CN106636009B - Secrete anti-wheat dwarf virus monoclonal antibody hybridoma cell strain and its monoclonal antibody application - Google Patents
Secrete anti-wheat dwarf virus monoclonal antibody hybridoma cell strain and its monoclonal antibody application Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
The invention discloses a kind of applications for secreting anti-wheat dwarf virus monoclonal antibody hybridoma cell strain and its monoclonal antibody.It is that BALB/c mouse is immunized in antigen with wheat dwarf wilt (WDV) the CP recombinant protein of prokaryotic expression, through cell fusion, screening, clone, the hybridoma cell strain 23F4 for passing on and secreting anti-WDV monoclonal antibody can be stablized by obtaining 1 plant, and deposit number is CGMCC No.13297.The odd contradictive hydroperitoneum indirect ELISA titer of hybridoma cell strain secretion reaches 10‑9, Antibody types and subclass are IgG1, kappa.The monoclonal antibody and WDV coat protein have specific reaction.It is ACP-ELISA the and dot-ELISA method that core establishes WDV in detection wheat plant using 23F4 monoclonal antibody, the sensitivity that both methods detects sick leaf respectively reaches 1:163 840 and 120 times of 1:5 dilutions (w/v, g/mL).The acquisition of the hybridoma cell strain and its monoclonal antibody and the detection, diagnosis and science bridle for being established as the virosis of Serology test provide substance and technical support.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of hybridization for secreting anti-wheat dwarf virus monoclonal antibody
The application of tumor cell strain and its monoclonal antibody.
Background technique
Wheat dwarf wilt (Wheat dwarf disease) is to endanger one of main virus disease of Wheat Production at present.
There is dwarfism in Czechoslovakia's discovery wheat in Vacke in 1961, he is it is thought that a kind of disease propagated by leafhopper
Caused by poison, and it is named as wheat dwarf virus (Wheat dwarf virus, WDV).It is earliest in Wheat in China dwarf wilt
It was found in 2007 in Hancheng Region, Shaanxi, onset area reached 10,000 mu at that time, and the morbidity field diseased plant rate that is averaged reaches 80%.Currently, should
Disease also occurs in succession in 13 Major Wheats production province in the China such as Hebei, Yunnan, Shanxi, Gansu, Henan.
Wheat dwarf wilt diseased plant is often in blocky appearance in field, classical symptom be plant seriously stunt, yellow, streak,
Tiller increases, heading is reduced or does not ear, and wheat yield is greatly lowered, and highest can 80% or more the underproduction.Wheat is pulling out
Being infected before section by WDV will cause plant and stunts, less serious case plant height 20cm, severe one only 10cm, and plant no longer increases；After jointing
Wheat, which is infected, will cause that tiller is different in size, and root generates numerous small tillers.The serious plant that falls ill is i.e. dead before jointing
It dies, though the plant energy jointing that morbidity is lighter, does not ear mostly, even if heading, seed are also not firm.
Wheat dwarf virus belongs to geminivirus infection section (Geminiviridae) Mastrevirus (Mastrevirus).
The twin graininess of virion presentation, particle diameter 18nm, 20 face bodies are symmetrical.WDV genome is by 2739-2750 core
The single-stranded cyclic DNA of thuja acid composition, is made of 4 code areas and 2 noncoding regions.The viral chain of WDV contains V1 and V2 two
Open reading frame is separately encoded motor protein (MP) and capsid protein (CP)；Antisense strand contains two open reading frame of C1 and C2,
It is separately encoded two replicase GAP-associated protein GAP enzymes (Rep and Rep A).Noncoding region is by short noncoding region (Short
Intergenic region, SIR) and long non-coding area (Long intergenic region, LIR) composition.
