ng/mL (namely 1.221ng per hole), and the method has good development and application prospects.
Drawings
FIG. 1 potency assay for AaA 1;
figure 2 reaction of AaA1 with different fungal antigens;
detection sensitivity determination of fig. 3 AaA 1;
FIG. 4 identification of type and subclass of AaA1 antibody;
FIG. 5 AaA1 antibody-bound Alternaria alternata target protein;
FIG. 6 shows the effect of the protein extract of Fritillaria thunbergii on the detection sensitivity of AaA 1.
Detailed Description
The invention is further explained below with reference to the figures and examples.
Example 1: preparation of hybridoma cell line
(1) Preparation of antigen: selecting single colonies of Alternaria alternata, respectively inoculating the single colonies into a potato glucose liquid culture medium, carrying out shake culture at 25 ℃ for 4-5 days, collecting spores and hyphae by using a 50mL centrifuge tube, centrifuging at 6000r/min for 20min, washing for 2 times by using PBS, carrying out ultrasonic crushing (power 200W, crushing for 2s and interval for 2s), centrifuging the crushed liquid at 6000r/min for 20min, collecting supernatant, measuring the content of albumin by using a Coomassie brilliant blue method, adjusting the protein concentration to 1000 mu g/mL, serving as an initial antigen for immune antigen and later detection, subpackaging a small amount of antigen liquid, and freezing and storing at-80 ℃. A small amount of the extract is stored at-20 deg.C before use.
(2) 3 healthy BaL b/c mice of 10-12 weeks old are selected, and an intraperitoneal injection method is adopted, wherein 200 mu L of antigen emulsified by equivalent Freund's complete adjuvant is injected into each mouse. The immunization was carried out 3 weeks later with 200. mu.L of antigen emulsified with an equal amount of Freund's incomplete adjuvant. After another 3 weeks, immunization was performed 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 the fusogenic agent, and the mouse myeloma cell line was SP 2/0. The whole process is operated under aseptic conditions. The mouse spleen cells and myeloma cells are put into a 50mL centrifuge tube, mixed and centrifuged (1200r/min, 2min), and then supernatant liquid is sucked up and the cells are homogenized by fingers. The centrifuge tube was placed in a water-filled beaker pre-warmed in a 37 ℃ water bath, and 0.7mL of cell fusion agent (50% PEG) was added slowly over 1 min. After standing for 1min, 40mL of RMPI-1640 medium, which had been pre-warmed to 37 ℃, was gradually added to dilute the PEG and lose its effect. After centrifugation (1000r/min, 2min), the supernatant was discarded. The precipitated cells were suspended in 40mL of HAT medium, and then distributed in 96-well cell culture plates previously supplemented with feeder cells, and subjected to 5% (V/V) CO2Incubate at 37 ℃ in an incubator. Half of HAT culture medium is changed after 3 days, half of HAT culture medium is changed after 5 days, and HT culture medium is used for continuous culture after 5 days, at the moment, parents die, and if cells grow, the hybridoma cells are obtained.
(4) When the cells in the small holes grow to cover one fourth of the area of the bottom of the holes, the supernatant can be sucked up and the antibody can be detected by indirect ELISA. Selecting strong positive cell strains which do not react with antigens of fusarium equiseti, botrytis cinerea, fusarium semitectum, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma phomopsis and phomopsis, performing multiple cloning culture by using a limiting dilution method until all the holes are positive to obtain a cell strain AaA1 secreting monoclonal antibodies, and further performing expanded culture on the cell strain AaA1 for preparing monoclonal antibody ascites and freezing and storing liquid nitrogen. The hybridoma cells are preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 17 days, and the preservation numbers of Wuhan university in Wuhan City are as follows: CCTCC No: C2019218.
example 2: generation of monoclonal antibodies
Taking BaL b/c mice of about 8 weeks old, injecting 0.3mL of pristane into abdominal cavity, injecting 5-10 x 10 of pristane into abdominal cavity after 7-10 days5And (3) carrying out injection on the hybridoma cells, wherein the abdominal cavity of the mouse obviously expands 7-10 days after injection, taking ascites, centrifuging at 2000r/min for 3min, and collecting supernatant, namely the ascites monoclonal antibody. Purifying monoclonal antibody by Protein A column chromatography, and storing at-80 deg.C. The AaA1 cell strain is prepared into monoclonal antibody, namely the monoclonal antibody which can specifically identify alternaria alternate.
