CN105367638A - Antigen and polyclonal antibody for identifying puccinia triticina and puccinia striiformis - Google Patents
Antigen and polyclonal antibody for identifying puccinia triticina and puccinia striiformis Download PDFInfo
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- CN105367638A CN105367638A CN201510827403.8A CN201510827403A CN105367638A CN 105367638 A CN105367638 A CN 105367638A CN 201510827403 A CN201510827403 A CN 201510827403A CN 105367638 A CN105367638 A CN 105367638A
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- 102000036639 antigens Human genes 0.000 title claims abstract description 19
- 108091007433 antigens Proteins 0.000 title claims abstract description 19
- 239000000427 antigen Substances 0.000 title claims abstract description 18
- 241001246061 Puccinia triticina Species 0.000 title abstract 11
- 241001123583 Puccinia striiformis Species 0.000 title abstract 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 26
- 230000002163 immunogen Effects 0.000 claims abstract description 18
- 230000003053 immunization Effects 0.000 claims abstract description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 14
- 210000004369 blood Anatomy 0.000 claims abstract description 13
- 239000008280 blood Substances 0.000 claims abstract description 13
- 230000000890 antigenic effect Effects 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 230000036039 immunity Effects 0.000 claims description 24
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 20
- 241000221300 Puccinia Species 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 13
- 241000233866 Fungi Species 0.000 claims description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 5
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- 239000012460 protein solution Substances 0.000 claims description 4
- 230000004044 response Effects 0.000 claims description 3
- 238000001262 western blot Methods 0.000 claims description 2
- 241000209140 Triticum Species 0.000 abstract description 6
- 235000021307 Triticum Nutrition 0.000 abstract description 6
- 229920001184 polypeptide Polymers 0.000 abstract description 5
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 5
- 239000007788 liquid Substances 0.000 description 15
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 241000221301 Puccinia graminis Species 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 238000002649 immunization Methods 0.000 description 7
- 238000010241 blood sampling Methods 0.000 description 6
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
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- 238000002474 experimental method Methods 0.000 description 4
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- 239000000725 suspension Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108010058846 Ovalbumin Proteins 0.000 description 3
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- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 229940092253 ovalbumin Drugs 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
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- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- 241000283977 Oryctolagus Species 0.000 description 2
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- 238000004587 chromatography analysis Methods 0.000 description 2
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- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 230000016571 aggressive behavior Effects 0.000 description 1
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- 238000013016 damping Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
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- 238000010438 heat treatment Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
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- 238000005374 membrane filtration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
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- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
-
- 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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- General Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention relates to an antigen and a polyclonal antibody for identifying puccinia triticina and puccinia striiformis. An antigenic polypeptide for preparing a polyclonal antibody for distinguishing Puccinia triticina from Puccinia triticina, which has an amino acid sequence shown in SEQ? ID? NO: 1 is shown. An immunogen for preparing a polyclonal antibody for distinguishing Puccinia triticina from Puccinia triticina, which is prepared by using the antigen polypeptide as a hapten. The polyclonal antibody for distinguishing the puccinia triticina and the puccinia triticina is characterized in that the immunogen is used for immunizing rabbits, and the immune rabbits with high titer are detected and screened; and (4) collecting whole blood of selected rabbits, and purifying to obtain the polyclonal antibody. The method for distinguishing the puccinia triticina from the puccinia triticina can simply, conveniently and effectively detect whether the puccinia triticina is infected in the early stage of the wheat.
Description
Technical field
The present invention relates to the antigen for differentiating puccinia triticinia and puccinia graminis bacterium and polyclonal antibody, belonging to field of plant disease control.
Background technology
Wheat rust (PucciniareconditaRob.exDesm.f.sp.triticiErikss.EtHenn.) is the fungal disease caused by Wheat rust fungus, this disease is the important disease of a class of China and even whole world man of all wheat producing countries, can cause the production loss of more than 15% during serious generation.
