CN113109562B - ELISA quantitative detection method of exogenous EPSPS protein in plant - Google Patents

Abstract

The invention discloses an ELISA quantitative detection method of exogenous EPSPS protein in plants, relating to the technical field of plant molecular detection and comprising the following steps: (1) preparing a polyclonal antibody; (2) preparing plant tissue extract; (3) adding the plant tissue extracting solution in the step (2) into a micropore plate for coating for 12-48h, then pouring out the coating solution, cleaning the micropore plate by using a PBST solution, and adding a PBS-millik solution for sealing; (4) and (3) cleaning the microporous plate after sealing, adding primary antibody working solution for incubation, then cleaning, adding secondary antibody working solution for incubation, adding substrate solution, terminating the reaction, measuring the absorbance value, and substituting the absorbance value into a standard curve to obtain the EPSPS protein content. The invention has the advantages that: the novel herbicide-resistant protein EPSPS is quantitatively detected, the used consumables are simple and easy to obtain, the used reagent does not need a complex preparation process, the cost is low, the implementation is easy, and the universality is realized.

Description

ELISA quantitative detection method of exogenous EPSPS protein in plant

Technical Field

The invention relates to the technical field of plant molecular detection, in particular to an ELISA quantitative detection method for exogenous EPSPS protein in plants.

Background

Glyphosate is one of the most widely used broad-spectrum herbicides in modern agriculture, an EPSPS gene is one of the most widely used herbicide-resistant genes, and the glyphosate resistance of crops is mainly controlled by an EPSPS mutant gene.

During the growth period of the soybeans and the rapes, the climate and the field environment are very suitable for the growth of farmland weeds, and the weeds and the soybeans compete for moisture, nutrients and sunlight to cause the reduction of the yield and the quality of the soybeans, so that the weeds are one of the main factors influencing the yield of the soybeans. The glyphosate is widely applied in China as a herbicide which has broad spectrum and high efficiency, low toxicity to animals, lower cost and capability of being degraded by soil microorganisms. The glyphosate and herbicide-resistant soybean varieties are applied together, and the method is the most direct and effective method for improving the yield and the quality of Chinese soybeans.

The metabolic pathway of shikimic acid in green plants is an important process in plant growth and development, and the metabolic process finally produces aromatic amino acid substances. The herbicide glyphosate finally influences the normal metabolism of protein in plants by inhibiting the metabolic pathway of shikimic acid in the plants, so that the plants die and the weeding purpose is achieved. The glyphosate-resistant plant can catalyze the metabolic step inhibited by the glyphosate due to the fact that the glyphosate-resistant EPSPS enzyme is contained, so that the normal operation of the shikimic acid metabolic pathway is ensured, and the normal growth of the plant is not influenced by the glyphosate.

The existing transgenic product has a large planting area, the aim of soybean transgenic is to improve the quality on one hand, and the aim of improving the stress resistance characteristics of soybean such as disease resistance, insect resistance, herbicide resistance and the like on the other hand, and common transgenic soybean exogenous genes comprise Vip series protein, pat genes, EPSPS herbicide resistance genes and the like. The research and application range of the herbicide-tolerant gene herbicide soybean is the most extensive and common.

The transgenic rape is the second largest transgenic agricultural product imported in China. The transgenic rape accounts for a considerable proportion in the global rape planting. The currently opened exogenous gene of rape is mostly herbicide-resistant and insect-resistant gene except the gene for improving the grease characteristic. As the number and variety of transgenic plants grown increases year by year in many countries, the impact of transgenic plants on human health and the environment is of increasing concern. Therefore, quantitative techniques for detecting transgenic proteins in transgenic plants are becoming one of the hot spots in research. For example, the patent publication No. CN103698418A discloses a quantitative detection method of transgenic protein CP4-EPSPS in plants, which is used for quantitatively detecting CP4-EPSPS gene in the prior art.

