CN105866413A - Colloidal gold rapid detection test paper for detecting transgenic protein g10-epsps and use method thereof - Google Patents
Colloidal gold rapid detection test paper for detecting transgenic protein g10-epsps and use method thereof Download PDFInfo
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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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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/9116—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
- G01N2333/91165—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1)
- G01N2333/91171—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1) with definite EC number (2.5.1.-)
- G01N2333/91182—Enolpyruvylshikimate-phosphate synthases (2.5.1.19)
Abstract
The invention discloses colloidal gold rapid detection test paper for detecting transgenic protein R1-EPSPS; the colloidal gold rapid test paper includes a sample pad, a gold labeled pad, a nitrocellulose membrane coating a quality control line and a detection line, and a sample sucking pad which are arranged on a bottom plate; the goal labeled pad is coated with a colloidal gold labeled anti-mouse g10-epsps monoclonal antibody, the detection line is coated with an anti-mouse g10-epsps monoclonal antibody, and the quality control line is coated with an anti-mouse IgG secondary antibody. The invention also discloses a use method of the colloidal gold rapid detection test paper, wherein the use method comprises the steps: vertically inserting the rapid detection test paper in a sample to be tested; 8-10 min later, observing; if only a C line appears, judging that the sample is negative; and if a T line and the C line appear at the same time, judging that the sample is positive. The hand-held type colloidal gold rapid detection test paper can be used for rapid, on-site and sensitive detection of the transgenic protein g10-epsps in the sample.
Description
Technical field
The present invention relates to transgene protein detection technique field, particularly relate to a kind of detection transgene protein g10-epsps
Hand-held gold colloidal speed test paper.
Background technology
Seminar's early-stage Study at inventor place is cloned into the EPSPS gene of antiweed from Pseudomonas alba
(g10-epsps).Epsp synthase (5-enolpyruvyl-shikimate-3-phosphatesynthase, EPSPS) is raw
Key enzyme during aromatic amino acid tryptophan, tyrosine, Phenylalanine biosynthesis in object；Glyphosate is
The synthesis being blocked aromatic amino acid by the activity of suppression epsp synthase ultimately results in subject plant death.But some
The activity of the epsp synthase of antibacterial is not suppressed by glyphosate, thus the expression of antibacterial EPSPS gene can make plant from
The injury of herbicide.Herbicide resistance gene EPSPS is proceeded to Semen sojae atricolor, it is intended to cultivate the transgenic of tool resistance glyphosate good characteristic
Semen sojae atricolor, improves the field weeding effect in Soybean production, reduces production cost.
This seminar utilizes the EPSPS gene of the antiweed of clone from Pseudomonas alba, constructs plant composing type
Expression vector pSOY19, is driven by 35S promoter, can be that transgenic plant provides glyphosate resistance.In pSOY19 plasmid only
Containing a kind of gene can expressed in plant cell: antibacterial EPSPS, it is positioned under the control of CaMV 35S promoter.Antiweed
Gene expression plasmid is 9557bp.Insertion sequence EPSPS full length gene 1321bp, encode one containing 440 amino acid whose many
Peptide.By agrobacterium-mediated transformation, EPSPS gene transformation Semen sojae atricolor China's spring No. 3 is turned EPSPS base with Jack kind acquisition
The Herbicide-Resistant Transgenic material of cause.
The common detection methods of transgene protein is PCR at present, but place, instrument, technical staff are wanted by PCR method
Asking comparison high, sample process is complicated, is not suitable for substantial amounts of selective mechanisms.There is partial transgenic albumen can be tested by speed
The method of paper and ELISA kit detects, but the transgene protein that g10-epsps is a new introduction, there is no at present
The detection method of this aspect.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of hand-held gold colloidal detecting transgene protein g10-epsps
Speed test paper and make usage, utilize the present invention can quickly, on-the-spot, detect the transgene protein g10-in sample delicately
epsps。
In order to solve above-mentioned technical problem, the present invention provides following a kind of gold colloidal detecting transgene protein R1-EPSPS
Speed test paper (for hand-held gold colloidal speed test paper), including the sample pad being arranged on base plate, gold mark pad (that is, label pad),
It is coated nature controlling line and the detection nitrocellulose filter of line, sample suction pad；The mouse-anti of colloid gold label it is coated with on described gold mark pad
G10-epsps monoclonal antibody, described detection line is for being coated with mouse-anti g10-epsps monoclonal antibody, and described nature controlling line is bag
There is anti-Mus IgG bis-anti-(the two of sheep anti-mouse igg resist).
