WO2023010682A1

WO2023010682A1 - Antibody capable of detecting nicotiana tabacum agglutinin protein, preparation method therefor and application thereof

Info

Publication number: WO2023010682A1
Application number: PCT/CN2021/123450
Authority: WO
Inventors: 袁清华; 黄振瑞; 马柱文
Original assignee: 广东省农业科学院作物研究所
Priority date: 2021-08-05
Filing date: 2021-10-13
Publication date: 2023-02-09
Also published as: CN113603772B; CN113603772A

Abstract

The present invention relates to the technical field of nocitiana tabacum agglutinin protein detection, and specifically discloses an antibody capable of detecting nocitiana tabacum agglutinin protein, a preparation method therefor, and an application thereof. The preparation method comprises the following steps: a preparation step for emulsion 1; a preparation step for emulsion 2; an immunization step, comprising: injecting the emulsion 1 into an animal body to carry out first immunization on the animal; once the first immunization has ended, injecting the emulsion 2 into the animal body to carry out second immunization on the animal; once the second immunization has ended, injecting the emulsion 2 into the animal body to carry out third immunization on the animal; and/or once the third immunization has ended, injecting the emulsion 2 into the animal body to carry out fourth immunization on the animal; and an antibody purification step. The antibody prepared by the method has high specificity for measuring nocitiana tabacum agglutinin protein, and can be used to detect the nocitiana tabacum agglutinin protein, thereby providing a novel way to detect nocitiana tabacum agglutinin.

Description

A kind of antibody capable of detecting tobacco lectin protein and its preparation method and application

technical field

The invention relates to the technical field of tobacco lectin protein detection, in particular to an antibody capable of detecting tobacco lectin protein and its preparation method and application.

Background technique

Tobacco lectin (Nictaba, Nicotiana tabacum agglutinin) was isolated from tobacco leaves for the first time in 2002, and it is an effective substance that can control various diseases and insect pests. Although there are very few related studies in China at present, a new research direction of using Nictaba as a carrier and using tobacco natural products for pest control is slowly being formed abroad. Tobacco lectins are distributed in various parts of the tobacco plant; the content of Nictaba is higher in the leaves, however, its presence is not detectable in all tobacco varieties.

In order to better use tobacco lectin for pest control; it is very necessary to detect whether tobacco lectin is contained in plants; but there is currently a lack of a specific method for detecting tobacco lectin.

Contents of the invention

In view of this, the present invention firstly provides a method for preparing an antibody capable of detecting tobacco lectin protein; the antibody prepared by the method has high specificity for detecting tobacco lectin protein, and can be used for detecting tobacco lectin protein, for The detection of tobacco lectins provides a new way.

Technical scheme of the present invention is as follows:

A method for preparing an antibody capable of detecting tobacco lectin protein, comprising the following steps:

Preparation steps of emulsion 1: mix antigen, Freund's complete adjuvant and PBS (specifically 0.01M PBS) to prepare antigen-containing emulsion 1;

Preparation steps of emulsion 2: mix antigen, Freund's incomplete adjuvant and PBS (specifically 0.01M PBS) to prepare antigen-containing emulsion 2;

Immunization step: the immunization step includes injecting the emulsion 1 into the animal to immunize the animal for the first time; after the first immunization, injecting the emulsion 2 into the animal to immunize the animal for the second time; After the immunization, inject the emulsion 2 into the animal to immunize the animal for the third time; and/or after the third immunization, inject the emulsion 2 into the animal to immunize the animal for the fourth time;

Antibody purification step: taking animal serum and purifying the antibody from the serum to obtain the antibody that can detect tobacco lectin protein;

Wherein, the amino acid sequence of the antigen in emulsion 1 and emulsion 2 is shown in SEQ ID NO: 1.

Preferably, in the preparation step of emulsion 1, the dosage ratio of antigen, Freund's complete adjuvant and PBS is 300-500ug: 1mL: 1mL;

In the preparation step of emulsion 2, the dosage ratio of antigen, Freund's incomplete adjuvant and PBS is 300-500ug:1mL:1mL.

Most preferably, in the preparation step of emulsion 1, the dosage ratio of antigen, Freund's complete adjuvant and PBS is 400ug:1mL:1mL.

Most preferably, in the preparation step of emulsion 2, the dosage ratio of antigen, Freund's incomplete adjuvant and PBS is 400ug:1mL:1mL.

