CN117106078A - Rice phytoplasma aurantia antigen membrane protein polyclonal antibody and application thereof - Google Patents
Rice phytoplasma aurantia antigen membrane protein polyclonal antibody and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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- G—PHYSICS
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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Abstract
The invention discloses a polyclonal antibody of a rice orange leaf phytoplasma antigen membrane protein and application thereof. According to the invention, the hydrophilic structural domain of the rice phytoplasma aurantiaca antigen membrane protein is recombined and expressed, and is used as an immunogen to prepare the ROLP-Amp-H polyclonal antibody for specifically detecting the rice phytoplasma aurantiaca, the polyclonal antibody has good detection specificity and high detection sensitivity, and the polyclonal antibody can be used for preparing a detection product of the rice phytoplasma aurantiaca. In addition, the invention also provides a Western blot detection method and a Dot-Elisa detection method for detecting the phytoplasma aurantiaca by using the ROLP-Amp-H polyclonal antibody, which not only provide a new method for detecting the phytoplasma aurantiaca, but also facilitate the development of products for detecting the phytoplasma aurantiaca more simply and effectively and the prevention and treatment of the rice orange leaf diseases.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and microorganism detection. More particularly relates to a polyclonal antibody of a rice orange leaf phytoplasma antigen membrane protein and application thereof.
Background
Rice orange leaf disease (Rice orange leaf disease, ROLD) is a disease of rice caused by phytoplasma aurantia (Rice orange leaf phytoplasma, ROLP) and is propagated in a persistent proliferative manner by electro-optic leafhoppers (Inazuma dorsalasis) and black tail leafhoppers (Nephotettix cinticeps). The rice plant is infected with orange She Binghou, the diseased plant turns yellow from the basal leaves, gradually develops upwards to yellow heart leaves, and finally the whole plant turns orange yellow and is died soon. In recent years, the damage of the disease in the south China rice district is increased year by year, and people pay attention.
Phytoplasma has no cell wall and is obligately parasitic in host vascular bundle phloem cells. At present, no control agent for phytoplasma exists, and the occurrence and transmission of phytoplasma diseases are controlled by blocking mediator insect bacteria in production. Therefore, the early diagnosis of the infected plants and the detection of the bacteria carrying rate of the mediator insects have very important significance in the prevention and control of phytoplasma diseases. Currently, the detection of the rice orange leaf phytoplasma mainly comprises two modes of field symptom observation and PCR detection. Wherein, the field symptom observation is often confused with the lack of element symptoms, so that the diagnosis is inaccurate; PCR detection is relatively accurate and specific, but it is time consuming and requires specific instrumentation and is not suitable for detection of large-scale samples in the field.
The serological detection is a detection method by utilizing antigen-antibody reaction, has the advantages of simple operation, strong specificity, suitability for large-scale detection and the like, provides a new way for detecting the rice orange leaf phytoplasma, and is beneficial to diagnosis and prevention of the rice orange leaf disease. However, no antibodies for specifically detecting the phytoplasma aurantiaca are currently available, and no report is made on how to prepare the antibodies for the phytoplasma aurantiaca.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the existing lack of antibodies for specifically detecting the phytoplasma aurantia, and provides a polyclonal antibody of antigen membrane proteins of the phytoplasma aurantia and application thereof.
The first object of the invention is to provide a polyclonal antibody of the antigen membrane protein of the phytoplasma aurantia.
The second object of the invention is to provide the application of the polyclonal antibody in detecting the phytoplasma aurantiaca or preparing a product for detecting the phytoplasma aurantiaca.
The third object of the invention is to provide a kit for detecting phytoplasma aurantia.
The fourth object of the invention is to provide a Western blot method for detecting phytoplasma aurantiaca.
The fifth object of the invention is to provide a Dot-Elisa detection method for detecting the phytoplasma aurantiaca.
A sixth object of the invention is to provide the polyclonal antibody, the application of the kit in diagnosing rice orange leaf disease or preparing a product for diagnosing rice orange leaf disease.
The above object of the present invention is achieved by the following technical scheme:
the invention obtains 14 candidate secretion proteins by software prediction based on a genome sketch of rice phytoplasma aurantia (ROLP) drawn in the earlier stage, wherein the protein comprises SRP5 protein encoded by ROLP_0194 genes, and the protein is rice phytoplasma aurantia antigen membrane protein (Amp). Based on the candidate secretory protein, the invention obtains the polyclonal antibody for specifically detecting the rice orange leaf phytoplasma through animal immunization by recombining and expressing partial amino acid sequences of the SRP5 protein, establishes a Western blot detection method and a Dot-Elisa detection method of the rice orange leaf phytoplasma, provides a new path for detecting the rice orange leaf phytoplasma, and is beneficial to diagnosis and prevention of rice orange leaf diseases.