Improvement as the gold colloidal speed test paper of the present invention:
Mouse-anti (specificity mouse-anti) g10-epsps monoclonal antibody is by with g10-epsps recombiant protein immunity BALB/
C mice, then prepares cell strain of monoclonal antibody by cell fusion, cloning screening, then prepares mouse ascites, warp
Cross purification to obtain.
Improvement as the gold colloidal speed test paper of the present invention: g10-epsps is to be passed through by the g10-epsps gene in G1
Being built into coli expression system after optimization, recombinant expressed purification obtains.
Further improvement as the gold colloidal speed test paper of the present invention: the mouse-anti g10-epsps Dan Ke of colloid gold label
The preparation method of grand antibody comprises the following steps:
1) gold colloidal of 20～30nm (preferably 25nm), is prepared；
2), antibody labeling:
Take the gold colloidal of 1ml, regulate pH to 8.0, add g10-epsps mouse monoclonal antibody (the mouse-anti g10-of 80 μ g
Epsps monoclonal antibody), mix homogeneously, room temperature reaction 30～50min；Add BSA to final concentration of 0.1% (that is, in 100ml
BSA containing 0.1g), stand 20～40min；
Then it is marked antibody purification, obtains the mouse-anti g10-epsps monoclonal antibody of colloid gold label.
The preparation method of the hand-held gold colloidal speed test paper of the transgene protein g10-epsps of the present invention includes following step
Rapid:
1) start from sample application zone be followed successively by the glass fibre membrane (i.e. sample pad) pasted side by side and be fixed with on base plate plate
Gold mark pad, nitrocellulose filter and the sample suction pad of colloidal gold labeled monoclonal antibody.
2) coated weight of the upper mouse-anti g10-epsps monoclonal antibody of gold mark pad is 5ng-50ng, Mus on nitrocellulose filter
The amount of anti-g10-epsps monoclonal antibody is 0.4 μ g-1.2 μ g.
3) test strips baking temperature is 37 DEG C, and the time is 30min.
The present invention provides the using method of above-mentioned gold colloidal speed test paper the most simultaneously: be inserted perpendicularly into be measured by speed test paper
In sample；
After 8～10min, it is observed；C line only occurs, it is judged that sample is negative；T line and C line there is simultaneously, it is determined that sample
Product are positive.
Remarks illustrate: detection line (T line), nature controlling line (C line).
The concrete detecting step of the hand-held gold colloidal speed test paper of the transgene protein g10-epsps of the present invention and principle
As follows:
First speed test paper is inserted perpendicularly in detection sample, less than the arrow locations in Fig. 5；Sample solution is along sample
Pad infiltrates into the label pad (gold mark pad) of the mouse-anti g10-epsps monoclonal antibody being coated with colloid gold label, if sample is molten
The g10-epsps Han transgene protein in liquid, then g10-epsps albumen by with the mouse-anti g10-epsps monoclonal in label pad
Antibodies, continues up, and the mouse-anti g10-epsps monoclonal antibody on NC film is combined, remaining unconjugated gold colloidal
Traget antibody and sheep anti-mouse igg two anti-binding on NC film, after 8-10min, can observe detection zone (that is, inspection in observation area
Survey line) color change, positive band i.e. occurs；If without transgene protein g10-epsps in sample solution, then detection
Qu Ze does not observes positive band.Regardless of whether the whether g10-epsps Han transgene protein in sample solution, when sample solution arrives
During nitrocellulose filter, the mouse-anti g10-epsps monoclonal antibody on gold label test strip all can anti-Mus IgG coated with control zone
Two anti-bindings, so that the C line colour developing on nitrocellulose filter.Otherwise, (this belongs to known normal to need to re-start experiment
Know).
Therefore, the result judgment rule of the hand-held gold colloidal speed test paper of transgene protein g10-epsps of the present invention is:
Negative findings (-), 1 C line only occurs；
Positive findings (+): T line and C line occur simultaneously.
