CN109536522A - A kind of preparation and application of the prokaryotic expression, polyclonal antibody of southern rice black-streaked dwarf virus P6 albumen - Google Patents
A kind of preparation and application of the prokaryotic expression, polyclonal antibody of southern rice black-streaked dwarf virus P6 albumen Download PDFInfo
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Abstract
The invention discloses the preparation method of a kind of prokaryotic expression of southern rice black-streaked dwarf virus P6 albumen, polyclonal antibody, this method includes extracting the RNA of southern rice black-streaked dwarf virus, reverse transcription cDNA, design primer to expand its P6 coat protein, construction recombination plasmid, escherichia coli prokaryotic expression, immunity inoculation, prepare polyclonal antibody.The invention also discloses a kind of southern rice black-streaked dwarf virus P6 protein polyclone antibodies in the application for detecting plant tissue SRBSDV viral level.Polyclonal antibody of the present invention has testing result reliable, quantitative detection can be carried out to the viral level in rice different tissues, so that the prediction and warning and science bridle for southern rice black-streaked dwarf disease provide technical support and theory support.
Description
Technical field
The present invention relates to the preparation of the prokaryotic expression, polyclonal antibody of a kind of southern rice black-streaked dwarf virus P6 albumen and
Using belonging to field of biotechnology.
Background technique
Southern rice black-streaked dwarf virus (Southern rice black-streaked dwarf virus, SRBSDV)
Belong to Reoviridae, Fijivirus category, be rice black-streaked dwarf virus (Rice black-streaked dwarf disease,
RBSDV) belong to virus.China found SRBSDV on the rice of Yangjiang city in 2001 first, then in China
Hainan, Jiangxi, Yunnan and Guizhou etc. area large area occur, the virosis cause when serious part field diseased plant rate up to 80% with
On, it causes large area to lose and receives.Southern rice black-streaked dwarf virus disease and rice black-streaked dwarf virus disease are drawn on the crops such as rice
It is all extremely similar in terms of the Disease symptoms, virion, geographical distribution and the host range that rise etc..Currently, south caused by SRBSDV
Square rice black-streaked dwarf virus disease has become a kind of ascending disease on rice, seriously affects rice safety production.
SRBSDV encodes 13 kinds of albumen, wherein 6 kinds of structural proteins (P1, P2, P3, P4, P8, P10) and 7 kinds of non-structural proteins
White (P5-1, P5-2, P6, P7-1, P7-2, P9-1, P9-2).Belong to viral sequence analysis and analysis with other Fijivirus: pushing away
Measure the function of 13 albumen.The structural proteins P1 size about 169kDa of S1 coding, has typical RdRp (RNA-
Dependent RNA Polymerase) gene order；The size about 141kDa virus core capsid protein P2 of S2 coding；S3 is compiled
The albumen P3 of code size about 135kDa, is a kind of capping enzyme；S4 encodes the albumen P4 of size about 132kDa, is virus coat Type B
Spike protein；The P5-1 albumen of S5-1 coding may participate in the formation of viroplasm；S6 encodes the albumen P6 of about 90 kDa of size,
The homology of the nucleotide of SRBSDV S6 and RBSDV S6 is that the P6 albumen of 70.6%, RBSDV S6 coding can interfere with DNA first
The system of defense of base and host shows that P6 is a kind of silencing suppressor；S7 is separately encoded about 40.9 kDa albumen P7-1 of size
With 36.2 kDa albumen P7-2；P7-1 can form the tubular structure of package virion.Utilize Sf9 baculovirus expression system
With BiFC experiments have shown that SRBSDV P7-1 albumen has the ability of independent induced synthesis Minute Tubule Structures；S8 encodes size about 68
The albumen P8 of kDa, for the core capsid albumen of virus；The outer layer capsid protein of S10 coding virus.
To understand and grasp rice pathogenesis situation, reinforce to the early monitoring of south China black streaked dwarf virus of rice and pre-
It is alert, it is badly in need of establishing the detection method for being directed to rice SRBSDV.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of south of a kind of high yield, easy purification, low cost
The prokaryotic expression method of square rice black-streaked dwarf virus P6 albumen.
The southern rice black-streaked dwarf virus P6 albumen that it is a further object of the present invention to provide a species specificity is good, potency is high
The preparation method of polyclonal antibody.