The invention provides a polyclonal antibody of a rice orange leaf phytoplasma antigen membrane protein, which is obtained by immunizing animals with the rice orange leaf phytoplasma antigen membrane protein as an antigen.
Specifically, the polyclonal antibody is obtained by immunizing animals with a hydrophilic structural domain of a phytoplasma aurantia antigen membrane protein as an antigen.
Specifically, the amino acid sequence of the hydrophilic structural domain of the rice phytoplasma aurantia antigen membrane protein is shown as SEQ ID NO. 1.
Specifically, the gene sequence of the hydrophilic structural domain of the rice phytoplasma aurantialba antigen membrane protein is shown in SEQ ID NO. 2.
Optionally, the animal is a rabbit.
The polyclonal antibody of the antigen membrane protein of the rice phytoplasma aurantiaca can be used for realizing the specific detection of the rice phytoplasma aurantiaca. Therefore, the invention claims the application of the polyclonal antibody in detecting the phytoplasma aurantiaca or preparing a product for detecting the phytoplasma aurantiaca.
Optionally, the product is a kit or a test strip.
The invention also provides a kit for detecting the phytoplasma aurantia of rice, which contains the polyclonal antibody.
The invention also provides a Western blot method for detecting the phytoplasma aurantia of rice, which comprises the following steps: protein of a sample to be detected is extracted for Western blot detection, and the polyclonal antibody is used during primary antibody incubation.
Specifically, goat anti-rabbit IgG-HRP antibody was used for the secondary antibody incubation.
Specifically, the dilution ratio of the antibody is 1:5000-30000 during primary antibody incubation.
Preferably, the dilution ratio of the antibodies is in the range of 1:5000 to 20000, see example 2.
Further preferably, the dilution ratio of the antibodies is 1:10000, see example 2.
The invention also provides a Dot-Elisa detection method for detecting the phytoplasma aurantiaca, which comprises the following steps: the proteins of the sample to be tested are extracted for Dot-Elisa detection, and the polyclonal antibody is used during primary antibody incubation.
Specifically, an AP-labeled goat anti-rabbit IgG antibody was used for the secondary antibody incubation.
Specifically, the dilution ratio of the antibody is 1:2000-10000 during the primary antibody incubation.
Preferably, the dilution ratio of the antibody at the time of primary antibody incubation is 1:2000, and the bands at this dilution ratio are clear, see example 3.
The polyclonal antibody or the kit can realize the specific detection of the rice orange leaf phytoplasma and can diagnose the rice orange leaf disease. Therefore, the invention also claims the polyclonal antibody, the application of the kit in diagnosing rice orange leaf disease or preparing a product for diagnosing rice orange leaf disease.
The invention has the following beneficial effects:
an effective preventive measure for controlling rice orange leaf disease is to detect the rate of ROLP in overwintering plants and leafhopper populations, thereby selecting a more appropriate planting time. In addition, the accurate detection of the bacteria carrying rate of ROLP in the field leafhopper population is also beneficial to timely spraying insecticide and inhibiting the propagation of rice orange leaf phytoplasma, so that the prevention and control of rice orange leaf diseases are realized.
According to the invention, the hydrophilic structural domain of the rice phytoplasma aurantiaca antigen membrane protein is expressed in a recombination mode, and the hydrophilic structural domain is used as an antigen to immunize rabbits, so that the ROLP-Amp-H polyclonal antibody capable of being used for specifically detecting the rice phytoplasma aurantiaca is prepared, the detection specificity of the polyclonal antibody is good, the sensitivity is high, and the polyclonal antibody can be used for preparing detection products of the rice phytoplasma aurantiaca. In addition, the invention also provides a Western blot detection method and a Dot-Elisa detection method for detecting the phytoplasma aurantiaca by using the ROLP-Amp-H polyclonal antibody, which not only provide a new method for detecting the phytoplasma aurantiaca, but also facilitate the development of products for detecting the phytoplasma aurantiaca more simply and effectively and the prevention and treatment of the diseases of the rice aurantiaca.
