CN105866414A - Transgenic protein g10-epsps quantitative detection method and used kit - Google Patents

Abstract

The invention discloses a g10-epsps quantitative detection kit, which comprises the following components: antibody pre-coated microplate plate; sample diluent, which is phosphate buffer; washing liquid, which is phosphate buffer containing Tween-20 ; reaction termination solution, H ₂ SO ₄ solution; V, enzyme-labeled antibody, horseradish peroxidase-labeled mouse anti-g10-epsps monoclonal antibody; VI, chromogenic reagent; VII, g10-epsps standard. The present invention also discloses a method for quantitatively detecting transgenic protein g10-epsps in plants using a kit, comprising the following steps: extracting protein from a sample to be tested to obtain a protein extract; using a g10-epsps quantitative detection kit to detect the protein The extract was tested; the concentration of the g10-epsps protein in the sample to be tested was calculated through the concentration-absorbance standard curve prepared with the g10-epsps protein standard.

Description

Quantitative detection method and kit used for transgenic protein g10-epsps

technical field

The invention relates to the technical field of transgenic plant detection, in particular to a method for obtaining and quantitatively detecting transgenic protein EPSPS.

Background technique

The inventor's research group previously studied the herbicide-resistant EPSPS gene cloned from Pseudomonas (g10). EPSP synthase (5-enolpyruvyl-shikimate-3-phosphatesynthase, EPSPS) is a key enzyme in the biosynthesis of aromatic amino acids—tryptophan, tyrosine, and phenylalanine in organisms; glyphosate is passed through Inhibiting the activity of EPSP synthetase to block the synthesis of aromatic amino acids eventually led to the death of the tested plants. However, the activity of EPSP synthase of some bacteria is not inhibited by glyphosate, so the expression of bacterial EPSPS gene can protect plants from herbicide damage. The herbicide resistance gene EPSPS is transferred into soybeans, aiming at cultivating transgenic soybeans with excellent glyphosate resistance characteristics, improving the field weeding effect in soybean production and reducing production costs.

The research group used the herbicide-resistant EPSPS gene cloned from Pseudomonas to construct a plant constitutive expression vector pSOY19, driven by the 35S promoter, which can provide glyphosate resistance to transgenic plants. The pSOY19 plasmid contains only one gene expressible in plant cells: bacterial EPSPS, under the control of the CaMV 35S promoter. The expression plasmid of herbicide resistance gene is 9557bp. Insertion sequence The full length of the EPSPS gene is 1321bp, encoding a polypeptide containing 440 amino acids. Through the Agrobacterium-mediated method, exogenous EPSPS genes have been transformed into soybean Huachun 3 and Jack varieties, and herbicide-resistant transgenic soybean materials with EPSPS genes have been obtained.

At present, the routine detection method of genetically modified proteins is PCR, but the PCR method has relatively high requirements on the site, equipment, and technical personnel, and the sample processing is complicated, so it is not suitable for a large number of screening tests. Some transgenic proteins can already be detected by rapid test strips and ELISA kits, but g10-epsps is a newly introduced transgenic protein, and there is no detection method for this aspect at present.

Contents of the invention

The technical problem to be solved by the present invention is to provide an accurate and reliable quantitative detection method for the transgenic protein g10-epsps and the used kit.

In order to solve the above technical problems, the invention provides a g10-epsps quantitative detection kit, comprising the following components:

Ⅰ. Antibody pre-coated microtiter plate;

Ⅱ. The sample diluent is phosphate buffer;

Ⅲ. The washing liquid is a phosphate buffer containing Tween-20;

Ⅳ. _The reaction termination solution is _H2SO4 solution;

Ⅴ. Enzyme-labeled antibody is a horseradish peroxidase (HRP)-labeled mouse anti-g10-epsps monoclonal antibody;

Ⅵ, chromogenic agent (for TMB chromogenic agent);

VII, g10-epsps standard.

