CN105671001B - Secrete anti-bean mosaic virus 4 monoclonal antibody hybridoma cell strain and its monoclonal antibody application - Google Patents
Secrete anti-bean mosaic virus 4 monoclonal antibody hybridoma cell strain and its monoclonal antibody application Download PDFInfo
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- CN105671001B CN105671001B CN201610096744.7A CN201610096744A CN105671001B CN 105671001 B CN105671001 B CN 105671001B CN 201610096744 A CN201610096744 A CN 201610096744A CN 105671001 B CN105671001 B CN 105671001B
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- C—CHEMISTRY; METALLURGY
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种分泌抗南方菜豆花叶病毒单抗杂交瘤细胞株及其单抗的应用。用纯化的南方菜豆花叶病毒(SBMV)粒子为抗原免疫BALB/c小鼠，经细胞融合、筛选、克隆，获得1株能稳定传代并分泌抗SBMV单克隆抗体的杂交瘤细胞株19H9，其保藏号为CGMCC No.12004。该杂交瘤细胞株分泌的单抗腹水间接ELISA效价达10‑7，抗体类型及亚类为IgG1、kappa轻链。该单抗与SBMV有特异性反应。利用19H9单抗建立检测豆科植物中SBMV的ACP‑ELISA和dot‑ELISA方法，这两种方法检测病叶的灵敏度分别达到1:81920和1:5120倍稀释(w/v,g/mL)。该杂交瘤细胞株19H9及其单抗的获得和血清学检测方法的建立为该病毒病的诊断、检测及科学防控提供物质和技术支撑。The invention discloses a hybridoma cell strain secreting anti-Southern Bean Mosaic Virus monoclonal antibody and the application of the monoclonal antibody. Purified Southern Bean Mosaic Virus (SBMV) particles were used as antigen to immunize BALB/c mice. After cell fusion, screening and cloning, a hybridoma cell line 19H9, which can be stably passaged and secreted anti-SBMV monoclonal antibody, was obtained. The deposit number is CGMCC No.12004. The indirect ELISA titer of monoclonal antibody ascitic fluid secreted by the hybridoma cell line reached 10-7 , and the antibody type and subclass were IgG1 and kappa light chain. The monoclonal antibody has a specific reaction with SBMV. ACP‑ELISA and dot‑ELISA methods for the detection of SBMV in leguminous plants were established by using 19H9 monoclonal antibody. The sensitivity of these two methods to detect diseased leaves reached 1:81920 and 1:5120 times dilution (w/v, g/mL) respectively . The acquisition of the hybridoma cell line 19H9 and its monoclonal antibody and the establishment of serological detection methods provide material and technical support for the diagnosis, detection and scientific prevention and control of the viral disease.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of secrete anti-MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS
The application of hybridoma cell strain and its monoclonal antibody.
Background technique
Bean mosaic virus 4 (Southern bean mosaic virus, SBMV) is Sobemovirus
(Sobemovirus) prototypical member includes mainly two strains of SBMV-B and SBMV-C, earliest by Zaumeyer and Harter
It was found that report.The virus is distributed mainly on tropical and subtropical region, and Temperate Region in China is also distributed, and can infect and endanger soybean, dish
The plants such as beans, pumpkin.Bean mosaic virus 4 disease generally occurs in field, and route of transmission is more, and harm is serious.Under natural situation
It can cause Kidney bean, cowpea, urd bean and the floral leaf of soybean, mottled disease, lead to flower, fruit, seed amount decline, yield reduces.
Some countries and regions such as Denmark, Australia, Egypt, East Africa are classified as quarantine object.China, which has, to be taken root, breeds, expanding
Scattered condition, once it is incoming, it has endless trouble.Currently, being included in two class quarantine harmful organisms of China, and from 1992
Put into practice by operation of law quarantine.
