CN108486065B - Secrete hybridoma cell strain and its monoclonal antibody application of anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody - Google Patents
Secrete hybridoma cell strain and its monoclonal antibody application of anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody Download PDFInfo
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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Abstract
The invention discloses a kind of applications of hybridoma cell strain and its monoclonal antibody for secreting anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody.It is that BALB/c mouse is immunized in antigen with Rice Gall Dwarf In Guangdong Province (RGDV) the CP recombinant protein of prokaryotic expression, through cell fusion, screening, clone, the hybridoma cell strain 11B6 for passing on and secreting anti-RGDV monoclonal antibody can be stablized by obtaining 1 plant, and deposit number is CGMCC No.14897.The odd contradictive hydroperitoneum indirect ELISA titer of hybridoma cell strain secretion reaches 10‑8, Antibody types and subclass are IgG1, kappa.The monoclonal antibody and RGDV coat protein have specific reaction.It is ACP-ELISA the and dot-ELISA method that core establishes detection RGDV using 11B6 monoclonal antibody, the sensitivity that both methods detects sick leaf respectively reaches 1:327 680 and 480 times of 1:20 dilutions (w/v, g/mL).The acquisition of the hybridoma cell strain and its monoclonal antibody and the detection, diagnosis and science bridle for being established as the virosis of Serology test provide substance and technical support.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of hybridization for secreting anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody
The application of tumor cell strain and its monoclonal antibody.
Background technique
Rice gall dwarf virus disease is a kind of rice caused by Rice Gall Dwarf In Guangdong Province (Rice gall dwarf virus, RGDV)
Virus disease.From 1979 after Thailand's discovery, rice gall dwarf virus disease is found on Malaysia, Japan, South Korea and other places in succession.1981
Year finds RGDV in China Guangdong for the first time, and rice gall dwarf virus disease Major Epidemic is in the Guangdong in China, Guangxi, Fujian, Hainan etc. at present
South Rice Region of China, serious field of falling ill even can not normally ear, and No kernels or seeds are gathered, as in a year of scarcity.RGDV host plant is also wrapped other than rice
The gramineous crops such as wheat, barley, oat, amur foxtail, wild rice and corn are included, but it does not infect barnyard grass.Infect RGDV rice
Classical symptom include: that the plant of morbidity stunts, tiller is reduced, and young leaves is short and narrow, and leaf color deepens, and blade back portion and leaf sheath have 0.1-
The circular white strumae of 0.2mm size, every leaf have 0-30.RGDV is by vector recilia dorsalis and rice green leafhopper to hold
Long proliferous type mode is propagated, and leafhopper once obtains poison, i.e., lifelong band poison simultaneously can transovarian transmission.RGDV belongs to Reoviridae
(Reoviridae), Phytoreovirus (Phytoreovirus).The virion of RGDV belongs to icosahedron, triple
Symmetrically, spherical, diameter is about 65-70nm.All measurement finishes the genome sequence of RGDV, forms containing 12 dsRNA,
Every RNA segment only encode an albumen, including six structural proteins (P1, P2, P3, P5, P6 and P8) and six it is non-structural
Albumen (Pns4, Pns7, Pns9, Pns10, Pns11 and Pns12).