The hand-held gold colloidal speed test paper of the transgene protein g10-epsps of the present invention to blade, seed and Bulk Grain
Deng complex matrices, there is preferable anti-jamming effectiveness, can be widely applied to the quick detection of transgene protein g10-epsps, have
The most peculiar advantage.
(1) simple to operate, it is not necessary to special instrument and equipment, can directly detect in scenes such as fields easily.
(2) the detection time short (5-8min)；Result criterion is unified, i.e. negative findings (-), 1 C line only occurs；Sun
Property (+): T line and C line occur simultaneously.
The hand-held gold colloidal speed test paper of the detection transgene protein g10-epsps of the present invention can be for blade, seed
Quick, on-the-spot and sensitive detection is carried out with transgene protein g10-epsps in the samples such as Bulk Grain.
Accompanying drawing explanation
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described in further detail.
Fig. 1 is the expression of SDS-PAGE detection g10-epsps albumen；
1: before induction；2: after induction (about 45KD).
Fig. 2 is the expression-form of SDS-PAGE detection g10-epsps albumen；
1: supernatant；2: inclusion body；3, full bacterium.
Fig. 3 is the purification effect of SDS-PAGE detection g10-epsps albumen；
1:250mM imidazole elution；2:150mM imidazole elution；
3:100mM imidazole elution；4:50mM imidazole elution.
Fig. 4 is the dialysis-effect of SDS-PAGE detection g10-epsps albumen；
1:50mM imidazole elution.
Fig. 5 is the structural representation of the transgene protein g10-epsps hand-held gold colloidal speed test paper of the present invention；
Wherein:
Figure A is the schematic front view of test paper；
Figure B is the schematic top plan view of figure A；
Figure C is the structural representation of the test paper of commercialization；
Figure D is the schematic side view of figure C.
Detailed description of the invention
The technical scheme of patent of the present invention below in conjunction with the accompanying drawings and is further illustrated by detailed description of the invention.
Embodiment 1, the expression and purification of EPSPS albumen
(1) lab scale of albumen is expressed
1, vector construction
According to protein sequence (g10-epsps), synthetic gene sequence, and it is built into carrier pET30a, obtains pET30a-
EPSPS positive plasmid.
2, actication of culture: utilize the commercial ET expression vector skeleton that EMD Biosciences company sells, construct
G10-epsps-E positive plasmid, converts BL21 (DE3), coating LB solid medium (blocking that concentration 50 μ g/mL).Next day, picking
Monoclonal bacterium colony accesses 5mL LB fluid medium (blocking that concentration 50 μ g/mL), cultivates 12h-14h for 37 DEG C.
3, lab scale is expressed: next day, and strain accesses 5mL LB fluid medium (blocking that concentration 50 μ g/mL) with 1:50 (v:v),
Cultivate to OD=0.4-0.6 for 37 DEG C, as inducing front comparison after drawing 1mL bacterium solution centrifugal treating.4mL bacterium solution adds concentration
The IPTG of 0.8mM, bacterium solution 8000rpm, 4 DEG C of centrifugal 1min after 25 DEG C of abduction delivering 6h, collect thalline.SDS-PAGE identifies albumen
Form, result shows that (Fig. 1) has the expression of obvious destination protein.
4, the qualification of protein expression form: the thalline (that is, the thalline collected by above-mentioned steps) of above-mentioned expression adds 1mL and breaks
Broken liquid carries out ultrasonic treatment.Cracking condition: temperature ice bath, power 240W, ultrasonic 2s, interval 2s, time 30min.To obtain
Full bacterium be centrifuged, 12000rpm, 4 DEG C of centrifugal 1min, cleer and peaceful inclusion body in collection.SDS-PAGE identifies protein expression form, knot
Fruit display (Fig. 2) destination protein is mainly expressed with soluble form.
" 2: inclusion body " in Fig. 2 i.e. refer to precipitation above；" 3: full bacterium " i.e. refer to above centrifugal before broken product
Thing.
(2) great expression of albumen and purification
1, actication of culture: on solid plate, picking pET30a-EPSPS monoclonal bacterium colony accesses 5mL LB fluid medium
(blocking that concentration 50 μ g/mL), cultivate 12h-14h for 37 DEG C.
