CN106636009A - Hybridoma cell strain capable of secreting wheat dwarf virus resisting monoclonal antibody and monoclonal antibody application of strain - Google Patents
Hybridoma cell strain capable of secreting wheat dwarf virus resisting monoclonal antibody and monoclonal antibody application of strain Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain capable of secreting a wheat dwarf virus resisting monoclonal antibody and a monoclonal antibody application of the strain. WDV (wheat dwarf virus) CP recombinant protein in prokaryotic expression is used as an antigen immune for BALB/c mice, the hybridoma cell strain 23F4 capable of stably going down to the future generation and secreting the WDV antibody is obtained after cell fusion, screening and cloning, and the collection number of the strain is CGMCC No. 13297. The abdominal dropsy indirect ELISA (enzyme-linked immuno sorbent assay) valence of the antibody secreted by the hybridoma cell strain reaches 10<-9>, and the antibody type and subclass are IgG1 and kappa chains. The antibody and WDV coat protein have specific reaction. The 23F4 antibody is used as a core for building ACP-ELISA and dot-ELISA methods for detecting WDV in a wheat plant, and diseased leaf detection sensitivity of the two methods reach 1:163840 and 1:5120 times of dilution (w/v, g/mL) respectively. Acquisition of the hybridoma cell strain and the antibody thereof and building of serological detection methods provide substantial and technical support for virus disease detection, diagnosis and scientific prevention and control.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of hybridization of the anti-wheat dwarf virus monoclonal antibody of secretion
The application of tumor cell strain and its monoclonal antibody.
Background technology
Wheat dwarf wilt (Wheat dwarf disease) is to endanger one of main virus disease of Wheat Production at present.
Vacke in 1961 has found that wheat occurs in that dwarfism in Czechoslovakia, and he is it is thought that one kind disease propagated by leafhopper
What poison caused, and it is named as wheat dwarf virus (Wheat dwarf virus, WDV).It is earliest in Wheat in China dwarf wilt
Found in Hancheng Region, Shaanxi in 2007, at that time onset area reached 10,000 mu, and the average diseased plant rate in morbidity field reaches 80%.At present, should
Disease also occurs in succession in 13 Major Wheats production province of the China such as Hebei, Yunnan, Shanxi, Gansu, Henan.
Wheat dwarf wilt diseased plant in field often in block appearance, its 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 the underproduction more than 80%.Wheat is being pulled out
Being infected by WDV before section can cause plant to stunt, the lighter's plant height 20cm, severe one only 10cm, and plant no longer increases；After jointing
Wheat is infected can cause tiller different in size, and root produces numerous little tillers.The serious plant of morbidity is i.e. dead before jointing
Die, though the lighter plant energy jointing of morbidity, does not ear more, even if heading, seed is also unreal.
Wheat dwarf virus belongs to geminivirus infection section (Geminiviridae) Mastrevirus (Mastrevirus).
Virion is presented twin graininess, and particle diameter is 18nm, and 20 face bodies are symmetrical.WDV genomes are by 2739-2750 core
The single-stranded cyclic DNA of thuja acid composition, is made up of 4 code areas and 2 noncoding regions.The viral chain of WDV contains V1 and V2 two
ORFs, is separately encoded motor protein (MP) and capsid protein (CP)；Antisense strand contains two ORFs 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 amboceptor of WDV is different husky leafhopper (Psammotettix alienus Dahlb), with the non-increasing of persistence
Grow mode to propagate in field.Lay eggs to survive the winter on various autumn crops and weeds during different husky leafhopper autumn, ovum starts in spring
Hatching.Wheat is turned green during the early spring, and the different husky leafhopper with poison endangers and propagate WDV in wheatland.After summer little wheat harvesting, adult adjourns to
On autumn crops and weeds.Now, weeds in field and various crop are both the host of WDV, are also the place that different husky leafhopper is lived,
Propagate for WDV and provide malicious source.Autumn weeds wither, and different husky leafhopper is transferred to the wheat paddock newly emerged, and wheat is emerged more early, leaf
Cicada endangers heavier to it, if now leafhopper band poison, wheat morbidity can be very serious.The environmental factors such as temperature are to affect different husky leafhopper
Key factor.When temperature is more than 15 DEG C, different husky leafhopper Population is dramatically increased, and autumn low temperature can reduce the biography of leafhopper
Malicious ability.