For a long time, the prediction of wheat rust mainly based on regularly, large-scale field Disease investigation is separated with the live body of indoor germ microspecies, culture & identification.Not only consuming timely to take a lot of work, and the actual demand that the accuracy of its result and confidence level also depend on that state of the art and the experience of personnel are observed and predicted in investigation to a great extent, are difficult to meet fast, high-throughput diagnostic detects.Particularly in the incobation stage of puccinia triticinia, the disease symptom of host is not easily observed, and is difficult to the period and the scale that define First aggression.Therefore, be necessary to utilize modern biotechnology, research and develop simple, sensitive and accurate puccinia triticinia quick diagnosis and detection means.
Summary of the invention
For meeting the demand in above-mentioned field, the invention provides a kind of antigen for detecting puccinia triticinia and polyclonal antibody.
The technical scheme of request protection of the present invention is as follows:
For the preparation of the antigenic peptide of polyclonal antibody distinguishing puccinia triticinia and red rust, its aminoacid sequence is as shown in SEQIDNO:1.
For the preparation of the immunogen of polyclonal antibody distinguishing puccinia triticinia and red rust, it is characterized in that, with antigenic peptide according to claim 1 for haptens preparation and obtaining.
Described immunogenic preparation method is: get the antigenic peptide 6mg described in claim 1, dissolves, obtain solution A with 1mLDMF; Get 15mgEDC and NHS, after fully dissolving with 0.2mLPH5.0MES, add in solution A, stirred at ambient temperature 24h, reaction solution B can be obtained; Take bovine serum albumin BSA50mg, make it fully to be dissolved in 3.8mL0.1MPH7.2PBS, reaction solution B is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 24h, at 4 DEG C, dialyse 3 days with 0.01mol/LPBS, change 3 dialyzates every day; Packing, be stored in-20 DEG C for subsequent use.
For distinguishing the polyclonal antibody of puccinia triticinia and red rust, it is characterized in that, with the immunogen described in Claims 2 or 3, immunity preparation being carried out to rabbit and obtaining.
The preparation method of polyclonal antibody according to claim 4, step is as follows:
(1) with the immunogen immune rabbit described in Claims 2 or 3, the immunizing rabbit of screening high-titer is detected;
(2) take whole blood to the rabbit chosen, purifying obtains polyclonal antibody.
The immune time of described immunizing rabbit is 7 times, and the described rabbit detecting screening immunity refers to that adopting elisa method to tire to rest fungus Detection of antigen reaches the immunizing rabbit of more than 2W.
Polyclonal antibody according to claim 4 is distinguishing the application in puccinia triticinia and red rust, it is characterized in that, react with polyclonal antibody according to claim 4 and testing sample, observing response result, if negative, then described sample is puccinia triticinia.
Described reaction comprises Elisa, Western-blot, immunofluorescent test.
The present invention is to the peculiar albumen (HAD-superfamilysubfamilyIIAhydrolase of puccinia triticinia, its aminoacid sequence is as shown in SEQIDNO:2) carry out the characteristics such as secondary structure, immunogenicity, hydrophilic and hydrophobic and analyze, have selected wherein one section, its aminoacid sequence as shown in SEQIDNO:1, synthetic.
The antigenic peptide of synthetic and BSA coupling are prepared immunogen.
Adopt the immunogen immune rabbit obtained, repeatedly immunity is through bioactivity, and selecting tires reaches the rabbit of ideal value, collects its antiserum(antisera).Polyclonal antibody is obtained with protein purification column purification antiserum(antisera).
To be at war with immune inhibition test to the antibody after purifying, test-results shows, and the polyclonal antibody of acquisition and natural puccinia graminis bacterium spore carry out immune response for positive, carries out immune response for feminine gender with natural leaf rust spore.
