CN102879574A - Bar/pat gene transfer plant and rapid test strip for derivative product of bar/pat transgenic plant - Google Patents
Bar/pat gene transfer plant and rapid test strip for derivative product of bar/pat transgenic plant Download PDFInfo
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- CN102879574A CN102879574A CN2012104044052A CN201210404405A CN102879574A CN 102879574 A CN102879574 A CN 102879574A CN 2012104044052 A CN2012104044052 A CN 2012104044052A CN 201210404405 A CN201210404405 A CN 201210404405A CN 102879574 A CN102879574 A CN 102879574A
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- antiweed
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Abstract
The invention discloses a specificity test strip using a bar/pat monoclonal antibody, the colloidal gold-labeled technology and a parallel movement immune affinity chromatography one-step method which belong to our independent intellectual property rights for rapid testing PAT proteins in samples. The clonal antibody is secreted by hybridoma cells 4C2 and is preserved with the number of CGMCC (China General Microbiological Culture Collection Center) No.6341. The test strip comprises high-protein affinity cellulose membranes, a sample pad, a colloidal gold pad, a water absorbing pad, a plastic substrate, double-faced and single-faced tapes, transparent protective tapes and color marked tapes, wherein the high-protein affinity cellulose membranes serve as system reaction vectors and display reaction results, the sample pad absorbs a to-be-tested sample and provides a proper reaction condition for a reaction system, the colloidal gold pad serves as a solid-phase vector of an colloidal gold-labeled antibody, the water absorbing pad absorbs the tested sample, the plastic substrate serves as a support board of the reaction system, the double-faced and single-faced tapes of different specifications are used for fixing the reaction membranes and water absorbing materials on the support board, the transparent protective tapes are used for fixing and protecting the sample pad and the colloidal gold pad, and the color marked tapes serve as feature identifiers. The immune colloidal gold test strip can provide direct proofs for judging whether detected samples contain transgenic plant samples (bar/pat gene transfer) and has wide application values for transgenic screening and transgenic safety evaluation.
Description
Technical field
The present invention relates to a kind of analysis test method, exactly is that a kind of immunoaffinity chromatography technology of utilizing is to turning
Bar/
PatThe method for quick of PAT albumen in gene plant and the converted products thereof.Belong to immunology and food safety detection technical field.
Background technology
Develop or transgenic product is screened or during safety evaluatio, often needed to carry out qualitative or semiquantitative determination to changing foreign gene over to genetically modified plants.
Bar/ pat gene is anti-herbicide gene, the same albumen-phosphinothricin acetyl transferase (PAT) of encoding.This gene is widely used in the anti-herbicide gene in the genetic engineering breeding, also is the marker gene in the genetic transformation, and it makes the herbicide of plant opposing take L-phosphiothricin (PPT) as active component.In recent years, genetically modified crops area and the ratio of whole world plantation increase year after year, and Resistant Herbicide Crops wherein accounts for 85% of global genetically modified crops.Because antiweed is marked at the commercialization genetically modified plants and is widely used, fast development along with enetically modified food, what turn the crop of PAT/bar gene and food abroad enters China in a large number, the food inspection technology that turns the PAT/bar gene is the important technological platform that transgenic foods safety is estimated, and its research has great importance.
Summary of the invention
Herbicid resistant in the plant is by external source
Bar/
PatThe expressed PAT albumen of gene produces, and this detection method is the PAT albumen for bar/pat gene expression in the genetically modified plants.The present invention adopts PAT albumen in the immunochromatographic method fast detecting sample, and whole process only needs 5-10 minute.The test strips steady quality, storage life can reach 1 year.The fast qualitative that is applicable to transgene component in the crops such as soybean, corn, cotton, rape, clover detects.
The purpose of this invention is to provide and a kind ofly have easy, accurate, quick, sensitivity and specificity is high, economical and practical, the Rapid detection test strip that turns herbicide resistant plants that is suitable for the characteristics such as Site Detection.