As an improvement of the g10-epsps quantitative detection kit of the present invention: the preparation method of the antibody pre-coated microtiter plate comprises the following steps:

(1) Dilute the specific mouse anti-g10-epsps monoclonal antibody with coating buffer to a concentration of 1.8-2.2 μg/ml, add it to a 96-well microtiter plate (100ul per well), and react at 37°C for 3h or 4h Stand overnight at ℃ (12h);

The coating buffer is a carbonate buffer;

(2) After drying the plate hole solution, add washing solution to wash, and then dry;

For example, it can be specifically: dry the plate well solution, add washing solution, soak for 5-10 minutes, dry the plate well solution, and pat dry on absorbent paper;

(3) Add the blocking solution to a 96-well ELISA plate (150ul per well), and react at 37°C for 2-3 hours;

(4) After drying (for example, drying the plate well solution, patting dry on absorbent paper, and drying with a freeze dryer); obtain an antibody pre-coated ELISA plate.

Remarks: The obtained antibody pre-coated microtiter plates are packed in aluminum foil bags with a vacuum packaging machine.

As a further improvement of the g10-epsps quantitative detection kit of the present invention:

The carbonate buffer is (preferably) Na ₂ CO ₃ -NaHCO ₃ buffer with a concentration of 0.1M and a pH of 9.6;

The coating concentration of the specific mouse anti-g10-epsps monoclonal antibody is (preferably) 1.9-2.1 μg/ml; more preferably 2 μg/ml;

The blocking solution is a carbonate buffer solution containing BSA; in the blocking solution, the concentration of BSA is 1%, and the carbonate buffer solution Na ₂ CO ₃ -NaHCO ₃ buffer solution has a concentration of 0.1M and a pH value of 9.6.

As a further improvement of the g10-epsps quantitative detection kit of the present invention:

The specific mouse anti-g10-epsps monoclonal antibody is obtained by immunizing BALB/c mice with g10-epsps recombinant protein, and then preparing monoclonal antibody cell lines through cell fusion and cloning screening, and then preparing mouse ascites and purifying .

As a further improvement of the g10-epsps quantitative detection kit of the present invention: g10-epsps is obtained by optimizing the g10-epsps gene in G1 and constructing it into an Escherichia coli expression system for recombinant expression and purification.

Further improvement as the g10-epsps quantitative detection kit of the present invention

In component V, the concentration of horseradish peroxidase (HRP)-labeled mouse anti-g10-epsps monoclonal antibody is 8-12 μg/ml (preferably 9-11 μg/ml, more preferably 10 μg/ml).

Remarks: The preparation method of horseradish peroxidase (HRP)-labeled mouse anti-g10-epsps monoclonal antibody is as follows:

1. Dissolve 5mg of HRP in pure water, add sodium periodate, and react at room temperature for 30 minutes;

2. Dialyze 5 mg of antibody against coupling buffer overnight;

3. Add HRP to the antibody solution and react at room temperature for 2 hours;

4. Add sodium borohydride to block the reaction site;

5. Dialyze with PBS overnight, add an equal amount of glycerol and store at -20°C.

As a further improvement of the g10-epsps quantitative detection kit of the present invention:

As the H ₂ SO ₄ solution of component IV, the concentration of sulfuric acid is 1.8-2.2M (preferably 1.9-2.1M, more preferably 2M).

The present invention also provides a method for quantitatively detecting transgenic protein g10-epsps in plants using the above kit, comprising the following steps:

(1) Extract protein from the sample to be tested to obtain protein extract;

(2) Use the g10-epsps quantitative detection kit to detect the protein extract, and the detection process includes sample addition, incubation, washing, enzyme addition, incubation, washing, color development, termination and reading;

(3) Calculate the g10-epsps protein concentration in the sample to be tested by using the concentration-absorbance standard curve prepared with the g10-epsps protein standard.

