Antibody against Botrytis cinerea ATP3 protein and its application

Abstract

The invention discloses an antibody against gray mold ATP3 protein and its application, and relates to the field of biotechnology. The antibody for detecting gray mold pathogens of the invention is obtained by immunizing animals with gray mold ATP3 protein as an antigen, and the amino acid sequence of the gray mold ATP3 protein is shown in SEQ ID NO.1. Based on the specific binding characteristics of antigen and antibody, the invention detects gray mold ATP3 protein by immunoblotting and immunomicrosphere technology, overcomes the disadvantages of traditional gray mold detection that the time is long and the requirements are high, greatly advances the detection time of gray mold, and achieves early, rapid and accurate detection effect of gray mold.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an antibody against Botrytis cinerea ATP3 protein and application thereof. Background Art

[0002] Botrytis cinerea is the pathogen of gray mold, which causes infection of crops such as grapes, resulting in quality decline, reduced yield, and even total crop failure. Under low temperature conditions, gray mold can spread and infect plants using conidia, and has the characteristics of latent infection and low temperature pathogenicity. In addition, gray mold has the characteristics of fast reproduction and large variation. In the greenhouse, gray mold spreads and infects faster, which can easily lead to large-scale outbreaks of gray mold. Once formed, the conidia of gray mold will float around the greenhouse, and the aerosols formed can easily adhere to the plants. The spores scattered in the soil are also infectious and can also be spread with rain. Gray mold has strong stress resistance, and its sclerotia, conidia, and mycelium overwinter in the soil with diseased plant tissues, and even directly form sclerotia on the plants to overwinter.

[0003] There are two methods for detecting gray mold: one is based on the typical symptoms of the plant when the disease occurs, combined with the morphology of hyphae and conidia; the other is based on nucleic acid detection of gray mold-specific genes or sequences. The disadvantage of the former method is that when gray mold is detected, the plant has already been infected and losses are inevitable. The advantage of the latter method is that the detection is high in accuracy and rapid, and it is suitable for samples such as soil, seeds, and plant tissues. However, this method requires special instruments and equipment for detection, and the operator needs professional training before operating it.

[0004] Therefore, it is an urgent problem for technicians in this field to establish a rapid and accurate detection method for the distribution and propagation characteristics of gray mold sclerotia, conidia and mycelium, which can be used for detection before the outbreak of gray mold to prevent and control it in advance, and prevent the losses caused by large-scale outbreaks of gray mold. Summary of the invention

[0005] In view of this, the present invention provides an antibody against Botrytis cinerea ATP3 protein and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] An antibody for detecting Botrytis cinerea pathogenic bacteria, obtained by immunizing animals with Botrytis cinerea ATP3 protein as an antigen.

[0008] Botrytis cinerea ATP3 protein participates in ATP biosynthesis and is indispensable for Botrytis cinerea. The sequence is specific and conservative and can effectively characterize Botrytis cinerea. The present invention uses ATP3 protein as an antigen to prepare antibodies. The antibodies can be used in combination with color immunomicrosphere labeling and immunoblotting techniques to specifically detect Botrytis cinerea from samples such as soil, seeds, and plant tissues, which has great advantages for the early diagnosis of Botrytis cinerea. Moreover, the method is a serological detection method, does not require expensive instruments and equipment, can be operated by ordinary technicians, and has the characteristics of simplicity, rapidity, and sensitivity.

[0009] As a preferred technical solution, the amino acid sequence of the Botrytis cinerea ATP3 protein is shown in SEQ ID NO.1.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned antibody for detecting gray mold pathogens, comprising constructing or synthesizing a recombinant plasmid containing gray mold ATP3 protein, cloning and expressing gray mold ATP3 protein, purifying gray mold ATP3 protein, and using the purified gray mold ATP3 protein as an antigen to immunize an animal, and the obtained immune serum is the antibody for detecting gray mold pathogens.

[0011] As a preferred technical solution, the plasmid used in constructing or synthesizing the recombinant plasmid containing Botrytis cinerea ATP3 protein is pET24a.

[0012] Another object of the present invention is to provide a kit for detecting Botrytis cinerea pathogens, wherein the kit comprises the above-mentioned antibody for detecting Botrytis cinerea pathogens, or comprises the antibody prepared by the above-mentioned preparation method.

[0013] As a preferred technical solution, the kit is an immunoblotting kit, which also includes a nitrocellulose blotting membrane, an HRP-labeled secondary antibody, a washing solution PBS, and an ECL chemiluminescent reagent.

[0014] As a preferred technical solution, the kit is a color immunomicrosphere kit, which also includes a nitrocellulose blotting membrane, a washing solution PBS, and the ATP3 antibody is labeled with a color immunomicrosphere.

[0015] Another object of the present invention is to provide the use of the above antibody for detecting gray mold pathogens, the antibody prepared by the above preparation method, or the above kit in the preparation of a product for detecting gray mold pathogens.

[0016] Another object of the present invention is to provide an immunological detection method for gray mold pathogens, comprising the following steps:

[0017] (1) A 1×1 cm square grid was drawn on the nitrocellulose blotting membrane through a protective film, and then the protective film was removed and 2 μl of the sample solution to be tested was aspirated and dotted on the square grid; the square grid was placed in a wet box, and filter paper moistened with PBS buffer was placed in advance, and incubated at 37°C for 2 h;

[0018] (2) Transfer the incubated nitrocellulose blot to a protein-free blocking buffer for blocking at 37°C for 2 h, then wash with 1× PBST and place on a decolorizing shaker for three washes at 40 rpm for 5 min each.

