Antibody capable of detecting tobacco lectin protein and preparation method and application thereof
阅读说明：本技术 一种可检测烟草凝集素蛋白的抗体及其制备方法和应用 (Antibody capable of detecting tobacco lectin protein and preparation method and application thereof ) 是由 袁清华 黄振瑞 马柱文 于 2021-08-05 设计创作，主要内容包括：本发明涉及烟草凝集素蛋白检测技术领域,具体公开了一种可检测烟草凝集素蛋白的抗体及其制备方法和应用。所述的制备方法包含如下步骤：乳剂1的制备步骤：乳剂2的制备步骤：免疫步骤：所述的免疫步骤包括,将乳剂1注射至动物体内对动物进行第一次免疫；第一次免疫结束后,将乳剂2注射至动物体内对动物进行第二次免疫；第二次免疫结束后,将乳剂2注射至动物体内对动物进行第三次免疫；和/或第三次免疫结束后,将乳剂2注射至动物体内对动物进行第四次免疫；以及抗体纯化步骤。由该方法制备得到的抗体对于测烟草凝集素蛋白的特异性高,可以用于检测烟草凝集素蛋白,为烟草凝集素的检测提供了一种新的途径。(The invention relates to the technical field of tobacco lectin protein detection, and particularly discloses an antibody capable of detecting tobacco lectin protein, and a preparation method and application thereof. The preparation method comprises the following steps: preparation of emulsion 1: preparation of emulsion 2: an immunization step: the immunization step comprises injecting the emulsion 1 into an animal body to carry out first immunization on the animal; after the first immunization is finished, injecting the emulsion 2 into an animal body to carry out second immunization on the animal; after the second immunization is finished, injecting the emulsion 2 into the animal body to carry out third immunization on the animal; and/or after the third immunization is finished, injecting the emulsion 2 into an 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 detecting the tobacco lectin protein, can be used for detecting the tobacco lectin protein, and provides a new way for detecting the tobacco lectin.)
1. A preparation method of an antibody capable of detecting a tobacco lectin protein is characterized by comprising the following steps:
preparation of emulsion 1: mixing the antigen, Freund's complete adjuvant and PBS to prepare an antigen-containing emulsion 1;
preparation of emulsion 2: mixing the antigen, Freund's incomplete adjuvant and PBS to prepare an antigen-containing emulsion 2;
an immunization step: the immunization step comprises injecting the emulsion 1 into an animal body to carry out first immunization on the animal; after the first immunization is finished, injecting the emulsion 2 into an animal body to carry out second immunization on the animal; after the second immunization is finished, injecting the emulsion 2 into the animal body to carry out third immunization on the animal; and/or after the third immunization is finished, injecting the emulsion 2 into an animal body to carry out fourth immunization on the animal;
antibody purification step: taking animal serum, and purifying the antibody from the serum to obtain the antibody capable of detecting the tobacco lectin protein;
wherein, the amino acid sequences of the antigens in the emulsion 1 and the emulsion 2 are shown as SEQ ID NO: 1 is shown.
2. The method of claim 1, wherein the antibody capable of detecting a tobacco lectin protein is produced by the method,
in the preparation step of the emulsion 1, the dosage ratio of the antigen, Freund's complete adjuvant and PBS is 300-500 ug: 1mL of: 1 mL;
in the preparation step of the emulsion 2, the dosage ratio of the antigen, Freund's incomplete adjuvant and PBS is 300-500 ug: 1mL of: 1 mL;
most preferably, the ratio of antigen, Freund's complete adjuvant and PBS in the preparation step of emulsion 1 is 400 ug: 1mL of: 1 mL;
most preferably, in the preparation step of emulsion 2, the antigen, Freund's incomplete adjuvant and PBS are used in a ratio of 400 ug: 1mL of: 1 mL.
