CN105543176B - Secrete resistant to PVY monoclonal antibody hybridoma cell strain and its monoclonal antibody application - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
The invention discloses the applications of a kind of secretion resistant to PVY (PVY) monoclonal antibody hybridoma cell strain and its monoclonal antibody.PVY virion using differential centrifugation purification is that BALB/c mouse is immunized in antigen, and through cell fusion, screening, clone, the hybridoma cell strain 3B2 for passing on and secreting anti-PVY monoclonal antibody can be stablized by obtaining 1 plant, and deposit number is CGMCC No.12001.The cell strain secretes odd contradictive hydroperitoneum indirect ELISA titer up to 10‑7, Antibody types and subclass are IgG1, kappa light chain, and the PVY coat protein of the monoclonal antibody and 30kDa have idiosyncrasy.ACP-ELISA, dot-ELISA and Tissue blot-ELISA detection method of PVY on detection crop is established using 3B2 monoclonal antibody, the sensitivity that wherein ACP-ELISA and dot-ELISA method detects sick leaf respectively reaches 1:81920 and 1:10240 times of dilution (w/v, g/mL).The preparation of anti-PVY monoclonal antibody and its diagnosis for being established as the potato virus disease and detection of detection method, epidemiological analysis and science bridle 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 resistant to PVY monoclonal antibody
The application of tumor cell strain and its monoclonal antibody.
Background technique
Marmor upsilon (Potato virus Y, PVY) is the important virus for endangering the crops such as potato, tobacco
One, in global wide-scale distribution and cause serious financial consequences.PVY is marmor upsilon section (Potyviridae) potato Y disease
Poison belongs to the line-up of delegates of (Potyvirus), is found for the first time in potato by Smith within 1931.Potyvirus is plant
Maximum category in virus, determines and possible kind there are about 200, and the virus of the category can infect Solanaceae, Chenopodiaceae, pulse family, Curcurbitaceae
Etc. various plants and result in significant economic losses.PVY is widely current in European potato seed growing area from nineteen fifty-three, 20th century
The seventies, the virus expanded to America, prevalence took place in Asia to the beginning of the nineties in last century virus, the virus exists at present
There is occurrence injury all over the world.The difference due to difference of Host Cultivars and virus strain of symptom caused by PVY, typically
Symptom includes heavy floral leaf, vein necrosis, leaf streak necrosis etc. of hanging down, and can cause potato yield loss up to 80%.Research hair
The primary source of infection of existing PVY is mainly Virus-infested, and cause of disease can be spread at a distance by potato seed allocation and transportation, in potato field PVY
By sick juice and malicious aphis propagation can also be taken.PVY particle is bending, and no coating, size is 730 × 11nm, gene
Group overall length about 10kb, 5 '-terminal covalent combination genomes connection virus protein (genome-linked viral protein,
VPg), 3 '-ends have Ploy (A).Genome only includes 1 big ORF, encodes the polyprotein of 1 about 360kDa, the poly
Albumen can be cleaved into 11 mature functional proteins, including the coat protein of a 29.8kDa.
The method that prevention and treatment PVY mainly uses Anti-virus Disease Breeding and produces nontoxic potato seed, and quick, accurate, sensitive, special
Virus detection techniques are the key that Anti-virus Disease Breeding and the nontoxic potato seed of production.It detects, divide with conventional indicator plant detection, Electronic Speculum
The methods of sub- Biological Detection is compared, serological method have many advantages, such as it is simple, economical, easy to operate, can detect on a large scale.At present
About the report of PVY Antibody preparation and serological method, but the detection of reported PVY antibody and serological method is sensitive
Spend it is not good enough, for this purpose, the present invention is prepared for 1 plant of anti-PVY of secretion by hybridoma technology using the PVY virion that purifies as antigen
Special, sensitive monoclonal antibody hybridoma cell strain, the high-throughput serology side of detection PVY is established using the monoclonal antibody of secretion as core
Method and kit, to be detection and diagnosis, the investigation of epidemiology, the production of nontoxic potato seed, the viral genome of China PVY
The analysis of function and its foundation of science bridle system 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 hybridization for secreting resistant to PVY monoclonal antibody
Tumor cell strain and its monoclonal antibody application.