[0019] (3) The incubated nitrocellulose blot membrane was transferred to the above-mentioned antibody for detecting gray mold pathogens diluted 10,000 times with PBS in advance, incubated at 37°C for 2 hours, and then washed with 1× PBST, placed on a decolorizing shaker and washed 3 times at a speed of 40 rpm, with each wash time of 5 minutes;

[0020] (4) Take out the washed nitrocellulose blot membrane and mark it according to the dilution, transfer it to HRP-labeled goat anti-rabbit secondary antibody diluted 5000 times, incubate it at 37°C for 2 hours, then wash it with PBST diluted 1×, place it on a decolorizing shaker and wash it 4 times at a speed of 40 rpm, each wash for 5 minutes;

[0021] (5) Use ultra-sensitive ECL chemiluminescent reagent to develop color and read the results.

[0022] Another object of the present invention is to provide an immunomicrosphere detection method for gray mold, comprising the following steps:

[0023] (1) Prepare blue immune microspheres, take 10 ml of NHS-labeled blue microspheres with a diameter of 1 μm, add 10 μl of the above-mentioned antibody for detecting gray mold pathogens at a mass ratio of 0.1‰ for cross-linking, cross-link for 10 h at 25°C in the dark, and rotate at 40 rpm; after cross-linking, add 250 μl of 2M Tris to a final concentration of 50 mM for blocking, and block for 10 h at 25°C in the dark at 40 rpm;

[0024] (2) The nitrocellulose blotting membrane was cut into 1×1 cm squares through a protective film, and then the protective film was removed and 2 μl of the sample solution to be tested was aspirated and dotted into the squares; the nitrocellulose blotting membrane was placed in a wet box, and filter paper moistened with PBS buffer was placed in advance, and incubated at 37°C for 2 h;

[0025] (3) Transfer the incubated nitrocellulose blot membrane to a protein-free blocking buffer for blocking at 37°C for 2 h, then wash with 1× PBST and place on a decolorizing shaker for 3 washes at 40 rpm for 5 min each. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0026] (4) Add the prepared blue immune microspheres onto the incubated nitrocellulose blotting membrane to evenly cover the surface; incubate at 25°C for 10 min, then wash with 1× diluted PBST, place on a decolorizing shaker and wash three times at a speed of 40 rpm, with each wash time of 2 min. After washing, color development is performed and the results are read.

[0027] Beneficial effects: Based on the specific binding characteristics of antigen and antibody, the present invention detects the ATP3 protein of Botrytis cinerea through immunoblotting and immunomicrosphere technology. The present invention overcomes the disadvantages of the traditional detection of Botrytis cinerea, which takes a long time and has high requirements, greatly advances the detection time of Botrytis cinerea, and achieves early, rapid and accurate detection of Botrytis cinerea. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 This is a schematic diagram of the preparation of Botrytis cinerea ATP3 protein according to an embodiment of the present invention, wherein MK: marker, A: purified ATP3 protein (arrow).

[0030] Figure 2 This is the titer detection of the anti-Botrytis cinerea ATP3 protein immune serum in the embodiment of the present invention.

[0031] Figure 3 This is the specific detection of the anti-Botrytis cinerea ATP3 protein immune serum in the embodiment of the present invention, wherein +, -, Blk are positive, negative, and blank samples, respectively, Fc and Ph are common Fusarium and Downy Phytophthora, respectively, and Bc1 and Bc2 are grape samples infected with Botrytis cinerea and Botrytis cinerea hyphae samples, respectively.

[0032] Figure 4 In the present embodiment, the anti-Botrytis cinerea ATP3 protein antibody is applied to immunoblotting to detect Botrytis cinerea samples.

[0033] Figure 5 To apply anti-Botrytis cinerea ATP3 protein antibody to blue immunomicrospheres to detect Botrytis cinerea samples.

[0034] Figure 6 In this embodiment of the present invention, the anti-Botrytis cinerea ATP3 protein antibody is applied to the accuracy of Western blotting to detect Botrytis cinerea samples. The upper line of text in the caption is the unblinding result, and the lower line of text is the blind test number, where + and - are positive and negative samples, respectively, and Fc, Bc, and Ph are common Fusarium, Botrytis cinerea, and Downy mildew, respectively.

[0035] Figure 7 In order to apply the anti-Botrytis cinerea ATP3 protein antibody to the blue immunomicrospheres to detect the accuracy of Botrytis cinerea samples, the upper line of text in the caption is the unblinding result, and the lower line of text is the blind test number, where + and - are positive and negative samples, respectively, and Fc, Bc, and Ph are common Fusarium, Botrytis cinerea, and Downy Phytophthora, respectively. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The sources of reagents shown in the embodiments of the present invention are only used to illustrate the technical solution of the present invention, and are not intended to limit the present invention, and can be replaced by products with the same functions.

[0038] The present invention provides an antibody against Botrytis cinerea ATP3 protein, wherein the antibody is obtained by immunizing New Zealand white rabbits with a vaccine prepared using the ATP3 protein. The amino acid sequence of the ATP3 protein is shown in SEQ ID NO.1:

[0039] MGSSHHHHHHSSGLVPRGSMLSRTARPALRAAVAASSRPVAPQAAGYATLREIEDRLKSIRNIEKITNTMKIVASTKLTRAQKAMRESRSYGESSNSVFEHAETKALESDKKRLIVVASSDKGLCGGIHSGLSKQVRRMLAADSNADIVVLGEKCKAQLS RSSGKNIVMSFAGVGKDVPTFADAQAIADQIVLLPTDYSSVQIVYNKFINATSYEATPIEAFSEEAIANSPNFSAFEIEDETLGNLREYALANSLFWALAEGHACEQSARRNAMDNASKNAGDMITKYQILFNRTRQAVITGELVEIITGAAASEE,SEQ ID NO.1.

