CN111983215A - Method and kit for targeted screening of active ingredients of traditional Chinese medicine - Google Patents
Method and kit for targeted screening of active ingredients of traditional Chinese medicine Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
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Abstract
The invention discloses a method and a kit for targeted screening of active ingredients of traditional Chinese medicines, wherein a targeted screening medium of the active ingredients of the traditional Chinese medicines used in the targeted screening method is based on ultrahigh affinity acting force between an E-coli protein E7 DNase inactive mutant CL7 and homologous immune protein Im7 thereof, so that one-step reversible immobilization of fusion of a CL7 label in cell lysate to GPCRs is realized, and therefore, the targeted screening method integrates the ultrahigh affinity between Im7/CL7 and the high specificity of a receptor recognition medicine, and can realize the high-specificity targeted rapid screening of the active ingredients of the targeted traditional Chinese medicines of the GPCRs.
Description
Technical Field
The invention relates to a method and a kit for targeted screening of active ingredients of traditional Chinese medicines, in particular to a method and a kit for targeted screening of active ingredients of traditional Chinese medicines by taking G Protein Coupled Receptors (GPCRs) in organisms as action targets, belonging to the technical field of targeted screening of active ingredients of medicines.
Background
At present, the commonly used preparation method of solid phase carriers for screening active ingredients of drugs is usually to fix cells, cell membranes and proteins on the surface of a solid matrix, and the adopted immobilization method mainly comprises the following steps: an arbitrary immobilization method, a directional immobilization method, and the like. After long-term research, the methods have the following common defects:
1. the surfaces of cells or cell membranes have various drug action targets, and the screened active ingredients are compounds, so the specific action targets of the screened active ingredients cannot be determined;
2. immobilized proteins are usually concentrated on soluble proteins (e.g. enzymes), and less have been studied for the family of G protein-coupled receptors that target the maximum in vivo effect of drugs;
3. the immobilized solid matrix can not be recycled, so that the immobilized solid matrix is difficult to realize large-scale preparation and application, and the raw material consumption is large.
Therefore, the preparation of novel drug active ingredient screening solid phase carriers is particularly important.
G Protein Coupled Receptors (GPCRs) are the largest drug action targets in organisms, have a seven-transmembrane alpha helical structure, and are closely related to chronic diseases seriously harming human health, such as cancer, diabetes, central nervous system diseases, cardiovascular system diseases and the like.
The existing GPCRs immobilization method mainly comprises the following steps: random immobilization methods and directional immobilization methods.
1. Method for random immobilization
The random immobilization method comprises two methods: physical adsorption method, chemical bonding method.
(1) Physical adsorption method
In the physical adsorption method, the protein is usually immobilized by immersing an immobilization substrate in a protein solution and performing physical adsorption.
The method is simple and easy to operate, the immobilized amount of the protein is large, but the specificity is poor, the interaction force between the protein and the immobilized matrix is weak, and the loss of the protein is easily caused due to the increase of the washing times in the use process, so that the effect is large.
(2) Chemical bonding method
Chemical bonding is usually carried out by chemically immobilizing a target protein using a functional group (e.g., amino group, thiol group, carboxyl group, etc.) in a protein molecule as a site of action.
When a target protein is immobilized by this method, there are problems that the orientation of the protein is random and inconsistent, and the active site of the protein is easily lost.
2. Directional immobilization method
The more commonly used method for directional immobilization of proteins comprises: affinity methods using specific tags for immobilization, click chemistry reactions, and the like.
Compared with the random immobilization method, when the directional immobilization method is adopted to immobilize the protein, the loss of the protein active site caused by steric hindrance can be obviously reduced, and the immobilized product has high activity and good stability, and can be used for screening the active ingredients of the medicine in a complex system and researching the interaction between the protein and the medicine.
The directional immobilization method has certain advantages in ensuring the bioactivity and stability of the immobilized protein, however, the method has the problem of large raw material consumption because the immobilized matrix can not be reused.
Disclosure of Invention
In order to solve the defects of the prior art, the first object of the present invention is to provide a medium for targeted screening of active ingredients of traditional Chinese medicines, the second object of the present invention is to provide a method for targeted screening of GPCRs targeted active ingredients of traditional Chinese medicines by using the medium for targeted screening of active ingredients of traditional Chinese medicines, and the third object of the present invention is to provide a kit applied to the method for targeted screening of active ingredients of traditional Chinese medicines.