Patent application publication No. CN 110699374A discloses a method for breeding glyphosate-resistant rape, which discloses applying 5-enolpyruvylshikimate-3-phosphate synthase gene in CN103834674A patent to the breeding of rape, and patent application publication No. CN112322631A discloses applying 5-enolpyruvylshikimate-3-phosphate synthase gene in CN103834674 patent to the breeding of soybean. The rape and the soybean can both keep the corresponding glyphosate-resistant herbicide characteristics, the position of the gene I.variabilis-EPSPS in the rape genome is different from that of any other glyphosate-resistant rape containing similar genes, and the gene I.variabilis-EPSPS is environment-friendly and has wide application prospect. The EPSPS protein involved in the plant is an improved EPSPS protein, although the function of the EPSPS protein is not changed, the sequence of the EPSPS protein is improved, and the expression quantity of the EPSPS protein in soybean and rape tissues cannot be detected by the original common EPSPS protein detection method.

Disclosure of Invention

The invention aims to provide a quantitative detection method for a novel herbicide-resistant protein EPSPS.

The invention solves the technical problems through the following technical means:

an ELISA quantitative detection method of exogenous EPSPS protein in plants comprises the following steps:

(1) preparation of polyclonal antibody: the peptide as shown in SEQ ID NO: injecting the EPSPS protein with the sequence shown in 1 into an immune rabbit for immunization, and then carrying out blood sampling and purification to obtain a polyclonal antibody;

(2) preparing a plant tissue extracting solution: grinding the plant tissue by using liquid nitrogen, leaching by using a PBS solution, centrifuging to obtain a plant tissue extracting solution, and heating the plant tissue extracting solution at the temperature of 100-105 ℃ for 1-3 min;

(3) adding the plant tissue extracting solution in the step (2) into a micropore plate for coating for 12-48h, then pouring out the coating solution, cleaning the micropore plate by using a PBST solution, and adding a PBS-millik solution for sealing;

(4) and (3) cleaning the microporous plate after sealing, adding primary antibody working solution for incubation, cleaning, adding secondary antibody working solution for incubation, fully cleaning, adding substrate solution, terminating the reaction, measuring the absorbance value, and substituting the absorbance value into a standard curve to obtain the EPSPS protein content.

Has the advantages that: the method can be used for quantitatively detecting the novel herbicide-resistant protein EPSPS, the used consumables are simple and easy to obtain, the used reagent does not need a complex preparation process, an additional protein extraction kit is not needed, the cost is low, the implementation is easy, and the universality is realized.

The invention uses the direct method to detect and optimize the coating time, so that the target protein in the plant tissue is adsorbed in the microporous plate to the maximum extent, the detection value is closer to the real value compared with the indirect method, and the detection limit is as low as 20 ng/mL.

The stress-resistant plant tissue extracting solution contains more peroxidases which often interact with a TMB substrate in the process of protein detection to cause nonspecific color development, so that the final result is influenced.

Preferably, the sequence of the EPSPS protein is synthesized by the upstream primer and the downstream primer which are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

Preferably, the plant tissue extract in step (2) is heated at 105 deg.C for 1 min.

Preferably, the pH value of the PBS solution in the step (2) is 7.4.

Preferably, the PBST solution in step (3) comprises a PBS solution and tween-20, and the added volume of tween-20 is 0.05% of the volume of the PBS solution.

Preferably, the PBS-mik solution in step (3) comprises a PBS solution and skimmed milk powder, wherein the skimmed milk powder is dissolved in the PBS solution at a volume ratio of 5%.

Preferably, the coating time in the step (3) is 12 h.

Preferably, the coating solution used for coating in step (3) is CBS coating solution or PBS solution.

Preferably, the primary antibody in the step (4) is a polyclonal antibody, and the secondary antibody is horseradish peroxidase-labeled mouse anti-rabbit IgG.

Preferably, the reaction time in the step (4) is 30min, and the stop solution is 2M diluted hydrochloric acid.

Preferably, the step (4) specifically comprises: and (3) cleaning the microporous plate after sealing, adding 100 mu L of primary antibody working solution for incubation, cleaning, adding 100 mu L of secondary antibody working solution for incubation, fully cleaning, adding 100 mu L of TMB substrate solution, reacting at room temperature for 30min, adding 50 mu L of 2M dilute hydrochloric acid to stop reaction, measuring the absorbance value, and substituting the absorbance value into a standard curve to obtain the EPSPS protein content.