[0040] Example 1

[0041] Preparation and purification of ATP3 protein from Botrytis cinerea and preparation of its antibody

[0042] 1. Preparation and purification of Botrytis cinerea ATP3 protein

[0043] Transformation expression and protein purification of the fully synthetic pET24a-ATP3 recombinant plasmid (synthesized by Suzhou Hongxun Biotechnology Co., Ltd., the constructed recombinant plasmid insert was inserted into the multiple cloning site, and its restriction endonuclease sites are NdeI and XhoI. The fully synthetic insert can be prepared as sticky ends through NdeI and XhoI and connected to the pET24a vector that has been digested with enzymes to prepare a recombinant plasmid)

[0044] Add 1 μl of recombinant plasmid to 100 μl of BL21 (DE3) competent bacteria, place on ice for 20 min; heat shock at 42°C for 90 sec, quickly place on ice for 3 min; add 500 μl of LB culture solution; shake and culture at 37°C, 220 rpm for 2 h, take 200 μl of bacterial solution and spread it on LB plate containing 50 μg / ml kanamycin, and invert and culture at 37°C overnight; the next morning, pick a single colony on the plate and inoculate it into a test tube containing 4 ml of LB culture solution containing 50 μg / ml kanamycin, and shake and culture at 37°C, 220 rpm until OD 600 The absorbance value was 0.6; the cells were inoculated into 1L LB culture medium containing 100 μg / ml kanamycin at a ratio of 1:250 (a total of 1L culture medium), and shaken at 37°C and 220 rpm until the bacterial OD 600 0.5 to 0.6 (about 4h); add inducer IPTG to each liter of culture medium to a final concentration of 0.1mM, and culture overnight at 220rpm and 16℃; centrifuge at 6000rpm for 5min to remove the supernatant and collect the fermented bacteria, which are then broken by ultrasound for purification.

[0045] Purification of Botrytis cinerea ATP3 protein, protein urea solution purification select affinity chromatography, 20mM Tris, 500mM NaCl, 2M urea (pH 7.4) as the equilibrium buffer, the target protein is present in the 250mM and 500mM imidazole eluents, such as attached Figure 1 As shown in the electrophoresis detection, the molecular weight of the ATP3 protein band is 34.16KDa. The buffer was replaced with a dialysis bag. The final protein buffer was 20mM Tris, 500mM NaCl, 2M urea (pH 7.4). After dialysis, the His tag was removed by enzyme digestion at 30℃ for 6 hours in a buffer system (500mM Tris-HCl, 500mM NaCl, 5mM EDTA, 10mM DTT, pH8.0) using a tag protein removal reagent (P2310M, TEVProtease (GST / His-tag), Shanghai Beyotime Biotechnology Co., Ltd.). Then, the His tag was removed by His tag protein purification agarose magnetic beads (P2239-10ml, BeyoMag TMThe His tag was removed by using agarose magnetic beads (IDA-Ni, Shanghai Bio-Tech Biotechnology Co., Ltd.) for purification of His-tagged proteins, and then concentrated and packaged at 1 mg / ml to obtain a 1 mg / ml ATP3 protein solution.

[0046] 2. Preparation of Antibodies against Botrytis cinerea ATP3 Protein

[0047] Specifically, there are two immunizations, with a one-week interval between the first immunization and the second immunization.

[0048] For the primary immunization, 600 μl of 1 mg / ml ATP3 protein solution (obtained in step 1) was emulsified with 600 μl complete Freund's adjuvant (Cat. No.: R19017-10ml, Shanghai Ruichu Biotechnology Co., Ltd.). After complete emulsification, 1 ml was drawn with a syringe and injected subcutaneously at multiple points in the neck of New Zealand white rabbits for immunization.

[0049] For the second immunization, 600 μl of 1 mg / ml ATP3 protein solution (obtained in step 1) was added with 600 μl of incomplete Freund's adjuvant (Cat. No.: R19016-10ml, Shanghai Ruichu Biotechnology Co., Ltd.) for emulsification. After complete emulsification, 1 ml was drawn with a syringe and injected subcutaneously at multiple points in the neck of New Zealand white rabbits for immunization.

[0050] One week after the second immunization, blood is collected from the ear vein to prepare serum and the titer is determined. When the titer reaches 1:10000 or above, all serum can be collected to obtain serum (antibody) against Botrytis cinerea ATP3 protein.

[0051] 3. Determination of Antibody Titer Against Botrytis Cinerea ATP3 Protein

[0052] The antibody titer against Botrytis cinerea ATP3 protein was detected by immunoblotting.

[0053] A nitrocellulose blotting membrane (Cat. No.: F619511-0005, 0.22 μm, 20×20 cm, Sangon Biotech (Shanghai) Co., Ltd.) was cut into 1×1 cm squares through the protective film, and then the protective film was removed and 2 μl of 1 mg / ml ATP3 protein solution (obtained in step 1) was aspirated and dotted on the squares. Placed in a wet box, the filter paper was wetted with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) in advance, and incubated at 37°C for 2 h. Two technical replicates were performed.