SBMV virion be etc. axisymmetric icosahedron (T=3), no coating, diameter be about 30nm.Its genome packet
Containing a molecule threadiness single stranded positive-sense RNA, it is about 4100-4500nt, accounts for about the 21% of virion weight.SBMV is in CsCl
Buoyant density is 1.36g/cm3, sedimentation coefficient 115S.The lethal temperature of virus is 90-95 DEG C, and dilution point of accumulation is 10-5-10-6,
(18-22 DEG C) of longevity in vitro is 1.65-20d.3 ' ends of viral RNA are both without Poly (A), also without similar to tRNA structure, 5 '
There is a VPg at end, it may be possible to necessary to virus infection, contain 4 ORF, ORF1 encodes the P1 albumen of a 21kDa, the egg
It is white related with viral transcellular movement；The 105kDa P2 albumen of ORF2 coding may be polymerase；ORF3 is located within ORF2,
A 18kDa albumen is encoded, function is also unknown；The coat protein of ORF4 coding 30kDa.
The virion is primarily present in host plant mesophyll cell, normal dispersed distribution or is gathered in cytoplasm and vacuole
In.Large stretch of virus crystal body is formed in the plant cell matter that SBMV infects, and generates many containing the special of thin fibrous material
Vesicle structure.Vesiculation, chloroplaset extrusion, but no discovery virus in chloroplaset and mitochondria is presented in infection cell
Particle.The host range of SBMV is very narrow, mostly leguminous plant, and natural host is Kidney bean and cowpea, and wherein SBMV-B strain can invade
Most of kinds of Kidney bean are contaminated, and strain SBMV-C can only infect cowpea.Its artificial infection host is relatively extensive, can infect big
23 kinds of plants of beans, mung bean, rde bean, scarlet runner bean, Madagascar bean, pea, semen viciae fabae, daghestan sweet clover etc. about 12 categories.Draw after infecting Kidney bean
The Disease symptom risen mainly includes mottled and floral leaf, but the symptom in different cultivars is widely different, and some is system morbidity, is had
It is partial onset, some symptoms are slight, and some is also with symptoms such as shrinkage and vein bandings.SBMV mainly passes through chrysomelid in field
It is propagated, juice mechanical system and seed dispersal can also be passed through.
Currently, the leguminous plants such as Kidney bean, cowpea are China's cultivated area is big, range is wide, and with foreign trade and kind
Mass transter is increasing, and the incoming chance of this virus also greatly increases, and in order to protect the production of China legume crop, prevents the disease
Poison should establish a set of easy, quick, effective quarantine detection method from the country is overseas passed to.Currently, for the mouth of SBMV
Bank detection, generally use Biological Detection, molecular Biological Detection and Electronic Speculum detection technique, these types of method low efficiency, not
It is suitble to batch samples detection.Serological method is easy to operate, and specificity is good, while can detecte extensive sample, but necessary
Viral monoclonal antibody dependent on specificity.Present invention is generally directed to the preparation of MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS and its detections
Application start work, is prepared for 1 plant of hybridoma 19H9 that can secrete anti-SBMV monoclonal antibody using hybridoma technology, and with its point
The monoclonal antibody secreted is that core establishes the serological method and detection kit for detecting the virus, to improve China to southern Braised Tofu with Vegetables
The detection level and speed of mosaic virus prevent the virus domestic from being overseas passed to.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of anti-bean mosaic virus 4 monoclonal of secretion
The application of the hybridoma cell strain and its monoclonal antibody of antibody.
The hybridoma cell strain 19H9 for secreting anti-MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS, in preservation on January 7 in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.12004, it can be secreted
The monoclonal antibody specific of anti-bean mosaic virus 4.
A kind of monoclonal antibody of the anti-bean mosaic virus 4 of the hybridoma cell strain 19H9 secretion, anti-south
The monoclonal antibody ascites indirect ELISA titer of adzuki bean mosaic virus is up to 10-7, Antibody types and subclass are that IgG1, kappa are light
Chain.It is found using ACP-ELISA method analysis, the sensitivity which detects sick leaf reaches 1:81920 times of dilution (w/v, g/
mL)。
There is spy in sick leaf texture of the monoclonal antibody of anti-bean mosaic virus 4 only with infection bean mosaic virus 4
Specific immunological reaction, and with infection Bean common mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus (TMV), cucumber mosaic
The sick leaf texture of virus and soybean, green soy bean, cowpea, pea, semen viciae fabae and the bean plant tissue of health do not occur any immune
Reaction.