Recilia dorsalis and rice green leafhopper are China is widely distributed, harmfulness is big, and it draws as passing virus mediator and propagate RGDV
The rice gall dwarf virus disease risen is serious in China's harm in recent years, causes Severe Reduction of Rice.And the detection technique of virus is the virus
The key of sick prevention and control.Field rice tumor dwarf virus is judged a situation arises to be still most important side according to Symptom Observation at present
Method, but virus infection rice early period does not have any symptom, and symptom caused by a variety of virosis is very much like, in addition, often ill
Malicious Combined Infection situation, therefore lack accuracy and science by the result that Symptom Observation obtains.Electronic Speculum observes virion
Expensive instrument is needed, and the size of somewhat different virion and form have similitude, it is caused only to examine as a kind of auxiliary
Survey means.The molecular detection technology of based on PCR method is although sensitive, but is unsuitable for the detection of the extensive sample in field.Serum
Method is simple to operation, quick, sensitive, cost is relatively low, and is suitable for the extensive detection of field sample, is ideal at present
The detection technique of plant virus, but serological method is necessarily dependent upon special, sensitive high-quality antibody.For this purpose, the invention patent
Preparation and its detection application mainly for Rice Gall Dwarf In Guangdong Province monoclonal antibody are carried out the work, and are prepared for using hybridoma technology
1 plant can secrete anti-RGDV monoclonal antibody hybridoma 11B6, and establish the serology side for detecting the virus with its monoclonal antibody secreted
Method, so that the detection, diagnosis and science bridle for China's Rice Gall Dwarf In Guangdong Province provide substance and technical support.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody of secretion
The application of hybridoma cell strain and its monoclonal antibody.
Anti- Rice Gall Dwarf In Guangdong Province monoclonal antibody hybridoma 11B6 is secreted, the specificity that it can secrete anti-Rice Gall Dwarf In Guangdong Province is single
Anti-, hybridoma cell strain 11B6 is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on December 15th, 2017
Object center, deposit number are CGMCC No.14897.
The monoclonal antibody ascites indirect ELISA titer of anti-Rice Gall Dwarf In Guangdong Province is up to 10-8, Antibody types and subclass are
The coat protein of IgG1, kappa, the monoclonal antibody and Rice Gall Dwarf In Guangdong Province 47kDa have specific immune response, utilize ACP-
ELISA and dot-ELISA method analysis finds that the sensitivity of monoclonal antibody detection infection RGDV rice disease leaf respectively reaches 1:327
480 times of 680 and 1:20 dilutions (w/v, g/mL).
The monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province only has a specific immune response with Rice Gall Dwarf In Guangdong Province, and with rice item
Line virus, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, rice stripe mosaic disease
It poison and healthy rice plants and non-take malicious recilia dorsalis and non-take malicious rice green leafhopper tissue any immune response does not occur.
Application of the monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province in Rice Gall Dwarf In Guangdong Province detection is built by core of monoclonal antibody
Vertical various immunological detection methods and immunological detecting kit.
The present invention has the advantages that the hybridoma cell strain 11B6 secretion water resistant rice 1) provided compared with prior art
Tumor dwarf virus monoclonal antibody specific, the immunological methods such as the dot-ELISA and ACP-ELISA that are established using the monoclonal antibody as core
And established with these methods kit energy high special, it is accurate, delicately detect Rice Gall Dwarf In Guangdong Province；2) institute of the present invention is utilized
The monoclonal antibody of preparation detects Rice Gall Dwarf In Guangdong Province, does not need the equipment such as expensive electron microscope, PCR instrument；3) institute of the present invention is utilized
The monoclonal antibody of preparation is effectively used for the detection and diagnosis of the internal water RGDV such as field rice and biography virus mediator recilia dorsalis, can also
For the epidemiological survey of the virosis, viral genome functional analysis, resistance breeding, science bridle etc..
Detailed description of the invention
Fig. 1 is the sensitivity analysis of dot-ELISA method detection rice RGDV；CK+ is that the rice disease leaf of infection RGDV is thick
Extract, CK- are healthy rice leaf crude extract.
Fig. 2 is the specificity analysis of dot-ELISA method detection RGDV: 2 points are 1 infection RGDV rice to 1 column up and down
2 biology of plant sample repeat；2, which arrange 2 biology that 2 points up and down are recombinant expression RGDVCP albumen, repeats；On 3 column
Lower 2 points are that 2 biology of 1 infection RSV rice plants sample repeat；2 points are 1 infection RBSDV rice to 4 column up and down
2 biology of plant sample repeat；2 points are that 2 biology of 1 infection RRSV rice plants sample repeat to 5 column up and down；
2 points are that 2 biology of 1 infection SRBSDV rice plants sample repeat to 6 column up and down；2 points are 1 RSMV to 7 column up and down
2 biology of rice plants sample repeat；2 points are that 2 biology of 1 healthy rice plants sample repeat to 8 column up and down.