Resistant to PVY monoclonal antibody hybridoma cell strain 3B2 is secreted, it can secrete resistant to PVY monoclonal antibody, hybridoma
Cell strain 3B2 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation on January 7th, 2016
Number be CGMCC No.12001.
A kind of resistant to PVY monoclonal antibody of the hybridoma cell strain secretion, between resistant to PVY odd contradictive hydroperitoneum
ELISA potency is connect up to 10-7, Antibody types and subclass are IgG1, kappa light chain, the coat protein with marmor upsilon 30KDa
There is specific immune response, finds that monoclonal antibody detects potato-infecting Y virus using ACP-ELISA and dot-ELISA method analysis
The sensitivity of sick leaf respectively reaches 1:81 920 and 240 times of 1:10 dilutions (w/v, g/mL).
The plant tissue of resistant to PVY monoclonal antibody and potato-infecting Y virus has a specific immune response, and with infection
Marmor solani, potato virus X, potato virus S, corium solani and health tobacco and potato leaf group
It knits and is not immunoreacted.
Application of the resistant to PVY monoclonal antibody on the viral diagnosis is the various immunologys inspection established using monoclonal antibody as core
Survey method and immunological reagent box.
The present invention has the advantages that the hybridoma cell strain 1) provided can a large amount of stably excretings compared with prior art
Anti- PVY is special, sensitive monoclonal antibody, ACP-ELISA, dot-ELISA and the Tissue established using the monoclonal antibody of secretion as core
The serological methods such as blot-ELISA and the kit energy high special established with these methods, it is accurate, delicately detect plant
In marmor upsilon；2) PVY is detected using monoclonal antibody prepared by the present invention, does not need expensive electron microscope, PCR instrument
Etc. equipment；3) using monoclonal antibody prepared by the present invention, it is effectively used for the detection and diagnosis of PVY in Field Plants, it is also possible to
In the epidemiological survey of the virosis, viral genome functional analysis, virus-free seed potato production, resistance breeding, science bridle etc.
Aspect.
Detailed description of the invention
Fig. 1 dot-ELISA method detects the specificity analysis of marmor upsilon.
The sensitivity analysis of Fig. 2 dot-ELISA method detection PVY.
Fig. 3 dot-ELISA method detects the result of PVY in Field Plants sample.
Result of Fig. 4 RT-PCR method to PVY in detection Field Plants sample.
The result of PVY in Fig. 5 Tissue blot-ELISA method Fields detection plant sample.
Biological deposits
It is commonly micro- that hybridoma cell strain 3B2 on January 7th, 2016 is preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101, deposit number CGMCC
No.12001, classification naming are secretion resistant to PVY (PVY) monoclonal antibody hybridoma.
Specific embodiment
Resistant to PVY monoclonal antibody hybridoma cell strain 3B2 is secreted, it can secrete resistant to PVY monoclonal antibody, hybridoma
Cell strain 3B2 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation on January 7th, 2016
Number be CGMCC No.12001.
A kind of resistant to PVY monoclonal antibody of the hybridoma cell strain secretion, between resistant to PVY odd contradictive hydroperitoneum
ELISA potency is connect up to 10-7, Antibody types and subclass are IgG1, kappa light chain, the coat protein with marmor upsilon 30KDa
There is specific immune response, finds that monoclonal antibody detects potato-infecting Y virus using ACP-ELISA and dot-ELISA method analysis
The sensitivity of sick leaf respectively reaches 1:81 920 and 240 times of 1:10 dilutions (w/v, g/mL).