Wheat directly concerns the people's livelihood as the second largest cereal crops of China, its yield.As one of main virosis of wheat class
Wheat dwarf virus endanger in recent years seriously, cause wheat crops Severe Reduction.Judge that field is little according to Symptom Observation at present
A situation arises remains topmost method for wheat dwarf wilt, but the symptom caused due to various virosis is very much like, and
And often with other virosis Combined Infections, therefore only rely on the result that Symptom Observation obtains and lack the degree of accuracy.Electron microscopic observation virus
Particle needs expensive instrument, and the virion size and form of different virus disease have similitude, cause it only as a kind of
Auxiliary detection means.The molecular detection technology of PCR-based method is although sensitive, but is unsuitable for Fields detection.Comparatively, 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 preferable plant virus, but serological method is necessarily dependent upon special, sensitive high-quality antibody.Therefore, this
Bright patent is carried out the work mainly for the preparation and its detection application of wheat dwarf virus monoclonal antibody, using hybridoma technology
Being prepared for 1 plant can secrete anti-WDV monoclonal antibodies hybridoma 23F4, and establish the detection viral serum with its monoclonal antibody secreted
Method and detection kit, so as to for the detection of China's wheat dwarf virus, diagnosis and scientific guidance prevention and control provide material and
Technical support.
The content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, there is provided the anti-wheat dwarf virus monoclonal antibody of one kind 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, preserving number is 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
IgG1, kappa, the monoclonal antibody has specific immune response with the coat protein of wheat dwarf virus 30kDa, using ACP-
ELISA and dot-ELISA method analyses find that the sensitivity of the monoclonal antibody detection infection WDV wheat disease leaves respectively reaches 1:163
840 and 1:5 120 times of 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 strains, luteovirus GAV strains, luteovirus PAV strains, Chinese wheat mosaic virus, Wheat Yellow
There is no any immune response in mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue.
Application of the monoclonal antibody of anti-wheat dwarf virus on wheat dwarf virus is detected is built by core of monoclonal antibody
Vertical various immunological detection methods and immunological reagent box.
The present invention has the advantage that compared with prior art：1) the hybridoma cell strain 23F4 for providing secretes anti-wheat
The immunological methods such as dwarf virus monoclonal antibody specific, the dot-ELISA set up as core with the monoclonal antibody and ACP-ELISA
And set up with these methods kit energy high special, accurately, delicately detect wheat dwarf virus；2) institute of the present invention is utilized
The monoclonal antibody detection wheat dwarf virus of preparation, it is not necessary to 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.
Description of the drawings
Fig. 1 is the sensitivity analysis that dot-ELISA methods detect wheat WDV；
Fig. 2 is the specificity analysis of dot-ELISA methods detection WDV：Upper and lower 2 of 1 row are selected as 1 infection WDV wheat plant
2 biology of thing sample repeat；Upper and lower 2 points of 2 row are that 2 biology of recombinant expressed WDV CP albumen repeat；3 row are upper and lower
2 points are 2 biology repetition of 1 infection WYMV wheat plant sample；Upper and lower 2 points of 4 row are 1 infection
2 biology of Chinese wheat mosaic virus wheat plant sample repeat；Upper and lower 2 points of 5 row are 1 infection luteovirus
2 biology of GAV strain wheat plant samples repeat；Upper and lower 2 points of 6 row are that 1 infection Barley yellow dwarf virus GPV strain is little
2 biology of wheat plant sample repeat；Upper and lower 2 points of 7 row are 1 infection luteovirus PAV strain wheat plant sample
2 biology of product repeat；Upper and lower 2 points of 8 row are 2 biology of 1 infection barley yellow mosaic virus wheat plant sample
Repeat；Upper and lower 2 points of 9 row are that 2 biology of 1 healthy wheat plant tissue sample repeat.
Fig. 3 is the representative result of WDV in the sample of dot-ELISA methods detection wheat field.