Fig. 3 is the representative result of RGDV in dot-ELISA method detection rice field sample.
Biological deposits
It secretes anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody hybridoma 11B6 and is preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101, preservation day is 2017
December 15, deposit number are CGMCC No.14897.
Specific embodiment
It secretes anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody hybridoma cell strain 11B6 and is preserved in Chinese science on December 15th, 2017
China Committee for Culture Collection of Microorganisms's common micro-organisms center, institute of microbiology, institute, deposit number CGMCC
No.14897, it can secrete the monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province.
The monoclonal antibody ascites indirect ELISA titer of anti-Rice Gall Dwarf In Guangdong Province is up to 10-8, Antibody types and subclass are
IgG1, kappa can have specific immune response with the 47kDa coat protein of Rice Gall Dwarf In Guangdong Province, using ACP-ELISA and
Dot-ELISA method analysis finds that the sensitivity of monoclonal antibody detection infection RGDV rice plants disease leaf respectively reaches 1:327 680
With 480 times of 1:20 dilutions (w/v, g/mL).
The monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province only has a specific immune response with Rice Gall Dwarf In Guangdong Province, and with rice item
Line virus, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, rice stripe mosaic disease
It poison and healthy rice plants and non-take malicious recilia dorsalis and non-take malicious rice green leafhopper tissue any immune response does not occur.
The monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province on the viral diagnosis application be using monoclonal antibody be core establish it is various be immunized
Learn detection method and immunological reagent box.
Hybridoma cell strain 11B6 provided by the invention can largely secrete the monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province, and its secretion
Monoclonal antibody potency height, high specificity, high sensitivity, stability are good.The detection RGDV's established using the monoclonal antibody as core is high-throughput
Serological method can be successfully applied to the detection of field RGDV, thus for the detection and diagnosis of China's Rice Gall Dwarf In Guangdong Province, early warning,
Breeding for disease resistance and science bridle provide substance and technical support.
Below with reference to embodiment and attached drawing, the invention will be further described.
One, the acquisition of hybridoma and its preparation of monoclonal antibody
1. immunogene and the preparation for detecting antigen
Specifically drawn according to the gene order (accession number: D13410.1) of reported RGDV coded housing albumen design a pair
Object: RGDV-CP-F:5 '-CTGGTTCCGCGTGGATCCATGTCGCGCCAAGCTT-3 ' and RGDV-CP-R:5 '-GATGCGGC
CGCTCGAGTCGACTTAGTTTACTGTGTAATACCTACCG-3 ', underscore part be BamH I, I restriction enzyme site of Sal, and by
The synthesis of Hangzhou Qing Ke Bioisystech Co., Ltd.Total serum IgE (the concrete operations of infection RGDV rice disease leaf are extracted with TRIzol reagent
Carried out referring to reagent specification), the CP base of the virus has been cloned from the rice total RNA of infection RGDV using RT-PCR method
Cause.
RT-PCR reaction: it is carried out according to the RNA reverse transcription reagent box specification of mill company, Japan as follows: in a centrifuge tube
The middle 1 μ L of total serum IgE that 5 μ L of deionized water, 4 × DN Master Mix, 2 μ L, 65 DEG C of metal baths are added and are denaturalized 5min, 37 DEG C of digestion
Genomic DNA 5min；Add 2 μ L 5 × RT Master MixII, 37 DEG C of reverse transcription 1h after mixing；After 98 DEG C of inactivation 5min
It is immediately placed on ice, sample can be reserved in -20 DEG C of refrigerators.PCR reaction system (50 μ L) is as follows: 5 μ L of dNTP, MgSO4 4μL、
5 μ L of KOD plus neo buffer, each 1 μ L of upstream and downstream primer, 1 μ L of cDNA template, 33 μ L of deionized water.PCR response procedures
It is as follows: 94 DEG C of initial denaturation 2min, 98 DEG C of denaturation 10sec, 55 DEG C of annealing 30sec, 68 DEG C of extension 40sec, 68 DEG C after 32 circulations
Extend 10min, 16 DEG C of 5min.After reaction, the detection of PCR product is carried out with agarose gel electrophoresis and be tapped and recovered.