The plant tissue of resistant to PVY monoclonal antibody and potato-infecting Y virus has a specific immune response, and with infection
Marmor solani, potato virus X, potato virus S, corium solani and health tobacco and potato leaf group
It knits and is not immunoreacted.
Application of the resistant to PVY monoclonal antibody on the viral diagnosis is the various immunologys inspection established using monoclonal antibody as core
Survey method and immunological reagent box.
Hybridoma cell strain provided by the invention can largely secrete anti-PVY monoclonal antibody, and the monoclonal antibody of cell strain secretion is special
Property is strong, potency is high, stability is good, high sensitivity.It can using the high-throughput serological method that the monoclonal antibody establishes detection PVY as core
It is successfully applied to the detection of PVY in Field Plants, to be the life of China's marmor upsilon disease detection and diagnosis, virus-free seed potato
It produces, science bridle provides substance and technical support.
Below with reference to embodiment and attached drawing, the invention will be further described.
One, hybridoma acquisition and its preparation of monoclonal antibody
1. immunogene and the preparation for detecting antigen
With following operating procedure purified virus particle:
1) tissue homogenizer is pre-chilled on ice；
2) the Tissues of Tobacco 200g of infection PVY is weighed, 200mL 0.5M phosphate buffer, that is, PB is added in every 100g tissue
Buffer (Na-EDTA containing 0.01M and 0.1% mercaptoethanol, pH 7.5) is homogenized 5-10min in tissue homogenizer, makes cigarette
The sufficiently homogenate of grass tissue filters homogenate with the double-deck cotton gauze, and 6 000rpm of filtrate is centrifuged 20min and removes plant residue；
3) resulting centrifuged supernatant adds to final concentration of 2.5%Triton X-100,4%PEG (molecular weight in stirring
000) and 0.1M NaCl for 6, then 4 DEG C of stirring 4h or more；
4) 11 000rpm are centrifuged 15min and remove supernatant；
5) it will be precipitated with the 0.5M PB (MgCl2 containing 0.01M and 0.5M urea) of 10mL pH 7.5 and sufficiently be suspended, 6
Supernatant is collected in beaker after 000rpm centrifugation 15min, is placed in 4 DEG C, precipitating settling flux, centrifugation are repeated 3 times；
6) above-mentioned centrifuged supernatant is merged, 33 000rpm ultracentrifugation 100min；
7) gained precipitating with 0.5M PB suspend after 8 000rpm be centrifuged 15min, collect supernatant, precipitating settling flux, from
The heart is repeated 3 times；
8) merging supernatant is added to and is surpassed from pipe, be added to and surpassed from pipe with syringe needle absorption 30% sucrose of 20mL
Sucrose cushions, 33 000rpm ultracentrifugation 100min are formed on bottom；
9) gained precipitating is suspended with the 0.01M PB (MgCl2 containing 0.01M) of appropriate pH 7.5,33 000rpm ultracentrifugations
100min removes supernatant；
10) gained precipitating is suspended with 0.01M PB, and gained suspension is Virus purification liquid；
11) Virus purification liquid is placed in JEOL JEM-1200EX electricity under the microscope after 3% phosphotungstic acid (pH 6.7) negative staining
It was found that the PVY particle of a large amount of high-purities.
2. immune animal
Be immunized 8 week old weight BALB/c female mices with the PVY virion of purification: 100 μ L/ of PVY virion only with
Isometric Freund's complete adjuvant mixing after fully emulsified, through subcutaneous abdomen multi-point injection 0.2mL every, be spaced 3 weeks, take and one
Exempt from equivalent amount of antigen and isometric incomplete Freund's adjuvant it is fully emulsified after, second is injected intraperitoneally 0.2mL every, after spending 3 weeks
It is injected intraperitoneally with the antigen of doubling dose, extracting spleen cell is merged after 3d.