[0054] Transfer the incubated nitrocellulose blot membrane to a protein-free blocking solution (Cat. No.: C510042-0500, Sangon Biotech (Shanghai) Co., Ltd.) for blocking. This step is performed in a wet box for ease of operation. Block at 37°C for 2 h, then wash with 1× diluted PBST (Cat. No.: C520004-0001, Sangon Biotech (Shanghai) Co., Ltd.), place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 to 10 min each time. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0055] Transfer the incubated nitrocellulose blot membrane to the serum (antibody) against Botrytis cinerea ATP3 protein (obtained in step 2) diluted 10,000 times, 100,000 times, and 200,000 times with PBS in advance. For the convenience of operation, this step is carried out in a 50ml centrifuge tube. Incubate at 37°C for 2 hours, then wash with 1× PBST, place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 minutes each time. During the washing process, prevent the membrane from sticking to the wall without buffer;

[0056] The washed nitrocellulose blot was taken out and marked with a ballpoint pen or cut corner according to the dilution, and transferred to HRP-labeled goat anti-rabbit secondary antibody diluted 5000 times (Cat. No.: SE134-0.1ml, Beijing Solebow Technology Co., Ltd.), incubated at 37°C for 2 hours, and then washed with 1× PBST, placed on a decolorizing shaker and washed 4 times at a speed of 40 rpm, each wash for 5 minutes. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0057] According to the instructions of the ultra-sensitive ECL chemiluminescent reagent (Cat. No.: C520045-0500, Sangon Biotech (Shanghai) Co., Ltd.), mix solution A and solution B at a volume ratio of 1:1 to prepare the immunoblotting solution. Place the membrane in the ECL chemiluminescent imager, add 50 μl of the solution to each square, and evenly cover the membrane surface. Adjust the exposure parameters to develop the color. After exposure and color development, the immunoblotting spots appear as black spots, as shown in the attached Figure 2 The titer of the immune serum against gray mold disease of the present invention is 1:100,000.

[0058] 4. Specificity detection of antibodies against Botrytis cinerea ATP3 protein

[0059] Mycelia of artificially cultured downy mildew (Phytophthora hibernalis, ATCC 60352, Shanghai Collection of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai), gray mold (Botrytis cinerea, ATCC58025, Shanghai Collection of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai), and common Fusarium (Fusarium commune, GDMCC NO.: 3.697, Guangdong Microbiological Culture Collection Center) were purified and cultured at 30°C using PDA medium (Product No.: HB0233, Haibo Biotechnology Co., Ltd.), and the pure culture was inoculated into potato glucose water medium (Product No.: HB0233-4, Haibo Biotechnology Co., Ltd.), and cultured at 30°C for 7 days to obtain mycelia. Collect grape samples infected with gray mold and artificially cultured hyphae and grind them to extract crude protein for detection. Add 1 ml PBS according to 50 mg, then add 10 zirconium oxide grinding beads with a diameter of 1 mm, grind at a speed of 10 m / s for 2 minutes to extract crude protein, and then centrifuge at 12000 rpm for 5 minutes to collect the supernatant to obtain the sample to be tested;

[0060] A nitrocellulose blotting membrane (Cat. No.: F619511-0005, 0.22 μm, 20×20 cm, Sangon Biotech (Shanghai) Co., Ltd.) was drawn into a 1×1 cm grid through the protective film, and then the protective film was removed, and 2 μl of the sample to be tested was drawn and dotted in the grid. Placed in a wet box, the filter paper was wetted with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) in advance, and incubated at 37°C for 2 h. 1 mg / ml bovine serum albumin was used as a negative sample, PBS buffer was used as a blank sample, and 1 mg / ml ATP3 protein solution was used as a positive sample. The positive, negative, and blank samples were repeated twice, and the artificially cultured mycelium and the grape samples infected with gray mold were repeated three times each.

[0061] Transfer the incubated nitrocellulose blot membrane to a protein-free blocking solution (Cat. No.: C510042-0500, Sangon Biotech (Shanghai) Co., Ltd.) for blocking. This step is performed in a wet box for ease of operation. Block at 37°C for 2 h, then wash with 1× diluted PBST (Cat. No.: C520004-0001, Sangon Biotech (Shanghai) Co., Ltd.), place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 to 10 min each time. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0062] Transfer the incubated nitrocellulose blot membrane to the serum (antibody) against Botrytis cinerea ATP3 protein diluted 10,000 times, 100,000 times, and 200,000 times with PBS (obtained in step 2). For ease of operation, this step is performed in a 50 ml centrifuge tube. Incubate at 37°C for 2 hours, then wash with 1× PBST, place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 minutes each time. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0063] The washed nitrocellulose blot was taken out and marked with a ballpoint pen or cut corner according to the dilution, and transferred to HRP-labeled goat anti-rabbit secondary antibody diluted 5000 times (Cat. No.: SE134-0.1ml, Beijing Solebow Technology Co., Ltd.), incubated at 37°C for 2 hours, and then washed with 1× PBST, placed on a decolorizing shaker and washed 4 times at a speed of 40 rpm, each wash for 5 minutes. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0064] According to the instructions of the ultra-sensitive ECL chemiluminescent reagent (Cat. No.: C520045-0500, Sangon Biotech (Shanghai) Co., Ltd.), mix solution A and solution B at a volume ratio of 1:1 to prepare the immunoblotting solution. Place the membrane in the ECL chemiluminescent imager, add 50 μl of the solution to each square, and evenly cover the membrane surface. Adjust the exposure parameters to develop the color. After exposure and color development, the immunoblotting spots appear as black spots, as shown in the attached Figure 3 As shown, only the grape leaves infected with Botrytis cinerea, Botrytis cinerea hyphae and the positive control were positive, indicating that the antibody against Botrytis cinerea ATP3 protein obtained in the present invention has specificity.

[0065] Example 2

[0066] Detection of Botrytis cinerea by using antibodies against Botrytis cinerea ATP3 protein (Western blotting)

[0067] Grape samples infected with gray mold (Botrytis cinerea, ATCC58025, Shanghai Collection Center of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai) were collected, and gray mold was isolated and purified by culture at 30°C using PDA medium (Article No.: HB0233, Haibo Biotechnology Co., Ltd.), and the pure gray mold culture was inoculated into potato glucose water medium (Article No.: HB0233-4, Haibo Biotechnology Co., Ltd.) and cultured at 30°C for 7 days to obtain mycelium. The mycelium was inoculated on grapes with 80-mesh woodworking sandpaper scratched in advance at a mass ratio of 1:50, and samples were prepared after culture at 30°C for 2 days (no mycelium growth visible to the naked eye). Artificially cultured gray mold hyphae and grape samples infected with gray mold were ground to extract crude protein for testing. 50 mg was added to 1 ml PBS, and then 10 zirconium oxide grinding beads with a diameter of 1 mm were added. The crude protein was extracted by grinding at a speed of 10 m / s for 2 minutes, and then centrifuged at 12000 rpm for 5 minutes to collect the supernatant, which was the sample to be tested.