Application of the anti-MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS in the detection of anti-bean mosaic virus 4 is with Dan Ke
Grand antibody is the various immunological detection methods and immunological reagent box that core is established.
The present invention has the advantages that the hybridoma cell strain 1,9H9 1) provided secretes anti-south compared with prior art
Adzuki bean mosaic virus monoclonal antibody specific, the immunologys such as the dot-ELISA and ACP-ELISA that are established using the monoclonal antibody as core
Method and the kit energy high special established with these methods, it is accurate, delicately detect bean mosaic virus 4；2) it utilizes
The detection bean mosaic virus 4 of monoclonal antibody prepared by the present invention does not need expensive electron microscope, PCR instrument etc. and sets
It is standby；3) the southern bean mosaic virus being effectively used for using monoclonal antibody prepared by the present invention in port and field crops
The detection of poison.
Detailed description of the invention
Fig. 1 is the sensitivity analysis of dot-ELISA method detection SBMV；
Fig. 2 is the specificity analysis of dot-ELISA method detection SBMV；
The 1 column Kidney bean disease leaf that 2 points are 2 infection SBMV up and down；2 points are 2 common floral leaves of infection Kidney bean to 2 column up and down
The Kidney bean disease leaf of virus；2 points are 2 healthy Phaseolus Leaves to 3 column up and down；2 points are 2 healthy soybean leaves to 4 column up and down；5
Column are selected up and down as 2 healthy green soy bean blades for 2；2 points are respectively the sick leaf and healthy cowpea for infecting cucumber mosaic virus to 6 column up and down
Beans blade；2 points are respectively to infect southern cowpea mosaic virus disease leaf and healthy pea leaves to 7 column up and down；8 arrange 2 points up and down
Respectively infection tobacco mosaic virus disease leaf and healthy Broad Bean Leaves.
Fig. 3 is the representative result of SBMV in dot-ELISA method detection port leguminous plant sample；
Fig. 4 is the representative result of SBMV in RT-PCR method detection port leguminous plant sample.
Biological deposits
It is common that hybridoma cell strain 19H9 on January 7th, 2016 is preserved in China Committee for Culture Collection of Microorganisms
Microorganism center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101, deposit number CGMCC
No.12004。
Specific embodiment
The hybridoma cell strain 19H9 of anti-MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS is secreted in preservation on January 7 in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica, deposit number is
CGMCC No.12004, it can secrete the monoclonal antibody of anti-bean mosaic virus 4.
A kind of monoclonal antibody of the anti-bean mosaic virus 4 of the hybridoma cell strain 19H9 secretion, anti-south
The monoclonal antibody ascites indirect ELISA titer of adzuki bean mosaic virus is up to 10-7, Antibody types and subclass are that IgG1, kappa are light
Chain.It is found using ACP-ELISA method analysis, the sensitivity which detects sick leaf reaches 1:81,920 times of dilutions (w/v, g/
mL)。
There is spy in sick leaf texture of the monoclonal antibody of anti-bean mosaic virus 4 only with infection bean mosaic virus 4
Specific immunological reaction, and with infection Bean common mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus (TMV), cucumber mosaic
The sick leaf texture of virus and soybean, green soy bean, cowpea, pea, semen viciae fabae and the bean plant tissue of health do not occur any immune
Reaction.
Application of the anti-MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS on the viral diagnosis is using monoclonal antibody as core
The various immunological detection methods and immunological reagent box established.