The present invention, for distinguishing the method for puccinia triticinia and red rust, operates very easy, only need get rest fungus spore and antibody of the present invention reacts under suitable reaction conditions, and if there is positive reaction, then this sample is red rust.Whole process is without the need to instrument, and technician can carry related reagent and consumptive material, just can complete the discriminating of two kinds of rust in the wild, can detect whether leaf rust early infection occurs simple and effectively.
Accompanying drawing explanation
Fig. 1. the preparation flow figure of polyclonal antibody,
Immunization time arranges: as shown in table 2, initial immunity carries out second time immunity after 15 days, third time immunity is carried out after 15 days, within 7 days, first time takes a blood sample detections afterwards, carries out the 4th immunity after 7 days, detection of taking a blood sample for the second time afterwards for 7 days, the 5th immunity is carried out after 7 days, within 7 days, third time takes a blood sample detection afterwards, by that analogy, often organizes all to complete at least 6 immunity and take a blood sample for 4 times and detects.Wherein, the experimental rabbit immune effect of E004 group is better, and be preferred group of this experiment, its immunization time arranges as shown in table 3.
Fig. 2. the polyclonal antibody of purifying gained detects figure,
Be followed successively by from left to right: Marker, serum sample, purifying sample, applied sample amount about 10 micrograms/hole, antibody size is between 150kDa ~ 180kDa.
Embodiment
Below by specific embodiment, the present invention is described in further detail, it is to be appreciated that following embodiment only illustratively and illustrate, and does not limit the scope of the invention in any form.
Table 1 the present embodiment bacterial classification used
Biological chemical reagent not specified in following examples is this area conventional reagent, can be prepared by this area ordinary method and or commercially available, specification is the pure level in laboratory.
Embodiment 1. is for differentiating the polyclonal antibody preparation of puccinia graminis bacterium
Reagent and solution:
Freund's complete adjuvant, Freund's incomplete adjuvant (Beijing Kwinbon Biotechnology Co., Ltd.)
Phosphate buffered saline buffer (PBS, 0.02M, pH7.2),
Coating buffer (phosphate buffered saline buffer, 0.05M, pH7.4),
Reaction diluent (PBST, pH7.4,0.03M ,+0.05% polysorbas20),
Wash operating solution (20 × concentrated cleaning solution being diluted by 1:19 volume ratio with deionized water);
Confining liquid (adding 0.05%BSA in 0.02mol/LPBST),
Substrate nitrite ion: be made up of A liquid and B liquid, A liquid is hydrogen peroxide, and B liquid is tetramethyl benzidine;
Stop buffer (2mol/LH
2sO
4),
ELIAS secondary antibody (the diligent nation in Beijing is biological): the goat-anti rabbit IGg antibody of horseradish peroxidase-labeled
Shown in table 14 kind of rest fungus, the suspension 1*10 of often kind of rest fungus
5spore/ml
Key instrument equipment:
Vacuum freeze drier, electro-heating standing-temperature cultivator, ProteinA/G post, electrophoresis chamber, electrophoresis apparatus, enzyme plate, microplate reader, low-temperature and high-speed whizzer, ProteinA chromatography column etc.
Laboratory animal: new zealand rabbit, purchased from Chinese military medicine academy of sciences.
1, Peptide systhesis
According to the peculiar albumen of puccinia triticinia (HAD-superfamilysubfamilyIIAhydrolase) sequence (SEQIDNO:
2) design polypeptide, screening obtains antigenic peptide, and its aminoacid sequence is: CGVFKGNKPEDA, entrusts Beijing Kwinbon Biotechnology Co., Ltd. to carry out chemosynthesis to above-mentioned antigen protein, obtains haptens.
2, immunogen and coating antigen preparation
(1) haptens and bovine serum albumin (BSA) coupling form immunogen
Get the haptens 6mg that step 1 obtains, dissolve with 1mLDMF, obtain solution A; Get 15mgEDC and NHS, after fully dissolving with 0.2mLMES (PH5.0), add in solution A, stirred at ambient temperature 24h, reaction solution B can be obtained.Take bovine serum albumin BSA50mg, make it fully to be dissolved in 3.8mL0.1MPBS (PH7.2), reaction solution B is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 24h, at 4 DEG C, to dialyse 3d with 0.01mol/LPBS, change 3 dialyzates every day.Packing, be stored in-20 DEG C for subsequent use.