[0068] A nitrocellulose blotting membrane (Article No.: F619511-0005, 0.22 μm, 20×20 cm, Sangon Biotech (Shanghai) Co., Ltd.) was drawn into a 1×1 cm grid across the protective film, and then the protective film was removed, and 2 μl of the sample solution to be tested was aspirated and dotted in the grid. Placed in a wet box, a filter paper moistened with PBS buffer (Article No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) was pre-padded, and incubated at 37°C for 2 h. The positive sample was gray mold ATP3 protein (obtained in step 1 of Example 1), the negative sample was PBS buffer, artificially cultured gray mold hyphae and crude protein extracts of grapes infected with gray mold were used as test samples, and each sample was repeated twice.

[0069] Transfer the incubated nitrocellulose blot membrane to a protein-free blocking solution (Cat. No.: C510042-0500, Sangon Biotech (Shanghai) Co., Ltd.) for blocking. This step is performed in a wet box for ease of operation. Block at 37°C for 2 h, then wash with 1× diluted PBST (Cat. No.: C520004-0001, Sangon Biotech (Shanghai) Co., Ltd.), place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 min each time. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0070] The incubated nitrocellulose blot membranes were transferred to the serum (antibody) against Botrytis cinerea ATP3 protein (obtained in step 2 of Example 1) diluted 10,000 times with PBS in advance. For the convenience of operation, this step was performed in a 50 ml centrifuge tube. Incubate at 37°C for 2 hours, then wash with PBST diluted 1×, place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, each wash for 5 minutes. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0071] The washed nitrocellulose blot membrane was taken out and marked with a ballpoint pen or cut corner according to the dilution, and transferred to the HRP-labeled goat anti-rabbit secondary antibody diluted 5000 times (Cat. No.: SE134-0.1ml, Beijing Solebow Technology Co., Ltd.), incubated at 37°C for 2 hours, and then washed with 1× PBST, placed on a decolorizing shaker and washed 4 times at a speed of 40 rpm, each washing for 5 minutes. During the washing process, prevent the membrane from sticking to the wall without buffer;

[0072] According to the instructions of the ultra-sensitive ECL chemiluminescent reagent (Cat. No.: C520045-0500, Sangon Biotech (Shanghai) Co., Ltd.), mix solution A and solution B at a volume ratio of 1:1 to prepare the immunoblotting solution. Place the membrane in the ECL chemiluminescent imager, add 50 μl of the solution to each square, and evenly cover the membrane surface. Adjust the exposure parameters to develop the color. After exposure and color development, the immunoblotting spots appear as black spots, as shown in the attached Figure 3 As shown, the positive samples, artificially cultured gray mold hyphae samples, and infected grape samples all showed color, while the negative samples did not.

[0073] Example 3

[0074] Detection of Botrytis cinerea by using antibodies against Botrytis cinerea ATP3 protein (immunosphere method)

[0075] Prepare blue immune microspheres, take 10 ml of NHS-labeled blue microspheres with a diameter of 1 μm (Jiangsu Zhichuan Technology Co., Ltd.), add 10 μl of anti-Botrytis cinerea ATP3 protein serum (obtained in step 2 of Example 1) at a mass ratio of 0.1‰ for cross-linking, cross-link for 10 h at 25°C in the dark, and rotate at 40 rpm. After cross-linking, add 250 μl of 2M Tris (pH 7.4) to a final concentration of 50 mM for blocking, and block for 10 h at 25°C in the dark at 40 rpm.

[0076] A nitrocellulose blotting membrane (Article No.: F619511-0005, 0.22 μm, 20×20 cm, Sangon Biotech (Shanghai) Co., Ltd.) was marked with a 1×1 cm grid across the protective film, and then the protective film was removed, and 2 μl of the sample solution was aspirated and dotted in the grid. Placed in a wet box, a filter paper moistened with PBS buffer (Article No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) was pre-padded, and incubated at 37°C for 2 h. The positive sample was the gray mold ATP3 protein prepared in step 1 of Example 1, and the negative sample was PBS buffer. The crude protein extract of grapes infected with gray mold in implementation 2 was used as the test sample, and each sample was repeated twice.

[0077] Transfer the incubated nitrocellulose blot membrane to a protein-free blocking solution (Cat. No.: C510042-0500, Sangon Biotech (Shanghai) Co., Ltd.) for blocking. This step is performed in a wet box for ease of operation. Block at 37°C for 2 h, then wash with 1× diluted PBST (Cat. No.: C520004-0001, Sangon Biotech (Shanghai) Co., Ltd.), place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 min each time. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0078] Add the prepared blue immune microspheres to the incubated nitrocellulose blotting membrane and evenly cover the surface. Incubate at 25°C for 10 minutes, then wash with 1× diluted PBST, place on a decolorizing shaker and wash 3 times at 40 rpm, 2 minutes each time. After washing, the color will appear as blue dots, as shown in the attached Figure 4 As shown, the positive samples and the grape samples infected with gray mold all showed color, while the negative samples did not show color.