Recombinant expression carrier: reaction system (10 μ L) are as follows: 5 × CE is constructed using the homologous recombination kit of Nuo Weizan company
2 μ L of II Buffer, 1 μ L of RGDV CP genetic fragment, 1.5 μ L of carrier, 1 μ L of Exnase II, ddH2O 4.5μL.According to above-mentioned
After evenly mixing, 37 DEG C of 30min are put on ice system later.Protokaryon table is inserted it into using the method for above-mentioned homologous recombination
Up to being built into recombinant prokaryotic expression vector pGEX-4T-3-CP in vector pGEX -4T-3.PGEX-4T-3- containing recombinant expression carrier
BL21-CodonPlus (DE3) Escherichia coli of CP obtain through IPTG induction expression protein, GST-beads affinity column protein purification
RGDV CP recombination fusion protein.BALB/c mouse is immunized by antigen of the recombinant protein of purifying.
2. immune animal
With the 8 week old BALB/c female mice of RGDV CP protein immunization of purifying.That is 80 μ L of RGDV purifying protein and 80 μ L good fortune
The mixing of family name's Freund's complete adjuvant, it is fully emulsified after through 160 μ L/ of intraperitoneal injection only, be spaced 3 weeks, take the antigen for exempting from difference equivalent with one and
After freund 's incomplete adjuvant is fully emulsified, intraperitoneal injection two exempt from, with 120 μ L/ antigens and 80 μ L/ after 3 weeks
Physiological saline containing mycillin mixes, and carries out three through intraperitoneal injection and exempts from, and three are repeated once after 2 weeks exempt from step and carry out reinforcing exempting from
Epidemic disease, extracting spleen cell is merged after 3 days.
3. cell fusion
Take above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) in 6:1 ratio serum-free RPMI-
It being mixed in 1640 culture mediums, 1 500rpm is centrifuged 5min and removes culture medium, and use 50%PEG (molecular weight 1 500) as fusion agent,
2min is merged in 37 DEG C of water-baths, 1 500rpm is centrifuged 5min, precipitating after terminating fusion with the RPMI-1640 culture medium of serum-free
It is suspended, is dispensed into 96 porocyte plates with HAT culture medium, be placed in 37 DEG C containing 5%CO2Cell incubator in cultivate.
4. hybridoma, the screening in positive hole and its clone
After cultivating 5d in cell incubator, is changed the liquid once with HAT culture medium, change liquid, Deng Daorong with HT culture medium within the 10th day
When closing cell covering bottom hole 10-30%, using RGDV CP as antigen coat elisa plate, it is single that secretion is screened with indirect ELISA method
80 positive holes are obtained in anti-positive hole altogether.Selection 8 cell holes in strong positive reaction carry out 3 limiting dilution assays clones,
Obtain 1 plant of hybridoma cell strain 11B6 that can secrete anti-RGDV specific monoclonal antibody.Through 6 months or more subculture in vitro separately and repeatedly freeze
After recovery, 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
(1) 8 week old or so BALB/c mouse is taken, 0.3mL norphytane, Intraperitoneal injection 6 × 10 after 7-10d is injected intraperitoneally5It is a
Hybridoma, the visible mouse web portion of 7-10d obviously expands after injection, and syringe needle takes ascites, and 3 000rpm are centrifuged 3min, collects
Supernatant is odd contradictive hydroperitoneum.