Biological deposits
Classification And Nomenclature：Secrete anti-wheat dwarf virus (WDV) monoclonal antibody hybridoma；It is preserved in Chinese microorganism strain guarantor
Hide administration committee's common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101, preservation
Day is on November 30th, 2016, and preserving number is CGMCC No.13297.
Specific embodiment
Secrete anti-wheat dwarf virus monoclonal antibody hybridoma cell strain 23F4 and be preserved in Chinese science on November 30th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology of institute, preserving number is 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 analyses find that the sensitivity of the monoclonal antibody detection infection WDV wheat plant disease leaves respectively reaches 1:163 840 Hes
1:5 120 times of 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 strains, luteovirus GAV strains, luteovirus PAV strains, Chinese wheat mosaic virus, Wheat Yellow
There is no any immune response in mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue.
Application of the monoclonal antibody of anti-wheat dwarf virus on the Viral diagnosis is the various immunity set up as core with monoclonal antibody
Learn detection method and immunological reagent box.
The hybridoma cell strain 23F4 that the present invention is provided can in a large number secrete the monoclonal antibody of anti-wheat dwarf virus, and its secretion
Monoclonal antibody potency height, high specificity, sensitivity height, good stability.The high-throughout blood of the detection WDV set up as core with the monoclonal antibody
Clear method and its detection kit can be successfully applied to the detection of field WDV, so as to for the detection of China's wheat dwarf virus,
Early warning and science bridle provide material and technical support.
With reference to embodiment and accompanying drawing, the invention will be further described.
First, the acquisition of hybridoma and its preparation of monoclonal antibody
1. the preparation of immunogene and detection antigen
According to the gene order (accession number of the WDV coded housing albumen reported：JQ836568) design a pair and specifically draw
Thing：
WDV-CP-F(5’-GGATCCATGGTGACCAACAAGGACTCCCGA-3 ', dashed part is BamHI digestions position
Point) and WDV-CP-R (5 '-AAGCTTTTATTGAATCCCAATGGATTTGA-3 ', dashed part is HindIII restriction enzyme sites),
And synthesized by Hangzhou Qing Ke Bioisystech Co., Ltd.The STb gene of the wheat disease leaf of infection WDV is extracted using conventional CTAB methods,
Enter performing PCR reaction amplification WDV CP genes with reference to the specification of TOYOBO companies, reaction system is：
PCR response parameters are as follows：98 DEG C of 2min of denaturation, 98 DEG C of 10s of denaturation, anneal 55 DEG C of 30s, extends 72 DEG C of 45s, follows
Circle amplification 30 times, last 68 DEG C of extensions 10min, last 16 DEG C of 5min terminate PCR reactions.By amplified production in 0.8% agarose
Gel carries out electrophoretic analysis, and reclaims DNA fragmentation with PCR gel reclaims kits (Axygen companies), and concrete operations are with reference to product
Product kit specification is carried out.A is added to be connected with cloning vector pMD-18T vector the PCR primer end of purifying, matter of recombinating
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, the recombinant plasmid to extracting enters performing PCR and double digestion identification, and is carried by sequence verification recombinant clone
CP gene orders entrained in body pMD18-T-CP and the correctness of reading frame, sequence analysis software is DNAstar, NCBI-
BLAST, database used is GeneBank etc..CP genetic fragments Jing BamH I and Hind in recombinant plasmid pMD18-T-CP
During the pET-32a expression vectors of the same digestions of Jing are inserted after III double digestions.PCR, digestion screening positive clone, and by sequencing
Whether entrained CP gene orders and reading frame are correct in checking recombinant prokaryotic expression vector pET-32a-CP.By original 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 bacterium colony
To the LB fluid nutrient mediums containing ampicillin, 37 DEG C of overnight incubations, by 1:It is blue or green that culture is inoculated in benzyl containing ammonia by 100 ratio
In the fresh LB of mycin, shaken cultivation to OD600Final concentration of 1mM IPTG abduction delivering 4h are added during=0.6-0.8,
Thalline is collected by centrifugation.Part thalline adds 2 × SDS sample-loading buffers to suspend, denaturation 5-l0min in boiling water, 12 000rpm centrifugation
After take 10 μ L of supernatant and carry out SDS-PAGE electrophoretic analysis, remaining thalline Jing ultrasonic disruptions collect supernatant according to product description
Carry out using Ni2+- NTA affinity columns (QIAGEN companies) purify destination protein.SDS-PAGE electrophoresis finds to be 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 week old BALB/c female mice of WDV CP protein immunizations six of purifying.That is the μ L/ of WDV purifying proteins 80 only with wait body
Product Freund's complete adjuvant mixing, it is fully emulsified after Jing back ofs the body subcutaneous abdomen multi-point injection 0.16mL/ only, be spaced 3 weeks, take and exempt from one
After amount antigen and isopyknic freund 's incomplete adjuvant are fully emulsified, second lumbar injection, with doubling dose after 3 weeks
Antigen carries out lumbar injection, and extracting spleen cell is merged after 3 days.
