CN111646898B - Chenopodium quinotoxin III hapten, artificial antigen, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a quinotoxins III hapten, an artificial antigen, and a preparation method and application thereof. The structure of the quinotoxins III hapten is as follows:

Description

Chenopodium quinotoxin III hapten, artificial antigen, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a quinotoxins III hapten, an artificial antigen, and a preparation method and application thereof.

Background

Ericaceae plants are widely distributed in temperate zone of northern and southern hemispheres and in subarctic zone of northern hemispheres, and comprise more than 50 genera and more than 1700 genera. China has more Ericaceae, and is characterized in that southwest mountainous areas are taken as centers and are distributed all over the country. The azaleaceae contains many toxic plants, up to hundreds of species, and is particularly concentrated in the genera Pimpinella, Mureau, Vaccinium, Rhododendron and Laurel. Once people and animals eat toxic azalea by mistake, poisoning symptoms such as vomiting, abdominal pain, dizziness and dyspnea often appear, and the severe people also have symptoms such as lethargy, angle bow swelling, respiratory paralysis, convulsion and the like. Modern scientific research shows that: toxic rhododendron usually contains a unique structure of the ligneous toxin, wherein the ligneous toxin III is taken as a representative substance. Although Ericaceae are highly toxic, it is not so many species that can directly constitute a hazard to humans and animals, since they are mostly shrubs, mostly growing in mountain forests. Poisoning in humans often occurs in some medicinal or edible rhododendrons, such as rhododendron album and gold leaves. In addition, as part of toxic rhododendrons belong to Honey source plants and can secrete a large amount of nectar in the blooming period, and as the nectar contains toxins such as the lignocerin III and the like, the toxic rhododendrons are collected by bees, migrate and are enriched in the Honey to form toxic Honey, namely 'Mad Honey'. The Honey poisoning incidents of Turkey's black sea area and Nepal area all year round are caused by eating ' Mad Honey '. How to effectively detect the content of the ligninotoxin in honey and edible azalea and ensure the life health of people becomes one of the problems to be solved urgently in the field of food safety.

At present, the detection method of the resveratrol toxin III is mainly based on instrument analysis methods such as high performance liquid chromatography or liquid tandem mass spectrometry. The instrumental analysis has the advantages of high accuracy, good sensitivity and the like, but usually needs a complicated sample pretreatment process and needs professional technicians to carry out detection and analysis in a laboratory, which greatly limits the application of the technology in practical situations. The immunoassay method developed based on antigen-antibody specificity recognition has the advantages of simplicity, convenience, rapidness, sensitivity, specificity and the like, and particularly, the developed colloidal gold test strip is very suitable for rapid detection on site. In the construction process of the immunoassay method, the acquisition of excellent antibodies is a key, and once the antibodies are obtained, different analysis strategies and modes can be selected to construct a plurality of immunoassay methods. Because the small molecular compound has small molecular weight and is difficult to stimulate the organism to generate antibodies, the design and the modification are the core for constructing the immunoassay method on the basis of the chemical structure of the target compound.

Disclosure of Invention

Aiming at the blank of the existing quinoa toxin III antigen synthesis technology, the invention provides a quinoa toxin III hapten, an artificial antigen, a preparation method and application thereof.

Specifically, in the first aspect, the first time, the structure of the semiantigen of the resveratrol III is shown as follows:

。

the invention discovers that a 4-carbon atom spacer arm is introduced into the C14 hydroxyl position of the quinugine toxin III, and a hapten with an active group of carboxyl is obtained under the condition that the structure of the quinugine toxin III is kept unchanged as much as possible. Under the structure, the quinugine toxin III can be used as an antigenic determinant and exposed on the surface of carrier protein, so that an organism can be stimulated to generate an immune response well, and a foundation is laid for the preparation of a high-sensitivity anti-quinugine toxin III antibody.

In a second aspect, the present invention provides a method for preparing the quinotoxins III hapten, which comprises: and (3) carrying out a light-resistant reaction on the chenopodium album toxin III and succinic anhydride at room temperature to obtain the veratrum album toxin III.