Preferably, the determination of the standard curve comprises the steps of: the peptide as shown in SEQ ID NO: 1 to 1 mu g/mL, then diluting step by step to obtain 100 mu L of each of 300ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 10ng/mL and 0ng/mL, incubating the EPSPS protein solution overnight, adsorbing the solution on the surface of an ELISA plate, adding a primary antibody working solution, a secondary antibody working solution and a substrate in sequence, and determining the OD450 value.

Preferably, the plant is soybean or canola.

The invention has the advantages that: the method can be used for quantitatively detecting the novel herbicide-resistant protein EPSPS, the used consumables are simple and easy to obtain, the used reagent does not need a complex preparation process, an additional protein extraction kit is not needed, the cost is low, the implementation is easy, and the universality is realized.

The invention uses the direct method to detect and optimize the coating time, so that the target protein in the plant tissue is adsorbed in the microporous plate to the maximum extent, the detection value is closer to the real value compared with the indirect method, and the detection limit is as low as 20 ng/mL.

The stress-resistant plant tissue extracting solution contains more peroxidases which often interact with a TMB substrate in the process of protein detection to cause nonspecific color development, so that the final result is influenced.

The present invention uses a 96-well plate to remove the standard curve and about 90 samples can be detected. For a total of about 110 units per plate. Wherein the ELISA plate is about 12 yuan, the standard protein is about 10 yuan, the antibody is about 5 yuan, the PBS is about 5-8 yuan, the PBST is about 5 yuan, the skim milk powder is about 0.5 yuan, the substrate is about 50 yuan, and the stop solution is about 20 yuan.

Compared with the kit, the cost can be greatly saved, the price of the kit is about 1800 + 2500, and most of the kits are non-quantitative or semi-quantitative.

Compared with the test strip, the test strip can only be qualitative, the cost of each sample is about 10 yuan, and the cost of 90 samples is about 900 yuan.

Drawings

FIG. 1 is a graph showing the results of expression identification of a protein in a small assay in example 1 of the present invention; in the figure, M is a protein Marker; 1, before induction; 2, after induction.

FIG. 2 is a graph showing the results of expression identification of a protein in a small assay in example 1 of the present invention; in the figure, M is a protein Marker; 1, supernatant fluid; 2, precipitating; and 2, solubilizing.

FIG. 3 is a graph showing the results of protein purification in example 1 of the present invention; in the figure, M is a protein Marker; 1, discharging liquid; 2 represents 50mM imidazole eluate; 3 represents 100mM imidazole eluate; 4 represents 200mM imidazole eluate; 5 represents 500mM imidazole eluate.

FIG. 4 is a diagram showing the identification of a pure protein in example 1 of the present invention; in the figure, M is a protein Marker; 1, pure protein.

Fig. 5 is a diagram of an experimental chart for optimizing the standard curve range in embodiment 2 of the present invention.

FIG. 6 is a standard curve in example 2 of the present invention.

FIG. 7 shows the principle of ELISA detection system.

FIG. 8 is a graph showing the effect of removing the background color of the extract solution of soybean tissue according to example 5 of the present invention.

FIG. 9 is a graph showing the effect of removing background color of the extract solution of Chinese cabbage tissue in example 6 of the present invention.

Fig. 10 is a diagram illustrating the packet coating time optimization effect in embodiment 7 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.

PBS Phosphate buffer (Phosphate buffer saline) containing 0.1mM Na2HPO4, 1.9mM NaH per liter₂PO₄And 145mM NaCl to pH7.4, PBS concentration of 1 mM.

PBST: namely Phosphate Tween buffer (Phosphate buffer saline-TWEEN20), 0.05% by volume of TWEEN20 was added to the PBS solution. PBS-milk: and (3) dissolving the skimmed milk powder in PBS (phosphate buffer solution) according to the volume ratio of 5%, and uniformly mixing to prepare the instant milk powder.