(2) haptens and ovalbumin (OVA) coupling form coating antigen
Get the haptens 4mg that step 1 obtains, dissolve with 1mLDMF, obtain solution A; Get the glutaraldehyde 200ul of 2.5%, add solution A, reaction 30min can obtain reaction solution B.Take ovalbumin 50mg, make it fully to be dissolved in 3.8mL0.1MCB liquid, reaction solution B is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 16h, then add sodium borohydride reduction and react 2 hours, then at 4 DEG C, to dialyse 3d with 0.01mol/LPBS, change 3 dialyzates every day.Packing, be stored in-20 DEG C for subsequent use.
3, how anti-preparation
Immunogen: immunogen step 2 obtained, to experimental rabbit carries out initial immunity after mixing with Freund's complete adjuvant by 1:1 volume ratio, and each immunity is later carried out after adopting Freund's incomplete adjuvant and immunogen to mix by 1:1 volume ratio.Prepare 7 immunogens altogether, carry out immunity respectively.
Immunizing dose: initial immunity 0.2mg/ time/, second time starts 0.1mg/ time/.
Immunization ways: subcutaneous multi-point injection.
Immune animal: new zealand rabbit, often organizes 2,7 groups, uses 14 altogether.
Immunization time arranges: as shown in table 2, initial immunity carries out second time immunity after 15 days, third time immunity is carried out after 15 days, within 7 days, first time takes a blood sample detections afterwards, carries out the 4th immunity after 7 days, detection of taking a blood sample for the second time afterwards for 7 days, the 5th immunity is carried out after 7 days, within 7 days, third time takes a blood sample detection afterwards, by that analogy, often organizes all to complete at least 6 immunity and take a blood sample for 4 times and detects.Wherein, the experimental rabbit immune effect of E004 group is better, and be preferred group of this experiment, its immunization time arranges as shown in table 3.
Table 2 immunization time arranges plan
Table 3 is group immunization time plan preferably
Note M: immunity, 1M: first time immunity, by that analogy; C: blood sampling, 1C: first time blood sampling, by that analogy.
Blood sampling test item: each blood sampling detects the response situation of itself and natural antigen leaf rust spore.
Detection method: indirect competitive, step is as follows:
1) main agents
The goat anti-rabbit antibody (Beijing Kwinbon Biotechnology Co., Ltd.) of HRP mark, wash operating solution, antibody diluent (phosphate buffered saline buffer), antigenic dilution (CB liquid), substrate A/B liquid, confining liquid;
2) main consumptive material
96 hole enzyme plates (Xiamen company of happy Jiamei)
3) laboratory operating procedures
A, wrapper sheet: dilute coating antigen (the mass volume ratio 1:1000 of coating antigen and diluent) and 4 grow wheat rest fungus suspensions (diluting 100 times) respectively with antigenic dilution, add enzyme plate respectively, every hole 100ul, hatch 2 hours for 37 DEG C, wash plate once, pat dry;
B, close: add confining liquid and close, every hole 150ul, hatch 2 hours, abandon liquid, pat dry for 37 DEG C, for subsequent use;
C, add antibody: the antibody dilution respective concentration (antibody dilution reacted with coating antigen 100,000 times, the antibody dilution reacted with rest fungus 9000 times) step 3 prepared with antibody diluent, every hole adds 50ul, hatches 30 minutes for 37 DEG C;
D, washing: wash plate 3-5 time, every minor tick 30 seconds, pats dry;
E, add ELIAS secondary antibody: every hole adds the goat anti-rabbit igg antibody 100ul of the HRP mark of dilution 1000 times, hatches 30 minutes for 37 DEG C;
F, washing; Wash plate 3-5 time, every minor tick 30 seconds, pats dry;
G, colour developing: add equivalent A/B substrate nitrite ion, every hole 100ul, hatches 15 minutes for 37 DEG C;
H, termination: add stop buffer termination reaction;
I, reading: use the wavelength of microplate reader for 450nm/630nm, read the OD value in each hole.