3. cell fusion
Take above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) in 5:1 ratio serum-free RPMI-
It is mixed in 1640 (Gibco) culture mediums, removes culture medium after 1 500rpm centrifugation 5min, centrifuge tube containing cell is in 37 DEG C of water
1mL 50%PEG (molecular weight 1500) fusion agent is added in bath, merges 2min, is terminated with the RPMI-1640 culture medium of serum-free
1 500rpm is centrifuged 5min after fusion, and precipitating is suspended with HAT culture medium after sucking supernatant, is dispensed into 96 porocyte plates, 37 DEG C,
5%CO2Cell incubator in cultivate.
4. hybridoma, the screening in positive hole and its clone
It is changed the liquid once after cultivating 5d in cell incubator with HAT culture medium, 10d changes liquid with HT culture medium, until fusion
When cell covers bottom hole 5%-20%, to infect the potato disease leaf crude extract of PVY as the conventional indirect ELISA side of envelope antigen
Method screens positive hole, obtains 298 positive holes altogether.Specificity and sensitivity analysis are carried out to 298 holes, filter out 8 it is special and
Sensitive cell hole carries out cell limiting dilutions clone, and the hybridoma of special, sensitive monoclonal antibody of anti-PVY can be secreted by obtaining 1 plant
Cell strain 3B2.After 4 months or more subculture in vitro separately and multiple cryopreservation resuscitation, cell strain can well be grown, and stably excreting is anti-
Body.After expanding culture, for odd contradictive hydroperitoneum preparation and Liquid nitrogen storage.
5. the preparation of monoclonal antibody ascites and purifying
0.3mL norphytane, Intraperitoneal injection 7 × 10 after 7-10d is injected intraperitoneally in 8 week old or so BALB/c mouse5A hybridoma
Cell, visible mouse web portion obviously expands after 7-10d, takes ascites with syringe needle, and 8 000rpm are centrifuged 3min, collects supernatant and is
For odd contradictive hydroperitoneum.
1 times of volume ascites is taken to add 2 times of volume 0.06M PH4.8 acetate buffer solution dilutions, it is stirring while adding plus pungent at room temperature
Sour (30 μ l/mL ascites), 4 DEG C of clarification 1h, 1 2000rpm are centrifuged 20min, collection supernatant, then with 50% saturated ammonium sulphate
Immunoglobulin, 4 DEG C of placements 2h, 3 000rpm are centrifuged 20min, and the PBS solution of 2 times of ascites volumes of precipitating dissolves, thoroughly at 4 DEG C
Analysis obtains the monoclonal antibody of purifying, -70 DEG C of preservations afterwards for 24 hours.
6. the 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 antibody
DAS-ELISA detection is carried out, testing result shows 3B2 monoclonal antibody type and subclass is IgG1, kappa light chain.With the PVY of purification
Virion is antigen, detects odd contradictive hydroperitoneum potency with conventional indirect ELISA method, analyzes the result shows that odd contradictive hydroperitoneum potency reaches
10-7。
7. the specific detection of monoclonal antibody
With potato-infecting A viral (PVA), potato virus X (PVX), potato virus S (PVS) and potato leaf roll
The sick leaf crude extract of viral (PLRV) is coated with elisa plate, makees negative control with the tobacco of health, potato leaf crude extract, with
The tobacco disease leaf crude extract for infecting PVY is positive control, with the specificity of conventional ACP-ELISA method analysis monoclonal antibody.Specificity
Analysis as a result, it has been found that, 3B2 monoclonal antibody has a specific immune response to infection PVY disease leaf texture, and with infection PVA, PVX, PVS and
The sick leaf and health tobacco of PLRV, potato tissue crude extract are without any reaction.