2, lab scale is expressed: next day, and strain accesses 800mLLB fluid medium with 1:50 (v:v) and (blocks that concentration 50 μ g/
ML), cultivating to OD=0.4-0.6 for 37 DEG C, adding concentration is the IPTG of 0.8mM, bacterium solution 8000rpm after 25 DEG C of abduction delivering 6h, 4
DEG C centrifugal 15min, collects thalline.
3, strain cracking: add 100mL and crush liquid and carry out ultrasonic treatment.Cracking condition: temperature ice bath, power 240W, super
Sound 2s, interval 2s, time 15min.12000rpm, 4 DEG C of centrifugal 15min, collect supernatant.
4, supernatant purification: the supernatant of collection utilizes high-affinity NI resin (that is, Ni-NTA affinity chromatograph resin (nickel post))
It is purified, carries out eluting with 50mM, 100mM, 150mM, 200mM imidazoles respectively；
Collection flows through liquid, eluent.SDS-PAGE detects purification effect, egg when result shows (Fig. 3) 50mM imidazoles eluting
Bai Chundu is optimal.Imidazoles dialysis in eluent being removed with the Tris-HCl buffer of 50mM, pH=8.0, SDS-PAGE examines
Survey dialysis-effect, result show (Fig. 4) albumen dialyse after purity and concentration the most feasible.After testing, final purpose purity of protein is big
In 90%, concentration 2mg/mL, protein content 10mg.
(3) result
The protein induced expression condition of pET30a-EPSPS is: IPTG concentration 0.8mM, inducing temperature 25 DEG C, induction time 6h.
Albumen is mainly expressed at supernatant, and supernatant removes imidazoles, final gained g10-epsps albumen: concentration 2mg/ through NI column purification, dialysis
ML, purity more than 90%, protein 10 mg.
Prepared by embodiment 2, antibody
(1) prepared by monoclonal antibody:
1, prepared by immunogen: by equal to the albumen of expression and purification and isopyknic Freund adjuvant, YOULONG adjuvant mixing and emulsifying
Even, become Water-In-Oil state, in case immune mouse.
2, immunization strategy: by 4 Balb/c mices of protein immunization, subcutaneous inoculation 3 times, be spaced 4 weeks, after examine through ELISA
Surveying, antiserum titre is as follows:
3, cell merges: last immune two weeks after, (specially g10-epsps protein expression is pure for lumbar injection antigen
The albumen changed) carry out booster immunization, carry out cell fusion after three days.The neck that broken by mice is put to death, and 70% soak with ethanol 30min disappears
Poison, cuts off abdominal cavity at super-clean bench, takes out spleen, grinds, and crosses 80 eye mesh screens, obtains splenocyte, adds SP2/0 myeloma cell,
Cell fusion is carried out under the effect of PEG4000.
4, screening is merged: spread the cell merged into 96 orifice plates, cultivate with HAT culture fluid, change liquid after three days, change
Cultivate with HT culture fluid.After 10 days, take cells and supernatant and detect.Indirect ELISA method is used to screen, OD value >
The hole that negative control hole is 2.1 times is judged as positive hole.
Indirect ELISA method mainly comprises the steps that
1), with the albumen of the carbonate buffer solution dilution expression and purification of 0.1M, pH=9.6 to 1ug/ml, 96 hole enzymes are added
Target, every hole 100ul, 37 DEG C reaction 3h or 4 DEG C stand overnight；
2), get rid of liquid in plate hole, add 250ul lavation buffer solution, stand 30s, get rid of liquid in plate, be repeated 3 times；
3), add detection sample, every hole 100ul, be simultaneously introduced positive control (taken positive mice serum in 2), feminine gender
Comparison (mice serum before immunity) and blank (being not added with mice serum) 37 DEG C reaction 45min；
4), step 2 is repeated)；
5), add HRP labelling sheep anti mouse ELIAS secondary antibody, every hole 100ul, 37 DEG C reaction 45min.
5, cloning with build strain: use limiting dilution assay that positive hole is carried out cloning, detect after 10 days, by positive colony
Continue limiting dilution assay and carry out cloning, until the clone obtained is positive, positive cell strain can be set up.Finally obtain the positive
Cell strain 5 strain.
6, amplification culture: the monoclonal cell amplification culture of strain will be built, and frozen in liquid nitrogen.