Take following technical scheme in order to reach purpose of the present invention: a kind of
Bar/
PatThe PAT protein I gG antibody fast test of gene expression, sample pad on plastic support board is pasted, one end of loading pad closely connects the collaurum pad of the PAT monoclonal antibody that contains colloid gold label, the other end of collaurum pad closely connects nitrocellulose filter, nitrocellulose filter is coated with detection line (T line) and the nature controlling line (C line) that is separated from each other, detection line is for being coated on the PAT monoclonal antibody on the nitrocellulose filter (NC film), nature controlling line is the sheep anti-mouse igg antibody that is coated on the nitrocellulose filter, drawing between detection line and nature controlling line and apart from detection line 2.5～3mm place has index line to be used to identify the application of sample position, the other end of nitrocellulose filter closely connects to be inhaled the sample pad and forms test strips, and test strips also can be packed into and be formed the cassette packing in the plastic clip.
Described PAT monoclonal antibody is secreted by hybridoma cell strain 4C2
PATProtein monoclonal antibody
,This cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 9th, 2012, and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6341.Should
PATThe protein monoclonal monoclonal antibody have high specific, with other transgenic lines or expressing protein no cross reaction, the height of tiring reaches 10
7Above.
Described loading pad is glass fibre membrane or nonwoven fabrics, and inhaling the sample pad is absorbent filter.
Bar/pat Gene Detecting test paper in the described plant, the method for coating that it is characterized in that described anti-PAT protein monoclonal antibody is: will resist PAT albumen monoclonal antibody to be mixed with the solution of 1.5mg/ml with 0.01MpH7.2 phosphate buffer (PBS), there is Membrane jetter to rule with the parameter of 1.3ul/cm in NC film bottom, coated T line, be coated with simultaneously sheep anti-mouse igg on NC film top as the C line, after the line with NC film at drying room (20～25 ℃ of temperature, humidity is less than 30%) dry 8～10 hours.
Described quick bar/PAT Protein Detection test strips, the method that it is characterized in that described PAT protein antibodies mark colloid gold particle is: prepare diameter as the colloidal gold solution of 30～50nm take gold chloride-trisodium citrate reduction method, get 100ml collaurum liquid after preparation is finished and be placed in the beaker, use 0.2MK
2CO
3Transfer to pH8.2, press the 100ml colloidal gold solution and add 0.5mg PAT monoclonal antibody, stirring at room 2 hours, add final concentration 0.1% casein-sodium, 0.1% PEG 20000 sealing 20min, 12000 left the heart 30 minutes, abandoned supernatant, redissolve to 66.7ml with the collaurum working fluid, press 1ml solution and spread 16cm
2Ratio be layered on equably on glass fibre membrane or the nonwoven fabrics, put again 20～25 ℃ of drying room temperature, humidity is made the collaurum pad less than 30% drying 2～4 hours, and is for subsequent use.
The assembly method of test strips is: 20～25 ℃ of dry indoor temperatures, humidity is less than 30%, get plastic support board, paste at the middle part that coated NC film is placed on plastic support board, apart from edge 1mm overlap joint collaurum pad, paste the loading pad at collaurum pad opposite side apart from edge 2mm overlap joint in NC film T line one side; Inhale the sample pad in NC film C line one side apart from edge 1.5mm place overlap joint.Then with cutter the plastic plate that posts is cut into 3mm or the wide test strips of 4mm.
Detection method: tested plant/material (1mg-100mg) is ground, add and be equipped with in the centrifuge tube of 1ml carbonate buffer solution, in the test strips partial insertion centrifuge tube for preparing, the submergence of solution highly is below the marking line, if contain the Bar gene antigen in the sample then by collaurum-PAT antibody capture, and be diffused on the NC film, form Ab-Ag-collaurum formation compound with the antibody on the NC, when captive colloidal gold composite reaches some, then form a macroscopic T line, the C line is as the quality control standard of reagent, and positive and negative sample all can occur when detecting.With the naked eye directly observe the appearance situation of C, T line in 10 minutes, and judge testing result.
Testing result according to loading, the optimum mark pH value of determining test paper is 8.2, the optimum mark amount is the every 100ml colloidal gold solution of 0.5mg, best collaurum working fluid is the Tris-citrate buffer solution of pH9.0, contain 0.2% casein, 5% sucrose, 0.2% BSA, 0.2% tween, the coated concentration of best anti-PAT protein monoclonal antibody is 1.5mg/ml.Sentence read result in 20 minutes.