3. The method for preparing the antibody capable of detecting the tobacco lectin protein according to claim 1, wherein the amount of the antigen injected in the first, second, third and fourth immunization processes is 40-60 ug;
most preferably, the amount of antigen injected during the first, second, third and fourth immunizations is 50 ug/mouse.
4. The method for preparing an antibody capable of detecting a tobacco lectin protein of claim 1, wherein the injection in the immunizing step is subcutaneous injection; the animal in the immunization step is a mouse.
5. The method for preparing the antibody capable of detecting the tobacco lectin protein according to claim 1, wherein the time for the first immunization is 2-3 weeks; the time of the second immunization is 2 weeks; the time of the third immunization is 1 week; the time for the fourth immunization was 1 week.
6. The method for preparing the antibody capable of detecting the tobacco lectin protein of claim 1, wherein the antibody purification step specifically comprises:
(1) washing the equilibrium chromatographic column by using PBS buffer solution with 8-12 times of column volume;
(2) mixing the supernatant obtained after the high-speed centrifugation of the animal serum with a PBS buffer solution, and then adding the mixture into a chromatographic column; the volume ratio of the supernatant to the PBS buffer solution is 1: 1-2;
(3) eluting with PBS buffer solution with the volume of 8-12 times of the column volume until no protein is detected in the effluent liquid, and closing the chromatographic column to stop eluting;
(4) adding 2-4 times of column volume of PBS buffer solution containing citric acid or PBS buffer solution containing citric acid and glycine, and standing for 5-10 min; and then eluting, collecting eluent, concentrating the eluent and freeze-drying to obtain the antibody capable of detecting the tobacco lectin protein.
7. The method for preparing an antibody capable of detecting a tobacco lectin protein of claim 6, wherein the citric acid-containing PBS buffer in step (3) is 0.1M citric acid-containing PBS buffer;
the PBS buffer solution containing citric acid and glycine is PBS buffer solution containing 0.7M citric acid and 0.3M glycine.
8. An antibody capable of detecting a tobacco lectin protein produced by the production method according to any one of claims 1 to 7.
9. The antibody capable of detecting a tobacco lectin protein of claim 8, wherein the antibody capable of detecting a tobacco lectin protein has an amino acid sequence as set forth in SEQ ID NO: 2 or/and SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof.
10. Use of the antibody capable of detecting a tobacco lectin protein of claim 8 for detecting a tobacco lectin protein.
Technical Field
The invention relates to the technical field of tobacco lectin protein detection, and particularly relates to an antibody capable of detecting tobacco lectin protein, and a preparation method and application thereof.
Background
Tobacco lectin (Nictaba, Nicotiana tabacum agglutinin) was first isolated from tobacco leaves in 2002, and is an effective substance for preventing and treating various plant diseases and insect pests. Although related researches at home are few at present, a new research direction for preventing and treating plant diseases and insect pests by using natural tobacco products and taking Nictaba as a carrier is gradually formed at abroad. The tobacco agglutinin is distributed at each part of the tobacco plant; the amount of Nictaba in the leaves is higher, however its presence is not detectable in all tobacco varieties.
In order to better use the tobacco agglutinin for pest control; the detection of whether the plant contains the tobacco agglutinin is necessary; however, a special method for detecting tobacco lectin is lacking at present.
Disclosure of Invention
In view of this, the invention firstly provides a preparation method of an antibody capable of detecting the tobacco lectin protein; the antibody prepared by the method has high specificity for detecting the tobacco lectin protein, can be used for detecting the tobacco lectin protein, and provides a new way for detecting the tobacco lectin.