Two, the foundation of PVY immunological method and its kit is detected
1. detecting the ACP-ELISA method of PVY
The step of 1.1ACP-ELISA method:
1) sick leaf and healthy leaves are put into mortar after weighing respectively, and ELISA is added in the ratio (w/v, g/mL) of 1:20
It grinds, be homogenized after coating buffer；5 000rpm of homogenate is centrifuged 3min；It is centrifuged 100 hole μ L/ of supernatant and is coated with polystyrene board, with sense
The Tissues of Tobacco for contaminating PVY is used as positive control, makees negative control with healthy tobacco, potato tissue, 37 DEG C of 2h or；4 DEG C of mistakes
Night
2) the PBST confining liquid that 250 μ L contain 3% skimmed milk power, 37 DEG C of envelopes are added in PBST board-washing 3 times, each 3min, every hole
Close 0.5h；
3) with suitably diluted 100 hole μ L/ of odd contradictive hydroperitoneum of confining liquid, 37 DEG C of 1-2h after PBST is washed 3 times；
4) appropriate diluted AP label 100 hole μ L/ rabbit anti-mouse igg secondary antibody (Sigma) of addition after PBST is washed 3 times, 37 DEG C
1-2h；
5) after PBST is washed 4 times plus PNPP substrate is in color development at room temperature 30-60min, visually observes substrate colors change, colour developing at
The sample in yellow green hole is that after positive or 2M sodium hydroxide terminates reaction, the OD of 405nm is surveyed with 680 type enzyme-linked immunosorbent assay instruments
Value, using P/N > 2.1 as positive judgment criteria.
The foundation of 1.2ACP-ELISA detection method:
The working concentration for determining antibody is tested using square matrix, i.e., is slightly mentioned with 1:20 times of diluted (w/v, g/mL) PVY disease leaf
Liquid is coated with polystyrene board as envelope antigen；Elisa plate is longitudinally separately added into from top to down with confining liquid from 1:100 to 1:12
The PVY monoclonal antibody of 800 doubling dilutions is laterally from left to right separately added into confining liquid from 1:1 000 to 1:512 000 doubling dilution
AP label sheep anti-mouse igg secondary antibody, the crude extract of healthy tobacco or the corresponding dilution of potato plants is as negative right
According to each processing sets 3 repetitions, using P/N > 2.1 as positive judgment criteria, determines the most suitable work of antibody in ACP-ELISA
Concentration.3B2 monoclonal antibody is diluted as the result is shown with 000 times of 1:5, and the sheep anti-mouse igg secondary antibody of AP label is diluted to again with 1:7 000
Most suitable working concentration establishes the ACP-ELISA method of detection PVY according to most suitable working concentration.
The sensitivity and specificity of 1.3ACP-ELISA method are analyzed
It will be measured after PVY disease leaf crude extract coating buffer doubling dilution with the ACP-ELISA method established, the results showed that,
The sensitivity that ACP-ELISA detects sick leaf reaches 81 920 times of dilutions (w/v, g/mL), illustrates the monoclonal antibody of preparation and the side of foundation
Method has good sensitivity.This method detect PVY disease leaf crude extract be in very strong positive reaction, detection infection PVA, PVX,
The sick leaf texture of PVS and PLRV and the tobacco of health, potato tissue are negative, and yin and yang attribute Comparative result difference is extremely aobvious
It writes, illustrates that the specificity of this method and monoclonal antibody is good.
The foundation and Fields detection application of 2.dot-ELISA method
Dot-ELISA detects the step of PVY method in plant: 1 is pressed after the plant tissue after weighing is ground in mortar:
10-30 ratio (w/v, g/mL) continues grinding, homogenate after 0.01M PBS (pH7.4) is added；5 000rpm of homogenate centrifugation
3min；It takes and is checked on nitrocellulose (NC) on 2.5 μ L, while the potato leaf crude extract of health and infection PVY is set
Respectively as negative and positive control；Drying at room temperature 10-20min；NC film is immersed in the PBST containing 5% skimmed milk power and (contains
The 0.01M PBS of 0.05%Tween-20) room temperature closes 30min in confining liquid；NC film is put into room temperature in the diluted monoclonal antibody of appropriateness
It is incubated for 30-60min；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 appropriateness
In, it 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) add
Enter in 10ml substrate buffer solution (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl, pH9.5), mix, film is put into bottom
It develops the color in thing liquid, visual results, tap water floats when positive control shows purple obviously and negative control does not have any colour developing
It washes film and terminates reaction, photograph to record result.
