CN110804092B

CN110804092B - Monoclonal antibody for recognizing alternaria tenuis and hybridoma cell strain AtA8 thereof

Info

Publication number: CN110804092B
Application number: CN201911167167.6A
Authority: CN
Inventors: 赵伟春; 李吉二; 徐云飞; 高佳丽
Original assignee: Zhejiang Chinese Medicine University ZCMU
Current assignee: Zhejiang Chinese Medicine University ZCMU
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2021-05-18
Anticipated expiration: 2039-11-25
Also published as: CN110804092A

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: c2019227 hybridoma cell strain secretion; the hybridoma cell strain is named as AtA8, 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: C2019227. 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

Monoclonal antibody for recognizing alternaria tenuis and hybridoma cell strain AtA8 thereof

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 AtA8 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 combined enzyme-linked immunosorbent assay (ELISA) for alternaria tenuissima disclosed by the invention has the characteristics of high sensitivity and strong specificity, and is suitable for detecting a large amount of field alternaria tenuissima, so that a foundation is laid for the identification and biological research of the alternaria tenuissima by applying the antibody and the dynamic monitoring 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 parts by weight: CCTCC No: c2019227 hybridoma cell strain secretion.

The invention also provides a hybridoma cell strain which is named as AtA8 and is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 17 days, wherein the preservation numbers of Wuhan university in Wuhan City are as follows: CCTCC No: C2019227.

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 fungi and phomopsis longissima 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 spores is 12.21ng/mL (namely 1.221ng per well).

(3) The monoclonal antibody is of the type IgG 3.

(4) Monoclonal antibodies bind to the protein of interest only: the monoclonal antibody binds only to an antigen protein of about 30kDa of Alternaria tenuissima.

(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 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 AtA 8;

FIG. 2 AtA8 reaction with different fungal antigens;

the detection sensitivity assay of FIG. 3 AtA 8;

FIG. 4 identification of the type and subclass of 4 AtA8 antibody;

FIG. 5 AtA8 antibody-bound Alternaria tenuis target protein;

FIG. 6 shows the effect of protein extract of Fritillaria thunbergii on the detection sensitivity of AtA 8.

Detailed Description

The invention is further explained below with reference to the figures and the examples.

Example 1: preparation of hybridoma cell line

(1) Preparation of antigen: 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. Standing for 1min, gradually adding 40mL of RMPI-1640 medium pre-warmed to 37 deg.C to dilute PEGEliminating the 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 AtA8 secreting the monoclonal antibody, and further performing expanded culture on a cell strain AtA8 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: C2019227.

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. AtA8 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 AtA8 times, adding 100 μ L of the diluted monoclonal antibody into each well, washing at 37 deg.C for 1h with PBST for three times, and adding the diluted monoclonal antibody according to the instructions100 μ L of a 5000-fold diluted horseradish peroxidase-labeled rabbit anti-mouse (Sigma Co.) was subjected to color development at 37 ℃ for 1 hour after PBST washing with OPD substrate developing solution, and 50 μ L of 2M H was used₂SO₄After the reaction was terminated, the OD was read with a microplate reader_490nmAnd 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 AtA8 is 2.56 multiplied by 10⁶Double, AtA8 dilution 40000 times was determined for other detection experiments (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 mouse monoclonal antibody subclass identification kit (Baiotton center for experimental materials, batch number: C0601) is adopted01-L) identifying the monoclonal antibody. 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 ascitic monoclonal antibody diluted by PBST 40000 times 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 for detecting Ig types and subclasses in kit, adding into each well, repeating for 2 times, incubating at 37 deg.C for 30min, washing with PBST for 5 times, adding OPD color developing solution, developing for 20min in dark place, and adding 50 μ L of H2M₂SO₄The 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 AtA8 was determined to be IgG3 (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 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 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 30kDa, relative molecular weight of Alternaria tenuis (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 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 AtA8 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 was consistent with the result without adding the Zhejiang fritillaria extract, indicating that the Zhejiang fritillaria extract had 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

1. A monoclonal antibody recognizing Alternaria tenuis, which is represented by the deposit number: CCTCC No: c2019227 hybridoma cell strain secretion.

2. A hybridoma cell strain is named as AtA8, and is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 17 months, with the preservation number as follows: CCTCC No: C2019227.