Example 1

Preparation of antigens

1. Construction of prokaryotic expression vector

Designing primers according to a target gene (5-enolpyruvylshikimate-3-phosphate synthase) sequence disclosed in a patent with the publication number of CN103834674A, wherein the upstream and downstream primers are respectively shown in SEQ ID NO: 2 and SEQ ID NO: 3, the EPSPS protein sequence is shown as SEQ ID NO: 1 is shown. And (3) carrying out PCR amplification on a target fragment, carrying out enzyme digestion, and connecting to a pET28a vector to obtain a recombinant expression vector.

2. Small test expression

1) Plasmid transformation and strain preservation: the recombinant vector is transformed into Escherichia coli BL21(B) E.coli BL21 Origami B, spread on LB solid medium containing 50mg/L antibiotic Kan, and cultured at 37 ℃ for 12-16 h. Selecting single colony, culturing in LB liquid culture medium containing 50mg/L Kan for 12-16h, and storing glycerol strain at-80 deg.C.

2) Activating strains: 100 mu L of transformed Escherichia coli BL21(B) strain is inoculated into liquid culture medium containing 50mg/LKAn, and cultured and activated at 37 ℃ overnight.

3) Inducing expression: the activated strain was identified as 1: 5mL of the suspension was subjected to 50-scale culture at 37 ℃ until OD600 became 0.4 to 0.6, and 2.5mL of the suspension was collected and treated as a pre-induction control. 1.0mM IPTG was added to the remaining 2.5mL of the bacterial solution, and the cells were induced at 37 ℃ for 4 hours, and then the cells collected by centrifugation were treated as post-induction samples, and SDS-PAGE was used to identify the expression of the protein. As shown in FIG. 1, the results show that there is significant expression of the protein of interest after IPTG induction.

3. Protein mass expression and purification

1) Strain activation

After the protein small experiment is successful, a proper amount of strains are taken again to be inoculated into a liquid culture medium containing 50mg/L antibiotic Kan, and the liquid culture medium is cultured and activated overnight at 37 ℃.

2) Inducible expression

The activated strain was identified as 1: 50 expansion culture 1000mL, 37 degrees C culture until OD600 ═ 0.4-0.6, to the bacterial liquid adding 0.8mM IPTG, 37 degrees C induced expression 4h, 8000rpm, 4 degrees C centrifugation 5min, collect the thalli.

3) Cell lysis

100mL of the disruption solution was added for ultrasonic lysis. The cracking conditions are as follows: the temperature ice bath, the power of 60 percent is more than 85 percent, the ultrasonic treatment is carried out for 2s, the interval is 2s, and the time is 45 min. Centrifuging at 12000rpm and 4 deg.C for 20min, and collecting supernatant and precipitate. SDS-PAGE detection is carried out to judge the expression form of the target protein. As shown in FIG. 2, the results showed that the target protein was mainly expressed in the form of inclusion bodies.

4) Protein purification

The protein is mainly expressed in the form of inclusion bodies, the inclusion bodies are purified by an NI column, and the purification effect is detected by SDS-PAGE. The protein effect obtained with 50mM imidazole eluate was best (FIG. 3). The final purified protein was identified as shown in figure 4.

Example 2

Preparation of antibodies

1. Animal immunization

2 female New Zealand white rabbits (2 months old) were immunized with the antigen prepared in example 1 for three times, and injected at multiple points into the thigh muscle. For each immunization, the immunization dose of antigen protein of each new Zealand white rabbit is 500 mu g, the primary immunization is emulsified by equivalent Freund complete adjuvant, the secondary immunization is emulsified by equivalent Freund incomplete adjuvant, the third immunization is mixed by equivalent physiological saline, and the mixture is injected into ear vein. Specific immunization protocols are shown in table 1.

TABLE 1 immunization protocol for polyclonal antibody preparation

The blood was collected intravenously 14 days after the second immunization, and the antibody titer was measured by an indirect method, thereby determining whether or not an antibody against the target was produced. Performing boosting immunization for the third time after the titer reaches the standard; if the titer does not reach the standard, continuing subcutaneous immunization by Freund incomplete adjuvant for the third time, and boosting immunization for the fourth time after 28 days. Blood was taken 14 days after the booster immunization.