The main propagation mediator of WDV is different husky leafhopper (Psammotettix alienus Dahlb), with the non-increasing of persistence
Mode is grown to propagate in field.It lays eggs when different sand leafhopper autumn on a variety of autumn crops and weeds with overwintering, ovum starts in spring
Hatching.Wheat is turned green when the early spring, and the different husky leafhopper with poison endangers in wheatland and propagates WDV.After summer small wheat harvesting, adult is adjourned to
On autumn crops and weeds.At this point, weeds in field and various crop are both the host of WDV and the place that different husky leafhopper is lived,
Malicious source is provided for WDV propagation.Autumn weeds are withered, and different sand leafhopper is transferred to the wheat paddock newly emerged, more early, the leaf of wheat emergence
Cicada endangers it heavier, if leafhopper band poison at this time, wheat morbidity can be very serious.The environmental factors such as temperature are to influence different husky leafhopper
Key factor.When temperature is more than 15 DEG C, different sand leafhopper Population is dramatically increased, and autumn low temperature can reduce the biography of leafhopper
Malicious ability.
Wheat is as the second largest cereal crops in China, and yield is directly concerning the people's livelihood.As one of main virosis of wheat
Wheat dwarf virus endanger in recent years seriously, cause wheat crops Severe Reduction.Judge that field is small according to Symptom Observation at present
A situation arises is still most important method for wheat dwarf wilt, but the symptom as caused by a variety of virosis is very much like, and
And it often therefore only relies on the result that Symptom Observation obtains with other virosis Combined Infections and lacks accuracy.Electronic Speculum observation virus
Particle needs expensive instrument, and the virion size of different virus disease and form have similitude, it is caused only to be used as one kind
Assist detection means.The molecular detection technology of based on PCR method is although sensitive, but is unsuitable for Fields detection.In contrast, blood
Clear method is simple to operation, quick, sensitive, cost is relatively low, and can be applied to the extensive detection of field sample, is current
The detection technique of ideal plant virus, but serological method is necessarily dependent upon special, sensitive high-quality antibody.Therefore, this hair
Bright patent is carried out the work mainly for the preparation and its detection application of wheat dwarf virus monoclonal antibody, utilizes hybridoma technology
Anti- WDV monoclonal antibody hybridoma 23F4 can be secreted by being prepared for 1 plant, and the serum for detecting the virus is established with its monoclonal antibody secreted
Method and detection kit, thus for the detection of China's wheat dwarf virus, diagnosis and scientific guidance prevention and control provide substance and
Technical support.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of anti-wheat dwarf virus monoclonal antibody of secretion
The application of hybridoma cell strain and its monoclonal antibody.
The hybridoma cell strain 23F4 of anti-wheat dwarf virus monoclonal antibody is secreted, it can secrete anti-wheat dwarf virus
Specific monoclonal antibody, hybridoma cell strain 23F4 is preserved in Chinese microorganism strain preservation conservator on November 30th, 2016
Meeting common micro-organisms center, deposit number are CGMCC No.13297.
The monoclonal antibody ascites indirect ELISA titer of anti-wheat dwarf virus is up to 10-9, Antibody types and subclass are
The coat protein of IgG1, kappa, the monoclonal antibody and wheat dwarf virus 30kDa have specific immune response, utilize ACP-
ELISA and dot-ELISA method analysis finds that the sensitivity of monoclonal antibody detection infection WDV wheat disease leaf respectively reaches 1:163
120 times of 840 and 1:5 dilutions (w/v, g/mL).
The monoclonal antibody of anti-wheat dwarf virus only has specific immune response with wheat dwarf virus, and yellow with barley
Dwarf virus GPV strain, luteovirus GAV strain, luteovirus PAV strain, Chinese wheat mosaic virus, Wheat Yellow
Any immune response does not occur for mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue.
Application of the monoclonal antibody of anti-wheat dwarf virus in wheat dwarf virus detection is built by core of monoclonal antibody
Vertical various immunological detection methods and immunological reagent box.
The present invention has the advantages that the hybridoma cell strain 2,3F4 1) provided secretes anti-wheat compared with prior art
Dwarf virus monoclonal antibody specific, the immunological methods such as the dot-ELISA and ACP-ELISA that are established using the monoclonal antibody as core
And established with these methods kit energy high special, it is accurate, delicately detect wheat dwarf virus；2) institute of the present invention is utilized
The monoclonal antibody of preparation detects wheat dwarf virus, does not need the equipment such as expensive electron microscope, PCR instrument；3) institute of the present invention is utilized
The monoclonal antibody of preparation is effectively used for the detection of wheat dwarf virus in the plants such as wheat.