As the improvement of the method for the quantitative detection of transgenic protein g10-epsps in plants of the present invention:

After adding the HRP-labeled anti-g10-epsps protein polyclonal antibody, the incubation temperature is 22-26° C. (preferably 25° C.), and the incubation time is 40-50 minutes (preferably 45 minutes).

In the present invention, the mouse anti-EPSPS used is obtained by immunizing BALB/c mice with EPSPS recombinant protein, then preparing a monoclonal antibody cell line through cell fusion, cloning and screening, and then preparing mouse ascites and purifying it. The EPSPS standard product is obtained by optimizing the EPSPS gene in G1, constructing it into an E. coli expression system, and obtaining it through recombinant expression and purification.

The present invention uses self-developed g10-epsps monoclonal antibody to prepare a g10-epsps quantitative detection kit, and uses double-antibody sandwich ELISA to detect protein content in transgenic materials.

The quantitative detection method of the transgenic protein EPSPS provided by the invention is accurate and reliable, and has the advantages of short operation time and low cost compared with the prior art.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is that SDS-PAGE detects the expression of g10-epsps protein;

1: before induction; 2: after induction (about 45KD).

Fig. 2 is the expression form of g10-epsps protein detected by SDS-PAGE;

1: supernatant; 2: inclusion body; 3, whole bacteria.

Fig. 3 is the purification effect of SDS-PAGE detection g10-epsps protein;

1: 250mM imidazole eluate; 2: 150mM imidazole eluate;

3: 100 mM imidazole eluate; 4: 50 mM imidazole eluate.

Fig. 4 is the dialysis effect of SDS-PAGE detection g10-epsps protein;

1: 50mM imidazole eluent.

Figure 5 is a standard curve for detecting EPSPS using the method of the present invention.

detailed description

The invention is described in more detail below by way of example.

Embodiment 1, expression and purification of EPSPS protein

(1) Small-scale expression of protein

1. Carrier construction

According to the protein sequence (g10-epsps), the gene sequence was synthesized and constructed into the vector pET30a to obtain the pET30a-EPSPS positive plasmid.

2. Strain activation: The g10-epsps-E positive plasmid was constructed using the commercial ET expression vector backbone sold by EMD Biosciences, transformed into BL21(DE3), and coated with LB solid medium (Kana concentration 50 μg/mL). On the next day, single-clonal colonies were picked and inserted into 5 mL of LB liquid medium (Kana concentration 50 μg/mL), and cultured at 37°C for 12h-14h.

3. Small test expression: the next day, the bacteria were inserted into 5mL LB liquid medium (Kana concentration 50μg/mL) at 1:50 (v:v), cultivated at 37°C until OD=0.4-0.6, and absorbed 1mL of the bacteria solution After centrifugation, it was used as the control before induction. Add 0.8mM IPTG to 4mL of bacterial liquid, induce expression at 25°C for 6h, and then centrifuge at 8000rpm and 4°C for 1min to collect the bacterial cells. The form of the protein was identified by SDS-PAGE, and the results showed (Figure 1) that there was obvious expression of the target protein.

4. Identification of the expression form of the protein: add 1 mL of disrupting solution to the expressed cells (ie, the cells collected in the above steps) for ultrasonic lysis. Cracking conditions: temperature ice bath, power 240W, ultrasonic 2s, interval 2s, time 30min. The obtained whole bacteria were centrifuged at 12,000 rpm and 4° C. for 1 min, and the supernatant and inclusion bodies were collected. SDS-PAGE identified the expression form of the protein, and the results showed (Figure 2) that the target protein was mainly expressed in a soluble form.

"2: Inclusion body" in Figure 2 refers to the above precipitate; "3: Whole bacteria" refers to the above broken product before centrifugation.

(2) Mass expression and purification of protein

1. Strain activation: pick pET30a-EPSPS monoclonal colony from a solid plate and insert it into 5 mL of LB liquid medium (Kana concentration 50 μg/mL), culture at 37°C for 12h-14h.