The technical scheme of the invention is as follows:
a preparation method of an antibody capable of detecting a tobacco lectin protein comprises the following steps:
preparation of emulsion 1: mixing antigen, Freund's complete adjuvant and PBS (specifically 0.01M PBS), and making into emulsion 1 containing antigen;
preparation of emulsion 2: mixing antigen, Freund's incomplete adjuvant and PBS (specifically 0.01M PBS), and making into emulsion 2 containing antigen;
an immunization step: the immunization step comprises injecting the emulsion 1 into an animal body to carry out first immunization on the animal; after the first immunization is finished, injecting the emulsion 2 into an animal body to carry out second immunization on the animal; after the second immunization is finished, injecting the emulsion 2 into the animal body to carry out third immunization on the animal; and/or after the third immunization is finished, injecting the emulsion 2 into an animal body to carry out fourth immunization on the animal;
antibody purification step: taking animal serum, and purifying the antibody from the serum to obtain the antibody capable of detecting the tobacco lectin protein;
wherein, the amino acid sequences of the antigens in the emulsion 1 and the emulsion 2 are shown as SEQ ID NO: 1 is shown.
Preferably, in the preparation step of the emulsion 1, the dosage ratio of the antigen, Freund's complete adjuvant and PBS is 300-500 ug: 1mL of: 1 mL;
in the preparation step of the emulsion 2, the dosage ratio of the antigen, Freund's incomplete adjuvant and PBS is 300-500 ug: 1mL of: 1 mL.
Most preferably, the ratio of antigen, Freund's complete adjuvant and PBS in the preparation step of emulsion 1 is 400 ug: 1mL of: 1 mL.
Most preferably, in the preparation step of emulsion 2, the antigen, Freund's incomplete adjuvant and PBS are used in a ratio of 400 ug: 1mL of: 1 mL.
Preferably, the amount of antigen injected during the first, second, third and fourth immunizations is 40-60 ug.
Most preferably, the amount of antigen injected during the first, second, third and fourth immunizations is 50 ug/mouse.
Preferably, the injection in the immunization step is subcutaneous injection.
Preferably, the animal in the immunizing step is a mouse.
Preferably, the time of the first immunization is 2-3 weeks; the time of the second immunization is 2 weeks; the time of the third immunization is 1 week; the time for the fourth immunization was 1 week.
The inventor shows through a large number of experimental studies that: the mice are immunized four times by adopting the emulsion 1 and the emulsion 2, so that the antibody which can detect the tobacco agglutinin protein with higher titer can be obtained.
Preferably, the antibody purification step specifically comprises:
(1) washing the equilibrium chromatography column (sepharose 4B chromatography column) with PBS buffer (specifically 0.01M PBS buffer) with the column volume of 8-12 times;
(2) mixing the supernatant obtained by high-speed centrifugation of animal serum with PBS (specifically 0.01M PBS), and adding into chromatographic column; the volume ratio of the supernatant to the PBS buffer solution is 1: 1-2;
(3) eluting with 8-12 times column volume of PBS buffer (specifically 0.01M PBS buffer), stopping eluting until no protein is detected in effluent liquid, and closing the chromatographic column;
(4) adding 2-4 times of column volume of PBS buffer solution (specifically 0.01M PBS buffer solution) containing citric acid or PBS buffer solution (specifically 0.01M PBS buffer solution) containing citric acid and glycine, and standing for 5-10 min; and then eluting, collecting eluent, concentrating the eluent and freeze-drying to obtain the antibody capable of detecting the tobacco lectin protein.
The inventor shows through a great deal of research that: the antibody which can detect the tobacco agglutinin protein and has high purity can be obtained by adopting the chromatographic column method.
Most preferably, the citric acid-containing PBS buffer in step (3) is 0.1M citric acid-containing PBS buffer;
the PBS buffer solution containing citric acid and glycine is PBS buffer solution containing 0.7M citric acid and 0.3M glycine.
The inventor shows through a great deal of research that: the composition of the PBS buffer plays an important role in preparing a high-purity antibody capable of detecting the tobacco lectin protein; when the elution is carried out by using PBS buffer solution containing 0.1M citric acid, the antibody which has high purity and high titer and can detect the tobacco agglutinin protein can be enriched. Further studies have shown that: when the elution is carried out by using PBS buffer solution containing 0.7M citric acid and 0.3M glycine, the antibody which has higher purity and higher titer and can detect the tobacco agglutinin protein can be enriched.