Pattern detection
Take ELISA reagent as contrast, 20 parts of positive antiweed materials comprise corn, soybean, paddy rice, the sample of different gm contents.This reagent detects positive 20 parts, and sensitivity is 0.1%.In 50 parts of negative sample, this reagent detects negative 50 parts, and specificity is 100%.
The present invention turns antiweed
Bar/patThe advantage of gene plant quick detection test paper:
1. high specificity, susceptibility is high.This colloidal gold chromatographic test strip is prepared from as the basis take the polyclonal antibody of colloid gold label high-affinity, form without covalent bond between gold grain and the antibody molecule in the gold labeling antibody, the two combines by the Van der Waals force between the charges of different polarity, the specificity of colloid gold label antagonist and affinity impact are very little, and have higher mark rate.And test paper of the present invention has been used the monoclonal antibody by the secretion of CGMCC No.6341 hybridoma cell strain of high specific and high-titer.Therefore, test strips has stronger specificity and higher susceptibility, detects minimum limiting the quantity of and can reach 0.25ng.
2. easy, quick, ageing strong.Use colloidal gold chromatographic test strip and test card, need not any other reagent and instrument, but execute-in-place, and test strips can be judged testing result after adding test sample liquid in 15 minutes.
3. the result shows image, directly perceived, accurate.Test strip is all to show that red line T line and C line are as the testing result positive and negative marker, namely when coated film shows a red line C line, be illustrated in and contain checking matter in the test sample, when showing two red line T lines and C line, be illustrated in and do not contain checking matter in the test sample.The result judges image, directly perceived, accurate, simple and clear, is not prone to the artificial erroneous judgement such as false positive and false negative.
4. cost saving, applied widely, be convenient to promote.Use test strip, decline to a great extent than the expense with instrumental analysis and ELISA kit.In addition, test strips applied widely can satisfy different levels personnel needs, comprises professional inspection, customs quarantine control, health quarantine, quality monitoring, processing enterprise and plant family etc., easy to utilize, have wide market outlook and obvious economical, societal benefits.
Description of drawings
Fig. 1 utilizes this monoclonal antibody to turn bar gene cotton Western blot and detects 1 negative cell line contrast; 2 for turning bar gene cotton total protein; 3 is that non-transgenic cotton total protein 4 is EPSPS transgenic corn seed total protein.
This monoclonal antibody of Fig. 2 is in the result of Elisa detection specificity.
This monoclonal antibody of Fig. 3 is detecting the different detection sensitivity results that turn the Bar component content.
Fig. 4 is the Rapid detection test strip structural representation that turns bar/pat gene plant and derived product thereof.
Fig. 5 test sample result judges schematic diagram.
Embodiment
The preparation of mouse-anti bar/PAT protein monoclonal antibody
Be 200 μ g purifying bar/PAT albumen with protein content, lumbar injection BALB/C small white mouse is used the Fu Shi Freund's complete adjuvant; After 15 days, carry out secondary phase with the dosage immunity, use freund 's incomplete adjuvant; Carry out again three immunity after 15 days, do not add adjuvant with the pure protein of doubling dose; Extracting spleen cell merges after 3 days.
With immune mouse spleen cell and murine myeloma cell (SP2/0) ratio in 10: 1, mixing in the DMEM of serum-free nutrient culture media, the centrifugal 5min of 1500rpm, remove nutrient culture media, as fusion agent, at 37 ℃ of lower 0.6ml that add, merge 2min with 50%PEG3350, after DMEM nutrient culture media termination fusion, the centrifugal 5min of 1500rpm, precipitation suspends with the HAT nutrient culture media, minute installs to 96 holes to contain in the cell plates of feeder cells, 37 ℃, cultivate in the cell culture incubator of 5%CO2.Cultivate in the cell culture incubator after 5 days, change liquid once with the HAT nutrient culture media, changed liquid with the HT nutrient culture media on the 10th day, when waiting until at the bottom of the fused cell coverage hole 10%-50%, conventional indirect ELISA method screens positive hole.
The positive hole of the high specificity that filters out obtains monoclonal antibody cell line 4C2 with conventional limiting dilution assay clone.Cell line 4C2 further enlarges cultivation, for the preparation of odd contradictive hydroperitoneum and liquid nitrogen cryopreservation.
