CN101735318B - Cell strain, monoclonal antibody and kit for detecting tobacco rattle virus - Google Patents
Cell strain, monoclonal antibody and kit for detecting tobacco rattle virus Download PDFInfo
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- CN101735318B CN101735318B CN 200810225905 CN200810225905A CN101735318B CN 101735318 B CN101735318 B CN 101735318B CN 200810225905 CN200810225905 CN 200810225905 CN 200810225905 A CN200810225905 A CN 200810225905A CN 101735318 B CN101735318 B CN 101735318B
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Abstract
The invention relates to a cell strain, monoclonal antibody and kit for detecting tobacco rattle virus, and belongs to the technical fields of inspection and quarantine and biological technology. The monoclonal antibody for detecting the tobacco rattle virus is excreted by a cell strain 3C3; and the preservation number of the cell strain is CGMCC No.2679. The invention has the advantages that: the invention initially prepares a TRV monoclonal antibody and establishes an antigen enveloped ELISA (ACP-ELISA) detection method. The TRV specific monoclonal antibody has the advantages of good specificity, good homogeneity, infinite production and the like, so an ACP-ELISA serology method established by the monoclonal antibody has the characteristics of strong accuracy, easy standardization, large-scale production and the like. The method can also be effectively used for detecting field TRV. The acquisition of the TRV monoclonal antibody and the establishment of the detection method provide technical support for rapid diagnosis of the virus disease and lay a foundation for researching a molecular mechanism of a TRV-infected host and gene silencing.
Description
Technical field
The present invention relates to a kind of cell strain, monoclonal antibody and test kit that detects Tobacco rattle virus, belong to inspection and quarantine field and biological technical field.
Background technology
(Tobaccorattle virus TRV) belongs to Tobravirus (Tobravirus) to Tobacco rattle virus.TRV propagates through the nematode (Trchodorus and Paratrichodours) that is born in soil.Nematode tends to find in the place of impeded drainage.TRV is the principal element that causes disease on this soil.Continue to exist at this local TRV, and propagate on the sensitive plant kind through the nematode of locality.The weeds of numerous species are the main locations of virus, and yam and some other farm crop are less important places.TRV distributes extensively, can infect a large amount of floristics, on the stem tuber of yam, tobacco, ornamental plant, can cause important financial loss.It before this virales one of quarantine biology of China.Tobacco rattle virus be one type of application relatively extensively and efficient and persistence virus vector preferably, can not bring the symptom of virus induction the mediated gene silencing while.Improved virus can promote the insertion of non-virus sequence and to the follow-up infection of plant, also can identify the gene of host plant vegetative point, so TRV has widely in plant gene function is identified and uses.
At present, the method that detects TRV mainly adopts import antiserum(antisera) detection kit to carry out enzyme-linked immunosorbent assay (ELISA) and with the virus particle in the Electronic Speculum direct viewing tissue.See from existing report; Both at home and abroad the serology detection method of TRV is all set up based on the TRV polyvalent antibody at present; Commodity ELISA detection kit also is a polyvalent antibody; Yet many anti-there are deficiencies such as non-specific height, accuracy and uniformity are poor, output is limited, are difficult to satisfy in the growing production of flowers and plants detection demand virus; The Electronic Speculum detection needs expensive equipment and can not popularization and application.Thereby press for and set up quick, accurate, easy detection method, for the diagnosis of this virus disease provides technical support.
Summary of the invention
First technical problem that the present invention will solve provides monoclonal antibody and the monoclonal cell strain of secreting this antibody of a kind of detection Tobacco rattle virus of highly sensitive, high specificity.
Another technical problem that the present invention will solve provides a kind of test kit that contains the detection Tobacco rattle virus of said monoclonal antibody.
For realizing above-mentioned purpose, the present invention takes following technical scheme:
Detect the monoclonal antibody of Tobacco rattle virus, obtained by cell strain 3C3 secretion, the preserving number of this cell strain is CGMCC NO.2679.
Monoclonal cell strain 3C3, preserving number are CGMCC NO.2679.
Monoclonal cell strain 3C3 is used to prepare the purposes of the reagent that detects Tobacco rattle virus.