Blood sampling detected result:
This experiment carries out immunity in strict accordance with immune flow process, and complete antibody titer and with the detection of natural antigen in conjunction with indexs such as situations, table 4 lists the Virus monitory result preferably organizing rabbit (E004 group).
Table 4 is group Virus monitory result preferably
Note: K represents " thousand ", W represents " ten thousand ", and E004 represents the antibody taking from preferably group rabbit, and E011 represents coating antigen;
E004-1W represents preferably group antibody dilution 10,000 times, by that analogy; E011-1K represents that coating antigen dilutes 1000 times; Four kinds of rest fungus-100 represent the shown 4 kinds of rest fungus of table 1, and the spore suspension of often kind of rest fungus dilutes 100 times, corresponding 4 data detected by OD value Range Representation; The spore suspension of puccinia graminis bacterium-100 × indication rod rest fungus dilutes 100 times.
E004 group antagonist after the 7th blood sampling is tired and is verified, can reach later experiments requirement, therefore acquires the whole blood of this group and carry out antibody purification.
4, the purifying of polyclonal antibody
Carry out purifying with GErProteinA affinity column to rabbit anteserum sample, wash-out gained antibody packing 50uL/ after the neutral PB dialysis of 0.05M manages, and totally 13 pipes spend the night freeze-drying, every Guan Yuehan antibody 0.39mg.Concrete steps are as follows:
A. the centrifugal 10min of sample 10000x to be purified, gets supernatant and dilutes with 2 times of volume binding buffer liquid (0.02MpH7.0PB), for subsequent use through 0.45um membrane filtration.
B. prepare AKTA chromatograph pipeline and affinity column is installed, pressure limiting 0.3MPa flow velocity 1.0mL/min is set; Steady to baseline with 5 times of column volume (CV) binding buffer liquid balance chromatography columns, UV returns to zero; Flow velocity 0.5mL/min is with 10CV binding buffer liquid loading to UV back to zero, and period collects stream and wears sample; Flow velocity 1.0mL/min rinses to UV back to zero with 5CV0.1MpH4.0 sodium citrate buffer solution, and period collects wash-out sample and adjusts pH to neutral with 1.0MpH9.0Tris-HCl damping fluid immediately; Detergent line.
C. measure purifying with NanoVue ultramicron ultraviolet spectrophotometer and respectively walk the protein concentration collecting component, result is as shown in table 5, purification yield=purifying sample/serum sample x100%=21.7%.
Table 5 antibody purification respectively walks the protein concentration collecting component
Result as shown in Figure 2, is followed successively by from left to right: Marker, serum sample, purifying sample, and applied sample amount about 10 micrograms/hole, antibody size is between 150kDa ~ 180kDa.
5, the detection of polyclonal antibody
According to the detection method in step 3,4 kinds of rest fungus shown in the antibody of purifying and table 1 are reacted, detect tiring and specificity of antibody purification.Result is as shown in table 6, and during antibody dilution 2.7 ten thousand times, puccinia graminis bacterium (34C2/21C3) and leaf rust (THT, PHT) can distinguish by antibody purification.
Criterion: tire down on an equal basis, the OD value of reacting with puccinia graminis bacterium is that more than 2.1 times of leaf rust can be distinguished.
Many anti-to puccinia graminis bacterium tire be 2.7 ten thousand, OD value about 1.9, be 800,000, OD value be 1.8 to tiring of polypeptide coating antigen, negative control (confining liquid) do not developed the color, develops the color more weak to leaf rust, tire 2.7 ten thousand time develop the color 0.3.
