Antibody against ATP1 protein of Downy Phytophthora and its application

Abstract

The present invention discloses an antibody against ATP1 protein of downy mildew and its application, and relates to the field of biotechnology. The antibody for detecting downy mildew of the present invention is obtained by immunizing an animal with ATP1 protein of downy mildew as an antigen, and the amino acid sequence of the ATP1 protein of downy mildew is shown in SEQ ID NO.1. Based on the specific binding characteristics of antigen and antibody, the present invention detects ATP1 protein of downy mildew by immunoblotting and immunomicrosphere technology, overcomes the disadvantages of long time and high requirements of traditional detection of downy mildew, greatly advances the detection time of downy mildew, and achieves early, rapid and accurate detection effect of downy mildew.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an antibody against ATP1 protein of Downy Phytophthora and its application. Background Art

[0002] Phytophthora hibernalis infects crops such as grapes and causes downy mildew, which makes it impossible to photosynthesize, resulting in quality decline, reduced yields, or even total crop failure. It is the main pathogen that harms agricultural production and also causes downy mildew in dicotyledonous crops. In the grape industry, the harm caused by downy mildew is very serious, especially in greenhouse cultivation, which is very prone to downy mildew outbreaks due to factors such as humidity and poor air circulation.

[0003] At present, the main detection methods for downy mildew are: one is to detect through etiology combined with symptom characteristics and morphology; the other is nucleic acid detection based on downy mildew-specific genes or sequences. However, the plant can only be detected using etiological methods after it shows typical characteristics after being infected by downy mildew. At this time, losses are inevitable, and the detection is time-consuming and labor-intensive, with poor accuracy. Nucleic acid detection requires special instruments and equipment for detection, and has requirements on the operator's technical level. It is difficult to master it without training. The above disadvantages limit the early diagnosis and treatment of downy mildew.

[0004] Therefore, it is an urgent problem for technicians in this field to provide an early, rapid and accurate method for detecting spores and mycelium of downy mildew, which can be used for detection before the outbreak of downy mildew for early prevention and control, and to prevent losses caused by large-scale outbreaks of downy mildew. Summary of the invention

[0005] In view of this, the present invention provides an antibody against Downy Phytophthora ATP1 protein and its application.

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

[0007] An antibody for detecting downy mildew disease, obtained by immunizing an animal with the ATP1 protein of downy mildew as an antigen.

[0008] The ATP1 protein of Downy Phytophthora is involved in ATP biosynthesis and is indispensable to Downy Phytophthora. The sequence is specific and conservative and can effectively characterize Downy Phytophthora. The present invention uses the ATP1 protein as an antigen to prepare an antibody. The antibody can be used in combination with color immunomicrosphere labeling and immunoblotting techniques to specifically detect Downy Phytophthora from samples such as soil, seeds, and plant tissues, which has great advantages for the early diagnosis of Downy Phytophthora. Moreover, this 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 Downy Phytophthora ATP1 protein is shown as SEQ ID NO.1.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned antibody for detecting downy mildew, comprising constructing or synthesizing a recombinant plasmid containing downy mildew ATP1 protein, cloning and expressing the downy mildew ATP1 protein, purifying the downy mildew ATP1 protein, and using the purified downy mildew ATP1 protein as an antigen to immunize an animal, and the obtained immune serum is the antibody for detecting downy mildew.

[0011] As a preferred technical solution, the plasmid used in constructing or synthesizing the recombinant plasmid containing the Downy Phytophthora ATP1 protein is pET24a.

[0012] Another object of the present invention is to provide a kit for detecting downy mildew, wherein the kit comprises the above-mentioned antibody for detecting downy mildew, 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 blue immunomicrosphere kit, which also includes a nitrocellulose blotting membrane, a washing solution PBS, and the ATP1 antibody is labeled with blue immunomicrospheres.

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

[0016] Another object of the present invention is to provide an immunological detection method for downy mildew, 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 blotted membranes were transferred to the antibody for detecting downy mildew of claim 1 or 2 which was diluted 10,000 times with PBS in advance, incubated at 37° C. for 2 h, then washed with PBST diluted 1×, and placed on a decolorizing shaker for 3 washes at a speed of 40 rpm, with each wash lasting 5 min;

[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 downy mildew, comprising the following steps:

[0023] (1) Prepare colored immunomicrospheres, take 10 ml of NHS-labeled colored microspheres with a diameter of 1 μm, add 10 μl of the antibody for detecting downy mildew described in claim 1 or 2 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, and rotate 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 ATP1 protein of downy mildew by immunoblotting and immunomicrosphere technology. The present invention overcomes the disadvantages of the traditional downy mildew detection, which takes a long time and has high requirements, greatly advances the detection time of downy mildew, and achieves early, rapid and accurate detection of downy mildew. 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 the Downy Phytophthora ATP1 protein according to an embodiment of the present invention, wherein MK: marker, A: purified ATP1 protein (arrow).