Detailed description of the invention
Fig. 1 is the sensitivity analysis of dot-ELISA method detection wheat WDV.
Fig. 2 is the specificity analysis of dot-ELISA method detection WDV: the 1st column are selected up and down as 1 infection WDV wheat for 2
2 biology of plant sample repeat；2nd, which arranges 2 biology that 2 points up and down are recombinant expression WDV CP albumen, repeats；3rd
2 points are that 2 biology of 1 infection wheat yellow mosaic virus wheat plant sample repeat to column up and down；4th arranges 2 points up and down
It is repeated for 2 biology of 1 infection of Chinese wheat streak mosaic virus wheat plant sample；2 points are 1 infection to 5th column up and down
2 biology of luteovirus GAV strain wheat plant sample repeat；2 points are that 1 infection barley is yellow to 6th column up and down
2 biology of dwarf virus GPV strain wheat plant sample repeat；2 points are 1 infection luteovirus to 7th column up and down
2 biology of PAV strain wheat plant sample repeat；8th column are selected up and down as 1 infection barley yellow mosaic virus wheat for 2
2 biology of plant sample repeat；2 points are 2 biology weights of 1 healthy wheat plant tissue sample above and below 9th column
It is multiple.
Fig. 3 is the representative result of WDV in dot-ELISA method detection wheat field sample.
Biological deposits
Classification naming: anti-wheat dwarf virus (WDV) monoclonal antibody hybridoma is secreted；It is preserved in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101, preservation
Day is on November 30th, 2016, and deposit number is CGMCC No.13297.
Specific embodiment
It secretes anti-wheat dwarf virus monoclonal antibody hybridoma cell strain 23F4 and is preserved in Chinese science on November 30th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center, institute of microbiology, institute, deposit number CGMCC
No.13297, it can secrete the monoclonal antibody of anti-wheat dwarf virus.
The monoclonal antibody ascites indirect ELISA titer of anti-wheat dwarf virus is up to 10-9, Antibody types and subclass are
IgG1, kappa can have specific immune response with the 30kDa coat protein of wheat dwarf virus, using ACP-ELISA and
Dot-ELISA method analysis finds that the sensitivity of monoclonal antibody detection infection WDV wheat plant disease leaf respectively reaches 840 He of 1:163
120 times of 1:5 dilutions (w/v, g/mL).
The monoclonal antibody of anti-wheat dwarf virus only has specific immune response with wheat dwarf virus, and yellow with barley
Dwarf virus GPV strain, luteovirus GAV strain, luteovirus PAV strain, Chinese wheat mosaic virus, Wheat Yellow
Any immune response does not occur for mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue.
The monoclonal antibody of anti-wheat dwarf virus on the viral diagnosis application be using monoclonal antibody be core establish it is various be immunized
Learn detection method and immunological reagent box.
Hybridoma cell strain 23F4 provided by the invention can largely secrete the monoclonal antibody of anti-wheat dwarf virus, and its secretion
Monoclonal antibody potency height, high specificity, high sensitivity, stability are good.Using the high-throughput blood for the detection WDV that the monoclonal antibody is established as core
Clear method and its detection kit can be successfully applied to the detection of field WDV, thus for China's wheat dwarf virus detection,
Early warning and science bridle provide substance and technical support.
Below with reference to embodiment and attached drawing, the invention will be further described.