Get BALB/C mice about 8 ages in week, lumbar injection 0.3-0.5ml norphytane, pneumoretroperitoneum injected 5-10 * 10 in 7-10 days
5Individual hybridoma, 7-10 days visible mouse web portions obviously expand after the injection, take ascites, and the centrifugal 3min of 2000rpm collects supernatant, is monoclonal antibody ascites.Sad-the ammonium sulfate salting-out process monoclonal antibody purification ,-70 ℃ of preservations.The 4C2 cell line is prepared into monoclonal antibody, be can be single-minded the antibody of identification PAT albumen.
The titration of monoclonal antibody
1) coated
The PAT albumen of separation and purification is diluted with coated damping fluid, add in the ELISA Plate (100ul/ hole), the coated concentration of PAT albumen is 10ug/ml; Hatch 2h, then wash ELISA Plate with lavation buffer solution for 37 ℃.
1g non-transgenic soybean leaves total protein is added in the coated damping fluid of 10ml, in mortar, grind, obtain negative controls, negative control is added (100ul/ hole) in the ELISA Plate, be negative control hole; Hatch 2h, then wash ELISA Plate with lavation buffer solution for 37 ℃.
Sealing
Every hole adds the phosphate buffer that 100ul contains 5% skimmed milk power, hatches 30min, then washes ELISA Plate with lavation buffer solution for 37 ℃.
3) every hole adds monoclonal antibody or its dilution (diluting with enzyme labelled antibody dilution buffer liquid) that the above-mentioned purifying of 100ul obtains, and hatches 2h, then washes ELISA Plate with lavation buffer solution for 37 ℃.
4) every hole adds 100ul ELIAS secondary antibody working fluid, hatches 2h, then washes ELISA Plate with lavation buffer solution for 37 ℃.
5) every hole adds the 100ul substrate solution, after the room temperature lucifuge is hatched 60min, with the absorption value under the microplate reader mensuration 450nm wavelength.
Hole more than the negative control wells twice of absorption value is judged as positive hole.
Tiring of antibody sees Table 1 behind Hybridoma Cell Culture supernatant, mouse ascites and the purifying.
Tiring of table 1 monoclonal antibody
3. monoclonal antibody of the present invention adopts the transfer printing western blotting method to detect Expressed in Transgenic Plant PAT albumen.
)The extraction of seed protein: turn bar gene cotton, non-transgenic cotton, turn each 1g of EPSPS corn seed, adopt HEPES buffer directly to extract albumen, concrete grammar is for adding extract 1 mL of precooling, leave standstill 3～4 h behind the suspendible on ice, 4 ℃, 12 000 r/min, centrifugal 20 min, supernatant place-80 ℃ of refrigerators for subsequent use.
)The SDS-PAGE protein electrophorese
3) transferring film
4) sealing and hybridization
Sealing: transferring film is carefully taken out gel and film after finishing, and can use coomassie brilliant blue staining to judge transfer case gel again.Film is put into TBST wash once, place again the Ponceaux dyeing liquor, the 5min that at room temperature dyes, washing film to water with distilled water, to become clear colourless protein band clear.Film is put into the container that fills confining liquid (5% skimmed milk power), room temperature shaking table sealing 1h.
Primary antibodie is hatched: according to the antibody instructions with proper proportion (1:1000) with 5% skimmed milk power dilution primary antibodie, every film is 4ml approximately.Pvdf membrane after the sealing is carefully put into hybridization bag, then adds the primary antibodie solution for preparing,, carefully get rid of bubble, shaking table slowly sways and hatches 1-3h under the room temperature.After hatching film is taken out, wash film 3 times with the TBST shaking table, each 5min is to remove residual primary antibodie.
Two anti-hatching: the skimmed milk power with 5% carefully places hybridization bag by two anti-instructions dilution proportion two anti-(1:4000) with pvdf membrane, and every film adds 4ml two anti-solution, avoids Bubble formation, seals the hybrid belt mouth, and the room temperature shaking table is hatched 2h.After hatching film is taken out, wash film 3 times with the TBST shaking table, each 5min.