[0079] Example 4

[0080] Sensitivity of antibody detection against Botrytis cinerea ATP3 protein

[0081] 1. Detection sensitivity of immunoblotting

[0082] On the nitrocellulose blotting membrane with squares marked in advance, the gray mold ATP3 protein solution was spotted with a concentration gradient, starting with a 1mg / ml solution and diluting it 2 times, with concentrations of 1mg / ml, 0.5mg / ml, 0.25mg / ml, 0.125mg / ml, and 0.0625mg / ml, respectively. 2μl of these dilution solutions were respectively drawn and spotted on the nitrocellulose blotting membrane grid. That is, the protein content of each spot was 2μg, 1μg, 0.5μg, 0.25μg, and 0.125μg. PBS buffer was used as a blank sample (0μg), and each concentration was repeated 3 times. Placed in a wet box, the filter paper moistened with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) was pre-padded, and incubated at 37°C for 2h.

[0083] Transfer the incubated nitrocellulose blot membrane to a protein-free blocking solution (Cat. No.: C510042-0500, Sangon Biotech (Shanghai) Co., Ltd.) for blocking. This step is performed in a wet box for ease of operation. Block at 37°C for 2 h, then wash with 1× diluted PBST (Cat. No.: C520004-0001, Sangon Biotech (Shanghai) Co., Ltd.), place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 min each time. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0084] The incubated nitrocellulose blotted membranes were transferred to the serum (antibody) against Botrytis cinerea ATP3 protein diluted 10,000 times with PBS in advance (obtained in step 2 of Example 1). For ease of operation, this step was performed in a 50 ml centrifuge tube. Incubated at 37°C for 2 hours, then washed with 1× PBST, placed on a decolorizing shaker and washed 3 times at a speed of 40 rpm, each washing for 5 minutes. During the washing process, the membrane was prevented from sticking to the wall without buffer.

[0085] The washed nitrocellulose blot was taken out and marked with a ballpoint pen or cut corner according to the dilution, and transferred to HRP-labeled goat anti-rabbit secondary antibody diluted 5000 times (Cat. No.: SE134-0.1ml, Beijing Solebow Technology Co., Ltd.), incubated at 37°C for 2 hours, and then washed with 1× PBST, placed on a decolorizing shaker and washed 4 times at a speed of 40 rpm, each wash for 5 minutes. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0086] According to the instructions of the ultra-sensitive ECL chemiluminescent reagent (Cat. No.: C520045-0500, Sangon Biotech (Shanghai) Co., Ltd.), solution A and solution B were mixed in a volume ratio of 1:1 to prepare an immunoblotting solution. The membrane was placed in an ECL chemiluminescent imager, 50 μl of the solution was added to each square, and evenly covered on the membrane surface. The exposure parameters were adjusted to develop the color. After exposure and color development, the immunoblotting spots appeared as black spots, and the pixel values ​​of the black spots were collected. The pixel values ​​of the blank samples (0 μg) were deducted from the pixel points with ATP3 protein content of 2 μg, 1 μg, 0.5 μg, 0.25 μg, and 0.125 μg, respectively, and the curve y=16.32x+267.32 was drawn with protein content and pixel value as the ordinate and abscissa, respectively. The reference sensitivity is commonly used in the detection limit (LOD, limit of detection) detection method LOD = 3σ / S, σ is the standard deviation of the 0.125μg detection value (2.06), S is the linear slope (16.32). The calculated LOD sensitivity is 0.379μg.

[0087] 2. Detection sensitivity of immunomicrosphere method

[0088] On the nitrocellulose blotting membrane with squares marked in advance, the gray mold ATP3 protein solution was spotted with a concentration gradient, starting with a 1mg / ml solution and diluting it 2 times, with concentrations of 1mg / ml, 0.5mg / ml, 0.25mg / ml, 0.125mg / ml, and 0.0625mg / ml, respectively. 2μl of these dilution solutions were respectively drawn and spotted on the nitrocellulose blotting membrane grid. That is, the protein content of each spot was 2μg, 1μg, 0.5μg, 0.25μg, and 0.125μg. PBS buffer was used as a blank sample (0μg), and each concentration was repeated 3 times. Placed in a wet box, the filter paper moistened with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) was pre-padded, and incubated at 37°C for 2h.

[0089] Transfer the incubated nitrocellulose blot membrane to a protein-free blocking solution (Cat. No.: C510042-0500, Sangon Biotech (Shanghai) Co., Ltd.) for blocking. This step is performed in a wet box for ease of operation. Block at 37°C for 2 h, then wash with 1× diluted PBST (Cat. No.: C520004-0001, Sangon Biotech (Shanghai) Co., Ltd.), place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 5 min each time. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0090] The blue immune microspheres prepared in step 1 of Example 3 were added dropwise to the incubated nitrocellulose blotting membrane to evenly cover the surface. Incubate at 25°C for 10 min, then wash with PBST diluted to 1×, place on a decolorizing shaker and wash 3 times at a speed of 40 rpm, 2 min each time, and color after washing to form blue dots, and collect blue dot pixel values. The pixel points with ATP3 protein content of 2μg, 1μg, 0.5μg, 0.25μg, and 0.125μg were deducted from the pixel values ​​of the blank samples (0μg), and the curve y=17.31x+149.23 was drawn with protein content and pixel value as ordinate and abscissa, respectively. Reference sensitivity is commonly used limit of detection (LOD, limit of detection) detection method LOD=3σ / S, σ is the standard deviation of the 0.125μg detection value (0.73), and S is the linear slope (17.31). The calculated LOD sensitivity is 0.126μg.