2. Small test expression: the next day, the bacteria were inserted into 800mL LB liquid medium (Kana concentration 50μg/mL) at 1:50 (v:v), cultured at 37°C until OD=0.4-0.6, and the concentration was 0.8 mM IPTG, induced expression at 25°C for 6h, and centrifuged at 8000rpm at 4°C for 15min to collect the cells.

3. Strain cracking: add 100mL disrupting solution for ultrasonic cracking. Cracking conditions: temperature ice bath, power 240W, ultrasound 2s, interval 2s, time 15min. Centrifuge at 12000rpm at 4°C for 15min, and collect the supernatant.

4. Purification of the supernatant: the collected supernatant was purified using a high-affinity NI resin (ie, Ni-NTA affinity chromatography resin (nickel column)), and eluted with 50mM, 100mM, 150mM, and 200mM imidazole respectively;

Collect the flow-through and eluate. The purification effect was detected by SDS-PAGE, and the results showed (Fig. 3) that the protein purity was the best when 50mM imidazole was eluted. The imidazole in the eluate was removed by dialysis with 50 mM Tris-HCl buffer solution at pH=8.0, and the effect of dialysis was detected by SDS-PAGE. The results showed ( FIG. 4 ) that the purity and concentration of the protein after dialysis were feasible. After testing, the final target protein purity was greater than 90%, the concentration was 2 mg/mL, and the protein amount was 10 mg.

(3) Results

The conditions for inducing expression of pET30a-EPSPS protein were: IPTG concentration 0.8mM, induction temperature 25°C, induction time 6h. The protein is mainly expressed in the supernatant, and the supernatant is purified by NI column and dialyzed to remove imidazole, and finally the g10-epsps protein is obtained: the concentration is 2 mg/mL, the purity is greater than 90%, and the protein is 10 mg.

Embodiment 2, antibody preparation

(1) Monoclonal antibody preparation:

1. Immunogen preparation: Mix and emulsify the expressed and purified protein with an equal volume of Freund's adjuvant and YOULONG adjuvant into a water-in-oil state for immunization of mice.

2. Immunization strategy: immunize 4 Balb/c mice with the protein, subcutaneously immunize 3 times with an interval of 4 weeks, and finally detect by ELISA, the antiserum titers are as follows:

3. Cell fusion: Two weeks after the last immunization, the antigen (specifically expressed and purified protein of g10-epsps protein) was injected intraperitoneally for booster immunization, and cell fusion was performed three days later. The mice were killed by neck dislocation, sterilized by soaking in 70% ethanol for 30 minutes, and the abdominal cavity was cut open on an ultra-clean table, the spleen was taken out, ground, passed through an 80-mesh sieve, and splenocytes were obtained, and SP2/0 myeloma cells were added to the effect of PEG4000 for cell fusion.

4. Fusion screening: spread the fused cells into a 96-well plate, culture with HAT medium, change the medium after three days, and use HT medium for culture. After 10 days, the cell culture supernatant was taken for detection. The indirect ELISA method was used for screening, and the wells with OD value > 2.1 times of the negative control wells were judged as positive wells.

The indirect ELISA method mainly includes the following steps:

1) Dilute the expressed and purified protein to 1ug/ml with 0.1M, pH=9.6 carbonate buffer, add to a 96-well ELISA plate, 100ul per well, react at 37°C for 3h or stand overnight at 4°C;

2) Shake off the liquid in the plate wells, add 250ul washing buffer, let stand for 30s, shake off the liquid in the plate, repeat 3 times;

3) Add test samples, 100ul per well, and add positive control (positive mouse serum taken in 2), negative control (pre-immunization mouse serum) and blank control (no mouse serum) at 37°C for 45min;

4), repeat step 2);

5) Add HRP-labeled goat anti-mouse enzyme-labeled secondary antibody, 100ul per well, and react at 37°C for 45min.

