CN107326038A - A kind of polyclonal antibody and its application for being used to detect Prospect on Kiwifruit Bacterial Canker bacterium PSA3 - Google Patents
A kind of polyclonal antibody and its application for being used to detect Prospect on Kiwifruit Bacterial Canker bacterium PSA3 Download PDFInfo
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- CN107326038A CN107326038A CN201710757543.1A CN201710757543A CN107326038A CN 107326038 A CN107326038 A CN 107326038A CN 201710757543 A CN201710757543 A CN 201710757543A CN 107326038 A CN107326038 A CN 107326038A
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- hopz5
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- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 4
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Classifications
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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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- 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
- C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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
- G01N33/56911—Bacteria
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- Engineering & Computer Science (AREA)
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- Urology & Nephrology (AREA)
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- General Health & Medical Sciences (AREA)
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- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The present invention provides purposes of the hopZ5 genes in detection Prospect on Kiwifruit Bacterial Canker bacterium PSA3, a kind of polyclonal antibody for detecting Prospect on Kiwifruit Bacterial Canker bacterium PSA3 based on hopZ5 genes is provided, and the immune colloid gold reagent box containing the polyclonal antibody and its application are provided.The PSA3 polyclonal antibodies of the present invention have the characteristics of detection specificity is high and applied widely;The Prospect on Kiwifruit Bacterial Canker bacterium PSA3 point immune responses of offer and immunity colloidal gold test paper strip, detection method are simple, a large amount of samples can be detected simultaneously, with the value in basic unit's promotion and application.
Description
【Technical field】
The present invention relates to immunoassay, more particularly to a kind of Anti-TNF-α for being used to detect Prospect on Kiwifruit Bacterial Canker bacterium PSA3
Body, the immunity colloidal gold test paper strip containing the antibody, and apply the Prospect on Kiwifruit Bacterial Canker bacterium PSA3 of antibody detection
Method.
【Background technology】
Prospect on Kiwifruit Bacterial Canker is first major disease in Kiwi berry producing region, is found first in shizuoka County, Japan in 1984, then
Reported successively in multiple countries such as China, South Korea, Italy, France, Portugal, Spain, New Zealand and Chile.It is Chinese main
The province such as Shaanxi, Sichuan, Henan, Guizhou is distributed in, at present, the morbidity of the ground such as Sichuan Dujiang weir, Yaan, Cangxi is more serious, a lot
Orchard has resulted in the garden of ruining.The germ for causing the disease is Pseudomonas syringae pv.actinidiae (Pseudomonas
Syringae pv.actinidiae, PSA), the bacterium wide adaptability, pathogenicity are strong, break with tremendous force, and when falling ill serious, whole strain is withered
Extremely, it can cause to ruin garden between several years.In recent years, with the continuous expansion of Prospect on Kiwifruit Bacterial Canker susceptible variety planting area, nursery stock is numerous
Educate and allocate and transport lack of standardization, prevent and treat the influence of the factors such as difficulty so that the generation of Prospect on Kiwifruit Bacterial Canker, propagate and spread further expansion
Greatly, it has also become the destructive disease of Kiwi berry, it is one of upper significant problem urgently to be resolved hurrily of production.
Research report in recent years confirms that Prospect on Kiwifruit Bacterial Canker bacterium has a key property --- latent infection.Therefore,
Field carry disease germs fruit tree early diagnosis be prevent and treat Prospect on Kiwifruit Bacterial Canker key;It is that its is climing for preventing and treating to the accurate detection for allocating and transporting nursery stock
The key prolonged.Set up it is quick, conveniently, accurate detection method, the preventing and treating for Prospect on Kiwifruit Bacterial Canker is significant.
At present, Prospect on Kiwifruit Bacterial Canker bacterium is mainly by molecular Biological Detection, and it is accurate, sensitive that wherein PCR detections have
Advantage.According to the specific spray bacterium phenomenon of bacterium, many bacteriosises are directly entered performing PCR by band hyphostroma soak and detected,
It is more convenient quick.Such as Chinese invention patent application CN 201611158141.1 discloses a kind of quantitatively detection Kiwi berry and burst
The real time fluorescent PCR method of ulcer germ, it is all together primer amplified, sequencing comparison is carried out to amplified production, according to sequence
Comparison result judgement sample infection conditions.
However, when Kiwi berry limb, blade as testing sample etc. is organized in immersion, various secondary metabolites are especially
It is that polysaccharide, polyphenol are soaked out from tissue, PCR reactions and other detection modes is interfered so that this simple sample preparation
Method detects inapplicable to Prospect on Kiwifruit Bacterial Canker bacterium.It can be seen that, the method for preparing sample of Chinese Gooseberry is whether subsequent detection result is steady
Fixed premise, optimization and simplified sample making course, are to improve the important step of current Prospect on Kiwifruit Bacterial Canker bacterium detection.In addition, in view of
The condition such as instrument and operating environment needed for PCR detections are limited to is miscellaneous, still exists for department of basic unit and limits, therefore, it is necessary to
Other detection methods are developed, and then suitable for different fields and department.