The invention also provides the antibody capable of detecting the tobacco lectin protein, which is prepared by the preparation method.
Preferably, the antibody capable of detecting a tobacco lectin protein has the amino acid sequence shown in SEQ ID NO: 2 or/and SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof.
The invention also provides application of the antibody capable of detecting the tobacco lectin protein in detection of the tobacco lectin protein.
Has the advantages that: the antibody capable of detecting the tobacco lectin protein is prepared for the first time; the antibody capable of detecting the tobacco lectin protein has higher specificity for the tobacco lectin protein; therefore, the antibody capable of detecting a tobacco lectin protein according to the present invention can be used for detecting a tobacco lectin protein; the antibody capable of detecting the tobacco lectin protein is successfully prepared, and a new detection way is provided for detection of the tobacco lectin protein; in addition, the antibody capable of detecting the tobacco lectin protein prepared by the method has higher purity and titer; the method is simple to operate and low in preparation cost.
Detailed Description
The present invention is further illustrated below with reference to examples, which are not intended to limit the invention in any way.
Example 1 preparation of antibody capable of detecting tobacco lectin protein
(1) Mixing antigen (amino acid sequence shown in SEQ ID NO: 1), Freund's complete adjuvant and PBS (specifically 0.01M PBS), and making into emulsion 1 containing antigen; wherein the dosage ratio of the antigen, Freund's complete adjuvant and PBS is 400 ug: 1mL of: 1 mL;
(2) mixing antigen (amino acid sequence shown in SEQ ID NO: 1), Freund's incomplete adjuvant and PBS (specifically 0.01M PBS), and making into emulsion 2 containing antigen; wherein the dosage ratio of the antigen, Freund's incomplete adjuvant and PBS is 400 ug: 1mL of: 1 mL;
(3) injecting the emulsion 1 into a Balb/c mouse body in a subcutaneous injection mode to carry out first immunization on the Balb/c mouse; after the first immunization is finished, injecting the emulsion 2 into a Balb/c mouse body in a subcutaneous injection mode to carry out second immunization on the Balb/c mouse; after the second immunization is finished, injecting the emulsion 2 into Balb/c mice in a subcutaneous injection mode to carry out third immunization on the animals; after the third immunization is finished, injecting the emulsion 2 into Balb/c mice in a subcutaneous injection mode to carry out fourth immunization on the animals;
wherein, the amount of the injected antigen is 50 ug/mouse in the first, second, third and fourth immunization processes; the time of the first immunization is 3 weeks; the time of the second immunization is 2 weeks; the time of the third immunization is 1 week; the time of the fourth immunization is 1 week;
(5) taking Balb/c mouse serum, and purifying an antibody from the serum to obtain the antibody capable of detecting the tobacco lectin protein; the specific steps for purifying antibodies from serum are as follows:
5.1) washing the equilibrium chromatography column (in particular sepharose 4B chromatography column) with 10 column volumes of PBS buffer (in particular 0.01M PBS buffer);
5.2) mixing the supernatant obtained after the high-speed centrifugation of the animal serum with a PBS (phosphate buffer solution) of 0.01M in particular) and then adding the mixture into a chromatographic column; the volume ratio of the supernatant to the PBS buffer solution is 1: 1;
5.3) eluting with 10 times column volume of PBS buffer (specifically 0.01M PBS buffer) until no protein is detected in the effluent, and closing the chromatographic column to stop elution;
5.4) adding 2 times of column volume of PBS buffer solution (specifically 0.01M PBS buffer solution) containing 0.7M citric acid and 0.3M glycine, and standing for 5 min; and then eluting, collecting eluent, concentrating the eluent and freeze-drying to obtain the antibody capable of detecting the tobacco lectin protein. The antibody capable of detecting the tobacco lectin protein has the amino acid sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
Example 2 basic potency assay-Indirect ELSIA
(1) Antigen coating: the antigen was diluted with the coating solution to a prescribed concentration (1 ug/ml without separate description), 100. mu.L/well was added to a polystyrene 96-well plate, and left overnight at 4 ℃.