In a preferred embodiment, the preparation method comprises the following steps:

dissolving the quinugine toxin III and the succinic anhydride respectively by using anhydrous pyridine, mixing the two solutions, and reacting for 12-13 h at room temperature at 220-240 rpm in a dark place; after the reaction system is quenched by adding water, the mixture is extracted by ethyl acetate, then washed by dilute hydrochloric acid (preferably with the concentration of 0.1M), and the organic phase is dried and concentrated to obtain a crude hapten. And then, separating and purifying the obtained crude product by adopting semi-preparative liquid chromatography to obtain the target veratryn III hapten.

In a third aspect, the invention provides an artificial antigen of the quinugine toxin III, which is obtained by coupling the hapten of the quinugine toxin III with carrier protein;

wherein the carrier protein is selected from Bovine Serum Albumin (BSA), Ovalbumin (OVA), keyhole limpet hemocyanin, thyroid protein, human serum albumin; BSA and OVA are preferred.

The artificial antigen of the quinoa toxin III can be used as immunogen or coating antigen, the immunogen can be quinoa toxin III-HS-BSA, and the coating antigen can be quinoa toxin III-HS-OVA.

Preferably, in the artificial antigen of the quinuproar toxin III, the molar ratio of the veratrum toxin III hapten to the carrier protein is 23-25: 1, and more preferably 23.7: 1.

In a fourth aspect, the invention provides a preparation method of the artificial antigen of the quinoa toxin III, wherein a carrier protein is coupled to the carboxyl carbon of the hapten of the quinoa toxin III by adopting an activated ester method.

In a preferred technical scheme, the preparation method of the artificial antigen of the quinuproot toxin III comprises the following steps:

(1) mixing veratryn III hapten NHS: (NDissolving DCC (dicyclohexylcarbodiimide) in a DMF (dimethyl formamide) solvent, and reacting at room temperature for 6-8 h to obtain the hapten activation solution.

(2) Dissolving carrier protein in PBS, and slowly dripping the hapten activating solution prepared in the step (1) into the carrier protein solution to obtain the protein activating solution.

(3) Transferring the protein activating solution prepared in the step (2) into a dialysis bag with the molecular weight cutoff of 7 KDa, and dialyzing the dialysis bag in PBS buffer solution at 4 ℃ for 2-4 days.

(4) Taking out the dialysis bag, centrifuging the liquid phase in the dialysis bag, and collecting supernatant, namely the artificial antigen solution of the chenopodium quinovosum toxin III.

In a fifth aspect, the invention provides the use of said quinoa toxin iii hapten or said quinoa toxin iii artificial antigen in any one of the following aspects:

the application of the quinoa toxin III specific antibody is prepared;

② the application in detecting the specific antibody of the veratryn III.

Furthermore, the invention provides a specific antibody of the chenopodium album toxin III, which is prepared by taking the artificial antigen of the chenopodium album toxin III as immunogen (such as Balb/c mice, New Zealand white rabbits, sheep and the like, preferably Balb/c mice) and immunizing animals.

The antibody is a polyclonal antibody or a monoclonal antibody, preferably a polyclonal antibody.

Preferably, the artificial antigen is obtained by coupling the veratryn III hapten and BSA.

In a preferred embodiment of the present invention, the method of immunizing Balb/c mice is: when in first immunization, the immunogen is emulsified by equivalent volume of Freund complete adjuvant and then 4 Balb/c mice of 6-8 weeks are immunized; in the boosting immunization, Balb/c mice were immunized after emulsification with the same dose of immunogen and an equal volume of Freund's incomplete adjuvant.

Wherein the dosage of the immunogen for the primary immunization and the boosting immunization is 100 mug/mouse, the concentration of the immunogen is 1 mg/mL (the diluent is 0.01M PBS), and the immune dosage of each Balb/c mouse after emulsification is 0.2 mL/mouse.

The number of booster immunizations was 2.

The boosting is specifically performed 4 weeks after each immunization.

The mouse antiserum is a mouse source polyclonal antibody, and blood is collected from an eyeball vein and separated to obtain serum after the second boosting immunization for one week.

In the detection of serum, the assay may be an enzyme linked immunosorbent assay (ELISA).