Historically, it is 5 monoids to be currently known PSA points.Wherein, PSA1 is distributed in Japan;PSA2 only exists South Korea;
PSA3 is widely distributed in the whole world, including the Italian Prospect on Kiwifruit Bacterial Canker broken out of 2008-2009, China, intelligence, New Zealand, South Korea
Deng PSA3 is the monoid for causing global Kiwi berry industry heavy losses;PSA4 is distributed in New Zealand and Australia.In addition,
Fujikawa etc. confirms PSA5 monoids using genomics, and at present only in Japan's discovery, and PSA4 and PSA5 belong to lower toxicity
Group.The present invention, which is directed in PSA, is distributed monoid PSA3 that is wide and causing serious harm, by analyzing each monoid virulence factor difference,
Including effector difference, expect to find out the only distinctive effector of PSA3 monoids, be prepared for a kind of PSA3 polyclonal antibodies, and provide
Simple Serological testing method.
【The content of the invention】
The purpose of the present invention is to overcome prior art defect there is provided a kind of distinctive effector of PSA3 monoids of acquisition, so that
A kind of polyclonal antibody for being used to detect Prospect on Kiwifruit Bacterial Canker bacterium PSA3 is obtained, and the fast of PSA3 monoids is realized by the antibody
Speed, conveniently, efficient serology instrument, and then the carry disease germs diffusion of nursery stock of early prevention and treatment for Prospect on Kiwifruit Bacterial Canker and limitation provides
Foundation.
To achieve these goals, inventor is to having reported that PSA genome sequencing results are excavated, and contrast PSA is each
The use of monoid gene difference, especially effector difference there is provided hopZ5 genes in detection Prospect on Kiwifruit Bacterial Canker bacterium PSA3
On the way.
Based on hopZ5 genes, the present invention also provides a kind of polyclonal antibody for being used to detect Prospect on Kiwifruit Bacterial Canker bacterium PSA3
Preparation method, the described method comprises the following steps:To Pseudomonas syringae pv.actinidiae (Pseudomonas
Syringae pv.actinidiae, PSA) hopZ5 genes expanded, after the sequencing of gained hopZ5 genetic fragments is correct, warp
Restriction Enzyme BamH I and Xho I digestions are crossed, digestion products are connected to prokaryotic expression carrier PET28, obtain and contain hopZ5 genes
Recombinant plasmid PET28a-hopZ5, with the restructuring mass transitions E. coli BL21, through IPTG inducible protein tables
Reach and purify, obtain albumen hopZ5;
New zealand white rabbit is immunized with prokaryotic expression protein hopZ5, the polyclonal antibody PAb-hopZ5 is prepared.
In the present invention, the primer expanded to hopZ5 genes is as follows:
Hopz5-BamH I-F:5’-CGCGGATCCATGGGACTTTGTGCATCA-3
Hopz5-Xho I-R:5’-CGGTCTCGAGTTAGGATTCTATCGCTTTTC-3’
Underscore is respectively BamHI and XhoI restriction enzyme site sequences.
Pcr amplification reaction system:The 2 μ L of μ L, DNA of PrimeSTAR HS DNA Polymerase 0.3, up and down primer (10
μM) 2 10 μ L of μ L, 10 × Buffer of each 1 μ L, dNTP (2.5mM), plus ddH2O supplies 25 μ L, mixes laggard performing PCR reaction.
PCR amplification conditions are:94 DEG C of 4min, 94 DEG C of 30sec, 53 DEG C of 30sec, 72 DEG C of 1min30sec, 33 circulations, 72
DEG C 10min, 12 DEG C of preservations.
Wherein, the digestion step includes:
Target gene double digestion system is the μ L of 10 × H Buffer 2;Hopz58μL;BamH I 1μL;Xho I1μL;With
ddH2O supplies 20mL.Above-mentioned system is added on ice in 200 μ L EP pipes, is put into after mixing in 37 DEG C of thermostat water baths,
Digestion is carried out to stay overnight.Purpose band is extracted after then taking out the agarose gel electrophoresis nucleic acid staining dye for 1%, according to
DNA glue reclaim kit specifications are reclaimed to purpose fragment, obtain purpose fragment.
