CN102156193B - Method for detecting target protein in plants and special SPR (selective posterior rhizotomy) biosensor for method - Google Patents
Method for detecting target protein in plants and special SPR (selective posterior rhizotomy) biosensor for method Download PDFInfo
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Abstract
The invention discloses a method for detecting target protein in plants and a special SPR biosensor for the method. The invention provides application of the following SPR biosensor for detecting whether a target protein exists in an organism in an assisting manner. The organism is a plant which is specifically cottons. The target protein is Cry1AC protein; the sensing chip is a CM5 (control memory) chip. Experiment of the invention proves that the SPR biosensor for covering Cry1AC monoclonal antibody is constructed in the invention; the biosensor can be used for detecting whether Cry1AC protein exists in a transgenic cotton or not; and the biosensor has the advantages of high sensitivity, high flux and automation; and the operations are simple.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method and special-purpose surface plasmon resonance biosensor thereof that detects target protein in plant.
Background technology
Along with the safety issue of genetically modified plants and product receives publicity day by day, set up accurately, reliably, easily the transgenosis detection technique and detection platform extremely important to the commercial detection of genetically modified plants.Surface plasma resonance (Surface plasmon resonance, SPR) biology sensor is the optical bio chemical detection technique developed rapidly in the world in recent years.Surface plasmon resonance biosensor is that the golden film surface that probe or part are fixed in to sensor chip forms unimolecular layer, when the liquid that contains the analyte that can act on is with it flowed through the vane surface, intermolecular generation specific binding also can cause the change of vane surface refractive index, by detecting the spr signal change, monitors intermolecular interaction.Surface plasmon resonance biosensor have without mark, sensitive and accurate, quick, can realize the detection characteristics such as on-line continuous and high flux, be particularly suitable for interacting between biomolecule, whole process by sensor chip Real-Time Monitoring different kind organism molecule as polypeptide, protein, oligonucleotides, oligosaccharide and little molecular signal interaction between substances, and can measure in solution and the fixing amount of substance of effect mutually by analysis software.
The method commonly used of identifying at present genetically modified plants by detecting target protein is enzyme linked immunosorbent assay (enzyme-linked immunosor-bent assay, ELISA), ELISA has possessed the high sensitivity of enzyme reaction and the specificity of antigen-antibody reaction, have easy, quick, the characteristics such as expense is low.EPSPS in the RR soybean sample that 38 use for laboratory ELISA of 13 countries of European Union are 2% to transgene component is detected, and accuracy rate, up to 99%, detects and is limited to 0.35%.But be prone to the too high problem of background, lack standardization, and can only detect unwrought product, and can only detect the genetically modified organism of limited kinds.Western blot is the authoritative method whether foreign gene gives expression to protein that detects in plant genetic engineering, have very high sensitivity, but its operation is more loaded down with trivial details, and expense is higher, is unsuitable for the detection of quick, a large amount of samples.And the SPR detection has its unique advantage aspect Protein Detection: fast and convenient, detect in real time, sample is without mark, the advantages such as high flux detection, in the transgenosis context of detection, sizable development potentiality is arranged, detect by the SPR method research that whether contains target protein in genetically modified plants at present and there is not yet report.
Example: Dipel (Bacillus thuringiensis, Bt) toxoprotein gene is most popular anti insect gene in current world wide, the Bt transgenic cotton against pests increases year by year at the planting area of China, data show, in the cotton of planting this year in Hebei province, 90% is the Insect Resistant Cotton kind, yet Insect Resistant Cotton may be with the problem of serving as other genetically modified crops, such as the insect insect resistace, strengthen, natural enemy quantity reduces, gene drifts about, bio-diversity is destroyed etc., therefore, the Bt transgenic cotton against pests is carried out to long term monitoring and be very important.
According to rough Statistics, 60 groups of Bt insecticidal crystalline genes have been had been found that at present, but according to the different rough segmentations of homology of their desinsection scopes and gene order, be can be divided into many subclass again in six each class of large class (CryI, CryII, CryIII, CryIV, CryV, Cyt), what proceed at present cotton has several Bt insecticidal crystalline genes that lepidoptera pest had to stronger resistance such as CryIAc, CryIAb, CryIAc and CryIAb fusion.