In order to achieve the first objective, the invention adopts the following technical scheme:
a traditional Chinese medicine active ingredient targeted screening medium is characterized by being prepared by the following method:
step 1: under the acidic condition of hydrochloric acid, reacting a solid matrix with sodium nitrite to generate diazonium salt, wherein the used solid matrix is hydroxylated, aminated, sulfhydrylated or carboxylated modified microspheres, a solid plane or a solid phase extraction microcolumn;
step 2: reacting the his-tag recombinant Im7 with diazonium salt generated by Step1 to generate an Im7 affinity matrix, wherein the diazonium salt generated by Step1 specifically reacts with the his-tag imidazolyl group in the his-tag recombinant Im7 to generate the diazonium salt;
step 3: changing a solution system in which the Im7 affinity matrix is located into a 5mM ammonium acetate buffer solution with the pH value of 7.0, adding a cell lysate containing GPCRs into the solution system, incubating the Im7 affinity matrix and the cell lysate containing the GPCRs for 0.5h, and washing the solid for multiple times by using the 5mM ammonium acetate buffer solution with the pH value of 7.0, wherein the solid is the traditional Chinese medicine active ingredient targeted screening medium.
The traditional Chinese medicine active ingredient targeted screening medium is characterized in that in Step1, the microspheres are resin microspheres or porous microspheres, and the solid plane is paper, silicon wafers or glass.
The medium for targeted screening of the active ingredients of the traditional Chinese medicine is characterized in that Step3, GPCRs contained in the cell lysate comprise: beta is a2-adrenoceptors, secretin receptor family, metabotropic glutamate receptors, fungal mating pheromone receptors, cyclic adenosine receptors and all members of the Frizzled/Smoothened family.
In order to achieve the second objective, the invention adopts the following technical scheme:
a targeted screening method for active ingredients of traditional Chinese medicines is characterized by comprising the following steps:
step 1: under the acidic condition of hydrochloric acid, reacting a solid matrix with sodium nitrite to generate diazonium salt, wherein the used solid matrix is hydroxylated, aminated, sulfhydrylated or carboxylated modified microspheres, a solid plane or a solid phase extraction microcolumn;
step 2: reacting the his-tag recombinant Im7 with diazonium salt generated by Step1 to generate an Im7 affinity matrix, wherein the diazonium salt generated by Step1 specifically reacts with the his-tag imidazolyl group in the his-tag recombinant Im7 to generate the diazonium salt;
step 3: by utilizing the ultrahigh affinity between CL7 and Im7, CL7 label fusion GPCRs and Im7 affinity matrix generated by Step2 react to generate a traditional Chinese medicine active ingredient targeted screening medium;
step 4: incubating traditional Chinese medicine extract to be screened and a traditional Chinese medicine active ingredient targeted screening medium generated by Step3, centrifuging at 4 ℃ after incubation is finished, removing supernatant, washing precipitate with 20mM ammonium acetate buffer solution with pH of 7.0, centrifuging washing solution and precipitate at 4 ℃, carrying out TEV protease fixed-point enzyme digestion on the obtained precipitate at 30 ℃ and pH of 7 to obtain high-purity unlabeled GPCRs and CL7 label modified Im7 affinity matrix;
step 5: in the presence of high-concentration guanidine hydrochloride, the Im7 affinity matrix modified by a CL7 label is subjected to denaturation treatment to obtain a CL7 peptide chain and an Im7 peptide chain modified solid matrix; step 6: the solid matrix is modified by Im7 peptide chain induced by low-concentration guanidine hydrochloride, and Im7 affinity matrix is obtained again by renaturation.
The method for targeted screening of the active ingredients of the traditional Chinese medicine is characterized in that in Step1, the microspheres are resin microspheres or porous microspheres, and the solid plane is paper, silicon wafers or glass.