The ELISA detection method comprises the following steps: detecting the serum titer of the chenopodium album toxin III antibody by an indirect ELISA method; indirect competitive ELISA method, determining the half Inhibitory Concentration (IC) of antibody₅₀) And specificity.

In a sixth aspect, the invention provides the use of the specific antibody against quinuproot III in any one of the following aspects:

(1) the application in detecting the veratryn III;

(2) the application in the preparation of an immunochromatographic test strip of the quinugine toxin III;

(3) the application in preparing an enzyme-linked immunoassay detection kit of the quinugine toxin III.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

(1) the invention provides a novel quinoa toxin III hapten, an artificial antigen and a preparation method thereof for the first time. The quinugine toxin III-HS and the antibody prepared by the same provide a new means for establishing a rapid, simple, cheap, sensitive and specific detection method for the quinugine toxin III.

(2) Chenopodium quinotoxin III polyclonal antibody and IC prepared by utilizing quinugine toxin III hapten₅₀The detection sensitivity is as low as 5.8 ng/mL, and the detection method has the unique advantages of high sensitivity, high practical value and the like. The ELISA kit prepared based on the antibody, the colloidal gold test strip and other immunoassay methods have good application prospects in food toxin detection.

Drawings

FIG. 1 is a mass spectrum of a quinoxyrin III (panel A) and a quinoxyrin III hapten (panel B) in example 1 of the present invention.

FIG. 2 is a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of immunolog veratrxin III-HS-BSA of example 2 of the present invention.

FIG. 3 is a standard curve diagram of detecting Xylaria chenopodii III by using antiserum Xylaria chenopodii III-HS-BSA-5 # in example 3 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1 preparation and characterization of a Chenopodium quinotoxin III hapten (Chenopodium quinotoxin III-HS)

This example provides a quinotoxins iii hapten, which is prepared by the following steps:

(1) dissolving the chenopodin III (25 mg) and the succinic anhydride (6.7 mg) by using 4.0 mL of anhydrous pyridine respectively, mixing the two solutions, placing the mixed solution in an oil bath magnetic stirrer, and reacting for 12 hours at room temperature and 240 rpm in a dark place to obtain a reaction product.

(2) Extraction was performed with 25 mL ethyl acetate, followed by washing with 0.1M dilute hydrochloric acid (5 mL), and the organic phase was concentrated by drying to obtain a crude hapten.

(3) And (3) separating and purifying the obtained crude hapten by using semi-preparative liquid chromatography to obtain 12.2 mg (the yield is 40%) of white target compound chenopoxin III hapten.

The purified hapten was identified by high resolution mass spectrometry and the results are shown in FIG. 1. Chenopodium album toxin III (A), MSm/z 415.23509 [M-H+HCOOH]^-The element composition is C₂₁H₃₅O₈The ions are adduct ions, and when the compound is subjected to electrospray ionization, a formic acid is added into the molecular structure; chenopodium album toxin III-HS (B picture) MSm/z469.24421 [M-H]^-The element composition is C₂₄H₃₇O₉. Compared with the chenopodium quinoa toxin III, the chemical structure of the chenopodium quinoa toxin III-HS is added with C₄H₄O₃The method just proves that succinic anhydride is successfully linked on the chemical structure of the veratrum toxin III, and the success in preparing the veratrum toxin III-HS is confirmed, so that the method can be used for preparing antibodies. Due to the existence of a plurality of hydroxyl sites in the chemical structure of the lignocerin III, the simple high-resolution mass spectrum data is difficult to clearly determine the combination of ester bonds of the hydroxyl in the target hapten. Therefore, the invention adopts nuclear magnetic resonance to obtain the carbon spectrum of the target resveratrol toxin III hapten (¹³C-NMR) and hydrogen spectrum (¹H-NMR) data (Table 1). As can be seen from the chemical structure of rhodojaponin III, hydroxyl groups at positions C5, C6, C10 and C16 have large steric hindrance, and the binding reaction is difficult to occur. Hydroxyl at C3 and C14 has small steric hindrance, and is easy to generate combination reaction with succinic anhydride, so that the semi-molecule is easy to generateThe antigen crude product is mainly composed of two mixtures. The linkage at the C14 position facilitates exposure of the functional group of the veratryn III compared to the bound hapten at C3, from the viewpoint of stimulating the body to produce excellent antibodies. For this purpose, the invention adopts semi-preparative chromatography to separate and purify the hapten of the ligninotoxin III bound at the C14 position. The data in Table 1 show a carbon spectrum of 84.2 ppm for Compound C14; the hydrogen spectrum was 4.31(d, J = 7.0). This correlates with a carbon spectrum 76.7 ppm at C14 of the murumotoxin III; the hydrogen spectra were 3.24 (m) and the 4.77(s) phase ratios were all significantly shifted, indicating that the succinic acid binding occurred at the hydroxyl group at C14. The carbon spectrum and hydrogen spectrum data of C3, C5, C6, C10 and C16 are not changed obviously, and the correctness of the inference is proved again.