Summary of the invention
An object of the present invention is to provide the application of a kind of surface plasmon resonance biosensor in auxiliary detection biological targets albumen.
The invention provides the application of a kind of surface plasmon resonance biosensor as described below in auxiliary detection biological targets albumen.
Described biology is plant; Described target protein is pest-resistant albumen.
Described plant is the plant that proceeds to the encoding gene of described target protein.
Described pest-resistant albumen is Cry1Ac albumen.
Described Cry1Ac albumen is following (a) or protein (b):
(a) protein that the amino acid sequence shown in sequence 2 forms in sequence table;
(b) by the amino acid sequence of sequence 2 through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and there is the derivative protein by sequence 2 of identical function.
The replacement of described one or several amino acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino acid residues.
The encoding gene of described Cry1Ac albumen is following 1) or 2) or 3) shown in gene:
1) DNA molecular shown in sequence 1 in sequence table;
2) under stringent condition with 1) DNA molecular with identical function albumen of the DNA sequence dna hybridization that limits and coding;
3) with 1) DNA sequence dna that limits has the DNA molecular that 90% above homology and coding have identical function albumen.
Described stringent condition can be at 6 * SSC, and in the solution of 0.5%SDS, under 68 ℃, hybridization, then use 2 * SSC, 0.1%SDS and 1 * SSC, and 0.1%SDS respectively washes film once.
Another object of the present invention is to provide a kind of surface plasmon resonance biosensor for auxiliary detection biology target protein to be measured.
A kind of surface plasmon resonance biosensor for auxiliary detection biology target protein to be measured provided by the invention, be coupled on sensing chip the surface plasmon resonance biosensor obtained in covalently bound mode for resisting target protein antibody.
Described anti-target protein antibody is monoclonal antibody or polyclonal antibody;
Described anti-target protein antibody specific is monoclonal antibody;
Described sensing chip is the CM5 chip;
Described target protein is Cry1Ac albumen;
The monoclonal antibody that described anti-target protein antibody is anti-Cry1Ac albumen;
Described Cry1Ac albumen is following (a) or protein (b):
(a) protein that the amino acid sequence shown in sequence 2 forms in sequence table;
(b) by the amino acid sequence of sequence 2 through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and there is the derivative protein by sequence 2 of identical function.
The encoding gene of described Cry1Ac albumen is following 1) or 2) or 3) shown in gene:
1) DNA molecular shown in sequence 1 in sequence table;
2) under stringent condition with 1) DNA molecular with identical function albumen of the DNA sequence dna hybridization that limits and coding;
3) with 1) DNA sequence dna that limits has the DNA molecular that 90% above homology and coding have identical function albumen.
Described coupling comprises the steps: the CM5 chip after the flow of solution of the monoclonal antibody of described anti-Cry1Ac albumen is activated, and it is combined with the CM5 chip surface, obtains surface plasmon resonance biosensor:
In order resisting, the monoclonal antibody of Cry1Ac albumen and pH are 4.5 to the solution of the monoclonal antibody of described anti-Cry1Ac albumen, concentration is 10mM NaAc aqueous solution, the solution obtained, the concentration of the monoclonal antibody of described anti-Cry1Ac albumen in the solution of described monoclonal antibody is 50 μ g/ml;
Described flow velocity of flowing through is 5 μ l/min;
The described time of flowing through is 3min.
Described biology is plant, and described plant is specially cotton.
Above-mentioned being applied as according to the method comprised the steps carried out:
1) extract the total protein of biological tissue to be measured;
2) by step 1) total protein that obtains adopts the surface plasmon resonance biosensor of the antibody that is coated with described target protein to carry out the SPR detection, with wild-type plant in contrast, if the difference of the response of described biology to be measured and described wild-type plant is more than or equal to 40RU, in described biology to be measured, contain target protein, if the difference of the response of described biology to be measured and described wild-type plant is less than 40RU, in described biology to be measured, do not contain target protein.
Described target protein is Cry1Ac albumen;
Described antibody is monoclonal antibody or polyclonal antibody;
Described antibody specific is the Cry1Ac monoclonal antibody.
