CN110804094B - Monoclonal antibody for identifying alternaria tenuissima and hybridoma cell strain AtD2 thereof - Google Patents
Monoclonal antibody for identifying alternaria tenuissima and hybridoma cell strain AtD2 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: c2019228 hybridoma cell strain is secreted; the hybridoma cell strain is named as a hybridoma cell strain AtD2, 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: C2019228. the monoclonal antibody is used for identification and dynamic monitoring of animal and plant diseases caused by infection of alternaria tenuissima 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 AtD2 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 tenuissima disclosed by the invention has the characteristics of high sensitivity, strong specificity and suitability for detecting alternaria tenuissima in a large amount in fields, and lays a foundation for applying the antibody to identification and biological research of the alternaria tenuissima and dynamically monitoring the occurrence of diseases infected by the alternaria tenuissima, 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: c2019228 is secreted.
The invention also provides a hybridoma cell strain which is named as hybridoma cell strain AtD2 and is 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: C2019228.
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 well).
(3) The monoclonal antibody is IgM.
(4) The monoclonal antibody-bound target protein is single: the monoclonal antibody only binds to an antigen protein of about 43kDa 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
FIG. 1 Titer test of AtD2;
FIG. 2 the reaction of AtD2 with different fungal antigens;
FIG. 3 detection sensitivity assay for AtD2;
FIG. 4 type and subclass identification of AtD2;
FIG. 5 detection of AtD2-bound target protein;
FIG. 6 shows the effect of the extractive solution of Fritillaria thunbergii on the detection sensitivity of AtD2.
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 2 s), 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 Balb/c mice of 10-12 weeks old are selected, and 200 mu L of antigen emulsified by equivalent Freund's complete adjuvant is injected into each mouse by an intraperitoneal injection method. 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 a 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 (1200 r/min,2 min), and then supernatant liquid is sucked out and the cells are homogenized by fingers. The centrifuge tube was placed in a water-containing beaker pre-warmed in a 37 ℃ water bath, and 0.7mL of the 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 (1000 r/min,2 min), 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) CO 2 Incubate at 37 ℃ in an incubator. Half HAT medium was changed after 3 days, and 5 days laterHalf HAT culture medium is replaced, and HT culture medium is used for further culture for 5 days, at this time, the parents are dead, 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 AtD2 secreting the monoclonal antibody, and further performing expanded culture on the cell strain AtD2 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: C2019228.
example 2: generation of monoclonal antibodies
Taking BaL b/c mice of about 8 weeks old, injecting 0.3mL of pristanane into the abdominal cavity, injecting 5-10 multiplied by 10 into the abdominal cavity after 7-10 days 5 And (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 monoclonal antibody is prepared from the AtD2 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 for 60min by using skimmed milk. Diluting the monoclonal antibody AtD2 times, adding into coated wells, washing with 100 μ L of water at 37 deg.C, 1H, PBST for three times, adding 100 μ L of water-insoluble horse radish peroxidase-labeled rabbit anti-mouse (Sigma) diluted 5000 times according to the instruction, washing with 37 deg.C, 1H, PBST, adding OPD substrate, developing, and developing with 50 μ L of 2M H 2 SO 4 After the reaction was terminated, the OD was read with a microplate reader 490nm Positive when the ratio of the positive to the negative is more than 2Sex determination of the titer of the monoclonal antibody ascites.
Through detection, the titer of the AtD2 is determined to be 2.56 multiplied by 10 dilution 6 Fold, determine AtD2 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 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 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 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. 1000. Mu.g/mL of Alternaria tenuissima antigen was diluted 1-fold with a carbonate coating solution 0 ～1:80×2 10 And 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 1000 mug/mL Alternaria tenuissima antigen obtained by detection is 80960 times, namely the detection sensitivity is as follows: 1000 μ g/mL ÷ 80960=12.21ng/mL, i.e. 1.221ng per well (fig. 3).
Example 6: type and subclass detection experiment of monoclonal antibody
The monoclonal antibody was identified by mouse monoclonal antibody subclass identification kit (Baiotong center for laboratory materials, lot 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. Collecting 100 μ L ascites form diluted 20000 times with PBSTAdding antibodies into each well of an ELISA plate, incubating for 30min at 37 ℃, washing for 5 times by PBST, taking 100 microliter of each enzyme-labeled antibody of 8 detection antibody Ig types and subclasses in the kit, respectively adding into each well, setting 2 times for each enzyme-labeled antibody, incubating for 30min at 37 ℃, washing for 5 times by PBST, adding OPD color development solution, developing for 20min in dark, and using 50 microliter of 2M H 2 SO 4 The reaction was terminated. Detection OD of enzyme-linked immunosorbent assay (OD) 490nm The type and subclass of antibody Ig corresponding to the positive hole are the type and subclass of the monoclonal antibody.
The antibody type of AtD2 was determined to be IgM by detection (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 gum and 10% (M/V) of the separation gum 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 pressure at 80V, changing the constant pressure to 120V after the bromophenol blue indicator enters the separation gel, and stopping electrophoresis when the bromophenol blue indicator moves to the lower opening of the gel plate by about 1 cm.
(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 blocked at room temperature for 1h.
(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 horseradish peroxidase-labeled antibody diluted with 3% (M/V) BSA and shaken at room temperature at 75r/min for 1h.
(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 bands developed on the membrane are specific proteins 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 43kDa of Alternaria tenuis (FIG. 5).
Example 8: experiment for influence of fritillaria thunbergii extracting solution on monoclonal antibody detection sensitivity
The influence of host plant Zhejiang fritillaria 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 200W, crushing for 2s, and interval for 2 s) for 10min at a speed of 10min at 6000r/min, taking supernate, and diluting by 20 times with coating liquid for later use. 1000 mug/mL of Alternaria tenuissima antigen is diluted by the thunberg fritillary bulb diluent for 1 0 ～1:40×2 11 And 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. 20000 times of monoclonal antibody dilution and 5000 times of enzyme-labeled secondary antibody dilution.
Through detection, the AtD2 has no cross reaction to the thunberg fritillary bulb protein extracting solution. After the thunberg fritillary bulb protein extracting solution is added, the maximum dilution multiple of 1000 mug/mL alternaria tenuissima antigen is 81920 times, namely, the detection sensitivity is as follows: 1000. Mu.g/mL 81920=12.21ng/mL, which indicates that the Zhejiang fritillaria extract has no effect on the detection result (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: c2019228.
2. A hybridoma cell strain is named as hybridoma cell strain AtD2, 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: C2019228.
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