3. cell fusion
Above-mentioned immune mouse spleen cell is taken with murine myeloma cell (SP2/0) by 6:1 ratio, in serum-free
Mix in RPMI-1640 culture mediums, 1 500rpm centrifugation 5min remove culture medium, are melted with 50%PEG (molecular weight 1 500) conducts
Mixture, adds 1mL so as to merge 2min in 37 DEG C of water-baths, is terminated 1 after fusion with the RPMI-1640 culture mediums of serum-free
500rpm is centrifuged 5min, precipitates and is suspended with HAT culture mediums, in being dispensed into 96 porocyte plates, is placed in 37 DEG C containing 5%CO2It is thin
Cultivate in born of the same parents' incubator.
4. hybridoma, the screening in positive hole and its clone
After cultivating 5 days in cell culture incubator, liquid is changed once with HAT culture mediums, change liquid, Deng Daorong with HT culture mediums within the 10th day
When closing cell covering bottom hole 10-30%, with WDV CP as antigen coat elisa plate, secreted with the screening of conventional indirect ELISA method
The positive hole of monoclonal antibody, obtains altogether 116 positive holes.3 cell holes in strong positive reaction are selected, 3 limiting dilution assays gram are carried out
It is grand, obtain 1 plant of hybridoma cell strain 23F4 that can secrete anti-WDV specific monoclonal antibodies.More than 6 months subculture in vitro separately of Jing and repeatedly jelly
After depositing recovery, cell line can well grow, and stably excreting antibody.Jing after Amplification Culture, prepare for ascites and liquid nitrogen is protected
Deposit.
5. monoclonal antibody ascites is prepared and purified
Take 8 week old or so BALB/c mouse, lumbar injection 0.3-0.5mL norphytanes (Sigma), 7-10d pneumoretroperitoneums injection 6
×105Individual hybridoma, the visible mouse web portions of 7-10d substantially expand after injection, and syringe needle takes ascites, 3 000rpm centrifugations
3min, collects supernatant and is monoclonal antibody ascites.Take 1 times of volume ascites and add 2 times of volume 0.06M pH4.8 acetate buffers
Liquid dilutes, and room temperature is stirred below side and added sad (30uL/mL ascites), 4 DEG C of clarification 1h, and 12 000rpm centrifugation 20min collect supernatant,
Again with 50% saturated ammonium sulphate immunoglobulin (Ig), 4 DEG C of placement 2h, 3 000rpm centrifugation 20min are precipitated with 2 times of volumes
PBS dissolves, and obtains the monoclonal antibody of purifying, -70 DEG C of preservations after flowing dialysis 24h in 4 DEG C of refrigerators.