Drawings
FIG. 1 shows the results of a soluble assay for His-Amp-H prokaryotic expression proteins; m in the figure is Marker; u is an uninduced protein sample; i is the induced protein sample; l is the supernatant of centrifugation; s is centrifugal precipitation.
FIG. 2 shows the Coomassie brilliant blue staining results of His-Amp-H purified proteins; m is Marker; u is an uninduced protein sample; i is the induced protein sample; purified is a Purified protein sample, diluted 2 respectively 0 ，2 1 ，2 2 ，2 3 ，2 4 ，2 5 Multiple times.
FIG. 3 shows the Western blot detection results of detecting His-Amp-H prokaryotic expression protein by using ROLP-Amp-H polyclonal antibody; 1 in the figure is an uninduced protein sample; 2 is the induced protein sample; 3 is a purified protein sample.
FIG. 4 shows Western blot detection results of detecting infected rice samples by using ROLP-Amp-H polyclonal antibodies in different dilution ratios; a in the graph is the detection result with the dilution ratio of 1:5000; b in the graph is the detection result with the dilution ratio of 1:10000; c in the graph is the detection result with the dilution ratio of 1:20000; d in the graph is the detection result with the dilution ratio of 1:30000; the exposure time was 30s.
FIG. 5 shows Western blot detection results of ROLP-Amp-H polyclonal antibody detection of infected rice samples diluted at different fold ratios.
FIG. 6 shows the Western blot detection results of ROLP-Amp-H polyclonal antibodies for detecting health and an electro-optic leafhopper sample carrying ROLP.
FIG. 7 shows Western blot detection results of ROLP-Amp-H polyclonal antibody detection of electro-optic leafhopper protein samples carrying ROLP diluted at different multiples.
FIG. 8 shows the Dot-Elisa detection results of ROLP-Amp-H polyclonal antibodies for detecting rice leaf disease patterns infected with different viruses.
FIG. 9 shows the Dot-Elisa test results of ROLP-Amp-H polyclonal antibody test dilution of rice leaf samples carrying ROLP at different fold ratios.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 recombinant Rice Phytoplasma aurantiaca antigen Membrane protein (His-Amp-H) acquisition and preparation of polyclonal antibody
The invention obtains recombinant rice phytoplasma aurantiaca antigen membrane protein (His-Amp-H, carrying His tag) by prokaryotic recombinant expression of 171 amino acids of hydrophilic structural domain (Amp-H) of the rice phytoplasma aurantiaca antigen membrane protein; the amino acid sequence of the Amp-H is shown as follows (SEQ ID No. 1):
DDKLDLSTLECKGALELTAADAADAEKVVKQWKVQNTSLNAKVTKDSVKVVVADNKVTVTPADGDAGKALSGSKVLNLVGVCELDKLTLGTDKKLTLTVKNGKVDAEAGLKALKEAGAKVPATVNKDDVTFTVGKDDDA
the gene sequence encoding Amp-H is shown below (SEQ ID No. 2):
gatgataaactagatttaagcactttagaatgtaaaggcgctcttgaacttactgctgctgatgctgctgatgcagaaaaagttgttaaacaatggaaagttcaaaacacttcattgaatgcaaaagtaacaaaagattctgtaaaagtagtggttgctgataataaagtaacagttacacctgcagatggtgatgctggaaaagctttatcaggttcaaaagtattaaatttagtaggcgtatgtgaattagataaattaactttaggcacagacaaaaaacttacacttacagttaaaaatggcaaagtagatgcagaagctggtttaaaagctttaaaagaagctggagctaaagttcctgcaactgtaaacaaagacgacgtaactttcacagttggtaaagacgacgatgct
the specific process is as follows:
1. construction of His-Amp-H recombinant expression vector
The vector used for recombinant expression is pET28a (+); extracting the total RNA of ROLP and reversely transcribing the total RNA into cDNA, and amplifying the cDNA as a template to obtain an Amp-H fragment, wherein the sequence of the primer is as follows:
pEF28a(+)-Amp-F：cagcaaatgggtcgcggatccGATGATAAACTAGATTTA；
pEF28a(+)-Amp-R：gcaagcttgtcgacggagctcgaTGTTTTGTACCAAGGTGTTT。
fragment amplification was performed using the Phanta Max Super-Fidelity DNAPolymerase kit, and the PCR system was as follows:
PCR reaction procedure: 95 ℃ for 5min;95℃30s,55℃30s,72℃1min,35 cycles; terminating the reaction at 72℃for 5min and at 12 ℃; the obtained PCR product is detected by gel electrophoresis to obtain a single band with the same size, the correct band is cut off, and the gel recovery is carried out by using a gel recovery kit (Axygen) to obtain a recovery product. The obtained recovery product and a blank expression vector pET28a (+) are respectively subjected to double enzyme digestion, and the enzyme digestion system is as follows:
the enzyme digestion reaction procedure is as follows: 37 ℃ for 3 hours; 65 ℃ for 5min; the product was stored at-20℃until use.