Step 1), in, described extracting method comprises the steps:
A, the tissue of biology to be measured, extract and PVPP are mixed, obtain mixed liquor;
B, the mixed liquor that steps A is obtained are centrifugal, get supernatant, obtain total protein;
The described extract of every 1L is for being prepared as follows: by 8g NaCl, 0.2g KCl, 1.44g Na
2hPO
4, 0.24g KH
2pO
4, 0.5ml Tween20 and deionized water mix, and obtains extract;
Step 2) in, the described surface plasmon resonance biosensor that is coated with the antibody of described target protein is prepared as follows: the Cry1Ac monoclonal antibody is coupled on the CM5 chip in covalently bound mode, obtains being coated with the surface plasmon resonance biosensor of the antibody of described target protein.
Step 1) in A,
The pH of described extract is 7.4;
Described incorporation time is 2 hours; The temperature of described mixing is 4 ℃;
The proportioning of the tissue of described biology to be measured, extract and PVPP is 1g: 4ml: 0.1g;
Step 1) in B,
Described centrifugal speed is 13000rpm, and described centrifugal radius is 82mm, and described centrifuging temperature is 4 ℃, and described centrifugation time is 20min;
Step 2) in,
The pH value of described coupling is 4.5;
The sample introduction flow velocity that described detection adopts is 30 μ l/min; Sample size is 60 μ l; The sample introduction kind is KINJECT;
Described detected temperatures is 25 ℃;
Described biology is plant, and described plant is specially cotton; The blade that is organized as cotton of described biology;
Described wild-type plant is cotton DP5415.
The present invention of experiment showed, of the present invention has built the surface plasmon resonance biosensor of coated Cry1Ac monoclonal antibody, can detect in transgene cotton whether contain Cry1Ac albumen by it, and it has advantages of high sensitivity, high flux, robotization, and simple to operate.
The accompanying drawing explanation
Fig. 1 is determining of Cry1Ac monoclonal antibody coupling pH value
Fig. 2 is determining of Cry1Ac monoclonal antibody coupling pH value
The cultivation that Fig. 3 is the vegetable lamb material
The clone that Fig. 4 is the Cry1Ac gene and the structure of prokaryotic expression carrier
Fig. 5 is the abduction delivering situation analysis in five kinds of host strains of Cry1Ac-GST albumen
Fig. 6 is the abduction delivering situation analysis in BL21 (DE3) and Transetta (DE3) host strain of Cry1Ac-GST albumen
Fig. 7 is the abduction delivering situation analysis of Cry1Ac-GST albumen under different temperatures
Fig. 8 is that Cry1Ac-GST protein purification process SDS-PAGE analyzes
Fig. 9 is Cry1Ac-GST protein molecular sieve purifying chromatogram
Figure 10 is that the SDS-PAGE after Cry1Ac-GST protein molecular sieve purifying analyzes
Figure 11 is that Cry1Ac-GST albumen Weastern Blotting analyzes and ELISA test strip
Figure 12 is that Cry1Ac-GST Protein S PR detects
Figure 13 is that BSA Protein S PR detects
Figure 14 is that the cotton total protein extracts the SDS-PAGE analysis
The SDS-PAGE that Figure 15 is the cotton total protein analyzes
Figure 16 is that cotton total protein sample SPR detects
Figure 17 is cotton total protein ELISA test strip
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The preparation of embodiment 1, surface plasmon resonance biosensor and checking
By Cry1Ac monoclonal antibody (Fitzgerald, 10R-B122A.) be coupled to chip surface, pass into testing sample, determine in sample whether contain Cry1Ac albumen according to the variation of RU (response), use BIAcore 3000 to carry out relevant test.
The principle that Cry1Ac Protein S PR detects: use the BIAcore3000 (GE company) based on surface plasma resonance (SPR) principle design to detect in sample whether contain Bt albumen, the chip of selecting is the CM5 chip, be characterized in having covered the carboxylated glucosan of one deck at chip surface, glucosan is water wettability, there is low, the high binding capacity of non-specific binding, be easy to carry out covalent bond, outstanding characteristics such as chemical stability, be the most frequently used sensing chip.The Cry1Ac monoclonal antibody is usingd to covalently bound mode and be coupled at the Dextran Chip surface of carboxylated processing as part (ligand), sample flows through chip surface with constant flow velocity and concentration, if contain Cry1Ac albumen in analysans (analyte), it will be done mutually with the antibody be coupled on chip, the quality of chip surface material will change, the change of corresponding response (RU) under instrument record; If there is no Cry1Ac albumen in analysans, instrument will not have the change of response.