In order to achieve the third objective, the invention adopts the following technical scheme:
the kit applied to the method for targeted screening of the active ingredients of the traditional Chinese medicine is characterized by comprising the following materials:
(1) im7 affinity matrix;
(2) the CL7 label is fused with GPCRs cell lysate;
(3) TEV protease;
(4) guanidine hydrochloride;
(5) 5mM ammonium acetate buffer solution at pH 7.0;
(6) 20mM ammonium acetate buffer solution at pH 7.0;
(7) ultrapure water;
wherein, the Im7 affinity matrix is prepared by the following method:
step 1: under the acidic condition of hydrochloric acid, reacting a solid matrix with sodium nitrite to generate diazonium salt, wherein the used solid matrix is hydroxylated, aminated, sulfhydrylated or carboxylated modified microspheres, a solid plane or a solid phase extraction microcolumn, the microspheres are resin microspheres or porous microspheres, and the solid plane is paper, silicon wafers or glass;
step 2: and (3) reacting the his-tag recombinant Im7 with the diazonium salt generated by Step1 to generate the Im7 affinity matrix, wherein the diazonium salt generated by Step1 is specifically subjected to the diazonium salt reaction with the his-tag imidazolyl in the his-tag recombinant Im 7.
The kit is characterized by comprising the following specific using methods:
step 1: adding a CL7 tag fused GPCRs cell lysate into Im7 affinity matrix at 4 ℃ for reaction for 0.5h, washing the precipitate for multiple times with a 5mM ammonium acetate buffer solution with pH 7.0, centrifuging at 4 ℃, removing the supernatant, and performing Step2 on the precipitate; step 2: adding 1mg/mL of the traditional Chinese medicine extract to be screened into the precipitate obtained in Step1, incubating at 20 ℃ for 10min, centrifuging at 4 ℃, removing the supernatant, washing the precipitate with 20mM ammonium acetate buffer solution with pH 7.0, centrifuging at 4 ℃ to separate a washing solution and the precipitate, carrying out active ingredient identification analysis on the washing solution, and carrying out Step3 on the precipitate;
step 3: adding TEV protease into the precipitate obtained in Step2 at 30 deg.C and pH 7, performing enzyme digestion, centrifuging at 4 deg.C, collecting supernatant containing a large amount of unlabelled target receptor, and performing Step4 on the precipitate;
step 4: adding ultrapure water and guanidine hydrochloride to the precipitate obtained in Step3 to give a final concentration of 7M guanidine hydrochloride, denaturing the protein, centrifuging at 4 deg.C, removing the supernatant, adding ultrapure water to the precipitate for renaturation, and recovering the Im7 affinity matrix.
The invention has the advantages that:
1. the traditional Chinese medicine active ingredient targeted screening medium provided by the invention can realize one-step directional immobilization of CL7 label fusion GPCRs based on the ultrahigh affinity acting force between the colicin protein E7 DNase inactive mutant CL7 and the homologous immunity protein Im7 thereof; in addition, based on the TEV protease enzyme cutting site between the CL7 label and GPCRs, the targeted screening medium can be reversibly prepared by TEV protease enzyme cutting and guanidine hydrochloride induced denaturation and renaturation, so that the targeted screening medium can be repeatedly used, and the problem of large raw material consumption in the conventional protein immobilization method is solved;
2. according to the method for targeted screening of the active ingredients of the traditional Chinese medicine, due to the use of the medium for targeted screening of the active ingredients of the traditional Chinese medicine, the ultrahigh affinity between Im7/CL7 and the high specificity of a receptor recognition medicine are integrated, and the high-specificity targeted rapid screening of the active ingredients of the traditional Chinese medicine targeted by GPCRs can be realized;
3. the kit provided by the invention contains Im7 affinity matrix and CL7 label fused with GPCRs cell lysate, and the two react to generate the traditional Chinese medicine active ingredient targeted screening medium, so the kit can be applied to screening of GPCRs targeted traditional Chinese medicine active ingredients, and has the advantages of simple and convenient operation, accurate result and reusability.
Drawings
FIG. 1 is a schematic diagram of a reaction for producing Im7 affinity microspheres from aminated resin microspheres;
FIG. 2 is a schematic diagram of the screening of GPCRs for targeting Chinese medicinal active ingredients using a screening medium;
FIG. 3 shows the results of the assay for identifying active ingredients.
Detailed Description
G Protein Coupled Receptors (GPCRs) are a general name of a large class of transmembrane proteins in organisms, are also the largest drug action targets in the organisms, have a seven-transmembrane alpha helical structure, and are closely related to various chronic diseases seriously harming human health, such as cancer, diabetes, central nervous system diseases, cardiovascular system diseases and the like.
The invention is based on GPCRs and aims to establish a method for screening the active ingredients of the GPCRs targeted traditional Chinese medicine.
The invention is described in detail below with reference to the figures and the embodiments.