(2) ascites preparation and purification
1, prepared by ascites: carries and injects mineral oil at mouse peritoneal the last week, by some (about 106) cell infusion enter
Mouse peritoneal, collects ascites in about 10 days, and 4000rpm is centrifuged, and obtains supernatant and is monoclonal antibody ascites.
2, monoclonal antibody-purified: ascites is centrifuged 15min (4000rpm, room temperature), takes supernatant 10ml, 4 DEG C stirring under by
Drip and be slowly added to saturated ammonium sulfate to semi-saturation, continue stirring 30min, centrifugal 30min (13000rpm, 4 DEG C), abandon supernatant；Heavy
Shallow lake is dissolved in about 10mlPBS (0.01M, pH7.4)；Under 4 DEG C of stirrings, dropwise it is slowly added to saturated ammonium sulfate to 33%, continues stirring
30min, centrifugal 30min (13000rpm, 4 DEG C), abandon supernatant；Precipitation is dissolved in appropriate 10mlPBS (0.01M, pH7.4), 4 DEG C of dialysis
Overnight, measuring antibody content ,-20 DEG C frozen standby.Continue after ammonium sulfate precipitation to use Protein G pillar to be purified, newly
Pillar first crosses post with 5ml ultra-pure water, then balances purification pillar with 5ml 0.4M PB buffer (pH 7.0)；Antibody crosses post, process
Middle requirement is slow crosses post, is preferably combined on binding site in the hope of antibody protein；Continue 10ml 0.4M PB buffer (pH
7.0) balance purification pillar；Antibody on 5ml 0.1M glycine-HCI buffer (pH 2.7) elution of bound site, and washing
De-liquid adds in 1MTris-HCl (pH 8.0) 0.5ml and glycine, make pH remain the neutrality that applicable antibody preserves.
Thus obtain mouse-anti g10-epsps monoclonal antibody.
Embodiment 3, the preparation method of mouse-anti g10-epsps monoclonal antibody of colloid gold label, specifically include following steps:
1. the preparation of 25nm gold colloidal:
1.1 prepare: put into after 500ml beaker, 20ml small beaker, rotor, brown bottle, Glass rod etc. being cleaned in acid cylinder
(potassium dichromate: concentrated sulphuric acid: ultra-pure water=120g:200ml:1000ml) soaks 24 hours.Take out and first rinse 3-4 with tap water
Secondary, then with ultrapure water 3-4 time, it is placed in dry for standby in 37 DEG C of baking ovens.
The configuration of 1.2 1%HAuCl4 aqueous solutions: weigh spoon with plastics and weigh 1g gold chloride powder in brown bottle, add
99ml ultra-pure water fully dissolves, and 4 DEG C keep in Dark Place.
Note: gold chloride powder easily deliquescence, wants the when of weighing quickly, and residual chlorine auric acid powder seals with aluminium foil bag and protects
Deposit.
The configuration of 1.3 1% trisodium citrate aqueous solutions: weigh 1g trisodium citrate and be dissolved in 99ml ultra-pure water, mixed
Even.
The preparation of 1.4 25nm gold colloidals: measure 99ml ultra-pure water in beaker, adds 1ml 1%HAuCl4 aqueous solution,
It is placed in stirring and evenly mixing in constant temperature blender with magnetic force, opens and be heated to solution boiling, be rapidly added freshly prepd 1% Fructus Citri Limoniae of 2.5ml
Acid three sodium water solutions, continue agitating heating, and solution gradually becomes black-and-blue, the most purple black, reheat and redness occur, continue boiling and boil
Occur transparent orange red, continue boiling and boil 7-10min, naturally cool to room temperature, add ultra-pure water and make solution to 100ml.Add one
Fixed PEG20000 to its final concentration of 0.02%--0.05%.Pouring brown bottle into, 4 DEG C keep in Dark Place.
2. antibody labeling:
The labelling of 2.1 antibody: take the gold colloidal that 1ml produces, with the K of 1%2CO3Regulation PH to 8.0, adds on 80 μ g
State the g10-epsps mouse monoclonal antibody that purification obtains, mix homogeneously, room temperature reaction 40min.Add BSA to final concentration of
0.1%, stand 30min.