It is tested with conventional square matrix and determines that the most suitable work of the dot-ELISA monoclonal antibody and ELIAS secondary antibody of detection potato disease leaf is dense
Degree, experiments have shown that the most suitable working concentration of 3B2 monoclonal antibody and ELIAS secondary antibody is respectively that 1:5 000 and 1:8000 times dilute.With above-mentioned
The most suitable working concentration of antibody establishes the dot-ELISA method of detection PVY.Specificity analysis shows, this method detection infection PVY
Plant be in strong positive reaction, and detect infection PVA, PVX, PVS, PLRV disease leaf and health tobacco and potato leaf are in yin
Property reaction (Fig. 1).Sensitivity analysis shows when PVY disease leaf crude extract is diluted to 1:10 240 times (w/v, g/mL), with foundation
Dot-ELISA detection the positive spots of purple are still presented, i.e. its sensitivity for detecting sick leaf reaches 240 times of 1:10 dilutions and (schemes
2)。
It is examined with the dot-ELISA method of foundation morbidity potato samples doubtful to the field for picking up from Yunnan for 2015
It surveys, as a result, it has been found that, there are 20 samples to produce purpuriferous positive spots (Fig. 3) in 30 potato test samples, sample is used simultaneously
RT-PCR method is analyzed, the results showed that the gene piece for arriving PVY specificity is expanded in all dot-ELISA positive samples
Section, and do not expanded in the negative sample of all dot-ELISA detection to genetic fragment (Fig. 4), PCR product nucleic acid sequencing shows
Positive sample infects PVY really.Illustrate that the dot-ELISA method can accurately and reliably be used for the detection of PVY in plant.
3. detecting the foundation and its field sample detection of the Tissue blot-ELISA method of PVY
3.1Tissue blot-ELISA operating procedure:
1) preparation of samples: being taken as the blade or stem of object, and tender leaf is rolled into cylindrical shape and cuts a cross section with blade, tender stem is straight
It connects and is cut into cross section；
2) point sample: cross section is pressed into 3sec on NC film, while using health and infecting PVY plant tissue as yin
Property and positive control, 37 DEG C of oven drying 5min；
3) close: the NC film of point sample is immersed in the PBST (0.01M containing 0.05%Tween-20 containing 5% skimmed milk power
PBS) room temperature closes 30-60min in confining liquid；
4) primary antibody is incubated for: NC film is put into appropriate diluted PVY monoclonal antibody and is incubated at room temperature 1h；
5) secondary antibody is incubated for: NC film is put into room temperature in appropriate diluted AP enzyme label sheep anti-mouse igg and incubates after washing film 3 times with PBST
Educate 1h；
6) PBST washes film 4-5 times, each 3min；
7) plus substrate develops the color: 10ml colorbuffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl, pH
9.5) 66 μ l NBT are added in and 33 μ l BCIP substrates (Promega) mix, film is immersed in developing solution and carries out chromogenic reaction,
Until positive colour developing is obvious, negative control does not have that film is rinsed to termination reaction in deionized water when background, record colour developing result.
The determination and its field sample detection of the most suitable antibody working concentration of 3.2Tissue blot-ELISA
The most suitable working concentration for determining monoclonal antibody and ELIAS secondary antibody in Tissue blot-ELISA is tested by conventional square matrix.