5. Cloning and strain establishment: Use the limiting dilution method to clone the positive wells, detect them after 10 days, and continue to clone the positive clones by the limiting dilution method until the obtained clones are all positive, and a positive cell line can be established. Finally, 5 positive cell lines were obtained.

6. Expansion culture: The monoclonal cells of the established strain were expanded and cultured, and frozen in liquid nitrogen.

(2) Preparation and purification of ascites

1. Preparation of ascites: Inject mineral oil into the peritoneal cavity of mice one week in advance, inject a certain number (about 10 ⁶ ) of cells into the peritoneal cavity of mice, collect ascites about 10 days, centrifuge at 4000 rpm, and obtain the supernatant as monoclonal antibody ascites.

2. Monoclonal antibody purification: centrifuge ascitic fluid for 15min (4000rpm, room temperature), take 10ml of supernatant, slowly add saturated ammonium sulfate dropwise under stirring at 4°C to half saturation, continue stirring for 30min, centrifuge for 30min (13000rpm, 4°C), Discard the supernatant; dissolve the precipitate in about 10ml PBS (0.01M, pH7.4); slowly add saturated ammonium sulfate to 33% drop by drop under stirring at 4°C, continue stirring for 30min, centrifuge for 30min (13000rpm, 4°C), discard the supernatant The precipitate was dissolved in an appropriate amount of 10ml of PBS (0.01M, pH7.4), dialyzed at 4°C overnight, the antibody content was determined, and stored at -20°C for later use. After the ammonium sulfate precipitation, continue to use the Protein G column for purification. The new column is first passed through the column with 5ml ultrapure water, and then 5ml of 0.4M PB buffer (pH 7.0) is used to equilibrate the purification column; column, in order to better bind the antibody protein to the binding site; continue to equilibrate the purification column with 10ml 0.4M PB buffer (pH 7.0); elute the binding site with 5ml 0.1M glycine-hydrochloric acid buffer (pH 2.7) Antibody, and 0.5ml of 1M Tris-HCl (pH 8.0) was added to the eluent to neutralize glycine to keep the pH neutral for antibody storage.

Thus, the mouse anti-g10-epsps monoclonal antibody was obtained.

Embodiment 3, the assembly of kit of the present invention:

1. Preparation of buffer

(1) Coating buffer, 0.1M carbonate buffer CB (Na ₂ CO ₃ -NaHCO ₃ buffer), pH 9.6;

(2) diluent, 0.01mM phosphate buffer saline PBS, pH value is 7.4;

(3) washing liquid, PBS containing 0.2% Tween-20;

That is, add 0.2 g of Tween-20 to 100 ml of PBS, the PBS is 0.01 mM phosphate buffer saline PBS, the pH value is 7.4;

(4) Blocking solution, CB containing 1% BSA;

That is, add 1 g of BSA to 100 ml of CB, which is 0.1 M Na ₂ CO ₃ -NaHCO ₃ buffer solution with a pH value of 9.6;

(5) Stop solution, 2M H ₂ SO ₄ .

2. Preparation of ELISA plate

(1) Coated

Pipette a certain amount of EPSPS antibody (mouse anti-g10-epsps monoclonal antibody obtained in Example 2) into the required volume of coating buffer to make the concentration about 2 μg/mL, and configure it as a coating working solution. Add 100 μL of the coating working solution to the microwells of the microplate, and let stand at 4°C overnight or react at 37°C for 3 hours (note that water evaporation should be prevented).

(2) closed

Add 1% BSA to CB buffer solution (pH9.6) to prepare a blocking working solution (blocking solution).

Use the washing solution to blot the microplate plate dry with a plate washer-wash twice (soak for 5-10 minutes during washing), and pat the strip dry on clean absorbent paper.