Detect the test kit of Tobacco rattle virus, form by following reagent:
TRV monoclonal antibody 0.1ml (concentration is 1mg/ml)
AP mark sheep anti-mouse igg 0.1ml (Sigma Company products, concentration are 1mg/ml)
P-nitrophenyl SULPHOSUCCINIC ACID ESTER (p-nitrophenyl phosphate, PNPP) 0.15g
Positive control (containing Tobacco rattle virus tobacco leaf juice) 2ml
Negative control (health tobacco leaf juice) 2ml
Above reagent all is stored under 4 ℃
10 of 96 hole ELISA Sptting plates
1 bottle of sample diluting liquid ELISA confining liquid (10 *) (quality-concentration of volume percent is 20% skim-milk solution, and skim-milk is bright Company products)
This research is through animal immune, cytogamy, screening, clone and the ascites preparation of TRV virus; Obtained 1 strain TRV specific monoclonal antibody, and with these monoclonal antibodies set up to TRV specific reaction is arranged and with all unresponsive ACP-ELISA detection methods of virus such as BBWV1, BBWV2, CarMV, CMV, PVX, PVY, SCMV, TMV, ToMV and TuMV.
Monoclonal cell strain 3C3 is stored in Chinese common micro-organisms preservation center, and preserving number is CGMCC NO.2679, preservation date on 09 26th, 2008, address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101.
Tobacco rattle virus (Tobacco rattle virus; TRV) the BALB/C mice spleen cell of immunity and SP2/0 rat bone marrow tumour cell merge; Through screening and cloning, obtain the hybridoma (3C3) that 1 strain can be stablized the anti-TRV monoclonal antibody of the justacrine that goes down to posterity (MAb), and prepare its odd contradictive hydroperitoneum.Monoclonal antibody ascites indirect ELISA titer reaches 10
-6, antibody type and subclass are IgG1, and the light chain of monoclonal antibody is the κ chain.The Western-blot analysis revealed, this strain monoclonal antibody can both combine with the 22.3KD coat protein subunit specificity of TRV.Utilize monoclonal antibody to set up the method that antigen coated indirect ELISA (ACP-ELISA) detects TRV.When sick leaf did that 1:400 doubly dilutes, purification TRV virus concentration is 18ng/mL (the viral absolute magnitude in every hole is 1.8ng), this method still can detect virus.
Advantage of the present invention is: the present invention has prepared the monoclonal antibody of TRV first, has set up antigen coated FLISA (ACP-ELISA) detection method.The TRV monoclonal antibody specific, have high specificity, uniformity good, advantage such as can endlessly produce, thereby make the ACP-ELISA serological method of setting up with monoclonal antibody have accuracy strong, be prone to characteristics such as stdn and scale operation.This method also can be effective to the detection of field TRV.Although rare bibliographical information TRV is in the generation of China, TRV is as quarantine venereal disease evil, and the preparation of TRV antibody has important practical significance.The acquisition of TRV monoclonal antibody and the foundation of detection method will provide technical support for the quick diagnosis of this virus disease, lay the first stone for research TRV infects in host's molecule mechanism and the gene silencing research simultaneously.
Below in conjunction with embodiment the present invention is described further; Be not the qualification to invention, according to prior art well known in the art, embodiment of the present invention is not limited to this; Therefore all this areas of having done according to present disclosure be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the TRV virus particle of purifying.
Fig. 2 is that TRV coat protein Western-blot detects, wherein 1: susceptible leaf sap; 2: healthy leaf sap.
Embodiment
Embodiment 1: the foundation of monoclonal antibody and screening
One. material and method
1. viral, substratum
TRV, preserve in this laboratory.
The sheep anti-mouse igg of RPMI-1640 substratum, HT, HAT, antibody type and subgroup identification test kit, alkali phosphatase enzyme mark is the Sigma Company products.
2. the purification of Tobacco rattle virus
Soak into the Ben Shi cigarette with the geneome RNA 1 of laboratory preservation TRV PpK20 strain system and the bacterium liquid phase of RNA2 altogether with the volume mixing, collect sick leaf behind the 10-14d, and with reference to (Hsu H T such as Hsu; Kim J Y; Lawson R H.Purificationof lily symptomless virus and detection of the virus in lilies.Plant Disease, 1995,79:912-916.; The purifying of lily asymptomatic virus and the virus in lily detect; Plant diseases, 1995, method of purification somewhat modified purified virus 79:912-916); Add 20% sucrose solution pad in the centrifuge tube bottom when being centrifugal, viral purification liquid is observed particle shape down through the JEM-1200EX transmission electron microscope of the rearmounted Japanese JEOL of 2% phospho-wolframic acid (pH6.7) negative staining company.