Carrying out homologous recombination on the digested glue recovery product and the linearization vector by adopting a construction strategy of homologous recombination to obtain a His-Amp-H recombinant expression vector; the homologous recombination reaction system is as follows:
the homologous recombination reaction procedure was: 37 ℃ for 30min; stored at 4℃or used directly for E.coli transformation.
2. Screening of His-Amp-H recombinant expression bacteria
The product obtained by homologous recombination is transformed into escherichia coli competent cells DH5 alpha, and the specific steps are as follows:
(1) Standing DH5 alpha competent cells on ice for 5min until the DH5 alpha competent cells melt;
(2) Adding 10 mu L of the product into competent cells, flicking, mixing uniformly, and standing on ice for 25min;
(3) Heat shock in a 42 ℃ water bath for 45s, and standing on ice for 2min;
(4) Adding 700 mu L of liquid LB culture medium, and carrying out shaking culture at 37 ℃ and 200rpm for 1h;
(5) Directly sucking 100 mu L of bacterial liquid, coating the bacterial liquid on a solid LB culture medium plate containing antibiotics, and culturing for 12-24 h at 37 ℃.
Then positive transformant screening is carried out, and the specific steps are as follows:
single colony on the flat plate is selected for colony PCR, so that the primer, the system and the reaction program are amplified with the Amp-H fragment, and after gel electrophoresis, the single colony with the same size and single strip is identified as a positive transformant; inoculating the strain into 5mL of liquid LB culture medium containing antibiotics, and carrying out shaking culture at 37 ℃ and 200rpm for 12-16 h; preserving glycerol bacteria in the shaken bacterial liquid, extracting plasmids and sequencing; extracting recombinant plasmid from positive transformant with correct sequence, transforming Rossetta strain with correct sequence, selecting positive colony for shaking and preserving glycerol bacteria; thus obtaining His-Amp-H recombinant protein expression bacteria.
3. Prokaryotic induction expression of His-Amp-H recombinant protein
(1) Taking out the transformed Rossetta glycerol bacteria from the refrigerator at the temperature of minus 80 ℃, inoculating the transformed Rossetta glycerol bacteria into 6mL of LB liquid medium, and carrying out shaking culture for 12-15 h at the temperature of 37 ℃ and at the speed of 200 r/min;
(2) The bacterial liquid is prepared by the following steps of: 200 to 1L LB liquid medium containing kanamycin resistance, and culturing at 37deg.C under shaking at 200r/min for about 4 hr;
(3) Waiting for OD 600 When the bacterial concentration is 0.6-0.8, 1mL of bacterial liquid is absorbed, the bacterial concentration is centrifuged for 1min at 12000r/min, the supernatant is discarded, 100 mu L of Lysis buffer is added, 25 mu L of 5×loading buffer is added after the uniform re-suspension, and the solution is boiled and then placed on ice to serve as an uninduced total protein control;
(4) Adding IPTG (final concentration is 0.5 mmol/L) into the bacterial liquid, shake culturing at 37 ℃ for 6h at 200r/min, and inducing protein expression;
(5) Sucking 1mL of the induced bacterial liquid 12000r/min, centrifuging for 1min, discarding the supernatant, adding 100 mu LLysis buffer, adding 25 mu L of 5×loading buffer after uniform re-suspension, boiling, and placing on ice as a total protein control after induction;
(6) Collecting the residual bacterial liquid, centrifuging for 10min at 12000r/min, discarding the supernatant, and crushing cells or storing at-80deg.C for the next day for subsequent operation;
(7) The cells were resuspended with 100mL of Lysis buffer;
(8) Cell ultrasonic disruption: regulating the cell disruption instrument to 300W, running for 5s, suspending for 10s, breaking by using an ice bath until bacterial liquid becomes clear from turbid, centrifuging for 30min at 4 ℃ and 12000r/min, and transferring the supernatant into a new centrifuge tube which is pre-cooled in advance;
(9) Sucking 100 mu L of supernatant into a 1.5mL centrifuge tube, adding 25 mu L of 5×loading buffer, boiling, and placing on ice to serve as a total protein control of the supernatant; to the pellet was added 3mL of Lysis buffer to resuspend the pellet, 100. Mu.L of pellet suspension was pipetted into a 1.5mL centrifuge tube, 25. Mu.L of 5×loading buffer was added, boiled and placed on ice as a total protein control for the pellet.