[0091] Example 5

[0092] Accuracy of antibody detection against Botrytis cinerea ATP3 protein

[0093] 1. Immunoblotting accuracy

[0094] Mycelia of artificially cultured Phytophthora hibernalis (ATCC 60352, Shanghai Collection of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai), Botrytis cinerea (ATCC 58025, Shanghai Collection of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai), and Fusarium commune (GDMCC NO.: 3.697, Guangdong Microbiological Culture Collection Center) were purified and cultured at 30°C using PDA medium (Product No.: HB0233, Haibo Biotechnology Co., Ltd.), and the pure culture was inoculated into potato glucose water medium (Product No.: HB0233-4, Haibo Biotechnology Co., Ltd.), and cultured at 30°C for 7 days to obtain mycelia. Add 1 ml PBS according to 50 mg, then add 10 zirconium oxide grinding beads with a diameter of 1 mm, grind and extract crude protein at a speed of 10 m / s for 2 min, then centrifuge at 12000 rpm for 5 min to collect the supernatant to obtain the sample to be tested;

[0095] A nitrocellulose blotting membrane (Cat. No.: F619511-0005, 0.22 μm, 20×20 cm, Sangon Biotech (Shanghai) Co., Ltd.) was drawn into a 1×1 cm grid through the protective film, and then the protective film was removed, and 2 μl of the sample to be tested was randomly dotted in the grid, 1 mg / ml ATP3 protein solution was used as the positive sample and randomly dotted twice, and PBS was used as the negative sample and randomly dotted twice. Place it in a wet box, padded with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) to wet the filter paper, and incubate at 37°C for 2 hours.

[0096] The incubated nitrocellulose blotted membranes were transferred to the serum (antibody) against Botrytis cinerea ATP3 protein diluted 10,000 times with PBS in advance (obtained in step 2 of Example 1). For ease of operation, this step was performed in a 50 ml centrifuge tube. Incubated at 37°C for 2 hours, then washed with 1× PBST, placed on a decolorizing shaker and washed 3 times at a speed of 40 rpm, each washing for 5 minutes. During the washing process, the membrane was prevented from sticking to the wall without buffer.

[0097] The washed nitrocellulose blot was taken out and marked with a ballpoint pen or cut corner according to the dilution, and transferred to HRP-labeled goat anti-rabbit secondary antibody diluted 5000 times (Cat. No.: SE134-0.1ml, Beijing Solebow Technology Co., Ltd.), incubated at 37°C for 2 hours, and then washed with 1× PBST, placed on a decolorizing shaker and washed 4 times at a speed of 40 rpm, each wash for 5 minutes. During the washing process, prevent the membrane from sticking to the wall without buffer.

[0098] According to the instructions of the ultra-sensitive ECL chemiluminescent reagent (Cat. No.: C520045-0500, Sangon Biotech (Shanghai) Co., Ltd.), solution A and solution B were mixed at a volume ratio of 1:1 to prepare the immunoblotting solution. The membrane was placed in the ECL chemiluminescent imager, 50 μl of the solution was added to each square, and evenly covered on the membrane surface. The exposure parameters were adjusted to allow color development. After exposure and color development, the immunoblotting spots appeared as black spots.

[0099] Unblind test of gray mold samples in random samples, such as the attached Figure 6 As shown, the random point gray mold sample is completely consistent with the color-developed black point, with an accuracy of 100%.

[0100] 2. Detection accuracy of immunomicrosphere method

[0101] Mycelia of artificially cultured Phytophthora hibernalis (ATCC 60352, Shanghai Collection of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai), Botrytis cinerea (ATCC 58025, Shanghai Collection of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai), and Fusarium commune (GDMCC NO: 3.697, Guangdong Microbiological Culture Collection Center) were purified and cultured at 30°C using PDA medium (Product No. HB0233, Haibo Biotechnology Co., Ltd.), and the pure culture was inoculated into potato glucose water medium (Product No. HB0233-4, Haibo Biotechnology Co., Ltd.), and cultured at 30°C for 7 days to obtain mycelia. Add 1 ml PBS according to 50 mg, then add 10 zirconium oxide grinding beads with a diameter of 1 mm, grind and extract crude protein at a speed of 10 m / s for 2 min, then centrifuge at 12000 rpm for 5 min to collect the supernatant to obtain the sample to be tested;

[0102] A nitrocellulose blotting membrane (Cat. No.: F619511-0005, 0.22 μm, 20×20 cm, Sangon Biotech (Shanghai) Co., Ltd.) was drawn into a 1×1 cm grid through the protective film, and then the protective film was removed, and 2 μl of the sample to be tested was randomly dotted in the grid, 1 mg / ml ATP3 protein solution was used as the positive sample and randomly dotted twice, and PBS was used as the negative sample and randomly dotted twice. Place it in a wet box, padded with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.) to wet the filter paper, and incubate at 37°C for 2 hours.

[0103] The incubated nitrocellulose blotted membranes were transferred to the serum (antibody) against Botrytis cinerea ATP3 protein diluted 10,000 times with PBS in advance (obtained in step 2 of Example 1). For ease of operation, this step was performed in a 50 ml centrifuge tube. Incubated at 37°C for 2 hours, then washed with 1× PBST, placed on a decolorizing shaker and washed 3 times at a speed of 40 rpm, each washing for 5 minutes. During the washing process, the membrane was prevented from sticking to the wall without buffer.

[0104] The blue immune microspheres prepared in step 1 of Example 3 were added dropwise to the incubated nitrocellulose blotting membrane to evenly cover the surface. The membrane was incubated at 25°C for 10 min, then washed with 1× diluted PBST, and washed 3 times on a decolorizing shaker at a speed of 40 rpm for 2 min each time. After washing, the membrane was colored as blue dots.

[0105] Unblind test of gray mold samples in random samples, such as the attached Figure 7As shown, the gray mold sample at the random point is completely consistent with the color-developed blue point, with an accuracy of 100%.

