CN110894233A

CN110894233A - Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaC2 thereof

Info

Publication number: CN110894233A
Application number: CN201911133983.5A
Authority: CN
Inventors: 赵伟春; 徐云飞; 李吉二; 陈义妃
Original assignee: Zhejiang Chinese Medicine University ZCMU
Current assignee: Zhejiang Chinese Medicine University ZCMU
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2020-03-20
Anticipated expiration: 2039-11-19
Also published as: CN110894233B

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: c2019221 hybridoma cell strain secretion; the hybridoma cell strain is named as AaC2, 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: C2019221. 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

Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaC2 thereof

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 AaC2 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 combined enzyme-linked immunosorbent assay (ELISA) for Alternaria alternata disclosed by the invention has the characteristics of high sensitivity, strong specificity and suitability for detecting Alternaria alternata in fields in large quantities, and lays a foundation for the identification and biological research of Alternaria alternata by using the antibody and the dynamic monitoring 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: c2019221 hybridoma cell strain secretion.

The invention also provides a hybridoma cell strain which is named as AaC2 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: C2019221.

the invention has the following beneficial effects:

(1) the monoclonal antibody has strong specificity: the monoclonal antibody has strong reaction with Alternaria alternata antigens, does not react with Alternaria alternata, Fusarium oxysporum, Fusarium solani, Fusarium equiseti, Fusarium semitectum, Botrytis cinerea, Phoma niponicum and phomopsis longipes antigens, 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 24.41ng/mL (namely 2.441ng per well), and the monoclonal antibody has good development and application prospects.

(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 62kDa 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 protein extracting solution is 24.41ng/mL (namely 2.441ng per hole), and the method has good development and application prospects.

Drawings

Potency assay of FIG. 1 AaC 2;

FIG. 2 AaC2 reaction with different fungal antigens;

the detection sensitivity assay of FIG. 3 AaC 2;

FIG. 4 identification of the type and subclass of 4 AaC2 antibody;

FIG. 5 AaC2 antibody-bound Alternaria alternata target protein;

FIG. 6 shows the effect of protein extract of Fritillaria thunbergii on the detection sensitivity of AaC 2.

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 cloning culture for multiple times 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 by 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: C2019221.

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. Protein A column layerSeparating and purifying the monoclonal antibody, and storing at-80 ℃. AaC2 the monoclonal antibody is prepared by the cell strain, namely the monoclonal antibody which can specifically identify the 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 the monoclonal antibody AaC2 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 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 AaC2 is 1.28 multiplied by 10 dilution⁶Double, AaC2 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 tenuis, fusarium oxysporum, fusarium solani, phoma and phomopsis, and the antigen (1000 mug/mL) is 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 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 multiple⁰～1:80×2¹⁰Coated on an enzyme label plate with concentrationFrom top to bottom 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 the alternaria alternate antigen of 1000 mug/mL is 40960 times by detection, namely the detection sensitivity is as follows: 1000 μ g/mL/40960 ═ 24.41ng/mL, i.e. 2.441ng 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 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 AaC2 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 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 ascitic 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 of about 62kDa, the 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 with diluted Bulbus Fritillariae Thunbergii solution without Alternaria alternata antigenThe panels served as negative controls. 40000 times of monoclonal antibody dilution, and 5000 times of enzyme-labeled secondary antibody dilution.

Detection shows that AaC2 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 24.41ng/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

1. A monoclonal antibody recognizing Alternaria alternata represented by the deposit number: CCTCC No: c2019221 hybridoma cell strain secretion.

2. A hybridoma cell strain is named as AaC2, 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: C2019221.