Another object of the present invention is to provide a kind of southern rice black-streaked dwarf virus P6 protein polyclone antibody and is detecting
The application of plant tissue SRBSDV viral level.
The technical scheme is that a kind of prokaryotic expression method of southern rice black-streaked dwarf virus P6 albumen, including
Following steps:
(1) RNA of southern rice black-streaked dwarf virus is extracted；
It (2) is cDNA by RNA reverse transcription；
(3) using cDNA as the P6 coat protein gene of template amplification SRBSDV, the amplified production of P6 coat protein gene is obtained
With prokaryotic expression carrier pET-28a；
(4) recombined pronucleus expression plasmid pET-28a-P6 is constructed
Amplified production and prokaryotic expression carrier pET-28a to P6 coat protein gene carry out digestion respectively, recycle digestion
Product, and digestion products are attached, connection product converts competent escherichia coli cell, and screening, identification are obtaining building just
True recombinant plasmid pET-28a-P6；
(5) expression and purifying of P6 albumen
By recombinant plasmid pET-28a-P6 convert Bacillus coli cells, screening positive clone, Fiber differentiation, lytic cell,
Precipitating is collected by centrifugation, washs to obtain thick leach protein, then through protein purification system purifying and desalination, obtain albumen after purification.
In step (3), using cDNA as template, the P6 coat protein gene of following primer amplification SRBSDV, primer are utilized
Sequence is as follows:
Upstream primer: CGGGATCCATGTCTACCAACCTCACGAACATA, wherein underscore is BamHI restriction endonuclease；
Downstream primer: GCTCTAGATTACTCTGAAATAAGTTGCCACAA, wherein underscore is Xho I restriction endonuclease；
Upstream and downstream primer is respectively as shown in SEQ ID NO.1,2.
In step (4), the amplified production and prokaryotic expression carrier pET-28a of P6 coat protein gene are respectively adopted
BamHI and Xho I enzyme carries out digestion.
The present invention also provides a kind of preparation methods of southern rice black-streaked dwarf virus P6 protein polyclone antibody, after purification
P6 protein immune animal, separate serum, obtain protein polyclone antibody.
Immune animal is new zealand rabbit.
The present invention also provides a kind of southern rice black-streaked dwarf virus P6 protein polyclone antibodies in detection plant group
Knit the application of SRBSDV viral level.
The beneficial effects of the present invention are: utilizing prepared by the preparation method of polyclonal antibody of the present invention more grams of SRBSDVP6
Grand antibody, ELISA detection show the potency of prepared SRBSDVP6 polyclonal antibody up to 1:160000, and testing result can
It leans on.The SRBSDV P6 protein polyclone antibody prepared in the present invention is capable of the SRBSDV in the detection rice of specificity, and can
With the viral level for detecting rice different tissues, therefore, have great importance to the Function Identification and research of albumen,
It can be used for the early warning that SRBSDV occurs, so that the prediction and warning and science bridle for southern rice black-streaked dwarf disease mention
For technical support and theory support.
Detailed description of the invention
Fig. 1 is pET-28a Vector map；
Fig. 2 is digestion result schematic diagram, and 1 is the Plasmid DNA of digestion, and 2 be BamHI and Xho I digestion as a result, 3 be DNA
Marker；
Fig. 3 is the albumen peak figure that SRBSDV P6 albumen is obtained by the purifying of AKTA protein purification system and desalination；
Fig. 4 is the electrophoresis result figure after SRBSDV P6 protein purification；
Fig. 5 is ELISA detection figure；
Fig. 6 is the SRBSDV virus used in SRBSDV P6 and HSP antibody test rice leaf and stem tissue respectively,
Control is the rice of health, and HS (Huishui), PT (Pingtang), LB (Libo) is the rice sample of morbidity.
Specific embodiment
With reference to the accompanying drawing and invention is described further in specific embodiment:
The building and identification of embodiment 1:pET-28a-P6 recombinant plasmid
Go bail for deposit infection SRBSDV rice sample, utilize virus RNA extraction kit (Tiangeng biochemical technology (Beijing)
Co., Ltd) total serum IgE is extracted, cDNA is obtained with reverse transcription reagent box (Promega) reverse transcription.
Design primer expands P6 using cDNA as template.