Hybridoma cell strain 19H9 provided by the invention can largely secrete anti-bean mosaic virus 4 monoclonal antibody, and it is secreted
Monoclonal antibody high specificity, potency is high, stability is good.Using the high-throughput serology side for the detection SBMV that the monoclonal antibody is established as core
Method and its detection kit can be successfully applied to the detection of port SBMV, thus to improve China to bean mosaic virus 4
Detection level prevents the virus from overseas incoming domestic offer 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
With following operating procedure purified virus particle:
1) big mortar and tissue mixer device are pre-chilled；
2) 500g infection SMBV Kidney bean disease leaf is weighed, the 0.5M phosphate buffer of pH 7.5 is added in every 100g disease leaf
(i.e. PB buffer) 200mL (Na-EDTA containing 0.01M and 0.1% mercaptoethanol) is filtered after being homogenized 2min with the double-deck cotton gauze,
Filtrate 6,000rpm are centrifuged 20min, remove plant tissue residue；
3) gained supernatant is added dropwise to final concentration 2.5%Triton X-100,4%PEG (molecular weight 6,000) while stirring
With 0.1M NaCl, 4 DEG C of stirring 4h or more；
4) 11,000r/min is centrifuged 15min and must precipitate；
5) the precipitating 0.5M PB (MgCl containing 0.01M of pH 7.52With 0.5M urea) sufficiently suspend, 6,000rpm centrifugations
Supernatant is sucked out after 15min and is placed in centrifuge tube, precipitating settling flux, centrifugation are repeated 3 times；
6) merge supernatant, 33,000rpm ultracentrifugation 100min, 8,000rpm after the 0.5M PB suspension of gained precipitating
It is centrifuged 15min, collects supernatant, precipitating settling flux, centrifugation are repeated 3 times；
7) it is added in the centrifuge tube of existing 30% sucrose cushions in bottom after merging supernatant, 33,000rpm ultracentrifugations
100min；
8) the gained precipitating 0.5M PB (MgCl containing 0.01M of pH 7.52) suspend, suspension is Virus purification liquid；
9) sample is purified with electron microscope observation, finds the bean mosaic virus 4 particle of a large amount of high-purities.2. exempting from
Epidemic disease animal
Six week old weight 18-20g BALB/c female mices are immunized with SBMV purified virus.That is SBMV purified virus particle
100 μ L/ are only mixed with isometric Freund's complete adjuvant, after fully emulsified, only through back subcutaneous abdomen multi-point injection 0.2mL/, interval
3 weeks, take with one exempt from equivalent amount of antigen and isometric freund 's incomplete adjuvant it is fully emulsified after, second is injected intraperitoneally 0.2mL/
Only, it is injected intraperitoneally after spending 3 weeks with the antigen of doubling dose, extracting spleen cell carries out cell fusion after 3 days.
3. cell fusion
Take above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) in 7:1 ratio serum-free RPMI-
It is mixed in 1640 (Gibco) culture mediums, 1,500rpm centrifugation 5min removes culture medium, 1mL 50% is added in 37 DEG C of water-baths
PEG (molecular weight 1500) simultaneously merges 2min, and 1,500rpm is centrifuged after terminating fusion with the RPMI-1640 culture medium of serum-free
5min, precipitating are suspended with HAT culture medium, are dispensed into 96 holes and contain in the cell plates of feeder cells, be placed in 37 DEG C, contain 5%CO2
Cell incubator in cultivate.
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 the Phaseolus Leaves and purified virus for infecting SBMV virus as antigen coat, between routine
137 positive holes are obtained in the positive hole for connecing ELISA method screening secretion monoclonal antibody altogether.Selection 5 cell holes in strong positive reaction,
Limiting dilution assay clone is carried out, the hybridoma cell strain 19H9 of specific monoclonal antibody of anti-SBMV can be secreted by obtaining 1 plant.Through 6 months
After the above subculture in vitro separately and multiple cryopreservation resuscitation, cell strain can well be grown, and stably excreting antibody.After expanding culture,
For ascites preparation and Liquid nitrogen storage.