Preferably, during the first, second, third and fourth immunization process, the amount of injected antigen is 40-60ug.

Most preferably, during the first, second, third and fourth immunizations, the amount of injected antigen is 50ug/bird.

Preferably, the injection mentioned in the immunization step refers to subcutaneous injection.

Preferably, the animals in the immunization step are mice.

Preferably, the time for the first immunization is 2-3 weeks; the time for the second immunization is 2 weeks; the time for the third immunization is 1 week; the time for the fourth immunization is 1 week.

The inventors have shown through a large number of experimental studies that: using Emulsion 1 and Emulsion 2 to immunize mice with the above steps four times, higher titers of antibodies that can detect tobacco lectin protein can be obtained.

Preferably, the antibody purification step specifically includes:

(1) Wash the equilibrium chromatography column (Sepharose 4B chromatography column) with 8 to 12 times the column volume of PBS buffer (specifically 0.01M PBS buffer);

(2) The supernatant after high-speed centrifugation of animal serum is mixed with PBS buffer (specifically 0.01M PBS buffer), and then added to the chromatography column; the volume ratio of the supernatant to PBS buffer is 1:1～2;

(3) Elute with 8 to 12 times the column volume of PBS buffer (specifically 0.01M PBS buffer), until no protein is detected in the effluent, close the chromatography column to stop the elution;

(4) Add 2 to 4 times the column volume of PBS buffer containing citric acid (specifically 0.01M PBS buffer) or a PBS buffer containing citric acid and glycine (specifically 0.01M PBS buffer), Stand still for 5-10 minutes; then perform elution, collect the eluate, concentrate the eluate and freeze-dry to obtain the antibody that can detect tobacco lectin protein.

The inventors have shown through a large number of studies that: high-purity antibodies that can detect tobacco lectin protein can be obtained by using the above chromatography column method.

Most preferably, the PBS buffer containing citric acid described in step (3) refers to the PBS buffer containing 0.1M citric acid;

The PBS buffer solution containing citric acid and glycine refers to the PBS buffer solution containing 0.7M citric acid and 0.3M glycine.

The inventors have shown through a large number of studies that: the composition of the PBS buffer plays a crucial role in the preparation of high-purity detectable tobacco lectin protein antibodies; when using PBS buffer containing 0.1M citric acid for washing When deactivated, high purity and high titer antibodies that can detect tobacco lectin protein can be obtained by enrichment. Further studies have shown that: when eluted with PBS buffer containing 0.7M citric acid and 0.3M glycine, antibodies that can detect tobacco lectin protein with higher purity and higher titer can be enriched.

The present invention also provides an antibody capable of detecting tobacco lectin protein prepared by the above preparation method.

Preferably, the antibody capable of detecting tobacco lectin protein has the amino acid sequence described in SEQ ID NO: 2 or/and SEQ ID NO: 3.

The present invention also provides an application of the above-mentioned antibody capable of detecting tobacco lectin protein in detecting tobacco lectin protein.

Beneficial effects: the present invention prepares for the first time an antibody that can detect tobacco lectin protein; the antibody that can detect tobacco lectin protein has high specificity for tobacco lectin protein; therefore, the present invention can be used The antibody that can detect tobacco lectin protein is used to detect tobacco lectin protein; the antibody that can detect tobacco lectin protein according to the present invention is successfully prepared, which provides a new detection approach for the detection of tobacco lectin protein; in addition, The antibody capable of detecting tobacco lectin protein prepared by the method of the present invention also has high purity and titer; and the method is simple to operate and low in preparation cost.

Detailed ways

The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

Example 1 Preparation of an antibody capable of detecting tobacco lectin protein

(1) Mix the antigen (the amino acid sequence of which is shown in SEQ ID NO: 1), Freund's complete adjuvant and PBS (specifically 0.01M PBS) to prepare antigen-containing emulsion 1; wherein the antigen, Freund's complete The dosage ratio of adjuvant and PBS is 400ug: 1mL: 1mL;

(2) Take antigen (its amino acid sequence is as shown in SEQ ID NO: 1), Freund's incomplete adjuvant and PBS (specifically 0.01M PBS) are mixed, and are prepared into antigen-containing emulsion 2; wherein, antigen, Freund's The dosage ratio of Shi's incomplete adjuvant and PBS is 400ug: 1mL: 1mL;