One, the acquisition of hybridoma and its preparation of monoclonal antibody
1. immunogene and the preparation for detecting antigen
Specifically drawn according to the gene order (accession number: JQ836568) of reported WDV coded housing albumen design a pair
Object:
WDV-CP-F(5’-GGATCCATGGTGACCAACAAGGACTCCCGA-3 ', dashed part are BamHI digestion position
Point) and WDV-CP-R (5 '-AAGCTTTTATTGAATCCCAATGGATTTGA-3 ', dashed part are HindIII restriction enzyme site),
And it is synthesized by Hangzhou Qing Ke Bioisystech Co., Ltd.The total DNA of the wheat disease leaf of infection WDV is extracted using conventional CTAB method,
PCR reaction amplification WDV CP gene, reaction system are carried out referring to the specification of TOYOBO company are as follows:
PCR response parameter is as follows: 98 DEG C of 2min of initial denaturation are denaturalized 98 DEG C of 10s, and anneal 55 DEG C of 30s, extend 72 DEG C of 45s, follow
Circle amplification 30 times, last 68 DEG C of extensions 10min, last 16 DEG C of 5min terminate PCR reaction.By amplified production in 0.8% agarose
Gel carries out electrophoretic analysis, and recycles DNA fragmentation with PCR gel reclaims kit (Axygen company), and concrete operations are referring to production
Product kit specification carries out.Add A to connect with cloning vector pMD-18T vector the PCR product end of purifying, recombinates matter
Grain is named as pMD-18T-CP, and is transformed into bacillus coli DH 5 alpha competent cell, and with plasmid extraction kit, (Axygen is public
Department) recombinant plasmid is extracted, PCR is carried out to the recombinant plasmid of extraction and double digestion is identified, and is carried by sequence verification recombinant clone
Entrained CP gene order and the correctness of reading frame, sequence analysis software DNAstar, NCBI- in body pMD18-T-CP
BLAST, database used are GeneBank etc..CP genetic fragment is through BamH I and Hind in recombinant plasmid pMD18-T-CP
It is inserted into the pET-32a expression vector through same digestion after III double digestion.PCR, digestion screening positive clone, and pass through sequencing
Whether correct verify entrained CP gene order and reading frame in recombinant prokaryotic expression vector pET-32a-CP.It will be former if correct
42 DEG C of thermal shocks of nuclear expression plasmid pET-32a-CP are transformed into E. coli expression strains BL21 (DE3), the inoculation of picking single colonie
To LB liquid medium with ampicillin, culture is inoculated in the blueness of benzyl containing ammonia in the ratio of 1:100 by 37 DEG C of overnight incubations
In the fresh LB of mycin, shaken cultivation to OD600Final concentration of 1mM IPTG inducing expression 4h is added when=0.6-0.8,
Thalline were collected by centrifugation.Part thallus is added 2 × SDS sample-loading buffer and suspends, and 5-l0min, 12 000rpm centrifugation are denaturalized in boiling water
After take 10 μ L supernatants to carry out SDS-PAGE electrophoretic analysis, remaining thallus collects supernatant according to product description through ultrasonic disruption
It carries out using Ni2+- NTA affinity column (QIAGEN company) purifies destination protein.The discovery of SDS-PAGE electrophoresis has been purified to size
The recombinant protein band of about 50kD, is consistent with expected results.Using the recombinant C P albumen of purifying as immunogene and detection antigen.
2. immune animal
With the six week old BALB/c female mice of WDV CP protein immunization of purifying.That is 80 μ L/ of WDV purifying protein only with equal bodies
Product Freund's complete adjuvant mixing is carried on the back subcutaneous abdomen multi-point injection 0.16mL/ only after fully emulsified, is spaced 3 weeks, takes and exempt from one
After amount antigen and isometric freund 's incomplete adjuvant are fully emulsified, second of intraperitoneal injection, after 3 Zhou Houyong doubling doses
Antigen is injected intraperitoneally, and extracting spleen cell is merged after 3 days.
3. cell fusion
Take above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) in the ratio of 6:1, in serum-free
It is mixed in RPMI-1640 culture medium, 1 500rpm is centrifuged 5min and removes culture medium, uses 50%PEG (molecular weight 1 500) as melting
1mL is added in 37 DEG C of water-baths in mixture, it is made to merge 2min, is terminated 1 after merging with the RPMI-1640 culture medium of serum-free
500rpm is centrifuged 5min, and precipitating is suspended with HAT culture medium, is dispensed into 96 porocyte plates, is placed in 37 DEG C containing 5%CO2It is thin
It is cultivated in born of the same parents' incubator.