One, the preparation of surface plasmon resonance biosensor
1, the selection of Cry1Ac monoclonal antibody coupling pH value
What test was selected is CM5 chip (GE company, BR-1000-14), before the Cry1Ac monoclonal antibody is coupled to chip surface, need carry out the screening of optimum pH, concrete operations are as follows: the Cry1Ac monoclonal antibody is used respectively to pH=5.5,5.0,4.5,4.0 10mM NaAc aqueous solution be diluted to 50 μ g/ml, carry out respectively binding analysis, concrete grammar is the Cry1Ac monoclonal antibody with PBS damping fluid (137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4pH=7.4) dissolved dilution is to 1mg/ml, get 10 μ l and add the 10mM NaAc aqueous solution of the 190 different pH values of μ l (the pH value is 4.0,4.5,5.0,5.5), the monoclonal antibody solution of each pH value is with the flow velocity of the 20 μ l/min chip surface of flowing through, with chip surface duration of contact be 120s, rinse 150s with the PBS damping fluid immediately, then advance next monoclonal antibody solution.As shown in Figure 1, during pH=4.5, the Cry1Ac monoclonal antibody is attached to the amount maximum on chip to result, and response reaches 27566RU (response), so the Cry1Ac monoclonal antibody selects the 10mM NaAc of pH=4.5 to be diluted.
2, the coupling of Cry1Ac monoclonal antibody
After coupling pH value is determined, next step carries out the coupling of albumen, need to activate chip surface before coupling, test adopts EDC/NHS activation (1 in Fig. 2), after having activated, add the Cry1Ac monoclonal antibody of 10mM NaAc dilution of part: pH 4.5 until reach target level (2 in Fig. 2), then with the monoethanolamine sealing unnecessary activation site (3 in Fig. 2) of chip surface.Whole process is specially: the flow velocity with 5 μ l/min injects 0.1M N-hydroxy-succinamide (NHS) and N-ethyl-N '-(diethyl-aminopropyl)-carbon diamides (EDC) activating solution mixed by 1: 1 (v/v), the glucosan of activation chip surface, injection length is 7min; Get 200 μ l and obtain with the 10mM NaAc of pH=4.5 dilution the Cry1Ac monoclonal antibody that concentration is 50 μ g/ml, with the low flow velocity of the 5 μ l/min chip of flowing through, be combined with the site of chip surface activation, until reach predetermined coupling level; Seal the not activation site of binding antibody of chip surface with the 1M monoethanolamine again, flow velocity is 5 μ l/min, and injection length is 7min.As shown in Figure 2,1 is activation to the whole process of coupling; 2 is ligand coupling; 3 for sealing, can find out, the coupling level is about 8000RU (the corresponding ordinate of the four-headed arrow in Fig. 2), obtains protein chip.
Two, the checking of protein chip
1, the cultivation of vegetable lamb material
Buy wild type cotton DP5415 seed and transgene cotton Nucotn33B seed from Monsanto Company, warm water (30 ℃ of left and right) seed soaking 8h, insulation vernalization 12h under 25 ℃, when seed expose white bud to bud reach seed long 1/2 the time be sown in soil, cultivate one month under 25 ℃ of conditions in greenhouse, obtain wild type cotton DP5415 and transgene cotton Nucotn33B (seeing Fig. 3).
2, the structure of the clone of Cry1Ac gene and prokaryotic expression carrier
Extract the genomic DNA of Nucotn33B cotton leaf, and as template, Auele Specific Primer BtFW and BtRV (table 1) with target gene, adopt high-fidelity DNA polymerase through PCR (PCR), amplification obtains PCR product, pcr amplification program: 94 ℃ of for 3min, 94 ℃ of for 30s, 54 ℃ of for 30s, 72 ℃ of for45s, 35 cycles; 72 ℃ of for 5min; 10 ℃ of for ever.Result as shown in Figure 4, obtains the 1780bp fragment.