(2) saturated ammonium sulfate method purified monoclonal antibody ascites (by taking 1mL as an example)
1) 5 000rpm of 1mL ascites, 4 DEG C of centrifugation 5min are taken, supernatant is taken, 2mL normal saline dilution is added；
2) pH value is added dropwise in the saturated ammonium sulfate solution 3mL of 7.0-7.4, stirring while adding, room temperature after being all added
Continue to stir 10min；4 DEG C of static 2-3h；
3) 12 000rpm, 4 DEG C of centrifugation 10min abandon supernatant, precipitating 2mL physiological saline solution；
4) antibody is packed into bag filter, and for 24 hours, during which every 2h changes a PBS solution for 4 DEG C of dialysis in 0.01M PBS；
5) antibody in bag filter, 12 4 DEG C of 000rpm centrifugation 10min are taken out, supernatant is the antibody purified；It takes on 1 μ L
IgG content is measured with NanoDrop ultraviolet spectrometer clearly, antibody is saved in -80 DEG C after measurement.
6. the type and subgroup identification and titer of ascites of monoclonal antibody measure
By the anti-BALB/c mouse IgG of standard of the monoclonal antibody of purifying and Sigma company kit1、IgG2a、IgG2b、IgG3、
IgM, κ and lambda light chain antibody carry out DAS-ELISA analysis, the results show that 11B6 monoclonal antibody type and subclass are IgG1, kappa.
To recombinate RGDV CP albumen as envelope antigen, odd contradictive hydroperitoneum potency is detected with conventional indirect ELISA method, the results showed that 11B6
Odd contradictive hydroperitoneum potency reaches 10-8。
7. the specific detection of monoclonal antibody
With infection rice stripe virus (RSV), rice black-streaked dwarf virus (RBSDV), southern rice black-streaked dwarf respectively
Viral (SRBSDV), rice sawtooth dwarf virus (RRSV), rice stripe mosaic virus (RSMV) sick leaf crude extract wrap respectively
By elisa plate, with the healthy leaves crude extract of rice, non-malicious recilia dorsalis and the non-homogenate for taking malicious rice green leafhopper taken for feminine gender
Control is to infect sick leaf crude extract, the RGDV of Rice Gall Dwarf In Guangdong Province and take malicious recilia dorsalis and take the homogenate of malicious rice green leafhopper
Positive control, with the specificity of ACP-ELISA method measurement monoclonal antibody.The specific steps of ACP-ELISA method are as follows: above-mentioned virus sense
Sick leaf liquid nitrogen grind into powder in mortar of dye is added 0.05M carbonate by 1:20 (w/v, g/mL) times and is coated with buffer
Continue grinding homogenate, supernatant is crude extract after 5 000rpm are centrifuged 3min；1 head lobe cicada is put into eppendorf centrifuge tube, adds
With toothpick homogenate is mashed after entering 150 μ L 0.05M carbonate coating buffer；Crude extract or 100 hole μ L/ of homogenate are coated with ELISA
Plate；4 DEG C of overnight or 37 DEG C of 2h, make it be adsorbed in ELISA polystyrene plate hole；PBST is sealed after washing 3 times with 3% skimmed milk power
Close 30-60min；Appropriate diluted 100 hole μ L/ of monoclonal antibody, 37 DEG C of 1-2h are added；Appropriate diluted alkali is added in PBST after washing 3 times
Property phospholipase (AP, be used for rice sample) or horseradish peroxidase (HRP is used for leafhopper sample) label sheep anti-mouse igg
100 hole μ L/ of secondary antibody (Sigma company), 37 DEG C of 1-2h；PBST is developed the color after washing 4 times with PNPP or tmb substrate, after terminating reaction
OD is read with microplate reader405Or OD450Value, with the OD value ratio of negative sample be greater than 2.1 for the positive.As a result, it has been found that 11B6
Monoclonal antibody to infection RGDV rice disease leaf texture crude extract, take malicious recilia dorsalis and carry rice green leafhopper tissue homogenate have specifically
Property reaction, and with infection RSV, RBSDV, RRSV, SRBSDV, RSMV and healthy rice plants tissue and it is non-take malicious recilia dorsalis and
The non-homogenate for taking malicious rice green leafhopper is without any immune response.