4. hybridoma, the screening in positive hole and its clone
It after being cultivated 5 days in cell incubator, is changed the liquid once with HAT culture medium, changes liquid, Deng Daorong with HT culture medium within the 10th day
When closing cell covering bottom hole 10-30%, using WDV CP as antigen coat elisa plate, is screened and secreted with conventional indirect ELISA method
116 positive holes are obtained in the positive hole of monoclonal antibody altogether.Selection 3 cell holes in strong positive reaction carry out 3 limiting dilution assays gram
It is grand, obtain 1 plant of hybridoma cell strain 23F4 that can secrete anti-WDV specific monoclonal antibody.Through 6 months or more subculture in vitro separately and multiple jelly
After depositing recovery, cell strain can well be grown, and stably excreting antibody.After expanding culture, protected for ascites preparation and liquid nitrogen
It deposits.
5. the preparation of monoclonal antibody ascites and purifying
8 week old or so BALB/c mouse is taken, is injected intraperitoneally 0.3-0.5mL norphytane (Sigma), Intraperitoneal injection 6 after 7-10d
×105A hybridoma, the visible mouse web portion of 7-10d obviously expands after injection, and syringe needle takes ascites, 3 000rpm centrifugation
3min, collecting supernatant is monoclonal antibody ascites.Take 1 times of volume ascites that 2 times of volume 0.06M pH4.8 acetate buffers are added
Liquid dilution, room temperature are stirred side below and are added sad (30uL/mL ascites), and 4 DEG C of clarifications 1h, 12 000rpm are centrifuged 20min, collect supernatant,
Again with 50% saturated ammonium sulphate immunoglobulin, 4 DEG C of placement 2h, 3 000rpm are centrifuged 20min, 2 times of volumes of precipitating
PBS dissolution, flowing dialysis obtains the monoclonal antibody of purifying, -70 DEG C of preservations afterwards for 24 hours in 4 DEG C of refrigerators.
6. the type and subgroup identification and titer of ascites of monoclonal antibody measure
By the anti-BALB/c mouse IgG of standard of the monoclonal antibody of purifying and Sigma company1、IgG2a、IgG2b、IgG3, IgM, κ and λ
Light chain antibody carries out DAS-ELISA analysis, the results show that 23F4 monoclonal antibody type and subclass are IgG1, kappa.With between routine
Connect ELISA method detection odd contradictive hydroperitoneum potency, the results showed that 23F4 odd contradictive hydroperitoneum potency reaches 10-9。
7. the specific detection of monoclonal antibody
With infection Barley yellow dwarf virus GPV strain, luteovirus GAV strain, luteovirus PAV strain, in
State's wheat streak mosaic virus, wheat yellow mosaic virus, barley yellow mosaic virus sick leaf crude extract be coated with elisa plate, with wheat
Healthy leaves crude extract makees negative control, using the sick leaf crude extract for infecting wheat dwarf virus as positive control, uses ACP-ELISA
The specificity of method measurement monoclonal antibody.The specific steps of ACP-ELISA method are as follows: the sick leaf liquid nitrogen of above-mentioned virus infection is in mortar
Middle grind into powder is added coating buffer by 1:30 (w/v, g/mL) times and continues grinding homogenate, after 5 000rpm are centrifuged 3min
Supernatant is taken, 100 holes μ L/ are coated with elisa plate, and 4 DEG C of overnight or 37 DEG C of 2h make it be adsorbed in ELISA polystyrene plate hole；PBST is washed
30-60min is closed with 3% skimmed milk power after washing 3 times；Appropriate diluted 100 hole μ L/ of monoclonal antibody, 37 DEG C of 1-2h are added；PBST is washed
100 hole μ L/ of sheep anti-mouse igg secondary antibody (Sigma) of appropriate diluted alkaline phosphatase lipase (AP) label of addition after washing 3 times, 37 DEG C
1-2h；PBST is developed the color after washing 4 times with PNPP substrate, reads OD with microplate reader after the termination reaction of 2M sodium hydroxide405Value, with
It is greater than 2.1 with negative OD value ratio for the positive.As a result, it has been found that 23F4 monoclonal antibody only has a specific reaction to WDV, and with barley yellow dwarf
Viral GPV strain, luteovirus GAV strain, luteovirus PAV strain, Chinese wheat mosaic virus, wheat chrysanthemum
Mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue are without any immune response.