3. mouse immune, cytogamy, screening and clone
With reference to (blue or green tinkling of pieces of jade such as the blue or green tinkling of pieces of jade; Wu Jianxiang; Qi Yijun, etc. [J] used in broad bean wilt virus Study of Monoclonal Antibodies and detection. and mikrobe journal .2000,40 (2): 166-173.) immune programme for children of report is with the TRV virus particle of the purifying BALB/C mice in immune 8 ages in week.With reference to (Wu Jianxiang such as Wu Jianxiang; Lin Fucheng, Li Debao, etc. Pyricularia oryzae Study of Monoclonal Antibodies and the influence [J] that appressorium is formed thereof. the mikrobe journal; 2000,40 (6): method 638645.) is with the splenocyte and the mouse SP2/0 cytogamy of immune mouse.After fused cell is cultivated 5d; Change liquid more once with the HAT substratum; 10d changes liquid with the HT substratum, by the time at the bottom of the fused cell coverage hole during 5%-30%, gets supernatant with indirect ELISA method screening antibody positive hole; Envelope antigen is respectively the TRV of purification, the sick leaf sap (1:30 dilution) of Ben Shi cigarette that TRV infects during screening, and makes negative control with healthy leaf sap.Select the hybridoma of strong positive reaction, clone 3 times continuously with limiting dilution assay, the hole gained cell strain of last clone back test positive is the hybridoma cell strain of secrete monoclonal antibody.Hybridoma is used for ascites preparation and liquid nitrogen and preserves after enlarged culturing.
4. ascites prepares and antibody purification
Monoclonal antibody ascites preparation is according to method (Shi Manling, Wu Jianxiang, the Guo Wei of Shi Manling etc.; Deng. [J] used in Brassica 2 et 4 MONOCLONAL ANTIBODIES SPECIFIC FOR and detection. mikrobe journal, 2004,44 (2): 185-188.) carry out; Adopt sad-ammonium sulfate method (Chen Baiquan, Wu Meiying, Ye Qunrui. the comparison [J] of several kinds of partial purification monoclonal anti body methods. viral journal; 1990,6 (2): 122-126.) purified monoclonal antibody ascites, the purified monoclonal antibody of acquisition is in-70 ℃ of preservations.
5. antibody type and subgroup identification and ascites titration
Adopt the sheep anti-mouse igg 1 of Sigma company, IgG2a, IgG2b, IgG3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line behind the 24h, judges the antibody type and the subclass of monoclonal antibody.(1 μ g/mL) encapsulates elisa plate with purified virus, and the ascites of doubling dilution is done one and resisted, and the sheep anti-mouse igg of alkali phosphatase enzyme mark is that two anti-ascites of carrying out indirect ELISA mensuration monoclonal antibody are tired.
6.Western-blot detect
With reference to the method for kingfisher etc. (in kingfisher, Wu Jianxiang, Zhou Xueping. TOMV MONOCLONAL ANTIBODIES SPECIFIC FOR and detect and use [J]. the mikrobe journal; 2002; 42 (4): 454-457.) carry out, carry out the sds gel electrophoresis of coat protein subunit, a running gel cutting back part is used for coomassie brilliant blue staining observes with purified virus; Another part carries out electrotransfer, and the nitrocellulose filter with the electrotransfer gained carries out the Western-blot analysis with different ascites monoclonal antibodies then.
7.TRV the ACP-ELISA detection method
With reference to (Jiang J X, Chen Z X, Zhou X P.Production of a monoclonalantibody to sugarcane mosaic virus and its application for virus detection inChina [J] .Journal of Phytopathology.2003 such as Jiang; 151:361-364., Jiang Junxi, Chen Zhengxian; Zhou Xueping, the MONOCLONAL ANTIBODIES SPECIFIC FOR of corn mosaic virus and detect in the virus of China and to use the plant pathology magazine; 2003,151:361-364) operation steps is carried out ACP-ELISA, with the sick leaf sap 100 μ L/ holes adding elisa plate of 30 times of dilutions of 0.05mol/L carbonate buffer solution (pH9.6); The positive contrast of TRV purified virus; The negative contrast of healthy leaf sap, 5000-10000 times of dilution odd contradictive hydroperitoneums are one anti-, and the sheep anti-mouse igg binding substances of 8000 times of dilution AP marks is two anti-; Nitro phosphoric acid salt (PNPP) is substrate, surveys OD with 680 enzyme-linked immunosorbent assay instruments (BIO-RAD)
405Value is with P/N>2.1 as positive judgement criteria.