2.2 traget antibody purification: be first centrifuged 15 minutes with low speed (1500r/min), discard and become by the gold size particle shape condensed
Precipitation.Then it is centrifuged 30 minutes with 10000r/min.Carefully sucking-off supernatant, precipitate 0.1ml contains the 0.1M of 1%BSA
PBS (PH7.4) redissolves, and adds 5% sodium azide extremely final concentration of 0.05%, 4 DEG C of preservations.Obtain the mouse-anti g10-of colloid gold label
Epsps monoclonal antibody.
Embodiment 4, the making of transgene protein g10-epsps immune detection card
Base plate 7 is pasted sample pad 1 the most side by side and is fixed with the specific monoclonal of colloid gold label
Gold mark pad 2, cellulose acetate membrane 3 and the sample suction pad 4 of antibody.Gold mark pad 2 is fixed 40ng mouse-anti g10-epsps monoclonal
Antibody, the detection zone in cellulose acetate membrane 3 is fixed 0.8 μ g mouse-anti g10-epsps monoclonal antibody and consolidates in control zone
Fixed anti-Mus IgG bis-resists 0.4 μ g (respectively as detection line 5 and nature controlling line 6).It is subsequently placed in 37 DEG C of vacuum drying 30min.
The effect assessment of embodiment 5, transgene protein g10-epsps immune detection card detection actual standard product
1. specimen cup adds 200 μ L blank sample liquid (pH 7.4,0.2mol/L PBS:8g sodium chloride, 3.35g 12
Hypophosphite monohydrate disodium hydrogen, 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, distilled water dissolves and is settled to 1L), speed test paper is inserted sample
Product cup, reaction observes colour developing result after 8 minutes in result observation area at ambient temperature.Result shows, in observation area, T line is not
Colour developing, C line develops the color.
2. the g10-epsps standard substance adding 100 μ L 0.1 μ g/ml in specimen cup (use PBS solution dilution ibid to join
Become), according to the operating procedure as blank sample.Result shows, in observation area, C line, T line develop the color simultaneously.
The acquisition methods of g10-epsps standard substance is:
Prokaryotic expression by the His-EPSPS (embodiment 2 gained) of affinity purification, N end band in E. coli
Having 6 His labels, its mother liquid concentration is 3mg/ml, and by diluted to 900ng/ml, 3mL Brown Glass Brown glass bottles and jars only adds 100 μ L
900ng/ml standard substance, freezer dryer is overnight drained and is obtained standard substance lyophilized powder, is g10-epsps standard substance.
Embodiment 6, transgene protein g10-epsps immune detection card detect the effect assessment of actual blade sample
1. specimen cup adds 200 μ L feminine gender blade sample extraction liquid, according to the operating procedure as standard substance.Result
Showing, only control line C line colour developing in observation area, detection line T line does not develops the color.
2. specimen cup adds 200 μ L positive blade sample extraction liquid, according to the operating procedure as negative sample.Knot
Fruit shows, in observation area, control line C line and detection line T line all develop the color.
Remarks illustrate:
1, blade sample extraction liquid is 0.1M PBS, and blade extracts according to the method that the industry is conventional.
2, above-mentioned negative blade sample confirms through RT-PCR method, and g10-epsps content cannot detect；And positive blade
In sample, g10-epsps is through RT-PCR test positive.
Embodiment 7, transgene protein g10-epsps immune detection card detect the effect assessment of actual seed specimen
1. specimen cup adds 200 μ L feminine gender seed specimen extracting solution, according to the operating procedure as mark product.Result table
Bright, only control line C line colour developing in observation area, detection line T line does not develops the color.
2. specimen cup adds 200 μ L positive seeds sample extraction liquid, according to the operating procedure as negative sample.Knot
Fruit shows, in observation area, control line C line and detection line T line all develop the color.
Remarks illustrate:
1, seed specimen extracting solution is 0.1M PBS.Seed extracts according to the method that the industry is conventional.
2, above-mentioned negative seed specimen confirms through RT-PCR method and ELISA method method, and g10-epsps content cannot detect
Arrive；And g10-epsps is through RT-PCR test positive in positive seeds sample, through ELISA method detection level at 100-500 μ
g/ml。
Embodiment 8, transgene protein g10-epsps immune detection card detect the effect assessment of actual Bulk Grain sample
1. specimen cup adds 200 μ L feminine gender Bulk Grain sample extraction liquid, according to the operating procedure as mark product.Result table
Bright, only control line C line colour developing in observation area, detection line T line does not develops the color.