Primer is as follows:
Upstream primer: CGGGATCCATGTCTACCAACCTCACGAACATA, as shown in SEQ ID NO.1, wherein lower stroke
Line is BamHI restriction endonuclease.
Downstream primer: GCTCTAGATTACTCTGAAATAAGTTGCCACAA, as shown in SEQ ID NO.2, wherein lower stroke
Line is Xho I restriction endonuclease.
PCR amplification system used in DNA cloning for vector construction is as follows: 5 × buffer, 10 μ L, FastPfu Fly
DNA Polymerase 1 μ L (Quan Shijin), 2.5mmol dNTPs4 μ L, 1 μ L of DNA profiling (cDNA that reverse transcription obtains), 10 μ
Each 32.5 μ L of 1 μ L, ddH2O of mol upstream and downstream primer.
The following are PCR amplification conditions: 95 DEG C of 2min, 35 circulation (95 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 1min, 72 DEG C
5min.After PCR product is purified, it is connected to pEASY carrier (Quan Shijin).
PCR product is returned with reference to Nanjing Vazyme Biotechnology Co., Ltd.'s Ago-Gel QIAquick Gel Extraction Kit specification
It receives.Product after the recovery is inserted into the pET-28a through same double digestion by DNA ligase after BamHI and Xho I double digestion
In expression vector (expression vector map is as shown in Figure 1), 16 DEG C are connected 16 hours, are constructed pET-28a-P6 recombinant plasmid, will be weighed
Group plasmid is transformed into coli strain, cultivates on 50ug/mL kanamycins solid plate in 37 DEG C, picking individual colonies are through enzyme
It is correct rear (digestion result is as shown in Figure 2) to cut identification, then passes through its sequence of Beijing Qing Ke Biotechnology Co., Ltd sequence verification
Correctness.Sequencing result shows that SRBSDVP6 coding sequence obtained fulfills the expectation.Illustrate construction of recombinant plasmid
Success is simultaneously named as pET-28a-P6.
Expression and purifying of the embodiment 2:pET-28a-P6 in Escherichia coli
1: obtaining the recombinant strains of expression SRBSDV P6
It chooses the successful monoclonal of sequencing to be inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C, 200rpm trains overnight
It supports, mentions pET-28a-P6 recombinant expression carrier according to Nanjing Vazyme Biotechnology Co., Ltd.'s small amount plasmid extraction kit
It takes out, takes recombinant expression carrier to convert e. coli bl21 (DE3) bacterial strain, be coated on the plate of kanamycins containing 50ug/mL
It is incubated overnight in 37 DEG C, picking single colonie is inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C, 200rpm mistake
50% glycerol of sterilizing is added in bacterium solution, the protokaryon for obtaining expression SRBSDV P6 in -80 DEG C of refrigerators is frozen after mixing for night culture
Express bacterial strain.
The expression and purifying of 2:SRBSDVP6 recombinant protein
(1) inducing expression of recombinant protein
Above-mentioned recombined pronucleus expression bacterial strain is inoculated with into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C, 200rpm mistake
Night cultivates activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to again in 50ug/mL kanamycins fluid nutrient medium, 37
When DEG C shake culture is to OD600=0.5, the IPTG of final concentration of 0.7mM is added, (takes and does not lure within Fiber differentiation 16 hours at 18 DEG C
The bacterium led makees negative control).4 DEG C after the completion of induction, 4000rpm is centrifuged 15min, collects thallus, bacterial precipitation and negative control
With 2mL pre-cooling PBS buffer (with preceding addition 1mM PMSF) suspension, clasmatosis is carried out with Ultrasonic Cell Disruptor, each 10sec,
Totally 10 times, every minor tick 30sec；4 DEG C, 15,000rpm centrifugation 20min take supernatant, carry out protein purification.