8.ACP-ELISA method is to the specific assay of TRV and different virus
BBWV1 (broad bean wilt virus I), BBWV2 (broad bean wilt virus II), CarMV (carnation mottle virus), CMV (cucumber mosaic virus), PVX (potato virus X), PVY (marmor upsilon), SCMV (corn mosaic virus), TMV (tobacco mosaic virus(TMV)), ToMV (TOMV) and TuMV (Brassica 2 et 4) (being Zhejiang University's biotechnology research preserves, provides) with purifying (1ug/mL) encapsulate the ELISA Sptting plate; With the positive contrast of immunizing antigen TRV; Make negative control to encapsulate damping fluid; With above-mentioned ACP-ELISA method, measure the specific reaction of monoclonal antibody.
9.ACP-ELISA the method detection sensitivity detects
After the TRV virus (5mg/mL) that the sick leaf sap of TRV is done 1:10～1:10240 doubling dilution, purification is made 1:1000～1:128000 doubling dilution; Respectively as antigen coated elisa plate, and with dilution strong leaf sap of correspondence and the negative contrast of strong leaf purification liquid.Do to utilize after 1:5000 doubly dilutes the ACP-ELISA method to measure detection sensitivity 3C3 monoclonal antibody (monoclonal antibody that final screening obtains) ascites.
Two. the result
1. the purification of Tobacco rattle virus
With the sick leaf sample of improved method purification Ben Shi cigarette, under Electronic Speculum, observe the virus particle that high density is arranged in the purification liquid, form is two kinds long, and is short different shaft-like, particle complete (Fig. 1).
2. the fusion of hybridoma, screening, clone
The BALB/C mice splenocyte of immunity and SP2/0 murine myeloma cell merge under 50%PEG in the ratio of 5～10:1, use the HAT screening of medium, and the fusion rate of 6 96 porocyte plates is 100%.Use the HT substratum instead after merging 10d, when at the bottom of the hybridoma coverage hole of every hole during 5%-30%, adopt indirect ELISA method to detect the secretion situation of antibody in the cells and supernatant, the performance positive reaction of 198 holes is arranged, positive rate is 35%.Select 8 cell holes that are strong positive reaction, carry out the limiting dilution assay clone, obtain the specific antibody hybridoma cell strain that 1 strain 3C3 can secrete anti-TRV at last.Through subculture in vitro separately more than 6 months with repeatedly behind the cryopreservation resuscitation, this strain cell strain can well be grown, and stably excreting antibody.Carry out preservation, and be used for following experiment.
3. ascites antibody preparation, antibody type and subgroup identification, titration
The BALB/C mice of injection monoclonal hybridoma, about 7-10d postabdomen expands, and gathers ascites, and get once later every day, every desirable 5-20mL ascites of mouse.Antibody type and subgroup identification result show that antibody type and the subclass of 3C3 are IgG1, and the light chain of monoclonal antibody is the κ chain; The odd contradictive hydroperitoneum indirect ELISA titer all reaches 10
-2(table 1).
The characteristic of table 1 TRV monoclonal antibody
4.Western-blot analyze
TRV to purifying carries out the Western-blot analysis revealed, and this strain monoclonal antibody can both combine (Fig. 2) with the 22.3KD coat protein subunit specificity of TRV, explains that prepared monoclonal antibody is the specific antibody to the 22.3KD coat protein subunit of TRV.
5.ACP-ELISA specific detection
ACP-ELISA detects and shows; This strain monoclonal antibody only has specific reaction to TRV; And with viruses all reactionless (table 2) such as BBWV1, BBWV2, CarMV, CMV, PVX, PVY, SCMV, TMV, ToMV and TuMV, explain that the ACP-ELISA method of setting up with this this strain monoclonal antibody has excellent specificity to TRV.
The specificity of table 2 TRV monoclonal antibody
* add substrate at the OD of room temperature reaction after 30 minutes
405Value.
6. detection sensitivity confirms
Sensitivity detects and shows; The ACP-ELISA method of setting up with the 3C3 monoclonal antibody all is positive to the sick leaf sap of 1:5～100 times dilution; Promptly can reach 1:100 to the sensitivity that detects the disease leaf; Detection sensitivity to pure virus can reach 18ng/mL, and the absolute detected level of the virus in every hole is 1.8ng.