This PCR product is sent to order-checking, and result has the nucleotide shown in sequence 1 in sequence table for this PCR product, and Cry1Ac gene base sequence (GenBank:Y09787) homology in spending with the transgenicCry1Ac cotton of having reported reaches 96.18%.The gene of this PCR product is Cry1Ac, and the albumen of its coding is Cry1Ac (Bt albumen a kind of), and the amino acid sequence of this albumen is the sequence 2 in sequence table.
The PCR product is cut to rear recovery fragment with restriction enzyme Xho I and Hind III enzyme, with the prokaryotic expression carrier pGEX-KG (Beijing DingGuo ChangSheng Biology Technology Co., Ltd of cutting through same enzyme, MCV030) the carrier large fragment connects, obtain connecting product, proceed in bacillus coli DH 5 alpha, obtain transformant, extract the plasmid of transformant, the carrier of this plasmid for obtaining between the Xho I by the 1 insertion pGEX-KG of sequence in sequence table and Hind III restriction enzyme site, by this plasmid called after pGEX-KG-Cry1Ac.
The Auele Specific Primer that table 1 is target gene
3, the expression of Cry1Ac albumen, purifying and evaluation
1), the prokaryotic expression of Cry1Ac protein gene
Bt toxalbumin Cry1Ac is soluble protein, and utilizing prokaryotic expression system to obtain can purifying and activated destination protein, considers as much as possible to obtain expressing protein from non-inclusion body.
Transform respectively five kinds of host strain: BL21 with the above-mentioned prokaryotic expression plasmid pGEX-KG-Cry1Ac built, BL21 (DE3), BL21 (DE3) plys S, TransB (DE3), Transetta (DE3) is (all purchased from Beijing Quanshijin Biotechnology Co., Ltd, catalog number is CD901, CD601, CD701, CD811, CD801), obtain recombinant bacterium BL21/pGEX-KG-Cry1Ac, BL21 (DE3)/pGEX-KG-Cry1Ac, BL21 (DE3) plys S/pGEX-KG-Cry1Ac, TransB (DE3)/pGEX-KG-Cry1Ac, Transetta (DE3)/pGEX-KG-Cry1Ac.Above-mentioned 5 kinds of bacterium are seeded in the LB nutrient culture media and cultivate, and adding final concentration is 0.4mM IPTG (isopropyl-β-D-sulfo-galactopyranoside, Merck, CB420322) induce, at 37 ℃, cultivate 4h, obtain culture, carry out the SDS-PAGE detection, take and do not add IPTG as contrast.
Culture is carried out to the SDS-PAGE electrophoresis, result as shown in Figure 5, in BL21 (DE3)/pGEX-KG-Cry1Ac and Transetta (DE3)/pGEX-KG-Cry1Ac, can find significantly by IPTG abduction delivering Cry1Ac-GST fusion (with the Cry1Ac albumen of GST label, 91.8KD, as shown by arrows), and do not have Cry1Ac-GST albumen under the condition of inducing through IPTG not occur, Transetta (DE3) is during as host strain, the expression of Cry1Ac-GST albumen is more, therefore is chosen to be the host of inducible protein in enormous quantities.
The culture of BL21 (DE3)/pGEX-KG-Cry1Ac and Transetta (DE3)/pGEX-KG-Cry1Ac is carried out to ultrasonication, centrifugal, collect supernatant and precipitation, supernatant and precipitation are carried out to the SDS-PAGE electrophoresis, result as shown in Figure 6, can find out that the albumen (91.8KD) through the IPTG abduction delivering concentrates in the precipitation of broken thalline basically, be in inclusion body, and destination protein not in the solution supernatant, and the expression of Cry1Ac-GST albumen in Transetta (DE3) host strain is more, but mainly be present in inclusion body (as shown by arrows).Thereby need to be optimized the condition of prokaryotic expression system.