The invention converts the successfully constructed prokaryotic expression vector pET28a (+) -Amp-H into a prokaryotic expression Rossetta (DE 3) strain, and explores the solubility of pET28a (+) -Amp-H recombinant protein. After the transformed strain is induced to express for 6 hours at 30 ℃ and 200r/min by IPTG, the expressed strain is collected, and the strain after induced expression is subjected to resuspension and ultrasonic disruption, supernatant and precipitate of ultrasonic disruption of the strain are obtained after centrifugation, and the obtained protein sample is analyzed by SDS-PAGE (solubility analysis of His-Amp-H prokaryotic expression protein), and the result is shown in figure 1. As can be seen from FIG. 1, the present invention successfully achieved prokaryotic recombinant induced expression of Amp-H (molecular weight 25 kDa) (FIG. 1, lane 2). In addition, his-Amp-H recombinant protein was detected in both supernatant and pellet, and the content of His-Amp-H recombinant protein in supernatant was higher (FIG. 1, lane 3, box), indicating that His-Amp-H recombinant protein was soluble protein.
4. Purification of His-Amp-H recombinant proteins
The protein is purified by adopting a Ni-NTA resin affinity chromatography method (the whole process is carried out on ice or in the environment of 4 ℃), and the operation steps are as follows:
(1) Inducing expression protein and ultrasonically crushing thalli, and collecting protein supernatant, wherein the specific operation steps are as above;
(2) Sucking 800. Mu.L of HisPur TM Ni-NTAResin into the column by using a tip removing gun head, balancing twice by using a Lysis buffer with 2 times of volume (1.6 mL), and centrifuging at 4 ℃ for 1min at 500rcf to remove ethanol in the filler; adding the extract into the protein supernatant, and incubating for 2 hours at 4 ℃ and 10 r/min;
(3) Slowly pouring the incubated protein supernatant into a column for column passing;
(4) After the protein supernatant was completely passed through the column, the column was washed with 100mL Washing buffer for 3 total washes;
(5) Eluting His tag protein with an emulsion buffer for 4 times, wherein 50 mu L of supernatant is absorbed into a 1.5mL centrifuge tube, 10 mu L of 5×loading buffer is added, and the mixture is boiled and then placed on ice to serve as a purified protein control;
(6) Adding the obtained purified protein into a 10kDa ultrafiltration centrifuge tube, concentrating 2-3 mL PBS solution for 2 times, 4 ℃ and at 8000rpm for 10min; and obtaining the purified protein after concentration.
To determine the efficiency and quality of protein purification, the purification effect of His-Amp-H recombinant protein was examined by protein electrophoresis and Coomassie brilliant blue staining, and the results are shown in FIG. 2. As can be seen from FIG. 2, the purified protein showed a single band, without non-specific impurity bands, indicating high purification quality and high purification efficiency even when the protein sample was diluted 2 5 After doubling, a clear single band of interest was still detected (FIG. 2), indicating that the purified recombinant protein was useful for polyclonal antibody production.
5. Preparation of polyclonal antibodies
4mg of the purified His-Amp-H recombinant protein was sent to Shanghai Aibi pharmaceutical technologies Co., ltd, and was entrusted to the preparation of polyclonal antibodies. Specifically, the purified His-Amp-H recombinant protein is injected into the rabbit body in batches; selecting 4-6 points by injection at two sides of the spine of the rabbit by adopting a back multipoint injection method, injecting 0.1mL each point, selecting different points for injection at the above parts after 2 weeks interval (adjacent points are not needed to be selected, otherwise ulcer healing is bad); the antigen amount of each immunization is about 100 mug, and the injection is carried out for 4 to 5 times; after one week of immunization, serum of the rabbit is obtained by adopting a heart blood sampling method, and then the serum is purified, so that 10mg of polyclonal antibody (ROLP-Amp-H) is finally obtained; the antibody titer is detected by an enzyme-linked immunosorbent assay, and finally the detection sensitivity (titer) is 1600K.