Embodiment 2: Tobacco rattle virus (Tobacco rattle virus, TRV) ACP-ELISA detection kit
One. the test kit staple:
TRV monoclonal antibody (behind the purifying) 0.1ml (IgG content 1mg/ml)
AP mark sheep anti-mouse igg 0.1ml (Sigma Company products, concentration are 1mg/ml)
P-nitrophenyl SULPHOSUCCINIC ACID ESTER (p-nitrophenyl phosphate, PNPP) 0.15g
Positive control (containing Tobacco rattle virus tobacco leaf juice) 2ml
Negative control (health tobacco leaf juice) 2ml
Above reagent all is stored under 4 ℃
10 of 96 hole ELISA Sptting plates
Sample diluting liquid is 1 bottle of ELISA confining liquid (10X) (quality-concentration of volume percent is 20% skim-milk, and skim-milk is bright Company products)
1. sample is prepared:
Usually after sample preparation, detect immediately, handling the used container of sample should not combine with sample, to reduce the loss of sample.
If when testing sample was the water of extracting, (the juice volume: the ratio coating buffer volume) was diluted with 1:10 please to use the ELISA coating buffer; Also can directly add ELISA coating buffer ground sample, ratio is 1:10-a 30 (example weight: the coating buffer volume).Should prepare to be slightly more than and detect required testing sample amount, to guarantee carrying out smoothly of application of sample process.
2. operation steps:
(1) sample encapsulates: the sample that dilution is good adds elisa plate with 100 μ l/ holes, under 37 ℃, hatches 2h, or spends the night under 4 ℃.
(2) washing: ELISA washings (0.01mol/L PBST contains the phosphate buffered saline buffer that volume-concentration of volume percent is 0.05% tween 20) is filled it up with in every hole after getting rid of the solution in the plate hole, outwells triplicate after leaving standstill 3min.Washed plate places the thieving paper arsis to do.
(3) sealing: ELISA confining liquid 200 μ l/ holes add elisa plate, hatch 1h under 37 ℃, and falling antigen with sealing does not have the bonded site.
(4) washing: with (2).
(5) one is anti-: after TRV monoclonal anti body and function ELISA confining liquid was doubly diluted with 1:5000,100 μ l/ holes added elisa plate, hatched 1h under 37 ℃.
(6) washing: with (2).
(7) ELIAS secondary antibody: after with the ELISA confining liquid AP mark sheep anti-mouse igg doubly being diluted with 1:1000,100 μ l/ holes add elisa plate, hatch 1h under 37 ℃.
(8) wash: clap after washing 4-6 times with PBST and do.
(9) PNPP colour developing: PNPP is dissolved in nitrophenyl phosphate (PNPP) substrate buffer solution with 1:1 (mg/ml), and 100 μ l/ holes add elisa plate, and 0.5-1h develops the color under the room temperature.
(10) the NaOH50 μ l/ hole of termination reaction: 3mol/L adds elisa plate.
3. the result judges
Range estimation elisa plate, the hole that obvious colour-change is arranged are that TRV is positive, and the hole that does not have obvious color to change is the TRV feminine gender; Also available enzyme-linked immunosorbent assay instrument reading, 405nm wavelength are surveyed the OD value down.Hole OD405 value/negative hole OD405 value is positive greater than 2.1 per sample, and is negative smaller or equal to 2.1.Detected result only positive findings occurs and negative control is effective when not having color reaction at positive control.
4. preserve and validity period
Keep in Dark Place validity period 12 months in 2～8 ℃.
5. buffer formulation:
The ELISA coating buffer:
Yellow soda ash 1.59g
Sodium hydrogencarbonate 2.93g
Sodiumazide 0.2g
PH to 9.6 is transferred in adding distil water 950 dissolving backs, is settled to 1000ml
Phosphate buffered saline buffer (PBS, 0.01mol/L, pH7.4):
NaCl 8g
KCl 0.2g
KH
2PO
4 0.2g
Na
2HPO
4·12H
2O 3g
Sodiumazide 0.2g
PH to 7.4 is transferred in adding distil water 950 dissolving backs, is settled to 1000ml
ELISA washings (0.01mol/L PBST):
1000ml0.01mol/L add 0.5ml Tween-20 among the PBS.
The ELISA confining liquid:
0.01mol/L add skim-milk to final concentration 5% (W/V, mass and size percentage concentration) among the PBST
Nitrophenyl phosphate (PNPP) substrate buffer solution:
Terepthaloyl moietie ammonium 48.5ml
Magnesium chloride 0.05g
Sodiumazide 0.1g
Adding distil water 400ml dissolving, HCl transfers pH to 9.8 back adding distil water to 500ml.4 ℃ of preservations.