Based on foregoing hopZ5 genes and polyclonal antibody, the present invention also provides immune containing above-mentioned polyclonal antibody
Colloidal gold strip, the test strips include sample pad, gold standard pad, nitrocellulose filter and the water suction being sequentially assembled on bottom plate
Paper, wherein, the gold standard pad contains colloidal gold antibody compound, and the colloidal gold antibody compound contains with good grounds claim 2
Polyclonal antibody and colloidal gold solution that described preparation method is obtained, the concentration of the polyclonal antibody are not less than 50 μ g/ml,
Then with 0.1mol/L HCl/water solution and 0.1mol/L K2CO3The aqueous solution is adjusted to pH value 7.2, is obtained the collaurum and is resisted
Nanocrystal composition.
According to one kind preferred embodiment, the preparation method of the colloidal gold solution is:Take a clean bottle will
1g gold chlorides are all dissolved in 100mL ultra-pure water, are made into 1% (g/mL) chlorauric acid solution, and 4 DEG C of preservations of lucifuge.By before
1% chlorauric acid solution stated is diluted to 0.01% (g/mL) chlorauric acid solution, takes 100mL 0.01% chlorauric acid solution
It is added in a clean beaker, heats while stirring, 1% trisodium citrate 1.5mL of 37 DEG C of preheatings is added after boiling,
Soft stirring, solution cools down room temperature, ultra-pure water is supplied by yellow blackening, purple, purplish red until continue to boil 2min after claret
To 100mL, in the vial for being fitted into cleaning, 4 DEG C are kept in dark place..
Based on foregoing hopZ5 genes and polyclonal antibody, the present invention also provides one kind and uses immunity colloidal gold test paper strip
The method for detecting Prospect on Kiwifruit Bacterial Canker bacterium PSA3, the described method comprises the following steps:
(1) by Kiwi berry sample comminution to be detected to diameter 1-2mm fragments, according to testing sample and the ratio of sample loading buffer
Example is 0.05~0.2g:250~400 μ l, 2-15s in sample loading buffer is immersed in by testing sample;
(2) buffer solution for having soaked testing sample of 200 μ L steps (1) is taken to be added on according to claim 5 be immunized
In the sample pad of colloidal gold strip, detection zone T lines and C lines that 5-10min observes immunity colloidal gold test paper strip are stopped;
(3) judge that testing sample infects product for Prospect on Kiwifruit Bacterial Canker bacterium PSA3 when T lines and C lines show as claret.
Based on foregoing hopZ5 genes and polyclonal antibody, the present invention also provides a kind of Prospect on Kiwifruit Bacterial Canker bacterium PSA3's
Point immunologic detection method, the described method comprises the following steps:
(1) by Kiwi berry sample comminution to be detected to diameter 1-2mm fragments, according to testing sample and the ratio of sample loading buffer
Example is 0.005~0.01g:20~50 μ l, 2-15s in sample loading buffer is immersed in by testing sample;
(2) buffer solution for having soaked testing sample of step (1) is taken, is added dropwise on nitrocellulose filter, then room temperature is done
It is dry;
(3) closing 1h is carried out to the nitrocellulose filter of step (2) with the aqueous solution of 5wt% skimmed milk powers;
(4) 1h is incubated with PAb-hopZ5 according to claim 1, then washed 3 times with phosphate buffer PBS;
(5) again with (buying from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) alkali phosphatase enzyme mark goat-anti of commercialization
Rabbit igg is incubated 1h, is then washed 3 times with phosphate buffer PBS;
(6) chromogenic reaction is carried out under light protected environment using BCIP/NBT alkaline phosphatase colour reagent boxes, develop the color sample
It is judged as that Prospect on Kiwifruit Bacterial Canker bacterium PSA3 infects product.
It is demonstrated experimentally that Prospect on Kiwifruit Bacterial Canker bacterium PSA3 effector gene hopZ5, by cloning complete sequence and building protokaryon
Recombinant vector PET28a-hopZ5 is expressed, the hopZ5 protein immunizations through prokaryotic expression prepare polyclonal antibody PAb-
HopZ5, the antibody can be distinguished for examination pseudomonas and other pathogenetic bacterias, can distinguish PSA3 and other monoids, have
Special high the characteristics of, therefore can be applied to Prospect on Kiwifruit Bacterial Canker bacterium PSA3 detections.
Detection of the polyclonal antibody available for artificial culture and with hyphostroma is obtained based on hopZ5 genes, including prepared
Into immunity colloidal gold test paper strip, or applied to an immune detection, the characteristics of having applied widely to testing sample can be achieved
PSA3 various Serologic detections, be at present with regard to Prospect on Kiwifruit Bacterial Canker bacterium, particularly with pathogenic extremely strong PSA3 monoids, except
The comparatively ideal antibody that can be applied to Serologic detection found first beyond Molecular Detection means, before good exploitation
Scape.