2. Purification of antibodies

1) Centrifuging the serum at room temperature at 4000rpm for 15min, and taking the supernatant;

2) slowly adding saturated ammonium sulfate dropwise under stirring at 4 deg.C to half saturation, stirring for 30mins, centrifuging at 4 deg.C and 13000rpm for 30min, and removing supernatant;

3) dissolving the precipitate in appropriate amount of PBS (0.01M, pH7.4), adding saturated ammonium sulfate dropwise and slowly to 33% under stirring at 4 deg.C, stirring for 30min, centrifuging at 4 deg.C and 13000rpm for 30min, and removing supernatant;

4) dissolving the precipitate in PBS (0.01M, pH7.4), and dialyzing at 4 deg.C overnight;

5) purifying by using a Protein G column, wherein 5mL of ultrapure water is used for passing through a new column, and then 5mL of 0.4M PB buffer solution (pH 7.0) is used for balancing the purification column;

6) antibody column passing: the antibody requires slow column passage for better binding of the antibody protein to the binding site;

7) the column was equilibrated with 10mL of 0.4M PB buffer (pH 7.0);

8) the antibody at the binding site was eluted with 5mL of 0.1M glycine-hydrochloric acid buffer (pH 2.7), and 1M Tris-HCl (pH 8.0) was added to the eluate to neutralize the glycine-hydrochloric acid buffer, thereby maintaining the pH at a neutral level suitable for antibody preservation.

Example 3

Drawing of standard curve

1. Selection of concentration ranges for standard curves

In order to ensure that the experimental result is accurate, the range of the standard curve is particularly important, and the standard curve is in a proper range, so that the construction of the standard curve can be facilitated, the result can be finally accurately calculated according to the standard curve, and the R of the standard curve²Above 0.98, the OD450 value of each scatter point should be between 2 and 0.08 to avoid exceeding the optimal reading range of the instrument.

If the concentration of the standard curve is too high, the standard curve is easy to be misjudged in the qualitative process, a better standard curve cannot be constructed when the concentration exceeds the optimal degree range of the instrument, the calculated result is also smaller, even lower than 0, for example, in fig. 5 a, if the concentration is set to be 1000ng/ml, 750ng/ml, 500ng/ml, 400ng/ml, 300ng/ml and 200ng/ml, the final OD450 degree result is always out of the standard curve, and a certain degree of deviation can be caused. As shown in C in FIG. 5, concentration gradients of 10ng/ml, 7.5ng/ml, 5ng/ml, 2ng/ml, 1ng/ml and 0.5ng/ml were set.

2. Drawing of standard curve

The standard protein obtained in example 1 was taken, dissolved, and then diluted to 1. mu.g/mL in a gradient, and the high concentration mother liquor was stored at-80 ℃. The solution was diluted stepwise with 1ug/mL of the mother liquor to give 100. mu.L each of 300ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 10ng/mL, and 0 ng/mL.

After the standard protein solution is incubated overnight and adsorbed on the surface of an ELISA plate, primary antibody, secondary antibody and TMB substrate are added in sequence according to the test operation steps, and the OD450 value is detected and recorded.

The experimental results are as follows: the standard curve is finally obtained as shown in FIG. 6.

With reference to the formula: y is 367.6x-26.6, wherein x is the value of OD450 after the color development of the sample. y is the concentration of the protein of interest in the sample in ng/mL.

Example 4

An ELISA quantitative detection method of exogenous EPSPS protein in soybean, figure 7 is ELISA detection system principle, which comprises the following steps:

(1) extracting the soybean tissue extracting solution: a. each material was prepared by taking 20 mixed samples (leaf or stem tissue of fresh soybean) and grinding into powder with liquid nitrogen; about 0.1g of the powder was put into a 1.5mL centrifuge tube and weighed out by dry weight. b. Adding 1mL PBS, shaking, standing for 5min, centrifuging at 12000g for 10-15mins, sucking supernatant, and heating the supernatant at 105 deg.C for 1min to remove background color. The soybean in this example is the glyphosate resistant transgenic soybean of CN112322631A patent application;

(2) coating: a. the soybean tissue extract was diluted to an appropriate fold with CBS coating solution. If not diluted, the stock solution using PBS as a solvent may be centrifuged, and the supernatant may be directly coated. b. Add 100. mu.L of the boiled extract into the wells of the microplate, incubate at 37 ℃ for 1h, and then keep overnight at 4 ℃.