5) exposure is identified;
In the darkroom, take out an X-ray film, according to the fluorescence strong and weak adjustment time shutter, be generally 1-5min.After the exposure, open magazine and take out X-ray film, development, photographic fixing also develop photographic film.
6) film is scanned, with the optical density value of Quantity One image analysis software evaluating objects band.Fig. 1 is that the cotton Western blot that utilizes this monoclonal antibody to turn the bar gene detects, and the result shows, this monoclonal antibody can be expressed PAT albumen in the specific recognition plant.
Monoclonal antibody of the present invention adopts double antibodies sandwich ELISA method, detects transgenic line.
Get the transgenic corns strain of different transgene components, Bt176(turns the bar gene corn), Mon810(turns Cry1Ab gene pest-resistant corn), GA21(EPSPS transgenic corn), T25(turns the pat gene corn) seed is mixed into specimen material with the non-transgenic corn respectively.The transgene component mass ratio is respectively 1%, 10%, 100% in the specimen material.Sample thief material 1g adds the extraction buffer 1mL of 10ml sample precooling respectively, and grinds.Leave standstill 3～4 h behind the suspendible on ice, 4 ℃, 12000r/min, centrifugal 20 min, supernatant place-80 ℃ of refrigerators for subsequent use.
The specificity identification of monoclonal antibody
1. how anti-rabbit PAT albumen preparation method be as follows:
Select about body weight 2.0kg, 3 of male, healthy rabbit, the ear edge vein exploitating blood separation of serum is as negative control before the immunity ,-80 ℃ save backup.Immune programme for children is as follows: head exempts from, and after antigen and equivalent Freund's complete adjuvant were fully emulsified, subcutaneous multi-point injection, 1mg/ were only.Two exempt from (the 15th day), carry out immunity with the antigen that adds incomplete Freunds adjuvant, and the position is two hind paws, and 500 μ g/ only.Three exempt from (the 30th day), and dosage is the same, and immune position is enlargement De lymphonodi poplitei.Booster immunization (the 37th day) does not add adjuvant, and dosage is the same.Booster immunization is heart blood sampling and separation of serum after one week, detects antibody horizontal with indirect ELISA.Tiring is 1:32000, the heart blood sampling, and separation of serum, the Sodium azide of adding 0.01%, filtration sterilization ,-80 ℃ save backup.
The anti-bar gene protein of caprylic acid-ammonium purification rabbit IgG hyper-immune serum with the centrifugal 30min of serum 3000rpm, is abandoned precipitation; Ratio in 1:2 is mixed the serum after centrifugal and acetate buffer (0.06M, pH=5.0), regulates pH value to 4.5 with 0.1M HCl.Add while stirring sad (every mL serum add 75 μ l sad) under 4 ℃, stir 30min, 4 ℃ of static 2h, the centrifugal 30min of 12000rpm abandons precipitation; Supernatant adds the saturated (NH of 4 ℃ of placements while stirring with 1M NaOH adjust pH to 7.4
4)
2SO
4, make its concentration reach 45%, 4 ℃ and leave standstill more than the 1h; 4 ℃ of centrifugal 30min of 12000rpm abandon supernatant, and precipitation is with a small amount of PBS (0.01M, pH=7.4) dissolving, at 0.01M, 4 ℃ of dialysed overnight in the pH=7.4 PBS solution, during change liquid 3-5 time, SO42-retains situation in the 5%BaCl2 detection dislysate; 4 ℃ of centrifugal 30min of 12000rpm draw supernatant in-20 ℃ of preservations.Thick pure thing is further carried out purifying with affinity chromatography, and step is as follows: balance: add initial damping fluid balance columns bed identical with eluate pH value to entering; Loading: sample filters through filter membrane, gets the 5mL upper prop, and the 5min that vibrates gently vertically places.After all suspension precipitations, the sucking-off supernatant; Wash-out: add initial damping fluid 5mL, the 5min that vibrates gently vertically places.After all suspension precipitations, the sucking-off supernatant.Repeatable operation 3 times.Add elution buffer 3mL, the 5min that vibrates gently vertically places.After all suspension precipitations, the sucking-off supernatant.Repeatable operation 2 times.Collect eluent, every milliliter of eluent adds 70 μ L neutralization buffer.