(2) Washing: the next day, the liquid in the wells was discarded and the wash was washed 1 time.
(3) And (3) sealing: add 200. mu.L/well blocking solution and stand at 37 ℃ for 2h.
(4) Washing: washed 3 times with the wash solution.
(5) The sample to be tested (antibody prepared in example 1) was added: the sample to be tested (the sample needs to be diluted, and is diluted proportionally) is added into each hole at 100ul, and the incubation is carried out for 1 h.
(6) Washing: the sample to be tested was discarded and washed 3 times with the wash solution.
(7) Adding an enzyme-labeled secondary antibody: HRP-labeled goat anti-mouse IgG (1:10000 diluted with enzyme) was added to the cells, and the cells were incubated at 37 ℃ for 40 min.
(8) Washing: washed 5 times with wash liquor.
(9) Color development: adding fresh substrate solution 90 μ L/well, standing at 37 deg.C in dark for 15min
(10) Terminating reaction and carrying out color comparison: add 50. mu.L/well stop solution. The color turns yellow; the absorbance of each well at 450nm was measured using a microplate reader.
The results show that: the light absorption value in the measuring hole of the sample to be detected is more than 3 times of the average value of the negative control holes, which indicates that the antibody capable of detecting the tobacco lectin protein prepared by the invention is positive; and has higher titer.
Example 3 Western Blot experiment
(1) Preparation of Polyacrylamide gels
1.1) preparing a 15% separating gel solution: 2.3ml of deionized water, 5ml of 30% acrylamide, 2.5ml of Tris (pH 8.8), 100. mu.L of 10% ammonium persulfate and 100. mu. L, TEMED 6. mu.L of 10% SDS (after TEMED is added, the separation gel immediately starts to polymerize, so the separation gel is quickly and uniformly mixed immediately);
1.2) rapidly adding the separating glue solution into the gap between the two glass plates, leaving the space for filling the concentrated glue, and carefully covering the separating glue solution with a layer of isopropanol;
1.3) after the separation gel is completely polymerized, draining the liquid on the gel as far as possible, and then completely sucking the residual liquid by using the edge of a paper towel;
1.4) preparing a concentrated glue solution with the concentration of 5 percent: 2.7ml of deionized water, 500 mu L of 30% acrylamide 670 mu L, PH 8.8.8 Tris, 40 mu L of 10% ammonium persulfate and 40 mu L, TEMED 6 mu L of 10% SDS are sequentially added (after TEMED is added, the separation gel immediately starts to polymerize, so the separation gel is quickly and uniformly mixed immediately);
1.5) pouring the concentrated gel directly on the polymerized separating gel, and inserting a clean comb (carefully avoiding air bubbles) into the concentrated gel solution immediately;
1.26) after the polymerization of the concentrated gel is complete, the comb is carefully removed and the protein electrophoresis buffer is added to the electrophoresis tank.