6. the type and subgroup identification of monoclonal antibody and titer of ascites are determined
By the monoclonal antibody of purifying and the anti-BALB/c mouse IgG of standard of Sigma companies1、IgG2a、IgG2b、IgG3, IgM, κ and λ
Light chain antibody carries out DAS-ELISA analyses, as a result shows, 23F4 monoclonal antibodies type and subclass are IgG1, kappa.With between routine
ELISA method detection odd contradictive hydroperitoneum potency is connect, as a result shows 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 strains, luteovirus PAV strains, in
State's wheat streak mosaic virus, WYMV, the sick leaf crude extract coating elisa plate of barley yellow mosaic virus, with wheat
Healthy leaves crude extract makees negative control, to infect the sick leaf crude extract of wheat dwarf virus as positive control, uses ACP-ELISA
Method determines the specificity of monoclonal antibody.ACP-ELISA methods are concretely comprised the following steps：The sick leaf liquid nitrogen of above-mentioned virus infection is in mortar
Middle grind into powder, by 1:30 (w/v, g/mL) times add coating buffer solution to continue to grind homogenate, after 5 000rpm centrifugation 3min
Take supernatant, 100 μ L/ holes coating elisa plate, 4 DEG C are overnight or 37 DEG C of 2h so as to be adsorbed in ELISA polystyrene plate holes；PBST is washed
Wash 3 times and close 30-60min with 3% skimmed milk power afterwards；Add the μ L/ holes of monoclonal antibody 100 of appropriate dilution, 37 DEG C of 1-2h；PBST is washed
Wash anti-(Sigma) the 100 μ L/ holes of sheep anti-mouse igg two for alkaline phosphatase lipase (AP) mark for adding appropriate dilution afterwards 3 times, 37 DEG C
1-2h；PBST washs 4 times and is developed the color with PNPP substrates afterwards, and OD is read with ELIASA after 2M NaOH terminating reactions405Value, with
It is the positive more than 2.1 with negative OD values ratio.As a result find, 23F4 monoclonal antibodies only have a specific reaction to WDV, and with barley yellow dwarf
Viral GPV strains, luteovirus GAV strains, luteovirus PAV strains, Chinese wheat mosaic virus, wheat chrysanthemum
Mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue are without any immune response.
2nd, the immunological method of WDV and its foundation of kit are detected
1. the ACP-ELISA methods of WDV are detected
The step of 1.1 ACP-ELISA methods：
1) liquid nitrogen grind into powder in mortar is used after plant tissue is weighed, by 1:30 ratios (w/v, g/mL) are added
0.05M carbonate buffer solutions (pH 9.6) continue to grind homogenate 3min, 5 000rpm centrifugation 3min, and the μ L/ holes of supernatant 100 add
In elisa plate, to infect WDV wheats disease leaf as positive control, healthy wheat leaf is negative control, and 37 DEG C 2h or 4 DEG C overnight；
2) PBST washs 3 times and closes 30min with 3% skimmed milk power afterwards；
3) odd contradictive hydroperitoneum after appropriate dilution, 100 μ L/ holes, 37 DEG C of incubation 1h are added；
4) PBST washings add afterwards for 3 times the sheep anti-mouse igg two of alkaline phosphatase lipase (AP) mark of appropriate dilution anti-
(Sigma), 100 μ L/ holes, 37 DEG C of incubation 1h；
5) the μ L/ holes of PNPP nitros Phosphate substrate 100, room temperature is added to place 30min after being washed with PBST；
6) detect by an unaided eye, substrate colors become yellowish green hole for the positive；Or with after 2M NaOH terminating reactions, use
Enzyme-linked immunosorbent assay instrument surveys OD405, with P/N>2.1 used as positive criterion.
The determination of 1.2 ACP-ELISA methods detection sensitivities and specificity
The most suitable working concentration for determining monoclonal antibody and ELIAS secondary antibody in ACP-ELISA methods, i.e. ELISA are tested with conventional square formation
Plate laterally adds the monoclonal antibody with confining liquid doubling dilution, longitudinal direction plus the sheep anti-mouse igg two marked with confining liquid doubling dilution AP resist into
Row ACP-ELISA.Test shows that 23F4 monoclonal antibodies and the most suitable working concentration of ELIAS secondary antibody are respectively 1:6 000 and 1:8 000 times
Dilution, in the ACP-ELISA methods that the most suitable working concentration of antibody sets up detection WDV.To WDV disease leaf crude extracts from 1:160 to
655 360 doubling dilutions, make negative control with corresponding dilution strong leaf crude extract respectively, analyze the inspection of ACP-ELISA methods
Survey sensitivity.As a result show ACP-ELISA methods to 1:The sick leaf crude extract of 163 840 times of dilutions (w/v, g/mL) is still positive
Reaction, i.e., can reach 1 to the detection sensitivity of sick leaf:163 840 (w/v, g/mL) times dilute, and show that ACP-ELISA methods have
There is very high sensitivity.With ACP-ELISA methods detection WDV, Barley yellow dwarf virus GPV strain, the luteovirus set up
GAV strains, luteovirus PAV strains, Chinese wheat mosaic virus, WYMV, barley yellow mosaic virus and
Healthy wheat plant tissue, testing result shows that the ACP-ELISA methods detection WDV of foundation is in strong positive reaction, and detects big
It is wheat yellow dwarf virus GPV strains, luteovirus GAV strains, luteovirus PAV strains, Chinese wheat mosaic virus, little
Wheat yellow mosaic virus, barley yellow mosaic virus and healthy wheat plant tissue are negative, and yin and yang attribute Comparative result is poor
Heteropole significantly, illustrates the specificity of the method very well.