[0030] Figure 2 This is the titer detection of the anti-downy mildew ATP1 protein immune serum in the embodiment of the present invention.

[0031] Figure 3 This is the specific detection of the anti-downy mildew ATP1 protein immune serum in the embodiment of the present invention, wherein +, -, Blk are positive, negative, and blank samples, respectively, Fc and Bc are common Fusarium and gray mold, respectively, and Ph1 and Ph2 are grape leaves infected with downy mildew and downy mildew hyphae samples, respectively.

[0032] Figure 4 In the present embodiment, an anti-downy mildew ATP1 protein antibody is applied to immunoblotting to detect downy mildew samples.

[0033] Figure 5 To apply the anti-Phytophthora ATP1 protein antibody to blue immunomicrospheres to detect Phytophthora samples.

[0034] Figure 6 In this embodiment of the present invention, the anti-downy mildew ATP1 protein antibody is applied to the accuracy of immunoblotting detection of downy mildew 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-Downymphytum ATP1 protein antibody to the blue immunomicrospheres to detect the accuracy of Downymphytum 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 Downymphytum, 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 ATP1 protein of Downy Phytophthora, wherein the antibody is obtained by immunizing New Zealand white rabbits with ATP1 protein as a vaccine. The amino acid sequence of the ATP1 protein is shown in SEQ ID NO.1:

[0039] , SEQ ID NO.1.

[0040] Example 1

[0041] Preparation and purification of ATP1 protein from Downy Phytophthora and preparation of its antibody

[0042] 1. Preparation and purification of Downy Phytophthora ATP1 protein

[0043] Transformation expression and protein purification of the fully synthetic pET24a-ATP1 recombinant plasmid (Suzhou Hongxun Biotechnology Co., Ltd., the constructed recombinant plasmid insert is inserted into the multiple cloning site, and its restriction endonuclease sites are BglII and XhoI. The fully synthetic insert can be prepared as sticky ends through BglII and XhoI and connected to the pET24a vector that has been digested with enzymes to prepare a recombinant plasmid)

[0044] Add 1 μl of pET24a-ATP1 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 an LB plate containing 50 μg / ml kanamycin, and invert and culture at 37°C overnight; the next morning, pick a single clone 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 Downy Phytophthora ATP1 protein, protein urea solution purification select affinity chromatography, 20mM Tris, 500mMNaCl, 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 1As shown in the electrophoresis detection, the molecular weight of the ATP1 protein band is 58.04KDa. The buffer was replaced with a dialysis bag, and 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, TEV Protease (GST / His-tag), Shanghai Biotech Biotechnology Co., Ltd.). Then, the His tag was removed by His tag protein purification agarose magnetic beads (P2239-10ml, BeyoMag TM The 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 ATP1 protein solution.

[0046] 2. Preparation of Antibodies Against Downy Phytophthora ATP1 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 ATP1 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 into the neck of New Zealand white rabbits at multiple points for immunization.

[0049] For the second immunization, 600 μl of 1 mg / ml ATP1 protein solution (obtained in step 1) was added with 600 μl of incomplete Freund's adjuvant (catalog number: 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 ATP1 protein of Downy Phytophthora.

[0051] 3. Determination of Antibody Titer Against Downy Phytophthora ATP1 Protein

[0052] The antibody titer against ATP1 protein of Downy Phytophthora 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 drawn into a 1×1 cm grid through the protective film, and then the protective film was removed, and 2 μl of 1 mg / ml ATP1 protein solution (obtained in step 1) was aspirated and dotted into the grid. Placed in a wet box, padded with filter paper moistened with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.), 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 blotted membrane to the serum (antibody) against ATP1 protein of Phytophthora rapae (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℃ for 2h, then wash with PBST diluted to 1×, place on a decolorizing shaker and wash 3 times at a speed of 40rpm, 5min 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 best immune serum titer against ATP1 protein of Downy Phytophthora obtained by the present invention is 1:100,000.