Firstly, preparing a targeting screening medium for active ingredients of traditional Chinese medicines
The traditional Chinese medicine active ingredient targeted screening medium provided by the invention can realize the targeted traditional Chinese medicine active ingredient screening of GPCRs, and is prepared by the following method:
step 1: reacting a solid matrix with sodium nitrite to generate diazonium salt under the acidic condition of hydrochloric acid, wherein the solid matrix is hydroxylated, aminated, sulfhydrylated or carboxylated modified microspheres (such as resin microspheres and porous microspheres), solid planes (such as paper, silicon wafers and glass) or solid-phase extraction microcolumns (specifically used is the matrix surface of the solid-phase extraction microcolumns);
step 2: reacting the his-tag recombinant Im7 with diazonium salt generated by Step1 to generate an Im7 affinity matrix, wherein the diazonium salt generated by Step1 specifically reacts with the his-tag imidazolyl group in the his-tag recombinant Im7 to generate the diazonium salt;
step 3: changing a solution system in which the Im7 affinity matrix is located into a 5mM ammonium acetate buffer solution with the pH value of 7.0, adding a cell lysate containing GPCRs into the solution system, incubating the Im7 affinity matrix and the cell lysate containing the GPCRs for 0.5h, and washing the solid for multiple times by using the 5mM ammonium acetate buffer solution with the pH value of 7.0, wherein the solid is the traditional Chinese medicine active ingredient targeted screening medium.
The reaction principle of reacting aminated resin microsphere with sodium nitrite to generate diazonium salt in Step1 and reacting diazonium salt with his-tag recombinant Im7 to generate Im7 affinity microsphere (i.e. Im7 affinity matrix) in Step2 is shown in fig. 1.
The generated Im7 affinity microsphere is incubated with cell lysate containing CL7 label fused GPCRs to obtain the target screening medium (Step 3) of the active ingredients of the traditional Chinese medicine.
As the colicin protein E7 DNase inactive mutant CL7 and the homologous immunity protein Im7 have ultrahigh affinity acting force, the traditional Chinese medicine active ingredient targeted screening medium designed by the invention can realize one-step directional immobilization of CL7 tag fusion GPCRs, and in addition, based on TEV protease enzyme cutting sites between the CL7 tag and the GPCRs, after TEV protease enzyme cutting is carried out on the targeted screening medium, reversible preparation can be realized through guanidine hydrochloride induced denaturation and renaturation, so that the medium can be repeatedly used, and the problem of large consumption of raw materials in the existing protein immobilization method is solved.
Example 1
The method for preparing the traditional Chinese medicine active ingredient targeted screening medium by taking the aminated resin microspheres as a solid matrix comprises the following steps:
step 1: adding 1g of aminated resin microspheres into 30mL of 20mM hydrochloric acid solution, slowly adding 10mL of 20mM sodium nitrite solution at 4 ℃ in an ice bath, reacting the sodium nitrite with the aminated resin microspheres under the acidic condition of hydrochloric acid to generate diazonium salt, and after reacting for 10min, replacing a solution system with 5mM phosphate buffer solution with the volume of 5mL and the pH value of 7.0; step 2: adding 10ml of his-labeled recombinant Im7 cell lysate into a reaction system, reacting the his-labeled imidazolyl group in the his-labeled recombinant Im7 with the diazonium salt generated in the previous step to generate an Im7 affinity matrix, performing suction filtration after reacting for 20min, and washing and precipitating for multiple times by using a 5mM phosphate buffer solution with the pH value of 7.0 to obtain a precipitate, namely the Im7 affinity microsphere (namely the Im7 affinity matrix);
step 3: taking 200mg Im7 affinity microspheres, placing the affinity microspheres in a 5mM ammonium acetate buffer solution with the volume of 10mL and the pH value of 7.0, then adding 10mL of cell lysate containing 1mg/mL GPCRs into the solution system, incubating the Im7 affinity microspheres and the cell lysate containing the GPCRs for 1h at 4 ℃, washing the solid for multiple times by using the 5mM ammonium acetate buffer solution with the pH value of 7.0, centrifuging at 12000rpm for 2min at 4 ℃, removing the supernatant, and finally obtaining the solid, namely the traditional Chinese medicine active ingredient targeted screening medium (hereinafter referred to as screening medium) required by the invention.
Method for screening GPCRs (GPCRs) targeted traditional Chinese medicine active ingredients by using screening medium prepared in the prior art
The principle of screening GPCRs targeting traditional Chinese medicine active ingredients by using the screening medium prepared in the invention is shown in figure 2.