Temperature while reducing the abduction delivering of Transetta (DE3)/pGEX-KG-Cry1Ac, set up 28 ℃ and 16 ℃ of two gradients, cultivate respectively detection, upper cleer and peaceful precipitation after the culture ultrasonication is carried out to the SDS-PAGE detection, the results are shown in Figure shown in 7, induce cultivation through 16 ℃, obtain the Cry1Ac albumen (Cry1Ac-GST) ((91.8KD) as shown by arrows with the GST label in the supernatant of broken thalline.Can be for the work of next step protein purification.
2), the purifying of Bt albumen and evaluation
The Cry1Ac protein band GST label of pGEX-KG-Cry1Ac gene expression, so the preferential affinity chromatography purifying of selecting during purifying, used the GSTrap FF affinity column of GE company to carry out correlation test:
In the Transetta (DE3) that is 0.6-0.8 to the OD value obtained through second incubation/pGEX-KG-Cry1Ac bacterium liquid, add IPTG to final concentration be 0.4mM, induce under 16 ℃ and cultivate 12h, 5000g is centrifugal, collect thalline, with PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4pH 7.4) resuspended rear low temperature ultrasonication, under 4 ℃ of conditions, the centrifugal 40min of 10000g, get supernatant, with 0.45 μ m membrane filtration, carry out purifying according to the instructions of GSTrap FF affinity column, the sample of collecting respectively after ultrasonication sample, purification of samples and super filter tube concentrate carries out the 10%SDS-PAGE electrophoresis detection.
Result as shown in Figure 8, M:Maker; Broken: the supernatant after centrifugal after the ultrasonication of bacterium liquid; Purifying: the protein sample of collecting after GST affinity column purifying; Concentrated: the sample that purifying protein is collected after super filter tube is concentrated, as seen from the figure, through the glutathione agarose affinity purification, the purity of Cry1Ac-GST fusion is greatly improved, but still have some foreign proteins, while detecting for SPR, may certain influence be arranged to the confidence level of its result, therefore the albumen after the affinity chromatography purifying having been carried out to further purifying---the gel filtration purifying claims again molecular sieve purification.
Molecular sieve purification is a kind of purification process it separated according to the molecular size range difference, and in detachment process, at first large molecule is separated from pillar, is and then smaller molecule successively.In addition, purge process can complete the exchange of desalination and damping fluid simultaneously.Use the Superdex 20010/300GL purification column of GE company to exist
cry1Ac-GST albumen after concentrated to super filter tube on the chromatography system has carried out secondarily purified, sample when under the condition that is 280nm at the chromatogram wavelength, the collection peak occurs, (ordinate is mAU to result as shown in Figure 9, the milli absorbance unit), collect eluent corresponding to peak occurred during 44min-53min, be purifying destination protein Cry1Ac-GST.
The purifying destination protein Cry1Ac-GST of collection is carried out to the SDS-PAGE electrophoresis, result as shown in figure 10, can find out, molecular weight is that 91.8kD is the purpose purifying protein, after molecular sieve purification, in the purpose purifying protein, foreign protein obviously reduces, and obtains the higher purpose purifying protein of purity, is Cry1Ac-GST.
Characteristics according to Cry1Ac albumen and GST formation fusion, utilize the monoclonal antibody (Novagen of GST, 71097) purpose purifying protein Cry1Ac-GST is carried out to Western blotting detection, be about to destination protein Cry1Ac-GST wet going on pvdf membrane after SDS-PAGE separates of purifying, and carry out immuning hybridization, the dilution ratio of the monoclonal antibody of GST is 1: 4000, take do not add that IPTG induces Transetta (DE3)/pGEX-KG-Cry1Ac is control strain, result is as shown in Figure 11 a, destination protein Cry1Ac-GST (deriving from expression strain Transetta (DE3)/pGEX-KG-Cry1Ac) has hybridization signal, and size and destination protein in the same size, and not reaction of control strain (CK).
By Bt-Cry1Ab/1Ac test strips (Agdia), purifying destination protein Cry1Ac-GST is detected to (instructions and reagent are arranged in kit), result is as shown in Figure 11 b, the p-wire appearance, interpret sample is positive, is purifying destination protein Cry1Ac-GST.
Above experiment all proves, obtains destination protein Cry1Ac-GST.