[0058] 4. Specificity detection of antibodies against ATP1 protein of Downy Phytophthora

[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. Grape leaf samples infected with winter downy mildew and artificially cultured mycelium were collected and ground to extract crude protein for detection. 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 the supernatant was collected by centrifugation at 12000 rpm for 5 minutes 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 ATP1 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 leaf samples infected with winter downy mildew 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 blotted membrane to the serum (antibody) against ATP1 protein of Phytophthora rapae (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℃ for 2h, then wash with PBST diluted to 1×, place on a decolorizing shaker and wash 3 times at a speed of 40rpm, 5min 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 grape leaves infected with Downy Phytophthora, Downy Phytophthora hyphae and the positive control were positive, indicating that the antibodies against Downy Phytophthora ATP1 protein obtained in the present invention have specificity.

[0065] Example 2

[0066] Detection of Peronophytum by Antibodies Against Peronophytum ATP1 Protein (Western Blotting)

[0067] Grape leaf samples infected with Phytophthora hibernalis (ATCC 60352, Shanghai Collection of Microorganisms, 8th Floor, No. 938, Shunda Road, Nanxiang Town, Jiading District, Shanghai) were collected, and the downy mildew fungus was purified and cultured at 30°C using PDA medium (Article No.: HB0233, Haibo Biotechnology Co., Ltd.), and the pure downy mildew 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 grape leaves that had been scratched with 80-mesh woodworking sandpaper in advance at a mass ratio of 1:50, maintained at 70% humidity, and cultured at 30°C for 2 days (no mycelium growth visible to the naked eye) before sample preparation. Artificially cultured Downy Phytophthora hyphae and grape leaf samples infected with Downy Phytophthora were ground to extract crude protein for testing. 50 mg was added to 1 ml PBS, and then 10 1 mm diameter zirconium oxide grinding beads were added. The crude protein was extracted by grinding at a speed of 10 m / s for 2 min, and then centrifuged at 12000 rpm for 5 min to collect the supernatant as the sample.

[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 was aspirated and dotted in the grid. It was placed in a wet box, padded with filter paper moistened with PBS buffer (Article No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.), and incubated at 37°C for 2 h. The positive sample was the ATP1 protein of Downy mildew (obtained in step 1 of Example 1), the negative sample was PBS buffer, artificially cultured Downy mildew hyphae and crude protein extracts of grapes infected with Downy mildew 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 blotted membranes were transferred to the serum (antibody) against ATP1 protein of Phytophthora parasitica diluted 10,000 times with PBS in advance (obtained in step 2 of Example 1). For the convenience of operation, this step was performed in a 50 ml centrifuge tube. Incubated at 37°C for 2 hours, then washed with PBST diluted 1×, 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.

[0071] 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.

[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 4 As shown, the positive samples, artificially cultured Downy Phytophthora mycelium samples, and infected grape samples all showed color, while the negative samples did not.

[0073] Example 3

[0074] Detection of Downy Phytophthora by Antibodies Against ATP1 Protein of Downy Phytophthora (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-downy mildew ATP1 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 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 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 Downy mildew ATP1 protein prepared in step 1 of Example 1, and the negative sample was PBS buffer. The crude protein extract of grapes infected with Downy mildew 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 5 As shown, the positive samples, grape samples infected with Downy Phytophthora, all showed color, while the negative samples did not show color.

[0079] Example 4

[0080] Sensitivity of antibody detection against ATP1 protein of Downy Phytophthora

[0081] 1. Detection sensitivity of immunoblotting

[0082] On the nitrocellulose blotting membrane with pre-marked squares, the ATP1 protein solution of Downy Pythium was spotted in a concentration gradient. The 1 mg / ml solution was diluted twice, and the concentrations were 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml, respectively. 2 μl of these dilution solutions were respectively applied to the local grid of the nitrocellulose blotting membrane. That is, the protein content of each spot was 2 μg, 1 μg, 0.5 μg, 0.25 μg, and 0.125 μg. PBS buffer was used as the blank sample (0 μg), and each concentration was repeated 3 times. Placed in a wet box, the filter paper was pre-padded with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.), and incubated at 37°C for 2 hours.