The method for screening the GPCRs targeted traditional Chinese medicine active ingredients by using the screening medium prepared in the invention specifically comprises the following steps:
step 1: incubating traditional Chinese medicine extract to be screened and the screening medium prepared previously, centrifuging at 4 ℃ after incubation is finished, removing supernatant, washing precipitate by using 20mM ammonium acetate buffer solution with the pH of 7.0, centrifuging and separating washing liquid and precipitate at 4 ℃, wherein the obtained washing liquid contains traditional Chinese medicine active ingredients which are specifically combined with GPCRs, performing active ingredient identification analysis on the washing liquid, and performing TEV protease fixed-point enzyme digestion on the obtained precipitate under the conditions of 30 ℃ and the pH of 7 to obtain high-purity unlabelled GPCRs and Im7 affinity matrix modified by CL7 labels;
step 2: denaturing the Im7 affinity matrix modified by the CL7 label in the presence of high-concentration guanidine hydrochloride to denature and separate CL7 and Im7 so as to obtain a CL7 peptide chain and Im7 peptide chain modified solid matrix;
step 3: the solid matrix is modified by Im7 peptide chain induced by low-concentration guanidine hydrochloride, and Im7 affinity matrix is obtained again by renaturation.
Because the screening method uses the prepared traditional Chinese medicine active ingredient targeted screening medium, the ultrahigh affinity between Im7/CL7 and the high specificity of a receptor recognition drug are integrated, and the high-specificity targeted rapid screening of the GPCRs targeted traditional Chinese medicine active ingredients can be realized.
Example 2
By beta2-adrenoceptor (. beta.)2-AR) is target receptor protein, and the method for screening the Chinese medicinal active ingredients for relieving cough and asthma by using the Chinese medicinal active ingredient targeted screening medium prepared in the embodiment 1 specifically comprises the following steps:
step 1: taking 50mg of the traditional Chinese medicine active ingredient targeted screening medium prepared in the example 1, mixing the medium with 0.5mL of 1mg/mL liquorice water extract, incubating at 20 ℃ for 10min, centrifuging at 12000rpm at 4 ℃ for 2min, removing supernatant, washing precipitate with 20mM ammonium acetate buffer solution with pH of 7.0, centrifuging at 12000rpm at 4 ℃ for 2min again to separate washing solution and precipitate, and leaving supernatant (namely washing solution) for active ingredient identification analysis (the active ingredient identification analysis result is shown in figure 3, and figure 3 shows that the active ingredient and beta in the liquorice water extract can be screened and obtained by the method2-the component to which AR binds specifically, which demonstrates: GPCRs target Chinese medicine active ingredient established by usThe screening method is simple and convenient, and the screening result is accurate and reliable), Step3 is executed on the sediment;
step 2: adding TEV protease into the precipitate obtained in Step1 at 30 deg.C and pH 7 for site-specific digestion, centrifuging at 12000rpm at 4 deg.C for 2min, collecting supernatant containing a large amount of unlabeled target receptor, and subjecting the precipitate (CL 7-labeled Im7 affinity matrix) to Step 3;
step 3: adding ultrapure water and guanidine hydrochloride into the precipitate obtained in Step2 to make the final concentration of guanidine hydrochloride be 7M (performing denaturation treatment on the Im7 affinity matrix modified by the CL7 label, performing denaturation separation on CL7 and Im 7), after protein denaturation, centrifuging at 12000rpm for 2min at 4 ℃, removing the supernatant, adding ultrapure water into the precipitate (Im7 peptide chain modified microspheres) to perform renaturation, and recovering the Im7 affinity microspheres.
Therefore, the method for targeted screening of the active ingredients of the traditional Chinese medicine provided by the invention does not need to target the protein beta2the-AR is separated and purified, and the beta can be realized by adopting cell lysate2High-activity one-step reversible directional immobilization of-AR, effectively avoiding beta2The loss of the activity of the AR has the characteristics of high specificity and high activity, and the active ingredient of the cough and asthma relieving medicine can be obtained as a target screening medium after immobilization.
In addition, the targeted screening method for the active ingredients of the traditional Chinese medicine also realizes the fusion of beta with CL7 label2The one-step reversible directional immobilization of the-AR can be realized, a large amount of target receptor proteins without labels can be obtained by performing TEV protease fixed-point enzyme digestion, and compared with the separation and purification of the traditional three-step column chromatography of the recombinant protein, the method has the characteristics of simplicity, convenience, rapidness and high efficiency.