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 after 7-10 days
8×105A hybridoma, 7-10 days visible mouse web portions obviously expand after injection, and injection needle takes ascites, and 8,000rpm
It is centrifuged 3min, collecting supernatant is odd contradictive hydroperitoneum.1 times of volume ascites is taken to add 2 times of 4.8 acetate buffer solutions of volume 0.06M pH
Dilution, at room temperature while stirring plus sad (30uL/mL ascites), 4 DEG C of clarifications 1h, 12,000rpm centrifugation 20min collect supernatant,
Again with 50% saturated ammonium sulphate immunoglobulin, 4 DEG C of placement 2h, 3,000rpm centrifugation 20min, 2 times of volumes of precipitating
PBS solution dissolution obtains the ascites antibody of purifying, -70 DEG C of preservations in 4 DEG C of flowing dialysis afterwards for 24 hours.
6. the type and subgroup identification and titer of ascites of monoclonal antibody measure
With the type and subclass of the DAS-ELISA kit identification monoclonal antibody for identifying monoclonal antibody type of Sigma company, knot
Fruit shows that 19H9 monoclonal antibody subclass is IgG1, kappa light chain.Odd contradictive hydroperitoneum potency is detected with conventional indirect ELISA method, as a result
Show that 19H9 odd contradictive hydroperitoneum potency reaches 10-7。
7. the specific detection of monoclonal antibody
With infection Bean common mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus (TMV), the disease of cucumber mosaic virus
Leaf texture and health soybean, green soy bean, cowpea, pea and bean plant tissue crude extract be coated with elisa plate, with healthy Kidney bean
Leaf extract makees negative control, to infect the Kidney bean juice of bean mosaic virus 4 as positive control, with ACP-ELISA method
Measure the specific reaction of monoclonal antibody.ACP-ELISA method specifically: the sick leaf of above-mentioned virus infection is added by 1:30 (w/v, g/mL)
The hole 100uL/ is coated with elisa plate after entering the homogenate of ELISA coating buffer, and 4 DEG C of overnight or 37 DEG C of 2h make it be adsorbed in ELISA polyphenyl second
Alkene plate hole；PBST washing closes 30-60min with 3% skimmed milk power afterwards three times；The appropriate hole diluted monoclonal antibody 100uL/ is added,
37℃1-2h；PBST washing is added appropriate diluted alkaline phosphatase lipase (AP) afterwards three times and marks sheep anti-mouse igg secondary antibody (Sigma
Company) hole 100uL/, 37 DEG C of 1-2h；After PBST is washed four times, developed the color with PNPP substrate whole with 2M sodium hydroxide after 30-60min
It only reacts, the value of OD405 is read with microplate reader, to be greater than 2.1 with negative OD value ratio for the positive.As a result, it has been found that 19H9 monoclonal antibody
Have a specific reaction to infection SBMV disease leaf, and with infection Bean common mosaic virus, southern cowpea mosaic virus, Tobacco mosaic
Soybean, green soy bean, cowpea, pea, semen viciae fabae and the bean plant tissue of virus, the sick leaf of cucumber mosaic virus and health are without any
Specific immune response.
Two, the foundation of SBMV immunological method and its kit is detected
1. detecting the ACP-ELISA method of SBMV
The operating procedure of 1.1ACP-ELISA method:
(1) sick leaf is added 0.05M carbonate buffer solution (pH 9.6) by 1:30 (w/v, g/mL) ratio and is homogenized, and 5,000rpm
Be centrifuged 3min, centrifugation the hole supernatant 100uL/ be coated with elisa plate, using SBMV disease leaf as positive control, be good for leaf be negative control, 37 DEG C
2h or 4 DEG C overnight；
(2) 30min is closed with 3% skimmed milk power after PBST washing；
(3) appropriate diluted odd contradictive hydroperitoneum, the hole 100uL/, 37 DEG C of 1h are added；
(4) the sheep anti-mouse igg secondary antibody of appropriate diluted alkaline phosphatase lipase (AP) label is added after PBST washing
(Sigma), the hole 100uL/, 37 DEG C of 1h；
(5) PNPP nitro Phosphate substrate, the hole 100uL/, room temperature 30min are added after being washed with PBST；
(6) it detecting by an unaided eye, substrate colors become the hole of yellow green as the positive, or after being terminated with 2M sodium hydroxide and reacting,
OD405 is surveyed with enzyme-linked immunosorbent assay instrument, using P/N > 2.1 as positive judgment criteria.