(3) And (3) sealing: a. taking out the incubated ELISA plate, adding 300 mu L PBST into each hole, rinsing for 3-4 times, 2mins each time, and lightly knocking the edge of the ELISA plate to clean more thoroughly; b. after washing, the residual liquid was blotted dry on absorbent paper, and then 300L of 5% PBS-mil (100mL PBS +5g skim milk powder) was added to each well, followed by incubation at 37 ℃ for 1 h.

(4) Primary antibody incubation: a. the well-sealed elisa plate was removed, PBST was washed as described above, and the residual liquid was patted dry. b. The primary antibody was diluted 2000-fold with 1mM PBS to a concentration of 0.1ug/ml, 100. mu.L/well and incubated at 37 ℃ for 1 h. The primary antibody in this example was the polyclonal antibody obtained in example 2.

(5) And (3) secondary antibody incubation: a. taking out the enzyme label plate combined with the primary antibody, washing the PBST by the method, and patting dry the residual liquid; b. the secondary antibody was diluted 2000-fold, 100. mu.L/well and incubated at 37 ℃ for 1 h. The secondary antibody in this example is horseradish peroxidase-labeled mouse anti-rabbit IgG.

(6) Substrate color development reaction: a. taking out the incubated enzyme label plate, washing the PBST, and patting the plate dry. b. 100 μ L of TMB substrate solution was added to each well. After 30min reaction at room temperature, 50. mu.L of stop solution 2M dilute hydrochloric acid was added. c. The OD450 of the resulting solution was measured using a microplate reader and substituted into the formula to calculate the protein concentration.

Example 5

On the basis of example 4, the interference of the background color of the soybeans is removed

Certain substances are present in the soy tissue extract which, in the final colour development step, can react with the substrate TMB to cause non-specific colour development. These substances are peroxidases which are ubiquitous in plants, especially in plants with good stress resistance, and which can undergo a chromogenic reaction with TMB. In view of this presumption, the present embodiment adopts the simplest and most efficient heat inactivation method.

The heating temperature was set to 100 ℃ to ensure that the specific binding ability of the target protein was not impaired. The heating time is optimized, a 0min control group and three experimental groups of 1min, 2mins and 3mins are set, and the heating time is selected to be 1min finally according to the test result of the graph 8.

As can be seen, the receptor material is darker in color before the background color is removed, and the content of the target protein in the transformant is not high, so that the color of the transformant does not show a difference from the color of the receptor, and the protein content cannot be calculated according to the color. After heating for 1min, the background color of the receptor material substantially disappears, and the transformant has a specific color reaction of the target protein. After a time longer than 1min, the color development of the sample gradually faded, so that the heating time was selected to be 1 min. The method ensures that the background color interference is completely removed, and simultaneously, the specific binding capacity of the target protein and the antibody is also kept. The method is also suitable for the condition of low content of target protein.

Example 6

On the basis of the embodiment 5, the interference of background colors of the rapes is removed

In the embodiment, the rape tissue extracting solution is used as a material, the heating time is optimized, a 0min control group and three experimental groups of 1min, 2mins and 3mins are set, and the heating is finally selected for 1min according to the test result of fig. 9.

As can be seen, the receptor material is darker in color before the background color is removed, and the content of the target protein in the transformant is not high, so that the color of the transformant does not show a difference from the color of the receptor, and the protein content cannot be calculated according to the color. After heating for 1min, the background color of the receptor material substantially disappears, and the transformant has a specific color reaction of the target protein. After a time longer than 1min, the color development of the sample gradually faded, so that the heating time was selected to be 1 min.