In addition, in the method for targeted screening of the active ingredients of the traditional Chinese medicine, the used medium for targeted screening of the active ingredients of the traditional Chinese medicine can recover the biological function again after the guanidine hydrochloride denaturation and renaturation processes, has the characteristic of reutilization, and solves the problem of large raw material consumption in the existing protein immobilization method.
Third, Chinese medicine active ingredient target screening reagent kit and its using method
The kit provided by the invention can be used for screening GPCRs targeted traditional Chinese medicine active ingredients, and comprises the following materials:
(1) im7 affinity matrix;
(2) the CL7 label is fused with GPCRs cell lysate;
(3) TEV protease;
(4) guanidine hydrochloride;
(5) 5mM ammonium acetate buffer solution at pH 7.0;
(6) 20mM ammonium acetate buffer solution at pH 7.0;
(7) ultrapure water.
Wherein, the Im7 affinity matrix is prepared by the following method:
step 1: under the acidic condition of hydrochloric acid, reacting a solid substrate with sodium nitrite to generate diazonium salt, wherein the used solid substrate is a hydroxylated, aminated, sulfhydrylated or carboxylated modified microsphere, a solid plane or a solid phase extraction microcolumn, the microsphere is a resin microsphere or a porous microsphere, and the solid plane is paper, a silicon wafer or glass;
step 2: and (3) reacting the his-tag recombinant Im7 with the diazonium salt generated by Step1 to generate the Im7 affinity matrix, wherein the diazonium salt generated by Step1 is specifically subjected to the diazonium salt reaction with the his-tag imidazolyl in the his-tag recombinant Im 7.
Example 3
The cough and asthma relieving traditional Chinese medicine active component targeted screening kit is specifically composed of the following materials:
(1) im7 affinity microspheres prepared by Step1 and Step2 of example 1, 200 mg;
(2) CL7 fusion of beta2-10 mL of AR cell lysate;
(3) TEV protease 10 KU;
(4) 5mL of guanidine hydrochloride, 10M;
(5) 50mL of 5mM ammonium acetate buffer solution with pH 7.0;
(6) 25mL of 20mM ammonium acetate buffer solution with pH 7.0;
(7) 100mL of ultrapure water.
The method for screening the cough and asthma relieving traditional Chinese medicine active ingredients by using the kit comprises the following specific steps:
step 1: 10mL of CL7 tag fusion beta was added to 200mg of Im7 affinity microspheres at 4 deg.C2-AR cell lysate is reacted for 1h, then the pellet is washed several times with 5mM ammonium acetate buffer solution at pH 7.0, followed by centrifugation at 12000rpm for 2min at 4 ℃, supernatant is removed, and Step2 is performed on the pellet;
step 2: adding 0.5mL of 1mg/mL licorice aqueous extract to the precipitate obtained in Step1, incubating at 20 deg.C for 10min, centrifuging at 12000rpm at 4 deg.C for 2min, removing the supernatant, washing the precipitate with 20mM ammonium acetate buffer solution having pH of 7.0, centrifuging at 12000rpm at 4 deg.C for 2min again to separate the washing solution and the precipitate, leaving the supernatant (i.e., washing solution) for active ingredient identification analysis (the result of the active ingredient identification analysis is consistent with that in FIG. 3), and performing Step3 on the precipitate;
step 3: adding TEV protease into the precipitate obtained in Step2 at 30 deg.C and pH 7 for site-specific digestion, centrifuging at 12000rpm at 4 deg.C for 2min, collecting supernatant containing a large amount of unlabeled target receptor, and subjecting the precipitate (CL 7-labeled Im7 affinity matrix) to Step 4;
step 4: adding ultrapure water and guanidine hydrochloride into the precipitate obtained in Step3 to make the final concentration of guanidine hydrochloride be 7M (performing denaturation treatment on the Im7 affinity matrix modified by the CL7 label, performing denaturation separation on CL7 and Im 7), after protein denaturation, centrifuging at 12000rpm for 2min at 4 ℃, removing the supernatant, adding ultrapure water into the precipitate (Im7 peptide chain modified microspheres) for renaturation, and recovering the Im7 affinity microspheres (realizing the reuse of the Im7 affinity microspheres).