Invention also improves the preparation means of detection sample, when detected sample, soak time is in sample loading buffer
At 2-15 seconds, a large amount of secondary metabolites such as polysaccharide polyphenol that can be prevented effectively from sample are discharged, so as to avoid influence from examining
Survey result, compared with prior art with sample preparation it is simple the characteristics of.In addition, by it was found that, by detected sample sample-adding
Soak more more stable than the effect detected after conventional sterilized water immersion in buffer solution.
The present invention detection method detection speed faster, compared to current other Prospect on Kiwifruit Bacterial Canker bacterium detection methods more
To be quick, wherein, immunity colloidal gold test paper strip can complete detection in 15 minutes;And putting immunologic detection method also has
The characteristics of sample sample preparation is simple.
【Brief description of the drawings】
Fig. 1 is the result that Western blot detect Prospect on Kiwifruit Bacterial Canker bacterium hopZ5 albumen;
Fig. 2 is PAb-hopZ5 point immune detection sample type analysis results;
Fig. 3 is that field sample detection result is immunized in PAb-hopZ5 points;
Fig. 4 is PAb-hopZ5 immunity colloidal gold test paper strip specific test results;
Fig. 5 is PAb-hopZ5 immunity colloidal gold test paper strips field sample tests.
【Embodiment】
Following examples are used to explain technical scheme without limitation.
In the present invention, unless otherwise specified, for representing that " % " of concentration is weight percentage, " part " is weight
Part.
The preparation of the polyclonal antibody of hopZ5 gene of the embodiment 1 containing PSA3
Pseudomonas syringae pv.actinidiae PSA3 collection, separation and identification:Burst from Dujiang weir Kiwi berry producing region
The ulcer disease morbidity orchard collection typical incidence of leaf of symptom, in laboratory, disease makees the separation of opportunistic pathogen.
Cut blade morbidity scab about 5mm sizes, first with aseptic water washing, then with 0.5% sodium hypochlorite soak 10min enter
Row surface sterilization, then with sterilized water immersion rinsing 3 times, each 10min.Blade after sterilization is smashed to pieces, be soaked in 200 μ l without
In bacterium water, oese picks sterilized water in culture of being rule on LB culture mediums.
With reference to Reese Gorge et al. reports of 2010, carried out using 16s rDNA primers and HopZ5 primers dual
PCR is expanded.
Primer is as follows:
PsaF1:5'-TTTTGCTTTGCA CACCCGATTTT-3';
PsaR2:5'-CACGCACCCTTCAATCAGGATG-3.
HopZ5-F:5'-TCACTCCTAGACTGGAATAC-3';
HopZ5-R:5'-GGCTATCATGAAGGCTG TCA-3'.
After double PCR amplification, amplification delivers to sequencing, strain CRAFRU14.08, NZ-47 and ICMP1884 with PSA
The homology of (accession number is respectively CP019732.1, CP017009.1 and CP011972) reaches 99-100%.Sequencing result shows
The bacterium that separation is identified is Pseudomonas syringae pv.actinidiae and monoid is PSA3.
According to the sequences Design hopZ5 of the strains of Pseudomonas syringae pv.actinidiae ICMP 18884
The specific primer of full genome is cloned:
Upstream sequence:Hopz5-BamHI-F:5’-CGCGGATCCATGGGACTTTGTGCATCA-3;
Downstream sequence:Hopz5-XhoI-R:5’-CGGTCTCGAGTTAGGATTCTATCGCTTTTC-3’.
Pcr amplification reaction system:The 2 μ L of μ L, DNA of PrimeSTAR HS DNA Polymerase 0.3, up and down primer (10
μM) 2 10 μ L of μ L, 10 × Buffer of each 1 μ L, dNTP (2.5mM), plus ddH2O supplies 25 μ L, mixes laggard performing PCR reaction.
PCR amplification conditions are 94 DEG C of 4min, 94 DEG C of 30sec, 53 DEG C of 30sec, 72 DEG C of 1min30sec, 33 circulations, 72 DEG C
10min, 12 DEG C of preservations.
After the sequencing of gained hopZ5 genetic fragments is correct, two kinds of Restriction Enzyme BamH I and Xho I share enzyme cutting buffering liquid
10 × H Buffer carry out double digestion, and digestion products are connected to prokaryotic expression carrier PET28, obtain the weight containing hopZ5 genes
Group plasmid recombinant plasmid PET28a-hopZ5, with the restructuring mass transitions E. coli BL21, egg is induced through IPTG
White expression and purifying, obtain albumen hopZ5;
Prokaryotic expression protein hopZ5 is sent to Suzhou You Nuoke bio tech ltd and carries out immune New Zealand great Bai
Rabbit, prepares the polyclonal antibody PAb-hopZ5.