TABLE 1 of semiantigens of murumotoxin III¹³C-NMR and¹H-NMR data

Example 2 preparation of artificial antigen of Xylori toxin III

(1) The (10 mg) of the (I) chenopoxin III hapten prepared in example 1 was weighed separately,N-hydroxysuccinimide (4.2 mg) and dicyclohexylcarbodiimide (8.4 mg) in a glass reaction flask, 1 mL DMF was added. And placing the glass bottle containing the reaction solution on a magnetic stirrer, and reacting for 7 hours at room temperature in a dark place.

(2) 30 mg of BSA was dissolved in 6 mL of PBS buffer to obtain a protein solution.

(3) Adding 100 μ L of hapten activating solution obtained in the step (1) into the BSA solution prepared in the step (2) slowly and dropwise, and placing the reaction solution in a magnetic stirrer 4^oC, reacting for 5 hours.

(4) Transferring the protein activating solution in the step (3) into a dialysis bag with the molecular weight cutoff of 7 KDa, and then placing the dialysis bag into PBS buffer solutionMiddle 4^oDialysis is performed for 3 days (fluid changes every 12 hours).

(5) And (4) after the step (4) is finished, taking out the dialysis bag, centrifuging the liquid phase in the dialysis bag for 5 min at 3000 rpm, and collecting supernatant, namely the artificial antigen chenopodium album toxin III-HS-BSA solution.

The coupling molar ratio of the obtained carrier protein BSA and the hapten piguey toxin III-HS is 23.7:1 by MALDI-TOF-MS identification of piguey toxin III-HS-BSA (figure 2), and the result proves that the immunogen is successfully synthesized and can be used for mouse immunization and monoclonal antibody preparation.

And (3) replacing the BSA in the step (2) with OVA to obtain the veratryn III-HS-OVA.

Example 3 preparation and characterization of a polyclonal antibody to Veratrin III

Preparation of Chenopodium quinotoxin III polyclonal antibody

The quinoxyrin III-HS-BSA prepared in example 2 is used as immunogen to immunize mice, and the quinoxyrin III-HS-OVA is used as coating antigen to detect mouse antiserum. The Bradford method is used for determining the concentration of complete antigen, and the concentrations of the quinugine toxin III-HS-BSA and the quinugine toxin III-HS-OVA which are determined are both 5.5 mg/mL.

Diluting the immunogen to 1 mg/mL (diluting with 0.01M PBS and pH 7.3) during the first immunization, taking the diluted immunogen, mixing the diluted immunogen with Freund's complete adjuvant in the same volume, fully emulsifying, and inoculating 6-8 weeks old Balb/c mice (immunizing 6 mice) with 100 mug of the immunogen and 0.2 mL of the injection at multiple points subcutaneously on the back of the neck. The booster was then performed every 4 weeks, at which time the immunogen was emulsified with an equal volume of incomplete Freund's adjuvant. The immunization dose of the immunogen is the same as that of the first immunization dose, and the number of times of boosting immunization is 2.

Second, detection of mouse polyclonal antibody

Blood is collected from the eyeballs of the mice one week after the 3 rd boosting immunization, 6 antiserum (namely polyclonal antibody) are obtained through centrifugation at 3000 rpm, and the antiserum is named sequentially according to the serial numbers of the quinupristin III-HS-BSA-1 # and the quinupristin III-HS-BSA-2 #. Antiserum titers were determined by the classical checkerboard method and antiserum sensitivity by the indirect competitive ELISA method.

