CN110922479B - Monoclonal antibody for recognizing alternaria tenuis and hybridoma cell strain AtH9 thereof - Google Patents
Monoclonal antibody for recognizing alternaria tenuis and hybridoma cell strain AtH9 thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody for identifying Alternaria tenuissima and a hybridoma cell strain thereof, wherein the monoclonal antibody for identifying the Alternaria tenuissima is prepared from the following components in parts by weight: CCTCC No: c2019230 hybridoma cell strain secretion; the hybridoma cell strain is named as hybridoma cell strain AtH9, is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 17 months, and has the preservation number as follows: CCTCC No: C2019230. the monoclonal antibody is used for identification and dynamic monitoring of animal and plant diseases caused by Alternaria tenuissima infection and biological research of Alternaria tenuissima, 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 tenuissima and a hybridoma cell strain AtH9 thereof.
Background
Alternaria tenuissima, 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 tenuissima is also a pathogenic bacterium of black spot of numerous crops, and causes serious loss 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 enzyme-linked immunosorbent assay (ELISA) for alternaria tenuis disclosed by the invention has the characteristics of high sensitivity and strong specificity, and is suitable for detecting a large amount of alternaria tenuis in fields, so that a foundation is laid for the identification and biological research of the alternaria tenuis by applying the antibody and the dynamic monitoring of diseases infected by the alternaria tenuis, such as thunberg fritillary black spot and the like.
Disclosure of Invention
The invention provides a monoclonal antibody for identifying alternaria tenuis, which is prepared from the following components in percentage by weight: CCTCC No: c2019230 hybridoma cell strain secretion.
The invention also provides a hybridoma cell strain which is named as hybridoma cell strain AtH9 and is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 17 days, wherein the preservation numbers are as follows: CCTCC No: C2019230.
the invention has the following beneficial effects:
(1) the monoclonal antibody has strong specificity: the monoclonal antibody has strong reaction with Alternaria tenuissima antigens, does not react with Alternaria tenuissima, Fusarium oxysporum, Fusarium solani, Fusarium equiseti, Fusarium semitectum, Botrytis cinerea, Phoma stem rot fungus and phomopsis stolonifera antigens, and can be used for detecting Alternaria tenuissima and thunberg fritillary black spot caused by the Alternaria tenuissima.
(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 alternaria tenuis hyphae and the spores is 12.21ng/mL (namely 1.221ng per hole), and the monoclonal antibody has good development and application prospects.
(3) The monoclonal antibody is IgM.
(4) The monoclonal antibody-bound target protein is single: the monoclonal antibody binds only to an antigen protein of about 50kDa of Alternaria tenuissima.
(5) The detection sensitivity of the fritillaria thunbergii extracting solution on the monoclonal antibody is not influenced: 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 tenuissima hypha and the spores, the detection sensitivity of the monoclonal antibody is not influenced by the thunberg fritillary bulb extracting solution, the content of the thunberg fritillary bulb protein extracting solution is 12.21ng/mL (namely 1.221ng per hole), and the method has good development and application prospects.
Drawings
Potency assay of FIG. 1 AtH 9;
FIG. 2 AtH9 reaction with different fungal antigens;
the detection sensitivity assay of FIG. 3 AtH 9;
type and subclass identification of FIG. 4 AtH 9;
FIG. 5 AtH9 detection of bound target protein;
FIG. 6 shows the effect of Bulbus Fritillariae Thunbergii extract on the detection sensitivity of AtH 9.
Detailed Description
The invention is further explained below with reference to the figures and the examples.
Example 1: preparation of hybridoma cell line
(1) Selecting single colonies of Alternaria tenuissima, 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 2s), centrifuging the crushed liquid at 6000r/min for 20min, collecting supernatant, measuring the content of supernatant protein by using a Coomassie brilliant blue method, adjusting the protein concentration to 1000 mu g/mL, using the supernatant 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, fusarium oxysporum, fusarium solani, phoma and phomopsis, performing cloning culture for multiple times by using a limiting dilution method until all the holes are positive to obtain a cell strain AtH9 secreting the monoclonal antibody, and further performing expanded culture on a cell strain AtH9 for preparing monoclonal antibody ascites and liquid nitrogen cryopreservation. 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: C2019230.