Embodiment 2PSA3 hopZ5 albumen Western-blot detections
SDS-PAGE electrophoresis:
(1) clean glass plate and dry, clip the glass plate for recording polyacrylamide gel;
(2) configuration of separation gel:The SDS-PAGE separation gels of preparation 12%, are sequentially added in beaker:30%ACR
The μ l of 4mL, 1.5mol/L Tris-HCL (pH8.8) 2.5mL, 10%SDS 100,10% ammonium persulfate 100,5 μ l of μ l, TEMED,
Sterile deionized water complements to 10ml, is added to after shaking up with pipettor in the gap of two pieces of glass plates;
(3) sterilized water is added on separation gel, 30min is stood, after gelling to be separated is solid, pours out sterilized water, use filter paper
Exhaust residual liquid;
(4) SDS-PAGE for preparing 5% concentrates glue, is sequentially added in beaker:30%ACR 670 μ l, 1mol/L
Tris-HCL (pH6.8) 1.25mL, 10%SDS 50 μ l, the μ l of 10% ammonium persulfate 50, the μ l of tetramethylethylenediamine (TEMED) 4, go out
Bacterium deionized water complements to 5mL, is added to after shaking up on separation gel, inserts comb;
(5) processing of sample:Embodiment 1 is obtained into polyclonal antibody PAb-hopZ5 according to volume ratio 5:1 ratio is added
5 × SDS-PAGE sample-loading buffers, are then placed in 100 DEG C of hot water and boil 3min, take out standby;
(6) comb is extracted after the gel of step (4) solidifies completely, be fixed in electrophoretic apparatus, plus electrophoretic buffer, directly
To loading wells is covered, the μ l of sample 10 treated with pipettor gun aspiration step (5) are in gel pore;
(7) electrophoretic apparatus is connected with power supply, voltage is transferred to 80V, when bromophenol blue electrophoresis to be instructed is to separation gel, by electricity
Pressure is transferred to 120V, is powered off when bromophenol blue to separation gel bottom, takes out polyacrylamide gel.
Transferring film:
(1) after electrophoresis terminates, excision concentration glue;
(2) clip filter paper and nitrocellulose filter, make it soak completely in transferring film liquid, cut off a corner as note
Number;
(3) place in the following order:Plastic splint (the black)-filter paper of foam-rubber cushion-3-gel-nitrocellulose
- 3 filter paper of filter membrane-foam-rubber cushion-plastic splint (white), often add one layer, it is necessary to align, and discharge bubble.After being well placed,
By Boards wall in electric turn trough, power supply is connected, the clearance position that ice bag is put into electric turn trough carries out electrotransfer, 100V, 1h.
Hybridization:
(1) closing of film:After electrotransfer terminates, take out nitrocellulose filter and be put into clean culture dish, add closing
Film is totally immersed into by liquid (PBS solution for containing 5% defatted milk), is lain against room temperature on horizontal shaker and is acted on 2h;
(2) film is washed:Nitrocellulose filter is put into a new culture dish, cleaning solution PBS is added, lies against shaking table
Upper room temperature shakes 5min, and repeated washing 3 times;
(3) primary antibody, i.e. polyclonal antibody PAb-hopZ5 are added:Nitrocellulose filter after washing is put into one newly
Culture dish in, by film be totally immersed into PAb-hopZ5 antibody-solutions (with containing 5% skimmed milk power PBS solution PAb-hopZ5
Antibody is diluted to 1:2000), lie against and 3h is reacted at room temperature on shaking table;
(4) film is washed:Nitrocellulose filter is washed into film with PBS 3 times, each 5min;
(5) company of the Zhong Shan Golden Bridge alkali phosphatase enzyme mark goat anti-rabbit igg secondary antibody of commercialization is added:Nitrocellulose is filtered
Film is put into a culture dish, and film is totally immersed into two corresponding anti-solution and (secondary antibody is diluted to 1 with the PBS solution containing 5% skimmed milk power:
2000), lie against and 2h is reacted at room temperature on shaking table;
(6) film is washed:Nitrocellulose filter is washed into film with PBS 3 times, each 5min;
(7) develop the color:Nitrocellulose filter is taken out, is put into substrate solution and (adds 100 μ l in 3.8ml substrate buffer solutions
NBT and 100 μ l BCIP), lucifuge reaction 10-15min, to positive colour developing
(8) terminating reaction:Solution is removed, with distillation washing film, terminating reaction observes result and preserves diaphragm.
Western blot results are as shown in figure 1, be capable of detecting when target stripe and single, and size is 39kD, it was demonstrated that institute
The antibody of preparation preferably, can be used in follow-up requirement of experiment.
Embodiment 3PSA3 hopZ5 point immune detections sample type analysis
HopZ5 is an effector for Prospect on Kiwifruit Bacterial Canker bacterium, and effector is one of common virulence factor of pathogen, because
Can this need checking hopZ5 antibody detect the PSA cultivated on culture medium and disease plant.