Example 2 Western blot detection method of ROLP
The invention establishes a Western blot detection method of ROLP by utilizing a ROLP-Amp-H polyclonal antibody, which comprises two steps of protein sample extraction and Western blot analysis, and specifically comprises the following steps:
1. extraction of rice or insect protein samples
(1) Taking about 0.2g of rice leaves to be tested or one end of insect to be tested into a 2mL centrifuge tube containing glass beads, putting into liquid nitrogen for quick freezing, and grinding into powder;
(2) Adding 200 mu L of protein extract, and shaking and mixing uniformly;
(3) Standing on ice for 30min, and shaking and mixing once every 5min;
(4) Adding 50 mu L of 5×loading buffer, uniformly mixing, boiling in boiling water for 10min, and standing on ice for 5-10 min;
(5) Centrifuging at 4deg.C for 20min at 12000r/min, and transferring the supernatant to a new 1.5mL centrifuge tube; the obtained protein sample can be used for Western blot analysis or stored at-20 ℃ for standby.
2. Western blot analysis
(1) Preparation of the protein adhesive:
first, 12% of separation gel is prepared, and the formula is as follows:
mixing the above mixed solution, pouring into a clamping groove in an electrophoresis tank, and covering the rubber surface with isopropanol (about 1 mL); standing at normal temperature, removing isopropanol when gel is completely coagulated, and pouring 4% concentrated gel. The formula of the 4% concentrated glue is as follows:
and (3) pouring the mixed solution of the concentrated gel into a clamping groove, inserting a sample comb into the clamping groove, standing at normal temperature, and completely polymerizing the concentrated gel to obtain the gel for protein electrophoresis.
(2) Transferring: after electrophoresis, taking down gel, cutting off concentrated gel, leaching with water, and placing in transfer membrane buffer solution; cutting a nitrocellulose membrane (PVDF membrane) with the same size as the gel, firstly soaking in methanol for about 2s, then soaking the nitrocellulose membrane and two pieces of thick filter paper with the same size in a membrane transferring buffer solution, sequentially stacking the nitrocellulose membrane, the filter paper, the gel, the PVDF membrane, the filter paper and the sponge on a bracket according to the sequence of the sponge, the filter paper, the PVDF membrane, the filter paper and the sponge (no bubbles can be reserved in a gap), and inserting the nitrocellulose membrane and the filter paper into a transfer tank after clamping; pouring a film-transferring buffer solution and putting ice cubes; gel-bonding (-) pole, PVDF film (+) pole, current intensity of 0.65mA/cm 2 Transferring the film for 1.5h at 4 ℃;
(3) Closing: after finishing transferring the film, washing the PVDF film for 3-5 times by using TBST buffer solution at room temperature for 90r/min, each time for 4-5 min, soaking the PVDF film in TBST sealing solution containing 5% of skimmed milk powder, and sealing for 1-2 h or overnight at 4 ℃ at room temperature;
(4) Washing the film: recovering the sealing liquid, adding a proper amount of TBST buffer solution, rinsing for 4-5 times at 90r/min for 4-5 min each time;
(5) Incubation resistance: soaking PVDF membrane in 20mL of primary antibody (ROLP-Amp-H polyclonal antibody) diluted by blocking solution, and incubating for 1-2H at room temperature and 60 r/min;
(6) Washing the film: recovering primary antibody, adding a proper amount of TBST buffer solution, rinsing for 4-5 times at 90r/min for 4-5 min each time;
(7) Secondary antibody incubation: soaking PVDF membrane in 20mL of secondary antibody (goat anti-rabbit IgG-HRP antibody) diluted by blocking solution, and incubating for 1h at room temperature and 60 r/min;
(8) Washing the film: pouring out the secondary antibody, adding a proper amount of TBST buffer solution, rinsing for 5 times at 90r/min for 4-5 min each time;
(9) Color development: sucking the residual TBST buffer solution on the surface of the PVDF membrane by using filter paper, and preparing chromogenic substrate (NBT: BCIP is prepared according to the ratio of 1:1); the color development liquid is evenly dripped on the PVDF film, and the result is photographed and recorded by a chemiluminescent imager.