(3) Inject Emulsion 1 into Balb/c mice by subcutaneous injection to immunize Balb/c mice for the first time; after the first immunization, inject Emulsion 2 into Balb/c mice by subcutaneous injection. c mice to immunize Balb/c mice for the second time; after the second immunization, inject Emulsion 2 into Balb/c mice by subcutaneous injection to immunize the animals for the third time; After the immunization, the emulsion 2 was injected subcutaneously into the Balb/c mice to immunize the animals for the fourth time;

Among them, during the first, second, third and fourth immunizations, the amount of antigen injected was 50ug/monkey; the time of the first immunization was 3 weeks; the time of the second immunization was 2 weeks; The time for the third immunization is 1 week; the time for the fourth immunization is 1 week;

(5) Get the serum of Balb/c mice, and purify the antibody from the serum to obtain the antibody that can detect the tobacco lectin protein; the specific steps for purifying the antibody from the serum are as follows:

5.1) Wash the equilibrium chromatography column (specifically Sepharose 4B chromatography column) with 10 times column volume of PBS buffer solution (specifically 0.01M PBS buffer solution);

5.2) The supernatant after high-speed centrifugation of animal serum is mixed with PBS buffer (specifically 0.01M PBS buffer), and then added to the chromatography column; the volume ratio of the supernatant to PBS buffer is 1 :1;

5.3) Elute with 10 times the column volume of PBS buffer (specifically 0.01M PBS buffer), until no protein is detected in the effluent, close the chromatography column to stop the elution;

5.4) Add 2 times column volume of PBS buffer containing 0.7M citric acid and 0.3M glycine (specifically 0.01M PBS buffer), let stand for 5min; then perform elution, collect the eluate, concentrate and elute The antibody that can detect tobacco lectin protein can be obtained by lyophilizing the liquid. The antibody capable of detecting tobacco lectin protein has the amino acid sequence described in SEQ ID NO:2.

Example 2 Basic Potency Detection-Indirect ELSIA

(1) Antigen coating: Dilute the antigen with coating solution to the specified concentration (1ug/ml if not specified separately), add 100 μL/well into a polystyrene 96-well reaction plate, and place at 4°C overnight.

(2) Washing: discard the liquid in the hole the next day, and wash once with the washing solution.

(3) Blocking: add 200 μL/well blocking solution, and place at 37°C for 2 hours.

(4) Washing: Wash 3 times with washing liquid.

(5) Add the sample to be tested (the antibody prepared in Example 1): add the sample to be tested (if the sample needs to be diluted, it should be diluted in proportion) at 100 ul per well, and incubate for 1 h.

(6) Washing: Discard the sample to be tested, and wash 3 times with washing solution.

(7) Add enzyme-labeled secondary antibody: add HRP-labeled goat anti-mouse IgG (1:10000, diluted with enzyme), 100 μl/well, and incubate at 37°C for 40 minutes.

(8) Washing: wash 5 times with washing liquid.

(9) Color development: Add 90 μL/well of freshly prepared substrate solution, and place in the dark at 37°C for 15 minutes

(10) Stop reaction, colorimetry: add 50 μL/well stop solution. The color turns yellow; use a microplate reader to measure the absorbance of each well at 450 nm.

The results show that: the absorbance value in the measurement well of the test sample is more than 3 times the average value of the negative control well, which shows that the antibody prepared by the present invention that can detect the tobacco lectin protein is positive; and has a higher titer.

Embodiment 3 Western Blot experiment

(1) Preparation of polyacrylamide gel

1.1) Prepare a separating gel solution with a concentration of 15%: add 2.3ml of deionized water, 5ml of 30% acrylamide, 2.5ml of Tris with a pH of 8.8, 100μL of 10% ammonium persulfate, 100μL of 10% SDS, and 6μL of TEMED (add TEMED After that, the separating gel starts to polymerize immediately, so it should be mixed immediately and quickly);

1.2) Quickly add the separating gel solution in the gap between the two glass plates, leave the space required for perfusion of the stacking gel, and carefully cover the separating gel solution with a layer of isopropanol;

1.3) After the separation gel is completely polymerized, drain the liquid on the gel as much as possible, and then use the edge of the paper towel to absorb the remaining liquid;

1.4) Prepare a stacking gel solution with a concentration of 5%: add 2.7ml of deionized water, 670μL of 30% acrylamide, 500μL of Tris with a pH of 8.8, 40μL of 10% ammonium persulfate, 40μL of 10% SDS, and 6μL of TEMED (after adding TEMED) , the separating gel starts to polymerize immediately, so it should be mixed immediately and quickly);

1.5) Quickly pour stacking gel directly on the polymerized separating gel, and immediately insert a clean comb into the stacking gel solution (be careful to avoid mixing air bubbles);

1.26) After the stacking gel is completely polymerized, carefully remove the comb and add protein electrophoresis buffer into the electrophoresis tank.