The determination of 1.2ACP-ELISA method detection sensitivity and specificity
The most suitable working concentration for determining monoclonal antibody and ELIAS secondary antibody in ACP-ELISA detection method, examination are tested with conventional square matrix
It tests and shows that the most suitable working concentration of 19H9 monoclonal antibody and ELIAS secondary antibody is respectively that 1:5,000 and 1:8,000 times dilute.Most with antibody
The ACP-ELISA method that suitable working concentration establishes detection SBMV carries out 1:20-1:655,360 doubling dilution to sick leaf crude extract
(w/v, g/mL), and negative control is made with the strong leaf crude extract of corresponding dilution respectively, it is examined with above-mentioned ACP-ELISA method
It surveys.The result shows that ACP-ELISA method, to 1:81, the sick leaf crude extract of 920 times of dilutions (w/v, g/mL) is still positive, i.e.,
1:81 is reached to the detection sensitivity of sick leaf, 920 times of dilutions (w/v, g/mL) show the side ACP-ELISA of the monoclonal antibody and foundation
Method has the sensitivity and reliability of height.It is in strong positive reaction, detection infection Kidney bean that this method, which detects SBMV disease leaf crude extract,
Mosaic viruses, southern cowpea mosaic virus, tobacco mosaic virus (TMV), the sick leaf of cucumber mosaic virus and healthy soybean, hair
Beans, cowpea, pea, semen viciae fabae and bean plant tissue are negative, and yin and yang attribute contrast difference is extremely significant, illustrate this method and
The specificity of monoclonal antibody is fine.
The foundation and Fields detection application of 2.dot-ELISA method
The operating procedure of the dot-ELISA method of SBMV in 2.1 detection leguminous plants
It is homogenized afterwards after Phaseolus Leaves are weighed with ratio addition 0.01M PBS (pH 7.4) by 1:30 (w/v, g/mL)；
Homogenate 5,000rpm is centrifuged 3min；It takes 3 μ L to be centrifuged to check on nitrocellulose (NC) film, while health and infection is set
SBMV Phaseolus Leaves tissue crude extract is respectively as negative and positive control；Drying at room temperature 10-20min；NC film is immersed in containing 5%
Room temperature closes 30min in PBST (the 0.01M PBS containing 0.05%Tween-20) confining liquid of skimmed milk power；NC film is put into appropriateness
30-60min is incubated at room temperature in diluted monoclonal antibody；Film is washed 3-4 times with PBST, each 3min；NC film is put into the diluted AP enzyme of appropriateness
30-60min is incubated at room temperature in label sheep anti-mouse igg secondary antibody；PBST washes film 4-5 times, each 3min；66 μ L NBT and 33 μ L
BCIP substrate (Promega) is added to 10mL substrate buffer solution (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl2、pH
9.5) it mixes, film, which is put into substrate solution, to react, visual results；It is obvious and negative without any colour developing to positive control colour developing
When in tap water rinsing terminate reaction, photograph to record result.