The Western blot detection result of the ROLP-Amp-H polyclonal antibody for detecting the His-Amp-H prokaryotic expression protein is shown in figure 3. In a Western blot test, the invention respectively detects an uninduced protein sample, an induced protein sample and a purified protein sample of prokaryotic expression Amp-H, and the results show that the induced protein and the purified protein have obvious hybridization bands (figure 3) at the positions of target proteins, so that the antibody can perform recognition reaction with antigen proteins, and the antibody can be used for the Western blot test.
In order to further verify whether the ROLP-Amp-H polyclonal antibody can be used for detecting whether a field rice sample carries ROLP or not and the optimal use concentration of the antibody in a Western blot experiment, the invention uses ROLP-Amp-H polyclonal antibodies with different dilution ratios to carry out the Western blot experiment, and respectively detects a healthy rice sample and a rice sample infected with ROLP, and the result is shown in figure 4. As can be seen from FIG. 4, the ROLP-Amp-H polyclonal antibody of the present invention can be used to detect whether rice samples are infected with ROLP, and in four proportions of antibody dilution, the infected rice samples have a target band at a position close to 25kDa, while healthy rice samples have no band. By contrast, the invention considers that in the Western blot experiment, the optimal dilution ratio of the antibody is 1:10000. meanwhile, the sensitivity of the antibody is detected by diluting the concentration of the rice protein sample in different proportions, and the result is shown in figure 5. As can be seen from FIG. 5, when the rice protein sample is diluted 2 4 After doubling, ROLP can still be clearly detected, which shows that the polyclonal antibody prepared by utilizing the His-Amp-H prokaryotic expression protein has strong specificity and high quality.
In nature, ROLP is parasitic in the body of mediator insects in addition to the phloem of plants. Therefore, the invention also uses the method to detect healthy electro-optic leafhopper samples and electro-optic leafhopper samples carrying ROLP, and the result is shown in figure 6. As can be seen from FIG. 6, the samples of the electro-optic leafhoppers carrying the ROLPs all had the target bands at the positions close to 25kDa, while the healthy samples of the electro-optic leafhoppers had no bands at the positions, which indicates that the polyclonal antibodies of the ROLPs-Amp-H of the present invention can be used for detecting the samples of the electro-optic leafhoppers. In addition, the sensitivity of the antibodies is detected by diluting the concentrations of the electro-optic leafhopper protein samples carrying ROLP in different ratios, and the result is shown in FIG. 7. As can be seen from FIG. 7, when the concentration of the electro-optic leafhopper protein carrying ROLP was diluted 4 times, ROLP was still detectable, and when diluted 8 times, ROLP was not detectable. The detection sensitivity of the electro-optic leafhopper sample is lower than that of the rice sample, probably because the electro-optic leafhopper sample is total protein extracted from single-head electro-optic leafhoppers, and the protein content in the sample is lower.
Example 3 Dot-Elisa detection method of ROLP
Although Western blot detection can specifically detect protein samples carrying ROLPs, the experiment is time-consuming and expensive, and is not suitable for rapid detection of a large number of rice samples in the field. Therefore, the invention also establishes a Dot-Elisa detection method for detecting ROLP by utilizing the ROLP-Amp-H polyclonal antibody. The method specifically comprises the following steps:
(1) Sample preparation: sucking 50 mu L PBS into a 1.5mL centrifuge tube, taking a proper amount (about 0.1 g) of rice sample or electro-optic leafhopper sample, and stamping with a blue gun head;
(2) Spotting: cutting an NC film with proper size, placing the NC film in a clean culture dish, and sucking 1 mu L of sample points to the center of a lattice drawn by the NC film; (3) drying: after finishing the spot film, drying for 10min at room temperature, adding 10mL TBST buffer solution, rinsing for 5min at room temperature and 90 r/min;
(4) Closing: adding 10mL of 5% skimmed milk powder sealing solution, sealing at room temperature for 30min at 60 r/min;
(5) Washing: recovering the sealing liquid, adding 5mL TBST buffer solution, washing for 4 times at room temperature and 90r/min for 3-5 min each time;
(6) Incubation resistance: 10mL of each diluted 1 with 5% nonfat dry milk blocking solution was added: 2000 ROLP-Amp-H polyclonal antibody, at room temperature, 60r/min, incubated for 40min;
(7) Washing: pouring out the primary antibody, adding 5mL TBST buffer solution, washing for 4 times at room temperature and 90r/min for 3-5 min each time;
(8) Secondary antibody incubation: adding 10mL of AP-labeled goat anti-rabbit IgG secondary antibody diluted by 5% skimmed milk powder blocking solution, and incubating at room temperature for 40min at 60 r/min;
(9) Washing: pouring out the secondary antibody, adding 5mL TBST buffer solution, washing for 5 times at room temperature and 90r/min for 3-5 min each time;
(10) Color development: the residual TBST buffer solution on the NC film surface is sucked by filter paper, placed in a new culture dish, and a chromogenic substrate (chromogenic working solution: 1mL chromogenic buffer solution+300×BCIP 3.3 μL+150×NBT 6.6 μL) is prepared, uniformly dropped on the NC film, and the result is observed by direct photographing at room temperature and in dark place for 20-30 min. The invention detects healthy rice protein samples by the method, and simultaneously takes homogenates of leaf crude extracts of infected Rice Stripe Mosaic Virus (RSMV), infected Southern Rice Black Streak Dwarf Virus (SRBSDV), infected Rice Stripe Virus (RSV), infected rice saw tooth dwarf virus (RRSV) and infected rice tumor dwarf virus (RGDV) identified by laboratory RCP as negative controls; the disease leaf crude extract infected by SRBSDV and ROLP, RSMV and ROLP, RGDV and ROLP alone is used as positive control.