With the PSA bacteria suspensions of culture, total protein, the tissue soak of susceptible tissue extraction are sample.Wherein Kiwi berry is burst
Ulcer disease reference culture (Pseudomonas syringae pv.actinidiae, PSA) is from Plant Pathology laboratory point
From and identify strain.The first generation strain for being stored in -70 DEG C is activated.
The preparation process of PSA bacteria suspensions is:The first generation strain after activation is taken to carry out setting-out training on LB solid mediums
Support, be then put in 25 DEG C of constant incubator cultures, by the sterile water elution of bacterium colony, be prepared as PSA bacteria suspensions.
It is susceptible tissue total protein extraction process be:Liquid nitrogen is fully ground material, according to 1g:2ml mass volume ratio, is added
Protein extract buffer (50mmol/L Tris.HCl (PH 6.8), 4%SDS, 6% beta -mercaptoethanol, 4mol/L urea, 10%
Glycerine).100 DEG C of warm bath 10min, then 12000g centrifugations 10min, takes supernatant as total protein.
Tissue soak preparation process be:Fine grained chippings will be cut into hyphostroma, be added into 0.005g sample fragments sterile
The μ l of water 20, soak 10 seconds, draw soak standby.
Three kinds of samples respectively take 8 μ L drops on nitrocellulose filter, are incubated and develop the color by closing, primary antibody and secondary antibody, colour developing
Reaction result is shown in Fig. 2.It can be seen that three kinds of samples can develop the color on nitrocellulose filter, illustrate tissue soak and the total egg extracted
Bai Xiangtong, is used equally for DIBA to detect, but the sample making course of tissue soak simplifies, and need to will be with hyphostroma chopping immersion only
Can;In addition, PSA bacteria suspensions are still using hopZ5 antibody tests.As a result illustrate the hopZ5 antibody of the present invention available for various
The detection of type of sample.
Field sample detection is immunized in embodiment 4PSA3 hopZ5 points
Chinese gooseberry garden is gathered to the field sample returned to be handled, sample is cut into fritter, is peeled off and put with scalpel
Enter in centrifuge tube, add 10-20 μ L sterilized water, 8 μ L soaks points are drawn after 10s seconds on nitrocellulose filter, are remained
Under sample be used for extract plant tissue STb gene and for PCR detection, two kinds of detection methods are compareed, repeatedly 15
Group.
As a result show, except sample 3 shows false positive, that is, put and binding tests DIBA colour developings are immunized and PCR fails detection
Go out result, show as two kinds of detection means acquired results inconsistent.Other samples in addition show as DIBA's and PCR
As a result it is consistent, as a result see Fig. 3.Illustrate that DIBA detection method can be used for detecting field sample, with higher confidence level, accurately
Rate reaches more than 90%.Also, compared with traditional detection method such as PCR method, using antibody PAb-hopZ5's of the invention
Point immunologic detection method is simple to operate, is adapted to large-scale detection sample.
Embodiment 5PSA3 hopZ5 immunity colloidal gold test paper strip specific tests
Sequentially immune colloid gold test paper is constituted in bottom plate over-assemble sample pad, gold standard pad, nitrocellulose filter and blotting paper
Bar.Wherein, gold standard pad contains colloidal gold antibody compound, and the colloidal gold antibody compound contains 50 μ g/ml polyclonal antibody
PAb-hopZ5 and colloidal gold solution, then with 0.1mol/L HCl/water solution and 0.1mol/L K2CO3The aqueous solution is adjusted to pH
Value 7.2, is used as colloidal gold antibody compound.
By Prospect on Kiwifruit Bacterial Canker bacterium, pseudomonas syringae tomato pvs oryzae and oryzicola, bacillus megaterium, pseudomonas putida,
Pseudomonas fluorescens, bacillus subtilis, PSA1 and PSA2 are prepared as 1 × 108Cfu/mL bacteria suspension, using clear water as control,
Sequentially number standby.
Take 200 μ l bacteria suspensions to be added in the sample pad of immunity colloidal gold test paper strip, stand 5-10min, observation T lines and C
Line.When T lines and C lines show that red line is the positive simultaneously, only T lines show that red line is feminine gender, and T lines and C lines do not show red line
For null result.The colour developing situation of 9 parts of samples is as shown in Figure 4.
Learnt from Fig. 4, hopZ5 immunity colloidal gold test paper strips only the PSA3 of No. 1 sample bacteria suspension is just shown as T lines with
C lines develop the color simultaneously, are judged as the positive according to colour developing result, are consistent with sample itself.For bacteria suspension (the sample 2- without PSA3
9) only T lines, are shown as and show red line, feminine gender is judged as according to colour developing result, is consistent with sample itself.It is therefore seen that, this
The specific effect of the immunity colloidal gold test paper strip of invention preferably, detection PSA3 that can be special.