Add 150 μL of blocking working solution to each well and bake at 37°C for 3 hours.

(3) Drain and seal

Discard the blocking solution, pat dry the strips on clean absorbent paper, put them in a freeze-vacuum dryer for 3 hours, and heat-seal them in vacuum.

3. Preparation of EPSPS standard freeze-dried powder

Prokaryotic expression in Escherichia coli E.coli and His-EPSPS (gained in Example 2) by affinity purification, N-terminus has 6 His tags, its mother solution concentration is 3mg/ml, is diluted to 900ng/ml with diluent, Add 100 μL of 900ng/ml standard substance into a 3mL brown glass bottle, and dry it in a freeze dryer overnight to obtain the freeze-dried powder of the standard substance, which is the EPSPS standard substance (g10-epsps standard substance) of component VII.

4. Configuration of enzyme-labeled antibody working solution

Preparation of enzyme-labeled antibody: Take the purified specific rabbit polyclonal antibody and couple it with HRP to obtain enzyme-labeled antibody.

Take a certain amount of enzyme-labeled antibody and add it to the diluent, mix thoroughly to make the final concentration 2 μg/mL, and store it in the dark at 2-8°C; it is used as the enzyme-labeled antibody of component V.

That is, the enzyme-labeled antibody is a mouse anti-g10-epsps monoclonal antibody labeled with horseradish peroxidase (HRP).

5. The sample diluent is phosphate buffered saline.

6. Assembling the kit

name quantity pre-coated microtiter plate 1 block Standard freeze-dried powder 2 bottles Enzyme-labeled antibody working solution 1 bottle sample diluent 2 bottles Reagent 1 bottle Terminator 1 bottle detergent 1 bottle manual 1 copy

The color developer is a TMB color developer.

Embodiment 4, use kit of the present invention to detect the transgenic protein EPSPS in the plant

(1) Prepare standards and samples of unknown concentration

1) Preparation of standard products:

Dissolve the freeze-dried powder of EPSPS standard with 1mL diluent, the concentration of this solution is 2ppb;

Take 300 μL of 2ppb EPSPS standard, add TBA buffer (0.1M Tris, 0.1M Na ₂ B ₄ O ₇ 10H ₂ O, 0.01M MgCl ₂ , 0.05% (v/v) Tween-20, pH 7.8) 300 μL, The concentration of this solution is 1ppb;

Take 300 μL of 1 ppb EPSPS standard substance, add 300 μL of TBA buffer solution, the concentration of this solution is 0.5 ppb;

Take 300μL of 0.5ppb EPSPS standard substance, add 300μL of TBA buffer solution, the concentration of this solution is 0.25ppb;

Take 300μL of 0.25ppb EPSPS standard substance, add 300μL of TBA buffer solution, the concentration of this solution is 0.125ppb;

2) Processing of unknown concentration samples:

(1) Leaf pretreatment method: Take a ^5-10mm2 leaf sample, put it into a 1.5mL centrifuge tube and mash it, add 250μL of TBA buffer, shake and mix for 5 minutes, centrifuge at 4000rpm for 3 minutes, take 100μL of the supernatant for use for analysis.

Seed pre-treatment method: Take 0.1g crushed seed sample, put it into a 1.5mL centrifuge tube, add 1mL TBA buffer, shake and mix for 5 minutes, centrifuge at 4000rpm for 3 minutes, take 100μL supernatant for analysis.

Bulk grain pretreatment method: Take 0.1g of crushed bulk grain sample, put it into a 1.5mL centrifuge tube, add 1mL of TBA buffer, shake and mix for 5 minutes, centrifuge at 4000rpm for 3 minutes, and take 100μL of supernatant for analysis.

(2) Add standard or sample with unknown concentration:

Add 100 μL of TBA buffer solution (blank control)/standard product/sample of unknown concentration into corresponding microwells, shake gently to mix, and react in a dark environment at 25°C for 45 minutes.