2 Preparation of samples

2.1) Culture the cells to 10 ⁷ , collect the cells into a 15ml centrifuge tube, centrifuge at 2000rpm for 5min, and remove the supernatant;

2.2) The culture medium was washed twice, centrifuged at 2000rpm for 5min, and the supernatant was removed;

2.3) After collecting the cells, add 1ml PBS, 0.1% NP40, and lyse on ice for 10 minutes;

2.4) Centrifuge at 10,000rpm for 5min, take the supernatant, add an equal volume of 2×loading buffer, cook in boiling water for 5min, and store at -20°C after aliquoting;

3 Electrophoresis

3.1) Mix the antibody sample (10 μL) prepared in Example 1 to be detected with 2× (or 6×) loading buffer (10 μL), slowly add 15 μL of the mixture to the sample tank with a pipette gun, and pre-stain the Maker Generally add 10μL;

3.2) Turn on the power, use a voltage of 200V, and perform electrophoresis until the bromophenol blue loading buffer migrates to the bottom of the gel in the gel;

3.3) Cut off the power, take out the gel, soak the gel with R250 Coomassie Brilliant Blue for staining, and cook in boiling water for 5 minutes;

3.4) Take out the stained gel, soak the gel in tap water for decolorization, boil it in boiling water for about 20 minutes, and observe the protein bands after decolorization;

4 transfer film

4.1) After taking the glue, cut the glue into an appropriate size and immerse it in the transfer buffer;

4.2) Soak the PVDF membrane in methanol for 1 min, then transfer it to the transfer buffer, and immerse the filter paper in the transfer buffer (both the PVDF membrane and the filter paper are cut to the same size as the glue);

4.3) Rinse the graphite electrode with transfer buffer, spread two pieces of filter paper, and drop a little transfer buffer;

Spread the film, drop a little transfer buffer; spread the glue, and then drop a little transfer buffer (be careful not to generate air bubbles);

4.4) Finally, spread two pieces of filter paper and drop a little transfer buffer;

4.5) Cover the electrode, adjust the voltage to the maximum, and transfer the membrane with a gel volume of 1.5mA/cm2 for 1.5h (the load voltage should not exceed 1V/cm2);

5 closed

5.1) Take out the membrane and wash it three times with PBST, each time for 5 minutes (shaking on a horizontal shaker);

5.2) Take out the membrane and immerse in blocking solution at 37°C for 2 hours or overnight at 4°C (blocking solution is 1% casein or 2% OVA)

6 binding antibodies

6.1) Take out the membrane and wash it three times with PBST, each time for 5 minutes (shaking on a horizontal shaker);

6.2) Take out the membrane, soak it in primary antibody diluent diluted with 1% casein, 37°C, 1h (general primary antibody dilution 1:1000);

6.3) Take out the membrane and wash it three times with PBST, each time for 5 minutes (shaking on a horizontal shaker);

6.4) Take out the membrane, soak it in secondary antibody diluent diluted with 1% casein, 37°C, 1h

(goat anti-mouse-HRP 1:3000-1:5000, goat anti-rabbit-HRP 1:15000);

7 exposure

7.1) Dilute and mix A and B luminescent liquids in equal proportions (500ml each), place the film on the plastic wrap, drop the AB mixture evenly onto the film, cover with the plastic wrap, and let stand for 1 min;

7.2) Open the plastic wrap, blot the residual liquid on the surface with filter paper, and fix the inside of the plastic wrap in the cassette

7.3) Put the cassette in the darkroom, take out the film and quickly place it on the inner membrane of the cassette, close the cassette, and expose according to the fluorescence intensity you see (the general exposure time is 1 min, if the band is weak, you can choose to press the film overnight);

7.4) Open the cartridge, take out the film and immediately immerse it in the developing solution for 1 min;

7.5) Take out the film, rinse it with deionized water and soak it in the fixer for 1 min;

7.6) Take out the film, rinse it with deionized water and dry it, calibrate the Marker, and analyze the experimental results.