(2) protein purification
Purifying and desalination are carried out using AKTA protein purification system, it is preferable that use AKTA purifier10 fast protein
Purification system carries out protein purification, and steps are as follows:
Since pET28a has 6 His coded sequences before terminator, so pET28a-P6 recombinant expression carrier obtains
Albumen can use Ni-NTA carry out affinitive layer purification.Affinity chromatography is with protein or large biological molecule and to be incorporated in Jie
Based on special affinity in matter between aglucon, because being connected to a NTA in the matrix of chromatography gel used in His-tags
[(nitrilotriacetic acid) nitrilotriacetic acid], can with Ni ions binding, and the 6 of Ni ion and fusion protein ×
Attraction is generated between His amino acid, so that the fusion protein with His-tags histidine tag be distinguished with other albumen
It comes, five yuan of imidazole rings of histidine residues are the key that albumen and Ni ionization, therefore with the imidazoles of high concentration
(Imidazole) solution elution when marquis (such as 400mM), imidazoles be convenient for protein His-tags imidazole ring competitive binding, most
Fusion protein is eluted from gel at last.
Concrete operations: taking out the thallus of -80 DEG C of preservations, and Lysis buffer is added and is resuspended, the bacterium solution after resuspension is in ice bath
Middle carry out ultrasonic disruption, power 350W, duty ratio 50%, each circulation 30s, total time 35min.It is broken even
Slurry, 4 DEG C, 12000rpm centrifugation 30min.Supernatant is adsorbed on Ni-NTA adsorption column with peristaltic pump, 5mL/min, repeats one
It is secondary, retain 20 μ L by the sample of adsorption column and carries out protein adhesive electrophoresis；Ni-NTA adsorption column is connected to fast protein purifying system
On system, eluted；Albumen is collected according to UV absorption curve, and samples carry out protein adhesive electrophoresis.
Fig. 3 is the albumen peak figure that SRBSDV P6 albumen is obtained by the purifying of AKTA protein purification system and desalination.SRBSDV
P6 purifying figure ultraviolet (UV) absorption value when being purifying P6 albumen, since SRBSDV P6 albumen is RNA silencing suppressor coding
Albumen is non-structural protein, therefore expression quantity is lower, causes in SRBSDV P6 purifying figure after appearance UV value with imidazole concentration
Increase and there is raising trend；SRBSDV P6 desalination is then UV absorption value when purifying obtained P6 albumen desalination.
Fig. 4 is the expression and purification P6 albumen result at 18 DEG C, IPTG concentration 0.7mM.SRBSDV P6 protein purification procedures
In, the distribution of albumen.1 is albumen marker, and 2 be full cell liquid after cell ultrasonication, and 3 be supernatant, and 4 be precipitating, and 5 be warp
Cross Ni column it is affine after efflux, 6 for purifying when appearance position efflux, 7 effluxes of appearance position when being desalination；Horizontal line institute in figure
The position that mark instruction SRBSDV P6 occurs.It is shown in upper figure, P6 albumen often has in supernatant under this condition, passes through
The P6 purity of protein that AKTA protein purification system obtains after purification reaches requirement, can be used for subsequent experimental.
The preparation of embodiment 3:SRBSDV P6 protein polyclone antibody and antibody titer ELISA detection
(1) preparation of polyclonal antibody
1. immune process:
The pET-28a-P6 albumen of 1.1 expression and purifications is stored in 4 DEG C as immunizing antigen, packing；
1.2 the 1st days, take 1ml antigen that 1ml Freund's complete adjuvant is added, emulsification (examines emulsification degree: 1 drop is emulsified antigen
Drop enters in physiological saline, if not scattering, shows to have reached requirement), the subcutaneous multiple spot of the nape of the neck (at least 8 points) injection is immunized 2
New Zealand White Rabbit；
1.3 the 15th days, take 1ml antigen that 1ml freund 's incomplete adjuvant is added, emulsification (examines emulsification degree: 1 drop is emulsified
Antigen liquid instills in physiological saline, if not scattering, shows to have reached requirement), the subcutaneous multiple spot of the nape of the neck (at least 8 points) injection is exempted from
2 New Zealand White Rabbit of epidemic disease；
1.4 the 29th days, take 1ml antigen that 1ml freund 's incomplete adjuvant is added, emulsification (examines emulsification degree: 1 drop is emulsified
Antigen liquid instills in physiological saline, if not scattering, shows to have reached requirement), the subcutaneous multiple spot of the nape of the neck (at least 8 points) injection is exempted from
2 New Zealand White Rabbit of epidemic disease；
1.5 the 43rd days, take 1ml antigen that 1ml freund 's incomplete adjuvant is added, emulsification (examines emulsification degree: 1 drop is emulsified
Antigen liquid instills in physiological saline, if not scattering, shows to have reached requirement), the subcutaneous multiple spot of the nape of the neck (at least 8 points) injection is exempted from
2 New Zealand White Rabbit of epidemic disease；
1.6 the 53rd days, arteria carotis took blood, and rabbit is put to death；
1.7 rabbit blood are stood overnight at 4 DEG C, and supernatant is collected in centrifugation (4 DEG C, 10000rpm) 30 minutes.