Before purifying and the anti-bar gene protein of the rabbit IgG hyper-immune serum Purity after purifying adopt common polyacrylamide gel electrophoresis (SDS-PAGE), and with the mensuration antibody concentration.
2. the anti-PPV IgG of rabbit and McAb best effort concentration determines
Determine with the square formation test the anti-bar gene protein of rabbit resists and McAb best effort concentration more.The general flow of monoclonal antibody double fastener heart ELISA: the anti-bar gene of rabbit IgG be coated with-washes plate-seal-wash plate-application of sample-wash plate-Jia McAb-and washes plate-Jia ELIAS secondary antibody-wash plate-colour developing-termination-result of determination.Concrete steps are as follows: be coated with coating buffer the anti-bar gene of rabbit 1gG is done 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400 dilution, 100 μ L/ holes, each dilutability delegation, behind 37 ℃ of coated 2h or 4 ℃ spend the night; Discard liquid in the hole, with cleansing solution washing 3 times, 5min/ time, pat dry; Sealing is made sealer with 3%BSA, 100 μ L/holes, and remove issuable bubble in each hole, and 37 ℃ of sealing 3h, the same washing three times pats dry; Application of sample, the PBS blank is established in 100 μ L/ holes simultaneously, 37 ℃ of effect 40min, the same washing three times pats dry; Add McAb and with dilution McAb is done 1:25,1:50,1:100,1:200,1:400,1:800,1:1600 dilution, add in the ELISA Plate 100 μ L/ holes, each dilutability is made two row, establishes simultaneously the PBS blank, 37 ℃ of effect 40min, the same washing three times pats dry; Add the HRP-sheep anti-mouse igg that ELIAS secondary antibody adds the 1:5000 dilution, 100 μ L/holes, 37 ℃ of effect 40min, the same washing three times pats dry; Colour developing and termination add the newly TMB substrate nitrite ion 100 μ L/holes of preparation, room temperature lucifuge colour developing 10-15min, and adding stop buffer (2M H2SO4 solution), OD450nm is read in microplate reader in 50 μ L/ holes.When maximum near 1, P/N value with positive hole OD value, the greatest dilution of the anti-PPV IgG of rabbit and McAb is its best effort concentration.The coated concentration of the anti-PPV IgG of rabbit that determines is 1:3200; The monoclonal antibody extension rate is 1:800.
Double antibodies sandwich Elisa detects this monoclonal antibody for the specificity of PAT albumen in the transfer-gen plant
Respectively with sample liquid Bt176, Mon810, GA21 and T25(1%, 10%, 100%) carry out following experiment as sample to be tested liquid: the vegetable protein of extraction (extract that comprises blade and seed), every hole 100 μ L are added on micro-reaction plate, 4 ℃ are spent the night coated, after the lavation buffer solution washing, add 3% BSA confining liquid with every hole 100 μ L, 37 ℃ of sealing 3h, the PBST washing, the Hybridoma Cell Culture supernatant, every hole 100 μ L are added on micro-reaction plate, and the SP2/0 cell culture supernatant is as negative control.37 ℃ of effect 60min, washing adds ELIAS secondary antibody, and 37 ℃ of effect 60min wash, and add the per 100 μ L of TMB substrate nitrite ion of new preparation, room temperature lucifuge colour developing 10-15min, 2mo1/L H
2SO
4Every hole 50 μ L cessation reactions, enzyme mark detector is measured the absorption value in each hole of OD450nm.The results are shown in Figure 2.
The above results explanation, cross reaction does not occur in the monoclonal antibody for preparing and the non-bar of turning gene plant, has stronger specificity to turning the bar gene plant.This monoclonal antibody only has specific reaction to PAT albumen, and with Bt albumen, EPSP without specific reaction, illustrate to have with this monoclonal antibody and detect the good specificity of bar/PAT albumen.
The sensitivity of monoclonal antibody is identified
Sample preparation: with Bt176(bar gene masculine material), with its non-transgenic corn parent seed, mix according to mass ratio, the preparation transgene component is respectively 100%, 10%, 5%, 1%, 0.1%, 0.01%, 0% testing sample.Select Mon810(not contain the transgenic corns strain of bar gene) negative contrast, the blending ratio of itself and non-transgenic corn is the same.