Two, the foundation of the immunological method and its kit of RGDV is detected
1. detecting the ACP-ELISA method of RGDV
The step of 1.1 ACP-ELISA method:
1) liquid nitrogen grind into powder in mortar is used after plant tissue weighing, is added in 1:20 ratio (w/v, g/mL)
0.05M carbonate buffer solution (pH 9.6) continues grinding homogenate 3min, and 5 000rpm are centrifuged 3min, and 100 hole μ L/ of supernatant is added
In elisa plate, to infect RGDV rice disease leaf as positive control, healthy Rice Leaf is negative control, and 37 DEG C 2h or 4 DEG C overnight；
2) 30min is closed with 3% skimmed milk power after PBST is washed 3 times；
3) odd contradictive hydroperitoneum after appropriate dilution is added, 100 holes μ L/, 37 DEG C of incubation 1h；
4) the sheep anti-mouse igg secondary antibody of appropriate diluted alkaline phosphatase lipase (AP) label, 100 μ are added after PBST is washed 3 times
The hole L/, 37 DEG C of incubation 1h；
5) 100 hole μ L/ of PNPP nitro Phosphate substrate is added after being washed with PBST, is placed at room temperature for 30min；
6) it detects by an unaided eye, substrate colors become the hole of yellow green as the positive；Or it with after the termination reaction of 2M sodium hydroxide, uses
Enzyme-linked immunosorbent assay instrument surveys OD405, using P/N > 2.1 as positive judgment criteria.
The foundation of 1.2 ACP-ELISA methods and its sensitivity and specificity determine
The most suitable working concentration for determining monoclonal antibody and ELIAS secondary antibody in ACP-ELISA method, i.e. ELISA are tested with conventional square matrix
Plate laterally plus with the monoclonal antibody of confining liquid doubling dilution, longitudinal plus sheep anti-mouse igg secondary antibody with confining liquid doubling dilution AP label into
Row ACP-ELISA.Experiments have shown that the most suitable working concentration of 11B6 monoclonal antibody and AP ELIAS secondary antibody is respectively 1:5 000 and 1:8 000
It dilutes again, the ACP-ELISA method of RGDV in detection rice plants is established with the most suitable working concentration of antibody.It is thick to RGDV disease leaf
Extract makees negative control respectively from 1:20 to 655 360 doubling dilutions with the healthy rice leaf crude extract of corresponding dilution, point
Analyse the detection sensitivity of ACP-ELISA method.The result shows that establish ACP-ELISA method to 680 times of 1:327 dilution (w/v,
G/mL sick leaf crude extract) is still positive, i.e., can reach 1:327 680 (w/v, g/mL) times to the detection sensitivity of sick leaf
Dilution shows that ACP-ELISA method has very high sensitivity.With the ACP-ELISA method of foundation detection RGDV, RSV,
The crude extract of RBSDV, SRBSDV, RRSV, RSMV and healthy rice plants tissue.The result shows that the ACP-ELISA method of foundation
Detection infection RGDV rice disease leaf crude extract be in strong positive reaction, and detect respectively infect RSV, RBSDV, RRSV, SRBSDV,
RSMV and healthy rice plants tissue crude extract are negative, and yin and yang attribute Comparative result difference is extremely significant, illustrates the party
The specificity of method is very well.