Embodiment 6PSA3 hopZ5 immunity colloidal gold test paper strips field sample test
Field Plants tissue is taken, is cut into small pieces, is put into sterile mortar, 1mL sample loading buffers are added, soaked 12 seconds,
Detected sample is used as using soak.Residue is organized to be used to extract plant genomic DNA, enters performing PCR detection, with immune colloid gold test paper
Bar result is compareed, and is repeated 32 times.
Take the immunity colloidal gold test paper strip of the present invention to lie against on desktop, draw 200 μ L field sample immersions to be detected
Liquid stands 10min in sample pad, observes testing result.
Immunity colloidal gold test paper strip detects field sample result as shown in figure 5, wherein, 2,8,14,21, No. 26 samples only show
Shown T lines, be feminine gender according to chromogenic reaction judged result, i.e., non-PSA3 infection.Compareed, examined according to PCR by the result with PCR
Survey confirm No. 14 and No. 26 samples it is actual to be positive, result is not consistent with developing the color, thus it is speculated that No. 14 samples are not by immune colloid gold
It is relatively low that the reason for test strips are correctly detected essentially consists in sample content of molds.And No. 26 samples not by immunity colloidal gold test paper strip just
Really it is the reason for detection, through follow-up identification, the infection type of the sample is PSA2 monoids.
In 32 parts of samples, the testing result of the immunity colloidal gold test paper strip of remaining sample is consistent with PCR detection, shows this
The PSA3 of invention hopZ5 immunity colloidal gold test paper strip testing results are relatively stable.
In summary, PSA3 polyclonal antibodies of the invention have the characteristics of detection specificity is high and applied widely;Carry
The Prospect on Kiwifruit Bacterial Canker bacterium PSA3 point immune responses of confession and immunity colloidal gold test paper strip, detection method are simple, can detect big simultaneously
Sample is measured, with the value in basic unit's promotion and application.
Sequence table
<110>Sichuan Agricultural University
<120>A kind of polyclonal antibody and its application for being used to detect Prospect on Kiwifruit Bacterial Canker bacterium PSA3
<130> 17155.1
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Hopz5- BamH I -F
<400> 1
cgcggatcca tgggactttg tgcatca 27
<210> 2
<211> 30
<212> DNA
<213> Hopz5-Xho I-R
<400> 2
cggtctcgag ttaggattct atcgcttttc 30
<210> 3
<211> 23
<212> DNA
<213> PsaF1
<400> 3
ttttgctttg cacacccgat ttt 23
<210> 4
<211> 22
<212> DNA
<213> PsaR2
<400> 4
cacgcaccct tcaatcagga tg 22
<210> 5
<211> 20
<212> DNA
<213> HopZ5-F
<400> 5
tcactcctag actggaatac 20
<210> 6
<211> 20
<212> DNA
<213> HopZ5-R
<400> 6
ggctatcatg aaggctgtca 20
Claims (8)
- Purposes of the 1.hopZ5 genes in detection Prospect on Kiwifruit Bacterial Canker bacterium PSA3.
- 2. a kind of preparation method for being used to detect Prospect on Kiwifruit Bacterial Canker bacterium PSA3 polyclonal antibodies, methods described includes following step Suddenly:To Pseudomonas syringae pv.actinidiae (Pseudomonas syringae pv.actinidiae, PSA) HopZ5 genes are expanded, after the sequencing of gained hopZ5 genetic fragments is correct, by Restriction Enzyme BamH I and Xho I digestions, Digestion products are connected to prokaryotic expression carrier PET28, the recombinant plasmid PET28a-hopZ5 containing hopZ5 genes are obtained, with institute Restructuring mass transitions E. coli BL21 is stated, expresses and purifies through IPTG inducible proteins, obtain albumen hopZ5;With institute HopZ5 protein immunization new zealand white rabbits are stated, polyclonal antibody PAb-hopZ5 is prepared.
- 3. preparation method according to claim 2, it is characterised in that the primer expanded to hopZ5 genes is as follows:Hopz5-BamH I-F:5’-CGCGGATCCATGGGACTTTGTGCATCA-3;Hopz5-Xho I-R:5’-CGGTCTCGAGTTAGGATTCTATCGCTTTTC-3’;Pcr amplification reaction system:The 2 μ L of μ L, DNA of PrimeSTAR HS DNA Polymerase 0.3, up and down primer (10 μM) The 10 μ L of μ L, 10 × Buffer of each 1 μ L, dNTP (2.5mM) 2, plus ddH2O supplies 25 μ L, mixes laggard performing PCR reaction;PCR amplification conditions are:94 DEG C of 4min, 94 DEG C of 30sec, 53 DEG C of 30sec, 72 DEG C of 1min30sec, 33 circulations, 72 DEG C 10min, 12 DEG C of preservations.