Selection detection sensitivity and the optimal monoclonal antibody of specificity and ELIAS secondary antibody dilution group are combined into the most suitable of Tissue blot-ELISA
Working concentration.As a result, it has been found that Tissue when the sheep anti mouse secondary antibody of monoclonal antibody 3B2 and AP label dilutes 1:5000 and 1:8000 times respectively
The detection sensitivity and specificity of blot-ELISA method are best, establish Tissue blot- according to the most suitable working concentration of antibody
ELISA method.With the Tissue blot-ELISA method of foundation morbidity potato sample doubtful to the field for picking up from Yunnan for 2015
Product are detected, as a result, it has been found that, there are 20 samples to produce purpuriferous positive spots (Fig. 5), sample in 30 potato test samples
Product are analyzed with RT-PCR method simultaneously, the results showed that PVY is arrived in amplification in all Tissue blot-ELISA positive samples
The genetic fragment of specificity, and do not expand in the sample of all Tissue blot-ELISA detection feminine gender to genetic fragment (figure
4), PCR product nucleic acid sequencing shows that positive sample infects PVS really.Illustrate Tissue blot-ELISA method can it is accurate,
It is reliably used for the detection of marmor upsilon in potato samples.
4. marmor upsilon dot-ELISA detection kit
1) kit main component:
The above reagent is stored in 4 DEG C
Nitrocellulose filter (NC) 10
2) operating procedure of potato samples is detected:
A. 0.01M is added in 1:10-30 ratio (w/v, g/mL) after the plant tissue after weighing being ground in mortar
Continue grinding, homogenate after PBS (pH7.4)；
B. 5 000rpm of homogenate is centrifuged 3min；
C. it takes 2.5 μ l supernatant point samples on NC film, while the potato tissue of health and infection PVY is set respectively as yin
Property 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, each 3min；NC film is put into the diluted AP enzyme of 1:3 000 label sheep anti-mouse igg secondary antibody
Middle incubation at room temperature 30-60min；
G.PBST washes film 4-5 times, each 3min；
H.66 μ l NBT and 33 μ l BCIP substrates be added to 10mL substrate buffer solution (0.1M Tris Cl, 0.1M NaCl,
0.025M MgCl, pH9.5) in, it mixes, film, which is put into substrate solution, carries out chromogenic reaction, visual results；
I. it develops the color obvious (purple) to positive control, and negative control does not have to rinse film end when any colour developing in tap water
It only reacts, 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 (0.01M PBS, pH7.4):
PH to 7.4 is adjusted after adding distilled water 950 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, g/mL).
Claims (5)
1. a kind of secretion resistant to PVY monoclonal antibody hybridoma cell strain 3B2, it is characterised in that resistant to PVY can be secreted
Monoclonal antibody, it is general that the hybridoma cell strain 3B2 on January 7th, 2016 is preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center, deposit number are CGMCC No.12001.
2. a kind of resistant to PVY monoclonal antibody of hybridoma cell strain 3B2 secretion as described in claim 1, it is characterised in that
The odd contradictive hydroperitoneum indirect ELISA titer is up to 10-7, Antibody types and subclass are IgG1, kappa light chain, with marmor upsilon
The coat protein of 30KDa has specific immune response, finds that monoclonal antibody detects using ACP-ELISA and dot-ELISA method analysis
The sensitivity of potato-infecting Y virus disease leaf respectively reaches 1:81920 and 1:10240 times dilutes, unit be gram/mL.
3. resistant to PVY monoclonal antibody as claimed in claim 2, it is characterised in that the monoclonal antibody and potato-infecting Y virus
Plant tissue has specific immune response, and rolls up with potato-infecting A virus, potato virus X, potato virus S, potato
The tobacco and potato leaf tissue of mosaic virus and health are not immunoreacted.
4. a kind of application of resistant to PVY monoclonal antibody as claimed in claim 2 on the viral diagnosis, it is characterised in that with
The monoclonal antibody establishes immunological detection method for core and detects the virus.
5. a kind of resistant to PVY monoclonal antibody as claimed in claim 2 is in the reagent of preparation immunology detection marmor upsilon
Application in box.
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| 马铃薯Y病毒单克隆抗体的制备及其检测应用;宋革等;《浙江大学学报（农业与生命科学版）》;20160918;第42卷(第5期);517-526 * |
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