The foundation and Fields detection application of 2.dot-ELISA method
2.1 dot-ELISA methods detect the operating procedure of RGDV in plant:
Liquid nitrogen grind into powder in mortar is used after the blade of rice plants sample is weighed, by 1:20's (w/v, g/mL)
Ratio continues grinding, homogenate after 0.01M PBS (pH7.4) is added；5 000rpm of homogenate is centrifuged 3min；It takes and is checked on 2.5 μ L
To on nitrocellulose filter (NC), while the rice leaf of health and infection RGDV are set respectively as negative and positive control；
Drying at room temperature 10-20min；NC film is immersed in the envelope of the PBST (the 0.01M PBS containing 0.05%Tween-20) containing 5% skimmed milk power
Close 37 DEG C of closing 30min in liquid；NC film is put into the diluted monoclonal antibody of appropriateness and is incubated at room temperature 40-60min；Film is washed 3-4 times with PBST,
Each 3min；NC film is put into the sheep anti-mouse igg secondary antibody of the diluted AP enzyme label of appropriateness and is incubated at room temperature 40-60min；PBST washes film
4-5 times, each 3min；66 μ L NBT and 33 μ L BCIP substrates (Promega) are added to 10mL substrate buffer solution (0.1mol/L
Tris Cl、0.1mol/L NaCl、0.025mol/L MgCl2, pH9.5) in mix, film is put into chromogenic reaction 10- in substrate solution
20min, visual results are obvious to positive control colour developing, and when negative no any colour developing, tap water rinse terminates anti-
It answers, photographs to record result.
The foundation of 2.2 dot-ELISA methods and its sensitivity and specificity determine
It is tested with conventional square matrix and determines the monoclonal antibody of RGDV and the most suitable working concentration of ELIAS secondary antibody in dot-ELISA method,
Experiments have shown that the most suitable working concentration of 11B6 monoclonal antibody and ELIAS secondary antibody is respectively 1:5 000 and 000 times of 1:8 dilution.With above-mentioned anti-
The most suitable working concentration of body establishes the dot-ELISA method of detection RGDV.Sensitivity analysis shows to be diluted to 1 when RGDV disease leaf:
When 20480 (w/v, g/mL) times, the positive spots of purple are still presented in the dot-ELISA method detection established with 11B6 monoclonal antibody, i.e.,
Its sensitivity for detecting sick leaf reaches 1:20480 times of dilution (w/v, g/mL) (Fig. 1).Specificity analysis shows, this method detection
The rice plants for infecting RGDV are in strong positive reaction, and detect and infect RSV, RBSDV, RRSV, SRBSDV, RSMV and health respectively
Rice plants tissue crude extract is negative (Fig. 2).
The application of 2.3 dot-ELISA methods
2017 are examined from 78 plants of rice samples that Rice In Guangdong field acquires with the dot-ELISA method of foundation
It surveys, as a result, it has been found that, there are 32 sample infection RGDV, disease incidence 41%, representative testing result such as Fig. 3 institute in 78 samples
Show.These samples are analyzed with RT-PCR method simultaneously, the results showed that in the positive sample that all dot-ELISA are detected
The genetic fragment for arriving RGDV specificity can be expanded, and is not expanded in all negative samples to any genetic fragment.PCR product core
Acid sequencing shows that positive sample infects RGDV really, illustrates that the dot-ELISA method can be accurately and reliably used in plant sample
The detection of Rice Gall Dwarf In Guangdong Province.
3. Rice Gall Dwarf In Guangdong Province dot-ELISA detection kit (detection rice sample)
1) kit main component:
The above reagent is stored in 4 DEG C
2) operating procedure of rice sample is detected:
A. liquid nitrogen grind into powder in mortar is used after the blade of rice plants sample being weighed, in the ratio (w/ of 1:20
V, g/mL) be added 0.01M PBS (pH7.4) after continue grinding, homogenate；
B. 5 000rpm of homogenate is centrifuged 3min；
C. take 2.5 μ L supernatant point samples on NC film, while the rice plants tissue that health and infection RGDV is arranged is made respectively
For negative 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 1:4 diluted RGDV monoclonal antibody and is incubated at room temperature 40-60min；
F. film is washed 3-4 times with PBST, each 3min；
G.NC film is put into the diluted AP enzyme of 1:5 000 label sheep anti-mouse igg secondary antibody and is incubated at room temperature 40-60min；
H.PBST washes film 4-5 times, each 3min；
I.66 μ LNBT and 33 μ LBCIP substrates be added to 10mL substrate buffer solution (0.1M Tris Cl, 0.1M NaCl,
0.025M MgCl2, pH9.5) in, mix caudacoria and be put into substrate solution and develop the color, visual results are obvious aobvious to positive control
Purple and negative control do not have tap water rinse film when any colour developing to terminate reaction, photograph 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:
A. phosphate buffer (PBS, 0.01mol/L, pH7.4): NaCl 8g, KCl 0.2g, KH2PO4 0.2g,
Na2HPO4·12H2O 3g adjusts pH to 7.4 after adding distilled water 950mL to dissolve, is settled to 1000mL.