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. AtH9 the monoclonal antibody is prepared by the cell strain, namely the monoclonal antibody which can specifically identify alternaria tenuissima.
Example 3: potency assay for monoclonal antibodies
The titer of the antibody was determined by indirect ELISA. 1000 mug/mL of Alternaria tenuissima antigen is diluted 1000 times by using a coating solution, then a whole piece of 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 skim milk for 60 min. Diluting the monoclonal antibody AtH9 times, adding into coated well, adding 100 μ L of the monoclonal antibody per well, washing at 37 deg.C for 1H, washing PBST for three times, adding 100 μ L of the well diluted 5000 times according to the instruction, washing at 37 deg.C for 1H, adding OPD substrate color development solution for color development, 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.
The detection proves that the potency of AtH9 is 5.12 multiplied by 10 dilution6Fold, determine AtH9 dilution40000 times was used for other assays (see FIG. 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 antibody by using an indirect ELISA method by using the coating solution as a blank control, wherein the monoclonal antibody is diluted by 40000 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 Alternaria tenuissima antigen have strong reaction, and whether the Alternaria tenuissima 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 1000 μ g/mL Alternaria tenuissima antigen with carbonate coating solution at a multiple ratio of 1:80 × 20~1:80×210And 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. 40000 times of monoclonal antibody dilution, and 5000 times of enzyme-labeled secondary antibody dilution.
The maximum dilution multiple of 1000 mug/mL Alternaria tenuissima antigen obtained by detection is 81920 times, 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). 1000. mu.g/mL of Alternaria tenuissima antigen was diluted 1000-fold with a coating solution and then coated on an enzyme plate at 100. mu.L/well, 16 wells were repeated, incubated at 37 ℃ for 2h or placed at 4 ℃ for 12h, and the coating solution was discarded and then washed 1 time with PBST. Adding 100 μ L of ascetic monoclonal antibody diluted 40000 times with PBST into each well of enzyme-linked plate, incubating at 37 deg.C for 30min, washing with PBST for 5 times, collecting 100 μ L of each of 8 enzyme-linked antibodies for detecting Ig type and subclass in the kit, and dividing into separate partsAdding 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 developing solution, developing in dark for 20min, and developing with 50 μ 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 type of antibody detected AtH9 was determined to be IgM (FIG. 4).
Example 7: detection assay for monoclonal antibody binding proteins
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 tenuissima 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 4000-fold ascites-type monoclonal antibody solution diluted with 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 having a relative molecular weight of about 50kDa of Alternaria tenuis (FIG. 5).
Example 8: experiment for influence of fritillaria thunbergii extracting solution on sensitivity of monoclonal antibody
The influence of host plant Fritillaria thunbergii of Alternaria tenuissima 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 tenuissima 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 Zhejiang fritillaria dilution without adding alternaria tenuissima antigen as negative control. 40000 times of monoclonal antibody dilution, and 5000 times of enzyme-labeled secondary antibody dilution.
Detection shows that AtH9 has no cross reaction to the protein extract of Fritillaria thunbergii. After the thunberg fritillary bulb protein extracting solution is added, the maximum dilution multiple of 1000 mu g/mL alternaria tenuissima antigen is 81920 times, namely the detection sensitivity is as follows: 1000 ug/mL/81920 ng/mL indicates that the Zhejiang fritillaria extract has no effect on the test results (FIG. 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 recognizing Alternaria tenuis, which is represented by the deposit number: CCTCC No: c2019230 hybridoma cell strain secretion.
2. A hybridoma cell strain is named as hybridoma cell strain AtH9, is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 17 days, and has the preservation number as follows: CCTCC No: C2019230.
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