Example 7

On the basis of embodiment 4, the coating time is optimized

The coating time applied in the embodiment is optimized through tests, and coating time tests 6h, 12h, 24h and 48h are set. As shown in FIG. 10, the final color development of 6h coating is slightly lighter than that of 12h, and the difference between the final color development result of 12h and the final color development concentration after 24h and 48h is no longer obvious. To schedule time reasonably for efficiency, the 12h coating step is typically scheduled as an overnight step.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Wuhan space Biotechnology GmbH, institute of agricultural sciences, Anhui province

<120> ELISA quantitative detection method for exogenous EPSPS protein in plant

<130> 2021

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Claims (9)

1. An ELISA quantitative detection method of exogenous EPSPS protein in plants is characterized in that: the method comprises the following steps:

(1) preparation of polyclonal antibody: the peptide as shown in SEQ ID NO: injecting the EPSPS protein with the sequence shown in 1 into an immune rabbit for immunization, and then carrying out blood sampling and purification to obtain a polyclonal antibody; the upstream primer and the downstream primer synthesized by the nucleotide sequence of the EPSPS protein are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification;

(2) preparing a plant tissue extracting solution: grinding plant tissue with liquid nitrogen, leaching with PBS solution, centrifuging to obtain plant tissue extractive solution, and heating at 100 deg.C for 1 min;

(3) adding the plant tissue extracting solution in the step (2) into a micropore plate for coating for 12-48h, then pouring out the coating solution, cleaning the micropore plate by using a PBST solution, and adding a PBS-millik solution for sealing;

(4) after sealing, cleaning the microporous plate, adding primary antibody working solution for incubation, cleaning, adding secondary antibody working solution for incubation, fully cleaning, adding substrate solution, terminating the reaction, measuring the absorbance value, and substituting the absorbance value into a standard curve to obtain the content of the EPSPS protein; the primary antibody in the primary antibody working solution is a polyclonal antibody, and the secondary antibody in the secondary antibody working solution is horse radish peroxidase-labeled mouse anti-rabbit IgG.

2. The method for the ELISA quantitative detection of the exogenous EPSPS protein in the plant according to claim 1, which is characterized in that: and (3) heating the plant tissue extracting solution in the step (2) at 105 ℃ for 1 min.

3. The method for the ELISA quantitative detection of the exogenous EPSPS protein in the plant according to claim 1, which is characterized in that: the pH value of the PBS solution in the step (2) is 7.4.

4. The method for ELISA quantitative detection of an exogenous EPSPS protein in a plant according to claim 3, wherein the method comprises the following steps: the PBST solution in the step (3) comprises a PBS solution and Tween-20, and the adding volume of the Tween-20 is 0.05 percent of the volume of the PBS solution.

5. The method for the ELISA quantitative detection of the exogenous EPSPS protein in the plant according to claim 1, which is characterized in that: the PBS-milk solution in the step (3) comprises a PBS solution and skimmed milk powder, wherein the skimmed milk powder is dissolved in the PBS solution according to the volume ratio of 5%.

6. The method for the ELISA quantitative detection of the exogenous EPSPS protein in the plant according to claim 1, which is characterized in that: the coating time in the step (3) is 12 h.

7. The method for the ELISA quantitative detection of the exogenous EPSPS protein in the plant according to claim 1, which is characterized in that: the reaction time in the step (4) is 30min, and the stop solution is 2M dilute hydrochloric acid.

8. The method for the ELISA quantitative detection of the exogenous EPSPS protein in the plant according to claim 1, which is characterized in that: the step (4) specifically comprises: and (3) cleaning the microporous plate after sealing, adding 100 mu L of primary antibody working solution for incubation, cleaning, adding 100 mu L of secondary antibody working solution for incubation, fully cleaning, adding 100 mu L of TMB substrate solution, reacting at room temperature for 30min, adding 50 mu L of 2M dilute hydrochloric acid to stop reaction, measuring the absorbance value, and substituting the absorbance value into a standard curve to obtain the EPSPS protein content.

9. The method for the ELISA quantitative detection of the exogenous EPSPS protein in the plant according to claim 1, which is characterized in that: the determination of the standard curve comprises the following steps: the peptide as shown in SEQ ID NO: 1 to 1 mu g/mL, then diluting step by step to obtain 100 mu L of each of 300ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 10ng/mL and 0ng/mL, incubating the EPSPS protein solution overnight, adsorbing the solution on the surface of an ELISA plate, adding a primary antibody working solution, a secondary antibody working solution and a substrate in sequence, and determining the OD450 value.

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