B.ELISA cleaning solution (0.01M PBST): add 0.5mL Tween-20 in 1000mL 0.01M PBS
C.ELISA confining liquid: skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v, g/mL).
4. Rice Gall Dwarf In Guangdong Province dot-ELISA detection kit (detection recilia dorsalis or rice green leafhopper sample)
1) kit main component:
The above reagent is stored at 4 DEG C
2) operating procedure:
A. sample preparation: single head leafhopper is put into 250 μ L eppendorf centrifugation, and 50 μ L PBS are added in every pipe, is mashed with toothpick
Leafhopper；
B. it point sample: takes a NC film to put to clean culture dish with tweezers, 3 μ L insect homogenates is taken to dub NC film
On, often take a sample to change a pipette tips；
C. dry: sample spot film is completed caudacoria and is dried at room temperature for, about 10-20min；
D. close: NC film is immersed in the closing of the PBST (the 0.01M PBS containing 0.05%Tween-20) containing 5% skimmed milk power
Room temperature closes 30min in liquid；
E. primary antibody is incubated for: NC film is put into 000 times of 1:3 diluted RGDV monoclonal antibody and is incubated at room temperature 40-60min；
F. it washs: washing film 3-4 times with PBST, each 3min；
G. secondary antibody is incubated for: NC film is put into the diluted HRP enzyme of 1:5 000 label sheep anti-mouse igg secondary antibody and is incubated at room temperature 40-
60min；
H. it washs: washing film 4-5 times with PBST, each 3min；Finally washed film 1 time with PBS；
I. it develops the color: being put into new culture dish after washed NC film is blotted with filter paper, appropriate TMB assay chromogenic substrate solution is added,
NC film is flooded, covers newspaper shading, visual results, obvious to the aobvious blue of positive control, feminine gender when any colour developing without remembering
Record testing result, developing time about 5-15min；
3) preservation and validity period:
It is kept in dark place in 2-8 DEG C, validity period 12 months.
4) buffer formulation:
A. phosphate buffer (PBS, 0.01mol/L, pH7.4): NaCl 8g, KCl 0.2g, KH2PO4 0.2g,
Na2HPO4·12H2O 3g adjusts pH to 7.4 after adding distilled water 950mL to dissolve, is settled to 1000mL.
B.ELISA cleaning solution (0.01M PBST): add 0.5mL Tween-20 in 1000mL 0.01M PBS
C.ELISA confining liquid: skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v, g/mL).
Claims (3)
1. a kind of hybridoma cell strain 11B6 for secreting anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody, it is characterised in that can secrete anti-
The specific monoclonal antibody of Rice Gall Dwarf In Guangdong Province, hybridoma cell strain 11B6 are preserved in Chinese microorganism strain on December 15th, 2017
Preservation administration committee common micro-organisms center, deposit number are CGMCC No.14897.
2. a kind of monoclonal antibody of the anti-Rice Gall Dwarf In Guangdong Province of hybridoma cell strain 11B6 secretion as described in claim 1.
3. a kind of application of monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province as claimed in claim 2 on the viral diagnosis,
It is characterized in that establishing various immunological detection methods and immunological detecting kit by core of the monoclonal antibody.
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| Hairpin RNA derived from the gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus, confers strong resistance to virus infection in transgenic rice plants;Takumi Shimizu等;《Journal of Biotechnology》;20120210;第157卷(第3期);第421-427页 * |
| 水稻瘤矮病毒P12 蛋白抗血清制备及其应用;孟媛等;《植物保护学报》;20131031;第40卷(第5期);第413-417页 * |
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