Adopt HEPES buffer directly to extract albumen, concrete grammar leaves standstill 3～4 h on ice for adding extract 1 mL of precooling behind the suspendible, and 4 ℃, 12 000 r/min, centrifugal 20 min, supernatant place-80 ℃ of refrigerators for subsequent use.
Just contain respectively 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 0% the seed that turns the bar gene element extracts the vegetable protein extract, and dilute concentration is 10 μ g/mL, crop sample liquid carries out Elisa test experience (step homospecificity test experience), the results are shown in Figure 3.This experimental result shows the monoclonal antibody of utilizing the present invention to obtain
5. the preparation of colloid gold label bar gene protein monoclonal antibody
1) colloid gold label
Adopt the trisodium citrate reduction method of improvement, its process is: under stirring condition, after 98 mL deionized waters are heated to 65 ℃, the gold chloride that adds 1.5 mL 1% continues heating, when treating that temperature rises to 95 ℃, the trisodium citrate that adds 2mL 1%, stopped heating when solution is claret, after the cooling, 4 ℃ keep in Dark Place.
Get colloidal gold solution, pH7.2～7.5, gold grain size 20～30nm.Bar gene protein monoclonal antibody 0. 5mg/mL1ul is added in the colloid 200ul gold solution, mix, namely get golden mark mouse-anti protein monoclonal antibody solution.Purified and concentrated, with for subsequent use after 4 times of the diluted.
2) assembling of test strips
Whole test-strips is by 2 layers of water adsorption glass fiber, 1 layer of nitrocellulose filter, and absorbent filter and white plastic backing plate form.Thieving paper, coated NC film (being coated with monoclonal antibody and goat anti-rabbit igg), sprayed glass fiber (golden labeling antibody) are fixed on the white plastic plate from top to bottom successively.
Cut the thieving paper of 40 mm * 5 mm, thieving paper, coated NC film, sprayed glass tunica fibrosa, sample pad are fixed on the PVC backboard with bonding agent from top to bottom successively, be cut into the wide test strips of 5mm, drying at room temperature is for subsequent use, test-strips.Ready-made test-strips is packed in the aluminium foil bag into sealed type storage with drying agent.As shown in Figure 3.
3) specimen detects
Before the detection, sample ground and add water or extract extracts, albumen is obtained in dissolving.During detection, first sample pad is immersed in the protein sample solution that has extracted, sample solution is flowed along the NC film to absorption pad by sample pad under the capillary suction effect.Specific protein in the sample solution when flowing through pad at first with the specific combination of the antibody generation Ag-Ab of colloid gold label wherein, flow through detect with the time antigen-antibody complex be fixed on detect with on the capture antibody combination, assemble forming macroscopic colloidal-gold strip band, show that testing result is positive.Excessive golden labeling antibody continue to flow be fixed on control with on second antibody form band in conjunction with assembling, show that the result who detects is effective.
4) sensitivity test
Antigen is detected by 0,0.25,0.5,1.0,5.0,10,20,40,80 ng/ ml dilute concentrations, but red minimum measured quantity, the i.e. for this reason sensitivity of reagent strip appear in detection line.When antigen concentration 〉=0.25, obvious positive red tape appears; 0.5, obvious strong positive band appears.Therefore the detection sensitivity of this test strips is 0.25ng.
5) stability test
Test-strips under not unpacking condition, detect respectively 37 ℃ deposited for 2 weeks, room temperature is deposited the test strips after 18 months.Result's demonstration, the detection of GMOs sensitivity to 0.1% has no significant effect.
Fig. 6 is application assembling test strips of the present invention, and the testing result that turns bar trans-genetic hybrid rice and negative paddy rice of carrying out.Show that this test strips can be used for immunochromatographyassay assay and turns bar gene plant material.
Claims (15)
1. the colloid gold test paper of antiweed transgene component in the fast detecting plant is characterized in that being comprised of base plate, adsorptive pads, nitrocellulose filter, monoclonal antibody gold mark pad, loading pad; Nitrocellulose filter is pasted at the base plate middle part, a detection line and a control line are arranged on the nitrocellulose filter, base plate one end is pasted monoclonal antibody gold mark pad and loading pad, the other end is pasted adsorptive pads, wherein, one end of monoclonal antibody gold mark pad one end lap nitrocellulose filter, an end of another end lap loading pad, another end lap adsorptive pads one end of nitrocellulose filter.