TRV monoclonal antibody: prepare according to embodiment 1 method.
Positive control: contain the juice refrigerated drying of Tobacco rattle virus tobacco leaf, add 2 milliliters of 0.01mol/LPBS during use.
Negative control: the lyophilize of health tobacco leaf juice adds 2 milliliters of 0.01mol/L PBS during use.
Embodiment 3: application is detected in field and laboratory sample
1. the ACP-ELISA method of setting up with the 3C3 monoclonal antibody; Tobacco rattle virus (the Tobacco rattle virus that utilizes this patent to produce; TRV) the sick leaf sap of ACP-ELISA detection kit 1:5～100 times dilution that Zhejiang University's biotechnology research is provided detects and all is positive, and promptly can reach 1:100 to the sensitivity that detects the disease leaf.
2. the ACP-ELISA method of setting up with the 3C3 monoclonal antibody; Tobacco rattle virus (the Tobacco rattle virus that utilizes this patent to produce; TRV) the sick leaf sap of ACP-ELISA detection kit 1:5～100 times dilution that China Agricultural University's agronomy and biotechnology institute virus test experience chamber is provided detects and all is positive, and promptly can reach 1:100 to the sensitivity that detects the disease leaf.
3. the ACP-ELISA method of setting up with the 3C3 monoclonal antibody; Tobacco rattle virus (the Tobacco rattle virus that utilizes this patent to produce; TRV) the sick leaf sap of ACP-ELISA detection kit 1:5～100 times dilution that biology department of Tsing-Hua University virus test experience chamber is provided detects and all is positive, and promptly can reach 1:100 to the sensitivity that detects the disease leaf.
4. the ACP-ELISA method of setting up with the 3C3 monoclonal antibody; Tobacco rattle virus (the Tobacco rattle virus that utilizes this patent to produce; TRV) the Ben Shi cigarette purified virus sample of ACP-ELISA detection kit infiltration morbidity that Zhejiang University's biotechnology research is provided; Detection sensitivity reaches 18ng/mL, and the absolute detected level of the virus in every hole is 1.8ng.
Claims (4)
1. detect the monoclonal antibody of Tobacco rattle virus, obtained by cell strain 3C3 secretion, the preserving number of this cell strain is CGMCC NO.2679.
2. monoclonal cell strain 3C3, preserving number is CGMCC NO.2679.
3. monoclonal cell strain 3C3 is used to prepare the purposes of the reagent that detects Tobacco rattle virus.
4. test kit that detects Tobacco rattle virus, form by following reagent:
TRV monoclonal antibody 0.1ml, IgG content 1mg/ml;
AP mark sheep anti-mouse igg 0.1ml, concentration is 1mg/ml;
P-nitrophenyl phosphoesterase 30 .15g;
Positive control: contain Tobacco rattle virus tobacco leaf juice 2ml;
Negative control: health tobacco leaf juice 2ml;
Above reagent all is stored under 4 ℃;
10 of 96 hole ELISA Sptting plates;
10X sample diluting liquid: 1 bottle of 20% skim-milk;
Said TRV monoclonal antibody is obtained by cell strain 3C3 secretion, and the preserving number of this cell strain is CGMCCNO.2679.
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| CN102586349B (en) * | 2012-02-24 | 2014-12-03 | 重庆邮电大学 | Preparation method of ethyl (R)-2-hydroxyl-4-phenylbutyrate by combining microbe reduction and chemical catalytic hydrogenation |
| GB201208942D0 (en) * | 2012-05-21 | 2012-07-04 | Vela Operations Pte Ltd | Extraction control for RNA |
| CN109254019B (en) * | 2018-09-21 | 2021-05-18 | 云南省农业科学院生物技术与种质资源研究所 | In-situ separation and fixation electron microscope diagnosis method for tobacco brittle fracture virus mitochondria |
| CN111397999B (en) * | 2020-05-09 | 2021-12-03 | 中国科学院植物研究所 | Simple method for enhancing contrast of micro-CT plant sample |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (1)
| Title |
|---|
| F. J. Legorburu等.Antigenic analysis of nematode-transmissible and non-transmissible isolates of tobacco rattle tobravirus using monoclonal antibodies.《Journal of General Virology》.1995,第76卷1497-1501. * |
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