[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 ATP1 protein of Phytophthora parasitica diluted 10,000 times with PBS in advance (obtained in step 2 of Example 1). For the convenience of operation, this step was performed in a 50 ml centrifuge tube. Incubated at 37°C for 2 hours, then washed with PBST diluted 1×, 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 ATP1 protein content of 2 μg, 1 μg, 0.5 μg, 0.25 μg, and 0.125 μg, respectively, and the curve y=18.75x+235.12 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 (1.83), S is the linear slope (18.75). The calculated LOD sensitivity is 0.292μg.

[0087] 2. Detection sensitivity of immunomicrosphere method

[0088] On the nitrocellulose blotting membrane with pre-marked squares, the ATP1 protein solution of Downy Pythium was spotted in a concentration gradient. The 1 mg / ml solution was diluted twice, and the concentrations were 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml, respectively. 2 μl of these dilution solutions were respectively applied to the local grid of the nitrocellulose blotting membrane. That is, the protein content of each spot was 2 μg, 1 μg, 0.5 μg, 0.25 μg, and 0.125 μg. PBS buffer was used as the blank sample (0 μg), and each concentration was repeated 3 times. Placed in a wet box, the filter paper was pre-padded with PBS buffer (Cat. No.: E607008-0001, Sangon Biotech (Shanghai) Co., Ltd.), and incubated at 37°C for 2 hours.

[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 the pixel values ​​of the blue dots. The pixel points with ATP1 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=19.15x+245.86 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 (1.87), and S is the linear slope (19.15). The calculated LOD sensitivity is 0.293μg.

[0091] Example 5

[0092] Accuracy of antibody detection against ATP1 protein of Downy Phytophthora

[0093] 1. Immunoblotting accuracy

[0094] 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. 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 ATP1 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 ATP1 protein of Phytophthora parasitica diluted 10,000 times with PBS in advance (obtained in step 2 of Example 1). For the convenience of operation, this step was performed in a 50 ml centrifuge tube. Incubated at 37°C for 2 hours, then washed with PBST diluted 1×, 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 random samples of downy mildew samples, such as the attached Figure 6 As shown, the random dotted Downy Phytophthora samples were completely consistent with the colored black dots, with an accuracy of 100%.

[0100] 2. Detection accuracy of immunomicrosphere method

[0101] 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. 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 ATP1 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 ATP1 protein of Phytophthora parasitica diluted 10,000 times with PBS in advance (obtained in step 2 of Example 1). For the convenience of operation, this step was performed in a 50 ml centrifuge tube. Incubated at 37°C for 2 hours, then washed with PBST diluted 1×, 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 random samples of downy mildew samples, such as the attached Figure 7As shown, the random point of the downy mildew sample 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. An antibody for detecting downy mildew, characterized in that: The ATP1 protein of Downy Phytophthora is used as an antigen to immunize an animal; the amino acid sequence of the ATP1 protein of Downy Phytophthora is shown in SEQ ID NO.1; The specific preparation method is as follows: A recombinant plasmid containing the Downy Phytophthora ATP1 protein is constructed or synthesized, the Downy Phytophthora ATP1 protein is cloned and expressed, the Downy Phytophthora ATP1 protein is purified, and the purified Downy Phytophthora ATP1 protein is used as an antigen to immunize an animal, and the obtained immune serum is an antibody for detecting Downy Phytophthora disease; wherein The plasmid is pET24a, and the animal is a New Zealand white rabbit.

2. A kit for detecting downy mildew, characterized in that: The kit comprises the antibody for detecting downy mildew according to claim 1.

3. The kit according to claim 2, 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, characterized in that The kit is a color immunomicrosphere kit, further comprising a nitrocellulose blotting membrane, a washing solution PBS, and the ATP1 antibody is labeled with a color immunomicrosphere.

5. Use of the antibody for detecting downy mildew according to claim 1 or the kit according to any one of claims 2 to 4 in the preparation of a product for detecting downy mildew.

6. An immunological detection method for downy mildew, 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 membranes were transferred to the antibody for detecting downy mildew of claim 1 which was diluted 10,000 times with PBS in advance, incubated at 37° C. for 2 h, then washed with PBST diluted 1×, and placed on a decolorizing shaker for 3 washes at a speed of 40 rpm, with each wash lasting 5 min; (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.

7. An immunomicrosphere detection method for downy mildew, 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 the antibody for detecting downy mildew described in claim 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 to a final concentration of 50 mM for blocking, and block for 10 h at 25°C in the dark at 40 rpm; (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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