2. serum purifies:
Serum obtains the antibody that specificity is directed to destination protein through ProteinA column purification
2.1 serum are through 0.45um membrane filtration；
2.2 take out proteinA column, are balanced with 10 times of column volume PBS, and after PBS drains off, serum loading crosses column, collect stream
Liquid out；
2.3 10 times of column volume PBS balances, after draining off, are added the pH2.7 glycine solution of the 0.1M of 5 times of column volumes, receive
Collect eluent, every pipe 1ml is previously added 90ul1M Tris solution (pH8.8) neutralization in collecting pipe；
2.4 are added 10 times of column volume PBS balances, and 20% ethyl alcohol of 2 times of column volumes, 4 DEG C of preservation pillars are added after draining off；
The light absorption value of the 2.5 eluent detection 280nm collected, merging of the light absorption value greater than 1.0, PBS dialysed overnight,
Elisa detects potency.
(2) antibody titer ELISA is detected
ELISA process:
3.1 coatings: antigen is diluted to 1ug/ml with CBS, every hole 100ul is added in ELISA Plate, and 4 DEG C overnight；
3.2 closings: coating buffer is discarded, and 200ul confining liquid (5% skimmed milk power) is added in every hole, and 37 DEG C of placements 1.5 are small
When；
3.3 addition samples: discarding confining liquid, be added sample (serum or antibody), every hole 100ul addition ELISA Plate, and 37
DEG C place 1 hour；
3.4 washings: it is rinsed 10 times, is patted dry with tap water；
3.5 addition secondary antibodies: enzyme mark goat-anti rabbit is diluted to working concentration with confining liquid, and every hole 100ul is added ELISA Plate, and 37 DEG C
It places 30 minutes；
3.6 washings: it is rinsed 10 times, is patted dry with tap water
3.7 are added TMB chromogenic substrate: ELISA Plate is added in every hole 100ul, and 37 DEG C are placed 15 minutes
3.8 are added terminate liquid: 4 50ul of 2M H 2SO is added in every hole
3.9 readings: microplate reader OD450nm reading.
ELISA testing result is as shown in Figure 5.
Embodiment 4: rice different tissues SRBSDV viral level is carried out using SRBSDV P6 protein polyclone antibody
Western blot quantitative analysis
(1) rice protein is extracted:
1. difference clip rice leaf and stem tissue are ground into powder in mortar rapidly after liquid nitrogen flash freezer；
2. addition 0.5mL rice protein extraction Buffer (50mM Tris PH 7.5,6M urea, 150mM NaCl,
0.1%NP40,1mM PMSF), 20min is placed on ice；
3.15000g, 4 DEG C of centrifugation 15min abandon precipitating；
4. taking 100 μ l supernatants that 6 × Loading Buffer is added, concussion is mixed, and -80 DEG C of remaining sample freeze；
5. boiling water boiling 5min, ice bath, 12000rpm are centrifuged 5min, draw 15 μ l of supernatant and carry out protein electrophoresis, remaining sample-
20 DEG C freeze；
6. prepare SDS-PAGE glue, first carry out electrophoresis 20min with 90V, when sample enters separation gel, with 150V voltage into
Row electrophoresis, until bromophenol blue stops electrophoresis when running out of separation gel.