The Dot-Elisa detection results of ROLP-Amp-H polyclonal antibody detection of ROLP are shown in FIG. 8. As shown in FIG. 8, the ROLP-Amp-H polyclonal antibody has specific reaction to crude extracts of rice leaf tissues infected by ROLP, SRBSDV and ROLP complex infection, RSMV and ROLP complex infection, RGDV and ROLP complex infection, and has no immune reaction with homogenates of SRBSDV, RSMV, RSV, RRSV, RGDV and healthy rice plant tissues, so that the ROLP-Amp-H polyclonal antibody can be used for Dot-Elisa detection of ROLP and has higher specificity, can realize specific detection of ROLP, can detect ROLP even complex infected plants, has good detection effect and is not easy to leak detection or false positive.
In addition, the detection sensitivity of the ROLP-Amp-H polyclonal antibody is detected by Dot-Elisa experiments through diluting the crude extract of the disease leaf sample from 1:20 to 20480 times, and the result is shown in FIG. 9. As can be seen from FIG. 9, the Dot-Elisa detection method constructed by utilizing the ROLP-Amp-H polyclonal antibody has a detection sensitivity of 1:1280 times of dilution, the detection still presents purple positive spots at the moment, and the detection sensitivity is higher.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The polyclonal antibody of the rice orange leaf phytoplasma antigen membrane protein is characterized in that the polyclonal antibody is obtained by immunizing animals with the rice orange leaf phytoplasma antigen membrane protein as an antigen.
2. The polyclonal antibody according to claim 1, wherein the polyclonal antibody is obtained by immunizing an animal with the hydrophilic domain of the antigen membrane protein of Phytoplasma aurantia as an antigen.
3. The polyclonal antibody according to claim 2, wherein the amino acid sequence of the hydrophilic domain of the rice mycoplasma neris antigen membrane protein is shown in SEQ ID NO. 1.
4. Use of a polyclonal antibody according to any one of claims 1 to 3 for the detection of phytoplasma aurantia or for the preparation of a product for the detection of phytoplasma aurantia.
5. A kit for detecting phytoplasma aurantia in rice, characterized in that the kit comprises the polyclonal antibody according to any one of claims 1 to 3.
6. A Western blot method for detecting rice orange leaf phytoplasma is characterized in that the method comprises the following steps: extracting protein of a sample to be detected for Western blot detection, wherein the polyclonal antibody of any one of claims 1-3 is used in primary antibody incubation.
7. The method of claim 6, wherein the secondary antibody is incubated with goat anti-rabbit IgG-HRP antibody.
8. A Dot-Elisa detection method for detecting rice orange leaf phytoplasma is characterized by comprising the following steps: the proteins of the sample to be tested are extracted for Dot-Elisa detection, and the polyclonal antibody of any one of claims 1 to 3 is used for primary antibody incubation.
9. The method of claim 8, wherein the second antibody is incubated with an AP-labeled goat anti-rabbit IgG antibody.
10. Use of the polyclonal antibody of claim 1, the kit of claim 5 for diagnosing rice orange leaf disease or for preparing a product for diagnosing rice orange leaf disease.
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