The kit contains Im7 affinity microspheres and CL7 fusion beta2The AR cell lysate has ultrahigh affinity acting force with colicin protein E7 DNase inactive mutant CL7, and the AR cell lysate can identify cough and asthma relieving drug active ingredients with high specificity, so the kit can be used for quickly and high-specificity targeted screening of cough and asthma relieving traditional Chinese medicine active ingredients, and has the advantages of simple and convenient operation, accurate result and reusability.
It should be noted that all the technical solutions obtained by means of equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
Claims (9)
1. The method for targeted screening of the active ingredients of the traditional Chinese medicine is characterized by comprising the following steps:
step 1: under the acidic condition of hydrochloric acid, reacting a solid matrix with sodium nitrite to generate diazonium salt, wherein the used solid matrix is hydroxylated, aminated, sulfhydrylated or carboxylated modified microspheres, a solid plane or a solid phase extraction microcolumn;
step 2: reacting the his-tag recombinant Im7 with diazonium salt generated by Step1 to generate an Im7 affinity matrix, wherein the diazonium salt generated by Step1 specifically reacts with the his-tag imidazolyl group in the his-tag recombinant Im7 to generate the diazonium salt;
step 3: by utilizing the ultrahigh affinity between CL7 and Im7, CL7 label fusion GPCRs and Im7 affinity matrix generated by Step2 react to generate a traditional Chinese medicine active ingredient targeted screening medium;
step 4: incubating traditional Chinese medicine extract to be screened and a traditional Chinese medicine active ingredient targeted screening medium generated by Step3, centrifuging at 4 ℃ after incubation is finished, removing supernatant, washing precipitate by using 20mM ammonium acetate buffer solution with pH of 7.0, centrifuging and separating washing liquid and precipitate at 4 ℃, wherein the obtained washing liquid contains traditional Chinese medicine active ingredients which are specifically combined with GPCRs, and carrying out TEV protease fixed-point enzyme digestion on the obtained precipitate at 30 ℃ and pH of 7 to obtain high-purity unlabelled GPCRs and CL7 label modified Im7 affinity matrix;
step 5: in the presence of high-concentration guanidine hydrochloride, the Im7 affinity matrix modified by a CL7 label is subjected to denaturation treatment to obtain a CL7 peptide chain and an Im7 peptide chain modified solid matrix;
step 6: the solid matrix is modified by Im7 peptide chain induced by low-concentration guanidine hydrochloride, and Im7 affinity matrix is obtained again by renaturation.
2. The targeted screening method for active ingredients of traditional Chinese medicine according to claim 1, wherein in Step1, the microspheres are selected from resin microspheres or porous microspheres.
3. The targeted screening method for active ingredients of Chinese herbs according to claim 1, wherein in Step1, the solid plane is paper, silicon wafer or glass.
4. The kit applied to the traditional Chinese medicine active ingredient targeted screening method of any one of claims 1 to 3, characterized in that the kit is composed of the following materials:
(1) im7 affinity matrix;
(2) the CL7 label is fused with GPCRs cell lysate;
(3) TEV protease;
(4) guanidine hydrochloride;
(5) 5mM ammonium acetate buffer solution at pH 7.0;
(6) 20mM ammonium acetate buffer solution at pH 7.0;
(7) ultrapure water;
wherein the Im7 affinity matrix is prepared by the following method:
step 1: under the acidic condition of hydrochloric acid, reacting a solid substrate with sodium nitrite to generate diazonium salt, wherein the used solid substrate is a hydroxylated, aminated, sulfhydrylated or carboxylated modified microsphere, a solid plane or a solid phase extraction microcolumn, the microsphere is a resin microsphere or a porous microsphere, and the solid plane is paper, a silicon wafer or glass;
step 2: and (3) reacting the his-tag recombinant Im7 with the diazonium salt generated by Step1 to generate the Im7 affinity matrix, wherein the diazonium salt generated by Step1 is specifically subjected to the diazonium salt reaction with the his-tag imidazolyl in the his-tag recombinant Im 7.