The results show that: the antibody prepared in Example 1 reacts with the tobacco lectin protein and does not react with the empty vector, and the blank control group does not react with the tobacco lectin protein; this shows that the antibody prepared in Example 1 has no effect on tobacco agglutinin. Proteins are specific.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention; those skilled in the art can change, modify, replace and Variations all belong to the protection scope of the present invention.

Claims

A method for preparing an antibody capable of detecting tobacco lectin protein, characterized in that it comprises the following steps:

The preparation steps of emulsion 1: take the antigen, Freund's complete adjuvant and PBS and mix to prepare the emulsion 1 containing the antigen;

Preparation steps of emulsion 2: mix antigen, Freund's incomplete adjuvant and PBS to prepare antigen-containing emulsion 2;

Immunization step: the immunization step includes injecting the emulsion 1 into the animal to immunize the animal for the first time; after the first immunization, injecting the emulsion 2 into the animal to immunize the animal for the second time; After the immunization, inject the emulsion 2 into the animal to immunize the animal for the third time; and/or after the third immunization, inject the emulsion 2 into the animal to immunize the animal for the fourth time;

Antibody purification step: taking animal serum and purifying the antibody from the serum to obtain the antibody that can detect tobacco lectin protein;

Wherein, the amino acid sequence of the antigen in emulsion 1 and emulsion 2 is shown in SEQ ID NO: 1.
The method for preparing an antibody capable of detecting tobacco lectin protein according to claim 1, characterized in that,

In the preparation step of emulsion 1, the dosage ratio of antigen, Freund's complete adjuvant and PBS is 300-500ug: 1mL: 1mL;

In the preparation step of emulsion 2, the dosage ratio of antigen, Freund's incomplete adjuvant and PBS is 300-500ug: 1mL: 1mL;

Most preferably, in the preparation step of emulsion 1, the dosage ratio of antigen, Freund's complete adjuvant and PBS is 400ug: 1mL: 1mL;

Most preferably, in the preparation step of emulsion 2, the dosage ratio of antigen, Freund's incomplete adjuvant and PBS is 400ug:1mL:1mL.
The preparation method of an antibody capable of detecting tobacco lectin protein according to claim 1, characterized in that, in the first, second, third and fourth immunization process, the amount of injected antigen is 40-60ug ;

Most preferably, during the first, second, third and fourth immunizations, the amount of injected antigen is 50ug/bird.
The method for preparing an antibody capable of detecting tobacco lectin protein according to claim 1, wherein the injection described in the immunization step refers to subcutaneous injection; the animal described in the immunization step is a mouse.
The preparation method of an antibody capable of detecting tobacco lectin protein according to claim 1, wherein the time of the first immunization is 2 to 3 weeks; the time of the second immunization is 2 weeks; the time of the third immunization is 2 weeks; The time is 1 week; the time of the fourth immunization is 1 week.
The method for preparing an antibody capable of detecting tobacco lectin protein according to claim 1, wherein the antibody purification step specifically comprises:

(1) Wash the equilibrium chromatography column with PBS buffer solution of 8 to 12 times the column volume;

(2) The supernatant after high-speed centrifugation of animal serum is mixed with PBS buffer, and then added to the chromatography column; the volume ratio of the supernatant to PBS buffer is 1:1-2;

(3) Elute with 8-12 times the column volume of PBS buffer until no protein is detected in the effluent, close the chromatography column to stop the elution;

(4) Add 2 to 4 times the column volume of PBS buffer containing citric acid or PBS buffer containing citric acid and glycine, and let it stand for 5 to 10 minutes; then perform elution, collect the eluate, and concentrate the eluate The antibody that can detect tobacco lectin protein can be obtained by freeze-drying.
The preparation method of an antibody capable of detecting tobacco lectin protein according to claim 6, characterized in that the PBS buffer containing citric acid described in step (3) refers to the PBS buffer containing 0.1M citric acid;

The PBS buffer solution containing citric acid and glycine refers to the PBS buffer solution containing 0.7M citric acid and 0.3M glycine.
The antibody capable of detecting tobacco lectin protein prepared by the preparation method according to any one of claims 1 to 7.
The antibody capable of detecting tobacco lectin protein according to claim 8, wherein the antibody capable of detecting tobacco lectin protein has the amino acid sequence described in SEQ ID NO: 2 or/and SEQ ID NO: 3 .
The use of the antibody capable of detecting tobacco lectin protein according to claim 8 in detecting tobacco lectin protein.