- 4. preparation method according to claim 2, it is characterised in that the digestion step includes:10 × H of enzyme cutting buffering liquid Buffer are shared by Restriction Enzyme BamH I and Xho I and carry out double digestion, target gene is double Digestion system:10×H Buffer 2μL;Hopz5 8μL;BamH I 1μL;Xho I 1μL;Use ddH2O supplies 20mL;On State system to be added on ice in 200 μ LEP pipes, be put into after mixing in 37 DEG C of thermostat water baths, carry out digestion and stay overnight;Then take Purpose band is extracted after going out the agarose gel electrophoresis nucleic acid staining dye for 1%, according to DNA glue reclaim kit specifications Purpose fragment is reclaimed, purpose fragment is obtained.
- 5. the immunity colloidal gold test paper strip of the polyclonal antibody obtained containing preparation method according to claim 1, described Test strips include sample pad, gold standard pad, nitrocellulose filter and the blotting paper being sequentially assembled on bottom plate, it is characterised in that described Gold standard pad contains colloidal gold antibody compound, and the colloidal gold antibody compound contains preparation side according to claim 2 Polyclonal antibody and colloidal gold solution that method is obtained, the concentration of the polyclonal antibody are not less than 50 μ g/ml, Ran Houyong 0.1mol/L HCl/water solution and 0.1mol/L K2CO3The aqueous solution is adjusted to pH value 7.2, is obtained the colloidal gold antibody and is combined Thing.
- 6. test strips according to claim 5, it is characterised in that the preparation method of the colloidal gold antibody compound is:(1) 10mL colloidal gold solutions are added in clean beaker, 0.1mol/L HCL and 0.1mol/L K is added while stirring2CO3 Colloidal gold solution is adjusted to pH value 7.2;(2) lasting stirring is lower adds 500 μ g PAb-hopZ5 antibody according to claim 2, stirs 1h;(3) gold labeling antibody for combining stabilization is sub-packed in 2mL centrifuge tube, 4 DEG C, 2000r/min, 20min discard cohesion The precipitation of gold grain formation;(4) red supernatant is taken to be transferred to new centrifuge tube, 4 DEG C, 15000r/min, 30min abandon supernatant;(5) the kermesinus precipitation of ttom of pipe flowing is gold labeling antibody, and the flowable kermesinus precipitation in collecting pipe bottom is slow with 1 × PBS Fliud flushing, which will be precipitated, to be resuspended in 1/10,4 DEG C of original volume and is kept in dark place.
- 7. a kind of use immunity colloidal gold test paper strip detection Prospect on Kiwifruit Bacterial Canker bacterium PSA3 method, methods described includes following step Suddenly:(1) it is according to the ratio of testing sample and sample loading buffer to diameter 1-2mm fragments by Kiwi berry sample comminution to be detected 0.05~0.2g:250~400 μ l, 2-15s in sample loading buffer is immersed in by testing sample;(2) buffer solution for having soaked testing sample of 200 μ L steps (1) is taken to be added on immune colloid according to claim 5 In the sample pad of gold test paper strip, detection zone T lines and C lines that 5-10min observes immunity colloidal gold test paper strip are stopped;(3) judge that testing sample infects product for Prospect on Kiwifruit Bacterial Canker bacterium PSA3 when T lines and C lines show as claret.
- 8. a kind of Prospect on Kiwifruit Bacterial Canker bacterium PSA3 point immunologic detection method, the described method comprises the following steps:(1) it is according to the ratio of testing sample and sample loading buffer to diameter 1-2mm fragments by Kiwi berry sample comminution to be detected 0.005~0.01g:20~50 μ l, 2-15s in sample loading buffer is immersed in by testing sample;(2) buffer solution for having soaked testing sample of step (1) is taken, is added dropwise on nitrocellulose filter, then drying at room temperature;(3) closing 1h is carried out to the nitrocellulose filter of step (2) with the aqueous solution of 5wt% defatted milks;(4) 1h is incubated with polyclonal antibody PAb-hopZ5 according to claim 2, then washed with phosphate buffer PBS Wash 3 times;(5) 1h is incubated with alkali phosphatase enzyme mark goat anti-rabbit igg again, is then washed 3 times with phosphate buffer PBS;(6) chromogenic reaction is carried out under light protected environment using BCIP/NBT alkaline phosphatase colour reagent boxes, colour developing sample judges Sample is infected for Prospect on Kiwifruit Bacterial Canker bacterium PSA3.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110904252A (en) * | 2019-12-16 | 2020-03-24 | 中国科学院武汉植物园 | Method for rapidly detecting canker of kiwi fruit pollen |
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