The determination of 2.2dot-ELISA method detection sensitivity and specificity
The most suitable working concentration for determining monoclonal antibody and ELIAS secondary antibody in dot-ELISA detection method is tested with conventional square matrix,
Test result shows that the most suitable working concentration of 19H9 monoclonal antibody and ELIAS secondary antibody is respectively 1:5,000 and 1:8,000 times of dilution.With
The most suitable working concentration of above-mentioned antibody establishes the dot-ELISA method of detection SBMV.Sensitivity analysis shows when Kidney bean disease leaf is dilute
It releases 1:5, when 120 times (w/v, g/mL), purple is still presented using the dot-ELISA method detection that 19H9 monoclonal antibody is established as core
Positive spots, i.e. its sensitivity for detecting sick leaf reach 1:5,120 times of dilutions (w/v, g/mL) (Fig. 1).Specificity analysis shows,
This method detection SBMV disease plant has strong specific positive reaction, and detects infection Bean common mosaic virus, south
Cowpea mosaic virus, tobacco mosaic virus (TMV), the sick leaf of cucumber mosaic virus and healthy soybean, green soy bean, cowpea, pea, semen viciae fabae
With (Fig. 2) negative when bean plant tissue.
The Field information of 2.3dot-ELISA detection method
The doubtful morbidity pulse family sample 2015 provided by Shanghai inspection and quarantine bureau with the dot-ELISA method of foundation into
Row detection, as a result, it has been found that, there are 23 samples to produce purpuriferous positive spots (Fig. 3) in 50 test samples, sample is simultaneously into one
Step is detected with RT-PCR, the results showed that expands the PCR product for arriving SBMV specificity in all dot-ELISA detection positive samples
(Fig. 4), the positive sample that PCR product nucleic acid sequencing shows infects SBMV really, and dot-ELISA detects RT- in negative sample
PCR is not expanded to any genetic fragment.Illustrate that the dot-ELISA method of the foundation can accurately and reliably be used for pulse family sample
The detection of middle bean mosaic virus 4.
3. bean mosaic virus 4 dot-ELISA detection kit
1) kit main component:
The above reagent is stored at 4 DEG C
Nitrocellulose filter (NC) 10 2) detection leguminous plant sample operating procedure:
A. it is even afterwards 0.01M PBS (pH 7.4) will to be added by 1:30 (w/v, g/mL) ratio after the weighing of pulse family sample blade
Slurry；
B. homogenate 5,000rpm is centrifuged 3min；
C. it takes and is checked on NC film on 3 μ L, while health and infection SBMV Phaseolus Leaves crude extract are set respectively as yin
Property and positive control, drying at room temperature 10-20min；
D.NC film is immersed in the PBST containing 3% skimmed milk power (the 0.01M PBS containing 0.05%Tween-20) confining liquid
Room temperature closes 30min；
E.NC film is put into 1:2,000 times of diluted monoclonal antibody and is incubated at room temperature 30-60min；
F. film is washed 3-4 times with PBST, each 3min；NC film is put into the diluted AP enzyme label sheep anti-mouse igg secondary antibody of 1:3,000
Middle incubation at room temperature 30-60min；
G.PBST washes film 4-5 times, each 3min；66 μ L NBT and 33 μ L BCIP substrates are added to 10mL substrate buffer solution
(0.1M Tris Cl、0.1M NaCl、0.025M MgCl2, pH 9.5) mix, film is put into substrate solution the 10-20min that develops the color；
H. visual results develop the color obvious (purple) to positive control, and tap water when negative no any colour developing
Rinsing terminates 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, pH 7.4):
PH to 7.4 is adjusted after adding distilled water 950 to dissolve, and is settled to 1,000mL
ELISA cleaning solution (0.01M PBST):
Add 0.5mL Tween-20 in 1,000mL 0.01M PBS
ELISA confining liquid:
Skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v).
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| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
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|---|---|---|---|---|
| CN1600865A (en) * | 2003-09-23 | 2005-03-30 | 北京金长河科技发展有限公司 | Gene chip for detecting plant virus |
| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
Non-Patent Citations (2)
| Title |
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| Monoclonal Antibodies as Structural Probes of Southern Bean Mosaic Virus;J.H.TREMAINE等;《VIROLOGY》;19851231;第144卷;第80-87页 * |
| 南方菜豆花叶病毒单克隆抗体的初步研究;宋淑敏等;《病毒学报》;19910630;第7卷(第2期);第176-179页，尤其是第177页第2段，第178页第2段 * |
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