2. negative Bulk Grain sample adds 0.2% positive seeds sample, obtain positive Bulk Grain sample, this sample is carried
Take, specimen cup adds 200 μ L positive Bulk Grain sample extraction liquid, according to the operating procedure as negative sample.Result shows,
In observation area, control line C line and detection line T line all develop the color.
Remarks illustrate:
1, seed specimen extracting solution is 0.1M PBS.Seed extracts according to the method that the industry is conventional.
2, above-mentioned negative Bulk Grain sample confirms through RT-PCR method (current existing detection method), g10-epsps expression
Cannot detect；And g10-epsps content is through RT-PCR test positive in positive Bulk Grain sample.
Finally, in addition it is also necessary to be only several specific embodiments of the present invention it is noted that listed above.Obviously, this
Bright it is not limited to above example, it is also possible to have many deformation.Those of ordinary skill in the art can be from present disclosure
The all deformation directly derived or associate, are all considered as protection scope of the present invention.
Claims (5)
1. detect transgene protein R1-EPSPS gold colloidal speed test paper, including the sample pad being arranged on base plate, gold mark pad,
It is coated nature controlling line and the detection nitrocellulose filter of line, sample suction pad；It is characterized in that: on described gold mark pad, be coated with gold colloidal mark
The mouse-anti g10-epsps monoclonal antibody of note, described detection line is for being coated with mouse-anti g10-epsps monoclonal antibody, described matter
Control line resists for being coated with anti-Mus IgG bis-.
Gold colloidal speed test paper the most according to claim 1, it is characterised in that:
Mouse-anti g10-epsps monoclonal antibody is by by g10-epsps recombiant protein immunity BALB/c mouse, then by thin
Born of the same parents are merged, cloning screening prepares cell strain of monoclonal antibody, then prepare mouse ascites, obtain through purification.
Gold colloidal speed test paper the most according to claim 2, it is characterised in that: g10-epsps is by the g10-in G1
Epsps gene is built into coli expression system after optimizing, and recombinant expressed purification obtains.
4. according to the gold colloidal speed test paper described in claim 1,2 or 3, it is characterised in that: the mouse-anti g10-of colloid gold label
The preparation method of epsps monoclonal antibody comprises the following steps:
1), the gold colloidal of preparation 20～30nm；
2), antibody labeling:
Taking the gold colloidal of 1ml, regulate pH to 8.0, add the g10-epsps mouse monoclonal antibody of 80 μ g, mix homogeneously, room temperature is anti-
Answer 30～50min；Add BSA to final concentration of 0.1%, stand 20～40min；
Then it is marked antibody purification, obtains the mouse-anti g10-epsps monoclonal antibody of colloid gold label.
5. the using method of the gold colloidal speed test paper as described in Claims 1 to 4 is arbitrary, it is characterised in that:
Speed test paper is inserted perpendicularly in testing sample；
After 8～10min, it is observed；C line only occurs, it is judged that sample is negative；T line and C line occur simultaneously, it is determined that sample is
Positive.
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| CN112485437A (en) * | 2020-11-10 | 2021-03-12 | 基因赛奥(大连)生物科技发展有限公司 | CP4EPSPS and Bt Cry1Ab/Ac double-protein rapid test paper card and preparation and use methods thereof |
| CN117230020A (en) * | 2023-11-14 | 2023-12-15 | 中国农业科学院生物技术研究所 | G10 EPSPS monoclonal antibody hybridoma cell strain, antibody produced by same and application thereof |
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| CN112485437A (en) * | 2020-11-10 | 2021-03-12 | 基因赛奥(大连)生物科技发展有限公司 | CP4EPSPS and Bt Cry1Ab/Ac double-protein rapid test paper card and preparation and use methods thereof |
| CN117230020A (en) * | 2023-11-14 | 2023-12-15 | 中国农业科学院生物技术研究所 | G10 EPSPS monoclonal antibody hybridoma cell strain, antibody produced by same and application thereof |
| CN117230020B (en) * | 2023-11-14 | 2024-01-26 | 中国农业科学院生物技术研究所 | G10 EPSPS monoclonal antibody hybridoma cell strain, antibody produced by same and application thereof |
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