Example 3: potency assay for monoclonal antibodies
The titer of the antibody was determined by indirect ELISA. The Alternaria alternata antigen of 1000 mug/mL is diluted by 1000 times by using the coating solution, then the whole enzyme label plate (namely 1 mug/mL) is coated, the temperature is kept overnight at 4 ℃, the enzyme label plate is adsorbed on a polystyrene plate hole, PBST is washed for three times, and then the enzyme label plate is sealed by using skimmed milk for 60 min. Diluting monoclonal antibody AaA1 times, adding into coated well, adding 100 μ L of antibody per well, 37 deg.C, 1H, washing PBST for three times, adding 100 μ L of well diluted 5000 times according to the instruction, labeling rabbit anti-mouse with horseradish peroxidase (Sigma company), washing PBST for 1H at 37 deg.C, adding OPD substrate developing solution, developing, and developing with 50 μ L of 2M H2SO4After the reaction was terminated, the OD was read with a microplate reader490nmAnd determining the titer of the monoclonal antibody ascites by taking the positive result that the negative ratio is more than 2.
Through detection, the titer of AaA1 is determined to be 6.40 multiplied by 10 dilution5Fold, determine AaA1 dilution 20000 times for other detection experiments (as in figure 1).
Example 4: specificity detection experiment of monoclonal antibody
The detection objects are respectively fusarium equiseti, botrytis cinerea, fusarium semitectum, alternaria alternata, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma and phomopsis, and the antigens (1000 mug/mL) are diluted to 1 mug/mL by using a coating solution and then coated on an enzyme label plate. And (3) detecting the specificity of the monoclonal antibody by using an indirect ELISA method by using the coating solution as a blank control, wherein the monoclonal antibody is diluted by 20000 times, and the enzyme-labeled secondary antibody is diluted by 5000 times.
The antibody and the antigen of the fungus do not have cross reaction through the detection, but the antibody and the antigen of the alternaria alternata have strong reaction, and whether the alternaria alternata antigen exists or not can be obviously judged (figure 2).
Example 5: sensitivity detection experiment of monoclonal antibody
The sensitivity of the monoclonal antibody is determined by an indirect ELISA method. Diluting Alternaria alternata antigen 1000 μ g/mL to 1:80 × 2 by carbonate coating solution multiple0～1:80×210And the enzyme is coated on an enzyme label plate, and the concentration is from top to bottom from high to low. Repeat 8 times, column 12 blank control, only coated with coating solution. 20000 times of monoclonal antibody dilution and 5000 times of enzyme-labeled secondary antibody dilution.
The maximum dilution multiple of the alternaria alternate antigen of 1000 mug/mL is 81920 times by detection, namely the detection sensitivity is as follows: 1000 μ g/mL/81920-12.21 ng/mL, i.e. 1.221ng per well (fig. 3).
Example 6: type and subclass detection experiment of monoclonal antibody
The monoclonal antibody is identified by a mouse monoclonal antibody subclass identification kit (Baiotton center for experimental materials, batch number: C060101-L). The ELISA plate was coated with 1000. mu.g/mL of Alternaria alternata antigen diluted 1000-fold with the coating solution, 100. mu.L/well, 16 wells were repeated, incubated at 37 ℃ for 2h or placed in a 4 ℃ device for 12h, and washed 1 time with PBST after the coating solution was discarded. Adding 100 μ L of monoclonal antibody diluted 20000 times with PBST into each well of ELISA plate, incubating at 37 deg.C for 30min, washing with PBST for 5 times, adding 100 μ L of each enzyme-labeled antibody of 8 detection antibody Ig types and subclasses in the kit, adding into each well, repeating each enzyme-labeled antibody for 2 times, incubating at 37 deg.C for 30min, washing with PBST for 5 times, adding OPD color developing solution, and developing in dark place for 20min, 50. mu.L of 2M H2SO4The reaction was terminated. Detection OD of enzyme-linked immunosorbent assay (OD)490nmThe type and subclass of the antibody Ig corresponding to the positive hole are the type and subclass of the antibody of the monoclonal antibody.
The antibody type of AaA1 was determined to be IgG2a (FIG. 4).
Example 7: detection experiment of monoclonal antibody binding protein
The detection of the antibody binding protein by SDS-PAGE and Western blot technique includes the following steps:
(1) 5% (M/V) of the concentrated gel and 10% (M/V) of the separation gel were prepared.
(2) 80 mu L of Alternaria alternata antigen and 20 mu L of 5 multiplied sample buffer solution are mixed in a 1.5mL centrifuge tube, and are immediately placed in an ice bath for 3min after being subjected to metal bath at 99 ℃ for 10min, and then are centrifuged for 5min at 10000 r/min.