2 preparation of samples
2.1) cell culture to 107Collecting cells into a 15ml centrifuge tube, centrifuging at 2000rpm for 5min, and removing supernatant;
2.2) washing the culture medium twice, centrifuging at 2000rpm for 5min, and removing the supernatant;
2.3) after collecting cells, adding 1ml PBS, 0.1% NP40, and cracking on ice for 10 min;
2.4) centrifuging at 10000rpm for 5min, taking the supernatant, adding an equal volume of 2 × loading buffer, boiling for 5min, subpackaging, and storing at-20 ℃;
3 electrophoresis
3.1) mixing the antibody sample (10. mu.L) prepared in example 1 to be tested with 2 × (or 6 ×) loading buffer (10. mu.L), slowly adding 15. mu.L of the mixture to the sample tank using a pipette, and pre-staining with 10. mu.L of a Maker;
3.2) turning on a power supply, and adopting a voltage of 200V to carry out electrophoresis until the bromophenol blue sample loading buffer solution migrates to the bottom of the gel in the gel;
3.3) cutting off the power supply, taking out the gel, soaking the gel in R250 Coomassie brilliant blue for dyeing, and boiling for 5 min;
3.4) taking out the dyed gel, soaking the gel in tap water for decoloring, boiling for about 20min, and observing a decolored protein band;
4-turn film
4.1) taking the glue, cutting the glue into a proper size, and immersing the glue into a film transfer buffer;
4.2) soaking the PVDF membrane in methanol for 1min, transferring the PVDF membrane into a membrane transfer buffer, and soaking filter paper into the membrane transfer buffer (the PVDF membrane and the filter paper are both cut into the same size as the glue);
4.3) leaching the graphite electrode by using a membrane transfer buffer, laying two pieces of filter paper, and dripping a little of the membrane transfer buffer;
laying a film, and dripping a little of film buffer; spreading glue, and dripping a little of film buffer (taking care not to generate bubbles);
4.4) finally laying two pieces of filter paper, and dripping a little of membrane buffer;
4.5) covering the electrode, adjusting the voltage to the maximum, and transferring the gel volume to the membrane for 1.5h by 1.5mA/cm2 (the load voltage is not more than 1V/cm 2);
5 sealing
5.1) the membrane was taken out and washed three times with PBST, 5min each time (shaking on a horizontal shaker);
5.2) the membrane was removed and immersed in the blocking solution at 37 ℃ overnight at 2h or 4 ℃ (1% casein or 2% OVA in blocking solution)
6 binding antibodies
6.1) the membrane was taken out and washed three times with PBST, 5min each time (shaking on a horizontal shaker);
6.2) the membrane was removed and soaked in primary anti-dilution with 1% casein at 37 ℃ for 1h (typical primary anti-dilution 1: 1000) (ii) a
6.3) the membrane was taken out and washed three times with PBST, 5min each time (shaking on a horizontal shaker);
6.4) the membrane was removed and soaked in a secondary antibody dilution diluted with 1% casein at 37 ℃ for 1h
(goat anti-mouse-HRP 1: 3000-1: 5000, goat anti-rabbit-HRP 1: 15000);
7 Exposure
7.1) diluting and mixing A, B luminous liquid in equal proportion (each 500ml), placing a film on a preservative film, uniformly dripping the AB mixed liquid on the film, covering the preservative film, and standing for 1 min;
7.2) opening the preservative film, absorbing residual liquid on the surface by using filter paper, and fixing the preservative film in a hidden box
7.3) placing the cassette in a dark room, taking out the film, quickly placing the film on the inner film of the cassette, closing the cassette, and exposing according to the intensity of the visible fluorescence (the exposure time is 1min generally, and if the strip is weak, the sheeting can be selected overnight);
7.4) opening the cassette, taking out the film and immediately and completely immersing the film into the developing solution for 1 min;
7.5) taking out the film, rinsing the film by deionized water, and immersing the film into a fixing solution for 1 min;
7.6) taking out the film, rinsing the film by using deionized water, drying the film in the air, calibrating a Marker, and analyzing an experimental result.
The results show that: the antibody prepared in example 1 reacts with tobacco lectin protein and does not react with empty carrier, while the blank control group does not react with tobacco lectin protein; this demonstrates that the antibody prepared in example 1 is specific for tobacco lectin protein.
While embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limiting the present invention; variations, modifications, substitutions and alterations of the above-described embodiments by those of ordinary skill in the art are intended to be within the scope of the present invention.
Sequence listing
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