Two, the foundation of the immunological method and its kit of WDV is detected
1. detecting the ACP-ELISA method of WDV
The step of 1.1 ACP-ELISA method:
1) liquid nitrogen grind into powder in mortar is used after plant tissue weighing, is added in 1:30 ratio (w/v, g/mL)
0.05M carbonate buffer solution (pH 9.6) continues grinding homogenate 3min, and 5 000rpm are centrifuged 3min, and 100 hole μ L/ of supernatant is added
In elisa plate, to infect WDV wheat disease leaf as positive control, healthy wheat leaf is negative control, and 37 DEG C 2h or 4 DEG C overnight；
2) 30min is closed with 3% skimmed milk power after PBST is washed 3 times；
3) odd contradictive hydroperitoneum after appropriate dilution is added, 100 holes μ L/, 37 DEG C of incubation 1h；
4) the sheep anti-mouse igg secondary antibody of appropriate diluted alkaline phosphatase lipase (AP) label is added after PBST is washed 3 times
(Sigma), 100 hole μ L/, 37 DEG C of incubation 1h；
5) 100 hole μ L/ of PNPP nitro Phosphate substrate is added after being washed with PBST, is placed at room temperature for 30min；
6) it detects by an unaided eye, substrate colors become the hole of yellow green as the positive；Or it with after the termination reaction of 2M sodium hydroxide, uses
Enzyme-linked immunosorbent assay instrument surveys OD405, using P/N > 2.1 as positive judgment criteria.
The determination of 1.2 ACP-ELISA method detection sensitivities and specificity
The most suitable working concentration for determining monoclonal antibody and ELIAS secondary antibody in ACP-ELISA method, i.e. ELISA are tested with conventional square matrix
Plate laterally plus with the monoclonal antibody of confining liquid doubling dilution, longitudinal plus sheep anti-mouse igg secondary antibody with confining liquid doubling dilution AP label into
Row ACP-ELISA.Experiments have shown that the most suitable working concentration of 23F4 monoclonal antibody and ELIAS secondary antibody is respectively 1:6 000 and 000 times of 1:8
The ACP-ELISA method of detection WDV is established in dilution with the most suitable working concentration of antibody.To WDV disease leaf crude extract from 1:160 to
655 360 doubling dilutions make negative control respectively with the strong leaf crude extract of corresponding dilution, analyze the inspection of ACP-ELISA method
Survey sensitivity.The result shows that the sick leaf crude extract that ACP-ELISA method dilutes (w/v, g/mL) to 840 times of 1:163 is still positive
Reaction can reach 1:163 840 (w/v, g/mL) times to the detection sensitivity of sick leaf and dilute, show that ACP-ELISA method has
There is very high sensitivity.WDV, Barley yellow dwarf virus GPV strain, luteovirus are detected with the ACP-ELISA method of foundation
GAV strain, luteovirus PAV strain, Chinese wheat mosaic virus, wheat yellow mosaic virus, barley yellow mosaic virus and
Healthy wheat plant tissue, testing result shows that the ACP-ELISA method detection WDV of foundation is in strong positive reaction, and detects big
It is wheat yellow dwarf virus GPV strain, luteovirus GAV strain, luteovirus PAV strain, Chinese wheat mosaic virus, small
Wheat yellow mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue are negative, and yin and yang attribute Comparative result is poor
It is heteropolar significant, illustrate the specificity of this method very well.