2. the colloid gold test paper of antiweed transgene component in the fast detecting plant according to claim 1, described antiweed transgenosis is to turn the bar/pat gene.
3. the colloid gold test paper of antiweed transgene component in a kind of fast detecting plant according to claim 1 and 2 is characterized in that control line is to make by the sheep anti mouse polyclonal antibody is coated.
4. the colloid gold test paper of antiweed transgene component in the fast detecting plant according to claim 1 and 2, it is characterized in that detection line is to make by the PAT protein monoclonal antibody is coated, this PAT protein monoclonal antibody is the hybridoma cell strain secretion of CGMCC No.6341 by preserving number.
5. the colloid gold test paper of antiweed transgene component in the described fast detecting plant according to claim 1 and 2, it is characterized in that gold mark pad is to make colloid gold label by the PAT protein monoclonal antibody, this PAT protein monoclonal antibody is the hybridoma cell strain secretion of CGMCC No.6341 by preserving number.
6. the colloid gold test paper of antiweed transgene component in the described fast detecting plant according to claim 4, it is characterized in that gold mark pad is to make colloid gold label by the PAT protein monoclonal antibody, this PAT protein monoclonal antibody is the hybridoma cell strain secretion of CGMCC No.6341 by preserving number.
7. the colloid gold test paper of antiweed transgene component in the fast detecting plant according to claim 1 is characterized in that thieving paper double-layer water absorbing paper.
8. the purposes of colloid gold test paper in the antiweed transgenic plant detection of antiweed transgene component in claim 1 or the 2 described fast detecting plants.
9. the purposes of the colloid gold test paper of antiweed transgene component in the transgenic plant detection of anti-herbicide take L-phosphiothricin as active component in claim 1 or the 2 described fast detecting plants.
10. the purposes of the colloid gold test paper of antiweed transgene component in enetically modified food detects in claim 1 or the 2 described fast detecting plants.
11. the purposes of the colloid gold test paper of antiweed transgene component in the enetically modified food of anti-herbicide take L-phosphiothricin as active component detects in claim 1 or the 2 described fast detecting plants.
12. the colloid gold test paper of antiweed transgene component turns in detection in claim 1 or the 2 described fast detecting plants
BarPurposes in the/pat gene plant.
13. the colloid gold test paper of antiweed transgene component turns in detection in claim 1 or the 2 described fast detecting plants
BarPurposes in the/pat gene food.
14. a transgenosis detection kit is characterized in that: the colloid gold test paper that comprises antiweed transgene component in claim 1 or the 2 described fast detecting plants.
15. detection kit as claimed in claim 14, described transgenosis is for turning
Bar/ pat gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN105717295A (en) * | 2016-01-15 | 2016-06-29 | 北京市农林科学院 | Test strip for rapidly detecting CP4-EPSPS transgenic plant and derivative thereof |
| CN105866413A (en) * | 2016-02-28 | 2016-08-17 | 浙江大学 | Colloidal gold rapid detection test paper for detecting transgenic protein g10-epsps and use method thereof |
| CN107513521A (en) * | 2017-09-02 | 2017-12-26 | 中国农业科学院生物技术研究所 | Hybridoma cell strain, secreted monoclonal antibody and its application in bar/PAT albumen is detected |
| CN110205300A (en) * | 2019-06-04 | 2019-09-06 | 中国农业科学院生物技术研究所 | Bar monoclonal antibody hybridoma cell strain, antibody of generation and preparation method thereof |
| CN112326969A (en) * | 2020-10-26 | 2021-02-05 | 中国农业科学院生物技术研究所 | Enzyme linked immunosorbent assay kit for quantitatively detecting herbicide-resistant protein PAT/PAT |
| CN112798793A (en) * | 2020-12-30 | 2021-05-14 | 中国农业科学院油料作物研究所 | Test strip and test card for detecting PAT/bar protein, and preparation method and application thereof |
| CN115094042A (en) * | 2022-05-30 | 2022-09-23 | 中国农业科学院生物技术研究所 | PAT/PAT monoclonal antibody hybridoma cell strain, antibody produced by same and application of antibody |
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