With Super-Bradford protein quantification kit, (Beijing health is the century bio tech ltd, CW0013) and Multiskan Mk3 microplate reader (Thermo Fischer Scient Inc., 51118160) measure the light absorption value of protein sample at the 595nm place, thereby the concentration that calculates destination protein Cry1Ac-GST is 73 μ g/ml.
3), the checking of protein chip
Detect in order to check the protein chip built whether to can be used for transgenosis, designed positive and negative contrast proved.At first pass into sample: obtain destination protein Cry1Ac-GST (concentration is 73 μ g/ml) after molecular sieve purification, flow velocity is 30 μ l/min, and sample size is totally 60 μ l.At first experiment passes into PBS damping fluid (137mM NaCl, 2.7mM KCl, 10mMNa
2hPO
4, 2mM KH
2pO
4, pH=7.4) rinse chip surface, when the RU value is steady, pass into testing sample 2min, analyte is combined with the part of chip surface, then passes into the PBS damping fluid and rinses 2min, and analyte is dissociated from chip surface, now corresponding RU value deducts RU value corresponding to PBS damping fluid when initial, be the amount of substance of chip surface combination, after this, with the NaOH of 10 μ l 50mM with the flow velocity of the 30 μ l/min chip surface of flowing through, make the RU value recover initial size, realize chip regeneration.
As shown in figure 12, the Cry1Ac-GST albumen of purifying and the Cry1Ac monoclonal antibody of chip surface occur to do mutually result, and the 8420RU (advance damping fluid) of response before by sample introduction increases to 9570RU (with PBS, rinsing the value after 2min), increased about 1150RU; With the NaOH regeneration chip of 10 μ l 50mM, find regeneration effect better (seeing Figure 12).
In addition, test design the specificity of negative contrast with verification system, pass into bovine serum albumin(BSA) (BSA) (Sigma, A7030), whether detect the Bt monoclonal antibody and can be combined with BSA, result as shown in figure 13, can find out that the monoclonal antibody of chip surface does not occur to do mutually with BSA, the specificity that shows chip is fine, can detect for transgenosis.
The SPR of embodiment 2, protein chip detects
One, the extraction conditions of cotton total protein is groped
After being pulverized, wild type cotton DP5415 plant leaf blade liquid nitrogen grinding is transferred in centrifuge tube, add respectively following two kinds of Extraction buffers, withdrawal ratio is 1: 4 (being that the 1g vegetable material adds the 4ml Extraction buffer), add water-insoluble PVPP (crospolyvinylpyrrolidone according to 10% of its fresh weight in addition, Sigma, P6755), mix rear 4 ℃ of vibrations 2 hours, then under 4 ℃ of conditions, 13000rpm, centrifugal 20min, get supernatant and can carry out Protein Detection,-80 ℃ of preservations after also can liquid nitrogen flash freezer, if also have impurity in supernatant, can be again centrifugal.
Following two kinds respectively of Extraction buffers:
PBST(137mM?NaCl，2.7mM?KCl，10mM?Na
2HPO
4，2mM?KH
2PO
4，0.05％Tween20，pH?7.4)；
CBS(100mM?Na
2CO
3，100mM?NaHCO
3，5mM?DTT，0.1％Tween20，pH?9.6)；
The SDS-PAGE result as shown in figure 14, can find out, when Extraction buffer is PBST, the clear resolution of electrophoretic band is high, the extracted amount of cotton total protein is many, and when Extraction buffer is CBS, the protein content of extraction is less, and complicated operation, therefore the Extraction buffer of final definite cotton total protein is PBST.
Two, the SPR of protein chip detects
1, the extraction of testing sample total protein
The Extraction buffer PBST buffer that adopts above-mentioned one method and optimize extracts the DNA of wild type cotton DP5415, transgene cotton Nucotn33B (purchased from Meng Shan Far East company limited) and the total protein of a kind of cotton to be detected (from Hebei province's Langfang City field acquisition), the DP5415 total protein, Nucotn33B total protein, the cotton total protein to be detected that obtain are analyzed with SDS-PAGE, result as shown in figure 15, can find out, the cotton total protein all proposes, can be used for SPR and detects.