(3) Plate washing:

Shake the liquid in the wells to dry, and use 250 μL of washing solution/well for each washing, wash thoroughly for 3 times with an interval of 1 min between each time, and pat dry with absorbent paper.

(4) Add enzyme-labeled antibody:

Add 100 μL/well of the enzyme-labeled antibody, shake gently to mix, react in a dark environment at 25°C for 45 minutes, take out and repeat step (3) to wash the plate.

(5) Color rendering:

Add 100 μL/well of chromogen, and place in a dark environment at 25°C for 15 minutes to react.

(6) Termination and determination:

Add 100 μL of stop solution/well, shake gently to mix, set the microplate reader to 450nm, and measure the OD value of each well (preferably use dual wavelength 450/630nm for detection, and read the data within 5min).

(7) Calculate:

Using statistical drawing software, draw a four-parameter fitting standard curve according to the OD value of the determined standard substance, as shown in Figure 5; the concentration of EPSPS in the unknown sample can be calculated by the standard curve and the OD value of the unknown concentration sample.

(8) Results: as shown in Table 1.

Table 1. Detection effect of spiked samples

As can be seen from the results in Table 1, using the method of the present invention to detect the transgenic protein g10-epsps in plants, its standard addition recovery rate is between 95%-120%, illustrating: the detection of the transgenic protein g10-epsps in plants of the present invention The method has better accuracy.

Embodiment 5, using the kit of the present invention to detect transgenic protein EPSPS in plants

1. Use the g10-epsps quantitative detection kit to detect the expression of EPSPS in transgenic soybeans;

Transgenic plants are endowed with herbicide resistance through the EPSPS gene, which can be tested with glyphosate; direct spraying of 200ml/acre glyphosate can kill all non-transgenic soybeans.

The top leaves of the positive lines screened by transgenic soybean herbicides were taken, and the expression level of the inserted sequence (bacterial EPSPS) in the transgenic positive plants was detected with the g10-epsps quantitative detection kit.

1.1 Transgenic soybean research materials: roots, stems, and leaf tissues of two transgenic soybean lines in the V1 stage (2-3 triple compound leaves) in Anhui experimental field.

1.2 Quantitative detection research results

Two transgenic soybean lines in three repeated experimental fields in Datian, Anhui Province, line 1 (L1), line 2 (L2) V1 stage (2-3 three-fold compound leaf development stage), top three compound leaves, stems, Roots, 100mg of fresh weight materials were taken, and the g10-epsps expression level was detected with the g10-epsps quantitative detection kit.

1.2.1 Total protein extraction of various tissues and organs

a. Fresh tissue: Weigh 100mg of fresh tissue with 1ml of TBA buffer (0.1M Tris, 0.1MNa ₂ B ₄ O ₇ ·10H ₂ O, 0.01M MgCl ₂ , 0.05% (v/v) Tween-20at pH 7.8 and 0.2% (w/v) L-ascorbic acid) were ground, filtered through a mirocloth filter membrane (Fisher Scientific, Pittsburgh, PA) and the supernatant was taken. Centrifuge at 12000rpm, and take the supernatant as soybean fresh tissue protein extract for later use.

b. Dry grains: take 100 mg of dry grain powder, add 1 ml of TBA buffer solution, centrifuge at 12000 rpm, and take the supernatant as soybean dry grain protein extract for later use.

G10-epsps quantitative detection kit was used to detect the protein content in the samples.

The above fresh tissue and dry grain protein extract samples were diluted in gradients of 10 ² , 10 ³ , and 10 ⁴ , and the optimal dilution was selected for quantitative analysis according to steps (2) to (6) in Example 4.

c. The peak value of EPSPS protein absorption is read in μg/g fresh weight (FW), and finally the arithmetic mean and standard deviation (SD) of EPSPS protein content in each tissue of each test point are calculated.