The foundation and Fields detection application of 2.dot-ELISA methods
The operating procedure of WDV in 2.1 dot-ELISA methods detection plant
Liquid nitrogen grind into powder in mortar is used after the blade of wheat plant sample is weighed, by 1:20 ratio (w/v,
G/mL 0.01M PBS (pH7.4)) are added to continue to grind afterwards, be homogenized；The 000rpm of homogenate 5 is centrifuged 3min；Take 2.5 μ L of supernatant points
To on nitrocellulose filter (NC), while arranging the wheat leaf blade of health and infection WDV respectively as negative and positive control；Room
Temperature is dried 10-20min；NC films are immersed in the PBST containing 5% skimmed milk power (the 0.01M PBS containing 0.05%Tween-20) closing
Room temperature closing 30min in liquid；NC films are put into the monoclonal antibody of appropriateness dilution and are incubated at room temperature 30-60min；Film 3-4 time is washed with PBST, often
Secondary 3min；NC films are put into the anti-middle incubation at room temperature 30-60min of sheep anti-mouse igg two of the AP enzymes mark of appropriateness dilution；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 be put into substrate solution react, develop the color 10-
20min, visual results treat that positive control colour developing is obvious, and negative without during any colour developing, and tap water rinse terminates anti-
Should, Taking Pictures recording result.
The determination of 2.2 dot-ELISA methods detection sensitivities and specificity
The monoclonal antibody and the most suitable working concentration of ELIAS secondary antibody for determining WDV in dot-ELISA methods, examination are tested with conventional square formation
Test and show that the most suitable working concentration of 23F4 monoclonal antibodies and ELIAS secondary antibody is respectively 1:5 000 and 1:8 000 times of dilutions.With above-mentioned antibody
Most suitable working concentration set up detection WDV dot-ELISA methods.Sensitivity analysis shows, when WDV disease leaves are diluted to 1:5
When 120 (w/v, g/mL) times, the positive spots of purple are still presented with the dot-ELISA methods detection that 23F4 monoclonal antibodies are set up, i.e., its
The sensitivity of the sick leaf of detection reaches 1:5 120 times of dilutions (w/v, g/mL) are (Fig. 1).Specific analysis shows, the method detection sense
Dye WDV wheat plant be in strong positive reaction, and with Barley yellow dwarf virus GPV strain, luteovirus GAV strains, barley
Yellow dwarf virus PAV strains, Chinese wheat mosaic virus, WYMV, barley yellow mosaic virus and healthy wheat plant
Organize negative (Fig. 2).
The application of 2.3 dot-ELISA methods
With the dot-ELISA methods set up 2016 are picked up from the doubtful morbidity sample in the fields such as Shaanxi Province, Qinghai Province is carried out
Detection, as a result finds, has 79 samples to produce purpuriferous positive spots in 128 detection samples, and representational testing result is such as
Shown in Fig. 3.These samples are analyzed with PCR method simultaneously, in as a result showing the positive that all dot-ELISA are detected
The genetic fragment to WDV specificity can be expanded, and is not expanded to any genetic fragment in all negative samples.PCR primer
Nucleic acid sequencing shows that positive infects WDV really, illustrates that the dot-ELISA methods can accurately and reliably be used for plant sample
The detection of middle wheat dwarf virus.