(2) transferring film
The pvdf membrane more somewhat larger than glue that cut with a paper cutter out (for pvdf membrane, is first impregnated 30 seconds with methanol, then in dH2O
Middle immersion 3-5min, is then placed in transferring film buffer) rubber plate clamp is put into electrophoretic blotting groove.Pour into transferring film buffer, 4 DEG C,
100V 90min。
(3) it closes
After the completion of transferring film, pvdf membrane is taken out, is put in the PBST solution with 5% skimmed milk power, 2h is closed on shaking table；
(4) primary antibody is incubated for
Confining liquid is discarded, the P6 antibody (1:5000) and rice HSP90 antibody (1:5000) prepared is added, shaking table is incubated for
12h；
(5) secondary antibody is incubated for
Primary antibody is outwelled, washs pvdf membrane 3 times with 1 × PBST, each 5min；Corresponding secondary antibody is added, and (P6 antibody is corresponding
Secondary antibody is goat-anti rabbit；The corresponding secondary antibody of HSP90 antibody is sheep anti-Mouse), shaking table is incubated for 1h；
(6) ECL exposes
Secondary antibody is abandoned, washs pvdf membrane 3 times with 1 × PBST, each 5min；HRP chemiluminescent substrate is added on pvdf membrane
Liquid can detect protein expression signal (as shown in Figure 6) on 5200 chemiluminescence detectors in day, as can be seen from the results rice stem
SRBSDV viral level in stalk is higher than the viral level in blade.
(7) conclusion
The above results show that the SRBSDV P6 protein polyclone antibody prepared in the present invention is capable of the detection water of specificity
Therefore SRBSDV in rice, and can be used for detecting the viral level of rice different tissues Function Identification to albumen and is ground
Study carefully and have great importance, can be used for the early warning that SRBSDV occurs.
Nucleotide series table:
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>a kind of preparation and application of the prokaryotic expression, polyclonal antibody of southern rice black-streaked dwarf virus P6 albumen
<160> 2
<210>1
<211>32
<212> DNA
<213>artificial sequence
<400>1
CGGGATCCAT GTCTACCAAC CTCACGAACA TA 32
<210>2
<211>32
<212> DNA
<213>artificial sequence
<400>2
GCTCTAGATT ACTCTGAAAT AAGTTGCCAC AA 32
Claims (6)
1. a kind of prokaryotic expression method of southern rice black-streaked dwarf virus P6 albumen, comprising the following steps:
(1) RNA of southern rice black-streaked dwarf virus is extracted；
It (2) is cDNA by RNA reverse transcription；
(3) using cDNA as the P6 coat protein gene of template amplification SRBSDV, the amplified production and original of P6 coat protein gene are obtained
Nuclear expression carrier pET-28a；
(4) recombined pronucleus expression plasmid pET-28a-P6 is constructed
Amplified production and prokaryotic expression carrier pET-28a to P6 coat protein gene carry out digestion respectively, recycle digestion products,
And be attached digestion products, connection product converts competent escherichia coli cell, and it is correct to obtain building for screening, identification
Recombinant plasmid pET-28a-P6；
(5) expression and purifying of P6 albumen
Recombinant plasmid pET-28a-P6 is converted into Bacillus coli cells, screening positive clone, Fiber differentiation, lytic cell, centrifugation
Precipitating is collected, washs to obtain thick leach protein, then through protein purification system purifying and desalination, obtain albumen after purification.
2. the prokaryotic expression method of southern rice black-streaked dwarf virus P6 albumen according to claim 1, it is characterised in that:
In step (3), using cDNA as template, using the P6 coat protein gene of following primer amplification SRBSDV, primer sequence is as follows:
Upstream primer: CGGGATCCATGTCTACCAACCTCACGAACATA, wherein underscore is BamHI restriction endonuclease；
Downstream primer: GCTCTAGATTACTCTGAAATAAGTTGCCACAA, wherein underscore is Xho I restriction endonuclease；
Upstream and downstream primer is respectively as shown in SEQ ID NO.1,2.
3. the prokaryotic expression method of southern rice black-streaked dwarf virus P6 albumen according to claim 1, it is characterised in that:
In step (4), BamHI and Xho are respectively adopted to the amplified production and prokaryotic expression carrier pET-28a of P6 coat protein gene
I enzyme carries out digestion.
4. a kind of preparation method of southern rice black-streaked dwarf virus P6 protein polyclone antibody, it is characterised in that: wanted with right
P6 protein immune animal after purification in 1 is sought, serum is separated, obtains protein polyclone antibody.
5. the preparation method of southern rice black-streaked dwarf virus P6 protein polyclone antibody according to claim 4, special
Sign is: immune animal is new zealand rabbit.
6. southern rice black-streaked dwarf virus P6 protein polyclone antibody as claimed in claim 4 is in detection plant tissue SRBSDV
The application of viral level.
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