2, the SPR of protein chip detects
With the flow velocity of 30 μ l/min successively to passing into testing sample (detection method is with above-mentioned) in the protein chip obtained by embodiment 1: the total protein of wild type cotton DP5415 total protein, transgene cotton Nucotn33B total protein and unknown cotton to be detected, sample size is 60 μ l, the NaOH regeneration chip of the complete rear use 10 μ l 50mM of each sample detection is tested at least triplicate at every turn.
As shown in figure 16,1 is transgene cotton Nucotn33B to test findings; 2 is unknown cotton to be measured; 3 is wild type cotton DP5415, can find out, response when 1,2,3 samples only pass into damping fluid before loading is 0, and the response after the PBS damping fluid rinses 2min is respectively 142,112,66; Show, in three samples, it is very strong that the antibody of transgene cotton Nucotn33B and cotton to be measured and chip surface is made signal mutually, and there are certain non-specific adsorption in wild type cotton DP5415 protein sample and chip surface, therefore, each experiment with wild type cotton DP5415 in contrast, the response of testing sample is more than or equal to 40RU than wild type, show in this testing sample to contain Cry1Ac albumen, if difference is less than 40RU, in testing sample, do not contain Cry1Ac albumen.
Total protein by Bt-Cry1Ab/1Ac test strips (Agdia) to wild type cotton DP5415 total protein, transgene cotton Nucotn33B total protein and cotton to be detected detects, result as shown in figure 17, the testing result of wild type cotton DP5415 is negative, in sample, there is no Cry1Ac albumen; And the testing result of unknown cotton to be detected and transgene cotton Nucotn33B is positive, in sample, contain Cry1Ac albumen.
This SPR testing result with protein chip is consistent, proves that this method is correctly feasible.
Claims (5)
1. the surface plasmon resonance biosensor for auxiliary detection biology target protein to be measured, be coupled on sensing chip in covalently bound mode for resisting target protein antibody, obtains surface plasmon resonance biosensor;
Described sensing chip is the CM5 chip;
Described target protein is Cry1Ac albumen;
The monoclonal antibody that described anti-target protein antibody is anti-Cry1Ac albumen;
Described Cry1Ac albumen is the protein that the amino acid sequence shown in sequence 2 forms in sequence table.
2. biology sensor according to claim 1 is characterized in that:
Described coupling comprises the steps: the CM5 chip after the flow of solution of the monoclonal antibody of described anti-Cry1Ac albumen is activated, and it is combined with the CM5 chip surface, obtains surface plasmon resonance biosensor:
In order resisting, the monoclonal antibody of Cry1Ac albumen and pH are 4.5 to the solution of the monoclonal antibody of described anti-Cry1Ac albumen, concentration is 10mM NaAc aqueous solution, the solution obtained, the concentration of the monoclonal antibody of described anti-Cry1Ac albumen in the solution of described monoclonal antibody is 50 μ g/ml;
Described flow velocity of flowing through is 5 μ l/min;
The described time of flowing through is 3min;
Described biology is plant.
3. biology sensor according to claim 2, it is characterized in that: described plant is cotton.
4. arbitrary described surface plasmon resonance biosensor application in target protein in the auxiliary detection biology in claim 1-3;
Described biology is plant; Described target protein is Cry1Ac albumen;
Described plant is the plant that proceeds to the encoding gene of described target protein;
Described Cry1Ac albumen is the protein that the amino acid sequence shown in sequence 2 forms in sequence table.
5. application according to claim 4 is characterized in that:
The encoding gene of described Cry1Ac albumen is the DNA molecular shown in sequence 1 in sequence table.
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| CN102901830B (en) * | 2012-10-15 | 2014-11-05 | 浙江大学 | Construction method for high-throughput micro-fluidic chip detecting system |
| CN103175962A (en) * | 2013-03-18 | 2013-06-26 | 桂林理工大学 | Method of determining bacillus thuringiensis toxic protein CrylAc |
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| CN104865225A (en) * | 2015-05-14 | 2015-08-26 | 北京化工大学 | Method for detecting protein content of small rubber particles in rubber plants and method for screening rubber plant lines by using method |
| CN117783538A (en) * | 2023-12-26 | 2024-03-29 | 无锡佰翱得生物科学股份有限公司 | SPR reversible biosensor chip based on IM-CL affinity system and preparation method and application thereof |
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