The foundation and Fields detection application of 2.dot-ELISA method
2.1 dot-ELISA methods detect the operating procedure of WDV in plant
Will wheat plant sample blade weigh after use liquid nitrogen grind into powder in mortar, in 1:20 ratio (w/v,
G/mL continue grinding, homogenate after 0.01M PBS (pH7.4)) is added；5 000rpm of homogenate is centrifuged 3min；It takes and is checked on 2.5 μ L
To on nitrocellulose filter (NC), while the wheat leaf blade of health and infection WDV are set respectively as negative and positive control；Room
The dry 10-20min of temperature；NC film is immersed in the closing of the PBST (the 0.01M PBS containing 0.05%Tween-20) containing 5% skimmed milk power
Room temperature closes 30min in liquid；NC film is put into the diluted monoclonal antibody of appropriateness and is incubated at room temperature 30-60min；Film is washed 3-4 times with PBST, often
Secondary 3min；NC film is put into the sheep anti-mouse igg secondary antibody of the diluted AP enzyme label of appropriateness and is incubated at room temperature 30-60min；PBST washes film 4-
5 times, each 3min；66 μ L NBT and 33 μ L BCIP substrates (Promega) are added to 10ml substrate buffer solution (0.1mol/L
Tris Cl, 0.1mol/L NaCl, 0.025mol/L MgCl, pH9.5) in mix, film, which is put into substrate solution, to react, develop the color 10-
20min, visual results are obvious to positive control colour developing, and when negative no any colour developing, tap water rinse terminates anti-
It answers, photographs to record result.
The determination of 2.2 dot-ELISA method detection sensitivities and specificity
It is tested with conventional square matrix and determines the monoclonal antibody of WDV and the most suitable working concentration of ELIAS secondary antibody in dot-ELISA method, examination
It tests and shows that the most suitable working concentration of 23F4 monoclonal antibody and ELIAS secondary antibody is respectively 1:5 000 and 000 times of 1:8 dilution.With above-mentioned antibody
Most suitable working concentration establish detection WDV dot-ELISA method.Sensitivity analysis shows to be diluted to 1:5 when WDV disease leaf
When 120 (w/v, g/mL) times, the positive spots of purple are still presented in the dot-ELISA method detection established with 23F4 monoclonal antibody, i.e., its
The sensitivity for detecting sick leaf reaches 120 times of 1:5 dilutions (w/v, g/mL) (Fig. 1).Specificity analysis shows, this method detection sense
Contaminate WDV wheat plant be in strong positive reaction, and with Barley yellow dwarf virus GPV strain, luteovirus GAV strain, barley
Yellow dwarf virus PAV strain, Chinese wheat mosaic virus, wheat yellow mosaic virus, barley yellow mosaic virus and healthy wheat plant
It organizes negative (Fig. 2).
The application of 2.3 dot-ELISA methods
The doubtful morbidity sample in the fields such as Shaanxi Province, Qinghai Province was picked up to 2016 with the dot-ELISA method of foundation to carry out
Detection, as a result, it has been found that, there are 79 samples to produce purpuriferous positive spots in 128 test samples, representative testing result is such as
Shown in Fig. 3.These samples are analyzed with PCR method simultaneously, the results showed that in the positive sample that all dot-ELISA are detected
The genetic fragment to WDV specificity can be expanded, and is not expanded in all negative samples to any genetic fragment.PCR product
Nucleic acid sequencing shows that positive sample infects WDV really, illustrates that the dot-ELISA method can accurately and reliably be used for plant sample
The detection of middle wheat dwarf virus.
3. wheat dwarf virus dot-ELISA detection kit
1) kit main component:
The above reagent is stored at 4 DEG C
Nitrocellulose filter (NC) 10
2) operating procedure of detection plant sample detection:
A. liquid nitrogen grind into powder in mortar is used after plant sample being weighed, and is added in the ratio (w/v, g/mL) of 1:20
Continue grinding, homogenate 3min after 0.01M PBS (pH7.4)；
B. 5 000rpm of homogenate is centrifuged 3min；
C. it takes and is checked on NC on 3 μ L, while the Wheat Tissue crude extract of health and infection WDV are set respectively as feminine gender
And positive control, drying at room temperature 10-20min；
D.NC film is immersed in the PBST containing 5% skimmed milk power (the 0.01M PBS containing 0.05%Tween-20) confining liquid
Room temperature closes 30min；
E.NC film is put into 000 times of diluted monoclonal antibody of 1:3 and is incubated at room temperature 30-60min；
F. film is washed 3-4 times with PBST, then NC film is put into the sheep of 000 times of 1:5 diluted AP enzyme label by each 3min
30-60min is incubated at room temperature in anti-mouse IgG secondary antibody；
G. film is washed 4-5 times with PBST, each 3min, 66 μ L NBT and 33 μ L BCIP substrates are added to 10mL substrate buffer
Liquid (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl2, pH9.5) in and mix, film, which is put into substrate solution, to develop the color, naked eyes
Observe result；
H. to the obvious purple of positive control colour developing, and negative control does not have to be terminated when any colour developing with tap water rinse NC film
Reaction, photographs to record result.