1.2.2 Quantitative test results

Table 2. Expression levels of EPSPS in leaves, stems, roots, and grains of transgenic soybean line 1 (L1)

Table 3, Transgenic soybean line 2 (L2) leaf, stem, root, grain EPSPS expression level

The applicant declares that the present invention is illustrated by the above-mentioned embodiments, but the present invention is not limited to the above-mentioned, that is, it does not mean that the present invention must rely on the above-mentioned to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (9)

1.g10-epsps quantitative detection kit is characterized in that comprising following components: Ⅰ. Antibody pre-coated microtiter plate; Ⅱ. The sample diluent is phosphate buffer; Ⅲ. The washing liquid is a phosphate buffer containing Tween-20; Ⅳ. _The reaction termination solution is _H2SO4 solution; Ⅴ. Enzyme-labeled antibody is a mouse anti-g10-epsps monoclonal antibody labeled with horseradish peroxidase; Ⅵ. Chromogenic agent; VII, g10-epsps standard. 2. the g10-epsps quantitative detection kit according to claim 1, is characterized in that: the preparation method of described antibody pre-coated microplate comprises the steps: (1) Dilute the specific mouse anti-g10-epsps monoclonal antibody with coating buffer to a concentration of 1.8-2.2 μg/ml, add it to a 96-well microtiter plate, react at 37°C for 3 hours or stand overnight at 4°C; The coating buffer is a carbonate buffer; (2) After drying the plate hole solution, add washing solution to wash, and then dry; (3) Add the blocking solution to the 96-well microtiter plate, and react at 37°C for 2-3 hours; (4) After drying, the antibody pre-coated microtiter plate was obtained. 3. g10-epsps quantitative detection kit according to claim 2, is characterized in that: The carbonate buffer solution is Na ₂ CO ₃ -NaHCO ₃ buffer solution with a concentration of 0.1M and a pH value of 9.6; The coating concentration of the specific mouse anti-g10-epsps monoclonal antibody is 1.9-2.1 μg/ml; The blocking solution is a carbonate buffer solution containing BSA; in the blocking solution, the concentration of BSA is 1%, and the carbonate buffer solution Na ₂ CO ₃ -NaHCO ₃ buffer solution has a concentration of 0.1M and a pH value of 9.6. 4. the g10-epsps quantitative detection kit according to claim 2 or 3, is characterized in that: The specific mouse anti-g10-epsps monoclonal antibody is obtained by immunizing BALB/c mice with g10-epsps recombinant protein, and then preparing monoclonal antibody cell lines through cell fusion and cloning screening, and then preparing mouse ascites and purifying . 5. The g10-epsps quantitative detection kit according to claim 4, characterized in that: g10-epsps is obtained by optimizing the g10-epsps gene in G1 and constructing it into an Escherichia coli expression system for recombinant expression and purification. 6. g10-epsps quantitative detection kit according to claim 1, is characterized in that: In component V, the concentration of horseradish peroxidase-labeled mouse anti-g10-epsps monoclonal antibody is 8-12 μg/ml. 7. g10-epsps quantitative detection kit according to claim 1, is characterized in that: As the H ₂ SO ₄ solution of component IV, the concentration of sulfuric acid is 1.8-2.2M. 8. The method for the quantitative detection of transgenic protein g10-epsps in plants utilizing the kit described in claims 1 to 7, is characterized in that it comprises the following steps: (1) Extract protein from the sample to be tested to obtain protein extract; (2) detect the protein extract with the g10-epsps quantitative detection kit; (3) Calculate the g10-epsps protein concentration in the sample to be tested by using the concentration-absorbance standard curve prepared with the g10-epsps protein standard. 9. the method for quantitative detection plant transgenic protein g10-epsps according to claim 8, is characterized in that: After adding the HRP-labeled anti-g10-epsps protein polyclonal antibody, the incubation temperature is 22-26° C., and the incubation time is 40-50 minutes.