3. wheat dwarf virus dot-ELISA detection kits
1) kit main component：
Above reagent is stored at 4 DEG C
Nitrocellulose filter (NC) 10
2) operating procedure of plant sample detection is detected：
A. liquid nitrogen grind into powder in mortar is used after plant sample is weighed, by 1:20 ratio (w/v, g/mL) is added
0.01M PBS (pH7.4) continue afterwards grinding, homogenate 3min；
B. the 000rpm of homogenate 5 is centrifuged 3min；
C. 3 μ L of supernatant points are taken on NC, while arranging the Wheat Tissue crude extract of health and infection WDV respectively as feminine gender
And positive control, drying at room temperature 10-20min；
D.NC films are 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 films are put into 1:30-60min is incubated at room temperature in the monoclonal antibody of 3 000 times of dilutions；
F. film 3-4 time is washed with PBST, then NC films are put into 1 by each 3min:The sheep of the AP enzymes mark of 5 000 times of dilutions
Incubation at room temperature 30-60min during anti-mouse IgG bis- is anti-；
G. film 4-5 time is washed with PBST, each 3min, 66 μ L NBT and 33 μ L BCIP substrates are added to 10mL substrate buffers
Liquid (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl2, pH9.5) in and mix, film be put into substrate solution develop the color, naked eyes
Observation result；
H. the obvious purple of positive control colour developing is treated, and negative control is terminated when not having any colour developing with tap water rinse NC films
Reaction, Taking Pictures recording result.
3) preserve and the term of validity
Keep in dark place in 2-8 DEG C, the term of validity 12 months.
4) buffer formulation：
Phosphate buffer (PBS, 0.01M, pH7.4)：
Plus adjust pH to 7.4 after distilled water 950mL dissolvings, it is settled to 1000mL
ELISA cleaning solutions (0.01M PBST)：
Add 0.5mL Tween-20 in 1000mL 0.01M PBS
ELISA confining liquids：
Skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v).
Claims (4)
1. the hybridoma cell strain 23F4 of the anti-wheat dwarf virus monoclonal antibody of a kind of secretion, it is characterised in that can secrete resist it is little
The specific monoclonal antibody of wheat dwarf virus, hybridoma cell strain 23F4 is preserved in Chinese microorganism strain guarantor on November 30th, 2016
Administration committee's common micro-organisms center is hidden, preserving number is CGMCC No.13297.
2. a kind of monoclonal antibody of the anti-wheat dwarf virus of hybridoma cell strain 23F4 as claimed in claim 1 secretion,
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
The coat protein of enough and wheat dwarf virus 30kDa has specific immune response, using ACP-ELISA and dot-ELISA methods
Analysis finds that the sensitivity of the monoclonal antibody detection infection WDV wheat disease leaves respectively reaches 1:163 840 and 1:5 120 times of dilutions.
3. the monoclonal antibody of the anti-wheat dwarf virus of hybridoma cell strain 23F4 as claimed in claim 2 secretion, it is special
It is that the monoclonal antibody only has a specific immune response with wheat dwarf virus to levy, and with Barley yellow dwarf virus GPV strain, barley yellow dwarf
Viral GAV strains, luteovirus PAV strains, Chinese wheat mosaic virus, WYMV, elimination of barley's yellow mosaic
There is no any immune response in poison and healthy wheat plant.
4. application of a kind of monoclonal antibody of anti-wheat dwarf virus as claimed in claim 2 on the Viral diagnosis, its
It is characterised by various immunological detection methods and the immunological detecting kit set up as core with monoclonal antibody.
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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 |
-
2016
- 2016-12-23 CN CN201611208960.2A patent/CN106636009B/en active Active
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 |
Non-Patent Citations (3)
| Title |
|---|
| J.SCHUBERT等: "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", 《PLANT PROTECTION SCIENCE》 * |
| XUN ZHANG等: "Detection of wheat dwarf virus (WDV) in wheat and vector leafhopper (Psammotettix alienus Dahlb.) by real-time PCR", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 王亚娇等: "小麦矮缩病毒外壳蛋白基因的原核表达、抗体制备及应用", 《植物病理学报》 * |
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