3) preservation and validity period
It is kept in dark place in 2-8 DEG C, validity period 12 months.
4) buffer formulation:
Phosphate buffer (PBS, 0.01M, pH7.4):
PH to 7.4 is adjusted after adding distilled water 950mL to dissolve, and is settled to 1000mL
ELISA cleaning solution (0.01M PBST):
Add 0.5mL Tween-20 in 1000mL 0.01M PBS
ELISA confining liquid:
Skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v).
Claims (3)
1. a kind of hybridoma cell strain 23F4 for secreting anti-wheat dwarf virus monoclonal antibody, it is characterised in that can secrete resist it is small
The specific monoclonal antibody of wheat dwarf virus, hybridoma cell strain 23F4 are preserved in Chinese microorganism strain guarantor on November 30th, 2016
Administration committee's common micro-organisms center is hidden, deposit number is CGMCC No. 13297.
2. a kind of monoclonal antibody of the anti-wheat dwarf virus of hybridoma cell strain 23F4 secretion as described in claim 1,
It is characterized in that the odd contradictive hydroperitoneum indirect ELISA titer is up to 10-9, Antibody types and subclass are IgG1, kappa, the antibody energy
Enough and wheat dwarf virus 30kDa coat protein has specific immune response, utilizes ACP-ELISA and dot-ELISA method
Analysis finds that the sensitivity of monoclonal antibody detection infection WDV wheat disease leaf respectively reaches 1:163 840 and 120 times of 1:5 dilutions；It should
Monoclonal antibody only has a specific immune response with wheat dwarf virus, and with Barley yellow dwarf virus GPV strain, luteovirus GAV
Strain, luteovirus PAV strain, Chinese wheat mosaic virus, wheat yellow mosaic virus, barley yellow mosaic virus and strong
Any immune response does not occur for health wheat plant.
3. a kind of application of monoclonal antibody of anti-wheat dwarf virus as claimed in claim 2 on the viral diagnosis.
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Citations (2)
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|---|---|---|---|---|
| WO2000001832A2 (en) * | 1998-07-02 | 2000-01-13 | Plant Bioscience Limited | Inducible promoters |
| CN103911349A (en) * | 2014-03-21 | 2014-07-09 | 浙江大学 | Hybridoma cell strain secreting monoclonal antibody against barley yellow dwarf virus GAV strain and application of monoclonal antibody thereof |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000001832A2 (en) * | 1998-07-02 | 2000-01-13 | Plant Bioscience Limited | Inducible promoters |
| CN103911349A (en) * | 2014-03-21 | 2014-07-09 | 浙江大学 | Hybridoma cell strain secreting monoclonal antibody against barley yellow dwarf virus GAV strain and application of monoclonal antibody thereof |
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| Title |
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| Detection of wheat dwarf virus (WDV) in wheat and vector leafhopper (Psammotettix alienus Dahlb.) by real-time PCR;Xun Zhang等;《Journal of Virological Methods》;20100804;第169卷;第416-419页 * |
| Investigation of Differences between Wheat and Barley Forms of Wheat Dwarf Virus and their Distribution in Host Plants of Wheat Dwarf Virus and their Distribution in Host Plants;J.SCHUBERT等;《Plant Protection Science》;20021231;第38卷(第特1期);第43-48页 * |
| 小麦矮缩病毒外壳蛋白基因的原核表达、抗体制备及应用;王亚娇等;《植物病理学报》;20131231;第43卷(第4期);第362-367页，尤其第363页左栏第2段，第363-364页方法部分，第365页第2段 * |
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