[0106] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0107] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of antibodies for detecting Botrytis cinerea pathogens in the preparation of products for detecting Botrytis cinerea pathogens, It is characterized in that The antibody for detecting gray mold pathogen is prepared by the following method: Construct or synthesize a recombinant plasmid containing Botrytis cinerea ATP3 protein, clone and express Botrytis cinerea ATP3 protein, purify Botrytis cinerea ATP3 protein, use the purified Botrytis cinerea ATP3 protein as an antigen to immunize an animal, and the obtained immune serum is an antibody for detecting Botrytis cinerea pathogens; The amino acid sequence of the Botrytis cinerea ATP3 protein is shown in SEQ ID NO.1; the plasmid is pET24a; and the animal is a New Zealand white rabbit.

2. A kit for detecting gray mold pathogens, It is characterized in that The kit includes antibodies for detecting gray mold pathogens prepared by the following method: Construct or synthesize a recombinant plasmid containing Botrytis cinerea ATP3 protein, clone and express Botrytis cinerea ATP3 protein, purify Botrytis cinerea ATP3 protein, use the purified Botrytis cinerea ATP3 protein as an antigen to immunize an animal, and the obtained immune serum is an antibody for detecting Botrytis cinerea pathogens; The amino acid sequence of the Botrytis cinerea ATP3 protein is shown in SEQ ID NO.1; the plasmid is pET24a; and the animal is a New Zealand white rabbit.

3. The kit according to claim 2, It is characterized in that The kit is an immunoblotting kit, and further comprises a nitrocellulose blotting membrane, an HRP-labeled secondary antibody, a washing solution PBS, and an ECL chemiluminescent reagent.

4. The kit according to claim 2, It is characterized in that The kit is a color immunomicrosphere kit, further comprising a nitrocellulose blotting membrane, a washing solution PBS, and the ATP3 antibody is labeled with a color immunomicrosphere.

5. An immunological detection method for gray mold pathogens, It is characterized in that The following steps are involved: (1) A 1×1 cm square grid is drawn on the nitrocellulose blotting membrane through a protective film, and then the protective film is removed and 2 μl of the sample solution to be tested is aspirated and spotted on the square grid; Place in a wet box, pad with filter paper moistened with PBS buffer in advance, and incubate at 37°C for 2 h; (2) Transfer the incubated nitrocellulose blot to a protein-free blocking buffer for blocking at 37°C for 2 h, then wash with 1× PBST and place on a decolorizing shaker for three washes at 40 rpm for 5 min each. (3) The incubated nitrocellulose blot membrane was transferred to the antibody for detecting gray mold pathogens diluted 10,000 times with PBS in advance, incubated at 37°C for 2 h, and then washed with 1× PBST, placed on a decolorizing shaker and washed 3 times at a speed of 40 rpm, with each wash lasting 5 min; The antibody for detecting gray mold pathogen is prepared by the following method: A recombinant plasmid containing Botrytis cinerea ATP3 protein is constructed or synthesized, the Botrytis cinerea ATP3 protein is cloned and expressed, the Botrytis cinerea ATP3 protein is purified, and the purified Botrytis cinerea ATP3 protein is used as an antigen to immunize an animal, and the obtained immune serum is an antibody for detecting Botrytis cinerea pathogens; the amino acid sequence of the Botrytis cinerea ATP3 protein is shown in SEQ ID NO.1; the plasmid is pET24a; and the animal is a New Zealand white rabbit; (4) Take out the washed nitrocellulose blot membrane and mark it according to the dilution, transfer it to HRP-labeled goat anti-rabbit secondary antibody diluted 5000 times, incubate it at 37°C for 2 hours, then wash it with PBST diluted 1×, place it on a decolorizing shaker and wash it 4 times at a speed of 40 rpm, each wash for 5 minutes; (5) Use ultra-sensitive ECL chemiluminescent reagent to develop color and read the results.

6. An immunomicrosphere detection method for gray mold pathogens, It is characterized in that The following steps are involved: (1) Prepare blue immune microspheres. Take 10 ml of NHS-labeled blue microspheres with a diameter of 1 μm, add 10 μl of antibodies for detecting Botrytis cinerea at a mass ratio of 0.1‰ for cross-linking, and cross-link for 10 h at 25°C in the dark at a rotation speed of 40 rpm; After cross-linking, add 250 μl of 2M Tris to a final concentration of 50 mM for blocking, and block for 10 h at 25°C in the dark at a rotation speed of 40 rpm; The antibody for detecting gray mold pathogen is prepared by the following method: A recombinant plasmid containing Botrytis cinerea ATP3 protein is constructed or synthesized, the Botrytis cinerea ATP3 protein is cloned and expressed, the Botrytis cinerea ATP3 protein is purified, and the purified Botrytis cinerea ATP3 protein is used as an antigen to immunize an animal, and the obtained immune serum is an antibody for detecting Botrytis cinerea pathogens; the amino acid sequence of the Botrytis cinerea ATP3 protein is shown in SEQ ID NO.1; the plasmid is pET24a; and the animal is a New Zealand white rabbit; (2) Use a nitrocellulose blotting membrane to draw a 1×1 cm grid through a protective film, then remove the protective film and pipette 2 μl of the sample solution to be tested into the grid; Place in a wet box, pad with filter paper moistened with PBS buffer in advance, and incubate at 37°C for 2 h; (3) Transfer the incubated nitrocellulose blot membrane to a protein-free blocking buffer for blocking at 37°C for 2 h, then wash with 1× PBST and place on a decolorizing shaker for 3 washes at 40 rpm for 5 min each. During the washing process, prevent the membrane from sticking to the wall without buffer. (4) Add the prepared blue immune microspheres onto the incubated nitrocellulose blotting membrane to evenly cover the surface; incubate at 25°C for 10 min, then wash with 1× diluted PBST, place on a decolorizing shaker and wash three times at a speed of 40 rpm, with each wash time of 2 min. After washing, color development is performed and the results are read.

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