(3) Samples were added to the sample wells with a microsyringe, 2 wells for each sample, and protein Marker was added simultaneously.
(4) Electrophoresis: and (3) firstly keeping the constant voltage of 80V, changing the constant voltage of 120V after the bromophenol blue indicator enters the separation gel, and stopping electrophoresis when the bromophenol blue indicator moves to about 1cm of the lower opening of the gel plate.
(5) Dividing the gel into two parts, soaking one half into staining solution, staining for 45min on a shaking table, and decolorizing with decolorizing solution until the background is clear.
(6) And transferring the other half of the gel to a nitrocellulose membrane by a semi-dry transfer method at a constant pressure of 25V for 30 min.
(7) The transferred membrane was washed 4 times with PBST for 5min each.
(8) The membrane was immersed in 3% (M/V) skim milk and sealed at room temperature for 1 h.
(9) The membrane was immersed in a 2000-fold diluted monoclonal antibody solution in 3% (M/V) skim milk, shaken at 75r/min for 1h at room temperature, and incubated overnight at 4 ℃.
(10) Washing with the step (7).
(11) The membrane was immersed in 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 with the step (7).
(13) The membrane was immersed in 10mL of a freshly prepared DAB color developing solution, slowly shaken away from light until color development was achieved, and the color development reaction was stopped with distilled water. The band developed on the membrane is the specific protein combined by the antibody, and the relative molecular weight of the combined protein is calculated according to the protein Marker.
The antibody was detected to specifically bind to a protein of about 37kDa, relative molecular weight of Alternaria (FIG. 5).
Example 8: experiment for influence of thunberg fritillary bulb on sensitivity of monoclonal antibody
The influence of host plant Fritillaria thunbergii of Alternaria alternata on the detection sensitivity of the monoclonal antibody is determined by adopting an indirect ELISA method. Taking fresh thunberg fritillary bulb plants (including bulbs), carrying out ultrasonic crushing (power of 200W, crushing for 2s, interval of 2s) for 10min, carrying out centrifugation at 6000r/min for 10min, taking supernate, and diluting the supernate by 20 times with coating liquid for later use. Diluting Alternaria alternata antigen 1000 μ g/mL with the Bulbus Fritillariae Thunbergii diluent at a ratio of 1:40 × 20～1:40×211And the enzyme is coated on an enzyme label plate, and the concentration is from top to bottom from high to low. Repeating for 8 times, and coating the enzyme label plate with Bulbus Fritillariae Thunbergii diluent without adding Alternaria alternata antigen as negative control. 20000 times of monoclonal antibody dilution and 5000 times of enzyme-labeled secondary antibody dilution.
Detection shows that AaA1 has no cross reaction to the protein extract of Fritillaria thunbergii. After the thunberg fritillary bulb protein extracting solution is added, the detection sensitivity of the alternaria alternate antigen is 12.21ng/mL, and the result is consistent with the result without the thunberg fritillary bulb extracting solution, which shows that the thunberg fritillary bulb extracting solution has no influence on the detection result (figure 6).
Finally, it should also be noted that the above list is only a specific implementation example of the present invention. It is obvious that the invention is not limited to the above examples of facts, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (2)
1. A monoclonal antibody for identifying Alternaria alternata is prepared from the monoclonal antibody with the preservation number of CCTCC No: c2019218 hybridoma cell strain secretion.
2. A hybridoma cell strain is named as AaA1 and is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 17 days, and the preservation number is as follows: CCTCC No: C2019218.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106591240A (en) * | 2017-01-16 | 2017-04-26 | 西南大学 | Hybridoma cell strain 4F4 and antibody thereof |
| CN106701688A (en) * | 2017-01-16 | 2017-05-24 | 西南大学 | Hybridoma cell strain 7G11 and antibody thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106591240A (en) * | 2017-01-16 | 2017-04-26 | 西南大学 | Hybridoma cell strain 4F4 and antibody thereof |
| CN106701688A (en) * | 2017-01-16 | 2017-05-24 | 西南大学 | Hybridoma cell strain 7G11 and antibody thereof |
Non-Patent Citations (2)
| Title |
|---|
| Characterization of New Alternaria alternata–Specific;Olivier Denis等;《Mycopathologia》;20110904;第173卷;第151-162页 * |
| 浙贝母主要病原真菌的分离鉴定及药物敏感性研究;王丹依;《中国优秀硕士学位论文全文数据库 农业科技辑》;20190415(第4期);第35页 * |
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