Directly carry out antibody titer after adopting whole blood and suppress to detect, purifying many anti-after again carry out tiring and suppress to detect, antibody titer before and after contrast purifying and suppression detect data, result shows, purified antibodies reaches 1:900000 to tiring of polypeptide, with change essentially no before purifying, be 2.7 ten thousand to tiring of bacterium, inhibition is remarkable.
Table 6 antibody purification detected result
Claims (8)
1., for the preparation of the antigenic peptide of polyclonal antibody distinguishing puccinia triticinia and red rust, its aminoacid sequence is as shown in SEQIDNO:1.
2. for the preparation of the immunogen of polyclonal antibody distinguishing puccinia triticinia and red rust, it is characterized in that, with antigenic peptide according to claim 1 for haptens preparation and obtaining.
3. immunogen according to claim 2, is characterized in that, described immunogenic preparation method is: get the antigenic peptide 6mg described in claim 1, dissolves, obtain solution A with 1mLDMF; Get 15mgEDC and NHS, after fully dissolving with 0.2mLPH5.0MES, add in solution A, stirred at ambient temperature 24h, reaction solution B can be obtained; Take bovine serum albumin BSA50mg, make it fully to be dissolved in 3.8mL0.1MPH7.2PBS, reaction solution B is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 24h, at 4 DEG C, dialyse 3 days with 0.01mol/LPBS, change 3 dialyzates every day; Packing, be stored in-20 DEG C for subsequent use.
4. for distinguishing the polyclonal antibody of puccinia triticinia and red rust, it is characterized in that, with the immunogen described in Claims 2 or 3, immunity preparation being carried out to rabbit and obtaining.
5. the preparation method of polyclonal antibody according to claim 4, step is as follows:
(1) with the immunogen immune rabbit described in Claims 2 or 3, the immunizing rabbit of screening high-titer is detected;
(2) take whole blood to the rabbit chosen, purifying obtains polyclonal antibody.
6. the preparation method according to right 5, the immune time of described immunizing rabbit is 7 times, and the described rabbit detecting screening immunity refers to that adopting elisa method to tire to rest fungus Detection of antigen reaches the immunizing rabbit of more than 2W.
7. polyclonal antibody according to claim 4 is distinguishing the application in puccinia triticinia and red rust, it is characterized in that, react with polyclonal antibody according to claim 4 and testing sample, observing response result, if negative, then described sample is puccinia triticinia.
8. application according to claim 7, described reaction comprises Elisa, Western-blot, immunofluorescent test.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101709087A (en) * | 2009-11-26 | 2010-05-19 | 中国农业科学院植物保护研究所 | Puccinia triticina f.sp.tritic monoclonal antibody and preparation method and application thereof |
| CN105001326A (en) * | 2015-06-24 | 2015-10-28 | 中国农业科学院植物保护研究所 | Colloidal gold test strip for detecting wheat leaf rust |
| CN105348371A (en) * | 2015-11-23 | 2016-02-24 | 中国农业科学院植物保护研究所 | Antigen and polyclonal antibody for identifying Tilletia controversa Kuhn and Tilletia foetida |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101709087A (en) * | 2009-11-26 | 2010-05-19 | 中国农业科学院植物保护研究所 | Puccinia triticina f.sp.tritic monoclonal antibody and preparation method and application thereof |
| CN105001326A (en) * | 2015-06-24 | 2015-10-28 | 中国农业科学院植物保护研究所 | Colloidal gold test strip for detecting wheat leaf rust |
| CN105348371A (en) * | 2015-11-23 | 2016-02-24 | 中国农业科学院植物保护研究所 | Antigen and polyclonal antibody for identifying Tilletia controversa Kuhn and Tilletia foetida |
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| 曹丽华等: "小麦叶锈菌的特异性分子诊断检测技术", 《植物保护学报》 * |
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