5. The kit according to claim 4, wherein the kit is used in the following manner:
step 1: adding a CL7 tag fused GPCRs cell lysate into Im7 affinity matrix at 4 ℃ for reaction for 0.5h, washing the precipitate for multiple times with a 5mM ammonium acetate buffer solution with pH 7.0, centrifuging at 4 ℃, removing the supernatant, and performing Step2 on the precipitate;
step 2: adding 1mg/mL of the traditional Chinese medicine extract to be screened into the precipitate obtained in Step1, incubating at 20 ℃ for 10min, centrifuging at 4 ℃, removing the supernatant, washing the precipitate with 20mM ammonium acetate buffer solution with pH 7.0, centrifuging at 4 ℃ to separate a washing solution and the precipitate, carrying out active ingredient identification analysis on the washing solution, and carrying out Step3 on the precipitate;
step 3: adding TEV protease into the precipitate obtained in Step2 at 30 deg.C and pH 7, performing enzyme digestion, centrifuging at 4 deg.C, collecting supernatant containing a large amount of unlabelled target receptor, and performing Step4 on the precipitate;
step 4: adding ultrapure water and guanidine hydrochloride to the precipitate obtained in Step3 to give a final concentration of 7M guanidine hydrochloride, denaturing the protein, centrifuging at 4 deg.C, removing the supernatant, adding ultrapure water to the precipitate for renaturation, and recovering the Im7 affinity matrix.
6. A traditional Chinese medicine active ingredient targeted screening medium is characterized by being prepared by the following method:
step 1: under the acidic condition of hydrochloric acid, reacting a solid matrix with sodium nitrite to generate diazonium salt, wherein the used solid matrix is hydroxylated, aminated, sulfhydrylated or carboxylated modified microspheres, a solid plane or a solid phase extraction microcolumn;
step 2: reacting the his-tag recombinant Im7 with diazonium salt generated by Step1 to generate an Im7 affinity matrix, wherein the diazonium salt generated by Step1 specifically reacts with the his-tag imidazolyl group in the his-tag recombinant Im7 to generate the diazonium salt;
step 3: changing a solution system in which the Im7 affinity matrix is located into a 5mM ammonium acetate buffer solution with the pH value of 7.0, adding a cell lysate containing GPCRs into the solution system, incubating the Im7 affinity matrix and the cell lysate containing the GPCRs for 0.5h, and washing the solid for multiple times by using the 5mM ammonium acetate buffer solution with the pH value of 7.0, wherein the solid is the traditional Chinese medicine active ingredient targeted screening medium.
7. The active ingredient targeted screening media of claim 6, wherein in Step1, the microspheres are selected from resin microspheres or porous microspheres.
8. The active ingredient targeted screening medium of claim 6, wherein in Step1, the solid plane is paper, silicon wafer or glass.
9. The active ingredient targeting agent as claimed in claim 6, wherein GPCRs contained in said cell lysate include, in Step 3: beta is a2-adrenoceptors, secretin receptor family, metabotropic glutamate receptors, fungal mating pheromone receptors, cyclic adenosine receptors and all members of the Frizzled/Smoothened family.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005028667A1 (en) * | 2003-09-19 | 2005-03-31 | Riken | Drug targeting g protein-coupled receptor expressed in human mast cells and method of screening the same |
| CN104844710A (en) * | 2015-04-13 | 2015-08-19 | 西北大学 | Preparation method and applications of oriented immobilized PEGA composite resin |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005028667A1 (en) * | 2003-09-19 | 2005-03-31 | Riken | Drug targeting g protein-coupled receptor expressed in human mast cells and method of screening the same |
| CN104844710A (en) * | 2015-04-13 | 2015-08-19 | 西北大学 | Preparation method and applications of oriented immobilized PEGA composite resin |
Non-Patent Citations (6)
| Title |
|---|
| LI QIAN 等: "Reliable Analysis of the Interaction between Specific Ligands and Immobilized Beta-2-Adrenoceptor by Adsorption Energy Distribution", 《ANALYTICAL CHEMISTRY》 * |
| SHUNTANG LI 等: "A Highly Efficient Indirect P. pastoris Surface Display Method Based on the CL7/Im7 Ultra-High-Affinity System", 《MOLECULES》 * |
| VASSYLYEVA MARINA N 等: "Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA》 * |
| ZHAO XINFENG 等: "Oriented immobilisation of histidine-tagged protein and its application in exploring interactions between ligands and proteins", 《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》 * |
| 刘嘉君 等: "受体色谱：中药靶向活性成分高效筛选技术", 《世界科学技术-中医药现代化》 * |
| 容蓉 等: "《仪器分析》", 31 August 2014, 北京：中国医药科技出版社 * |
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