1. Determination of antiserum titres

The antibody titer was detected by indirect ELISA. The indirect ELISA procedure was as follows:

(1) coating: the antigen of example 2 was diluted in 0.05M, pH 9.6.6 carbonate buffer starting from 10. mu.g/mL in a fold ratio of 100. mu.L/well, 37^oC, reacting for 2 hours.

(2) Washing: the plate was decanted, spun-dried and washed 3 times with washing solution for 3 min each time.

(3) And (3) sealing: after patting dry, 200. mu.L/well blocking solution, 37^oC, reacting for 2 hours. And drying after washing for later use.

(4) Sample adding: antiserum was diluted in multiples starting at 1:1000 and added to coated wells at various dilutions, 100. mu.L/well, 37^oC, reacting for 1 h; after washing well, HRP-goat anti-rabbit IgG diluted 1:3000, 100. mu.L/well, 37 was added^oC, reacting for 1 h.

(5) Color development: the ELISA plate was removed, washed thoroughly, and 100. mu.L of TMB developing solution was added to each well, and the reaction was carried out at 37 ℃ in the dark for 15 min.

(6) Stopping and measuring by adding 100. mu.L of a stopping solution to each well to stop the reaction, and then measuring the OD of each well by using a microplate reader₄₅₀The value is obtained.

(7) And (4) interpretation of results: by OD₄₅₀The highest dilution factor of the serum corresponding to the value which is more than or equal to 2.1 times of that of the negative control hole (namely P/N is more than or equal to 2.1) is the ELISA titer of the serum.

2. Polyclonal antibody IC₅₀Measurement of (2)

(1) The coating and sealing processes are the same as above.

(2) Adding standard substances and antibodies: mu.L of the quinoxyrin III standard solution and 50. mu.L of the antibody dilution (diluted according to the antibody titer in Table 2) were added to each well, incubated at 37 ℃ for 30 min, then washed 3 times with PBST solution and patted dry. The solvent of the standard solution was PBS buffer, and the concentrations of the standard were 0, 0.125, 0.25, 0.5, 1, 2, 4 and 8 ng/mL solutions, three in parallel.

(3) Adding enzyme labeled secondary antibody, developing, terminating and reading.

The measured data are plotted as-Log 10 (competitor) value on the abscissa and OD₄₅₀The value is a vertical coordinate, a four-parameter equation of Origin 8.0 is used for fitting, and a standard curve is established to obtain IC₅₀The value is obtained. IC of 6 polyclonal antibodies of formula (I)₅₀The values are shown in Table 2, and the data in Table 2 show that the IC of the quinuproot toxin III-HS-BSA-5 #₅₀And the lowest concentration is 5.8 ng/mL. The standard curve chart of the detection of the quinagolide III by the quinagolide III-HS-BSA-5 # antiserum is shown in figure 3, and the figure shows that the linear detection range (IC) of the quinagolide III-HS-BSA-5 # to the quinagolide III₂₀-IC₈₀) 2.4-14.5 ng/mL, IC₅₀It was 5.8 ng/mL.

The result shows that the hapten structure chenopodin III-HS designed in the invention is used as an antigenic determinant, which can stimulate a mouse to generate a high-sensitivity antibody well.

TABLE 2 characterization of antiserum Properties

Note: IC (integrated circuit)₅₀The detection standard substance during the determination is the veratryn III.

3. Detection of polyclonal antibody specificity

Selection of antiserum IC₅₀And (4) carrying out specificity determination on the lowest quinuproot toxin III-HS-BSA-5 #. The detection method is the same as above. The concentration of the quinoa toxin III structural analogues (quinoa toxin I, rhodojaponin I and rhodojaponin III) is adjusted to 0, 1, 3, 9, 27, 81, 243 and 729 ng/mL, 3 replicates are set, and the specificity of the antibody is determined. The specificity of the antibody is expressed by cross-reactivity (CR) and is calculated by the following formula:

CR(%) =IC₅₀ (quinotoxin III)/IC₅₀(structural analogue) × 100%

The results are shown in Table 3, the veratryn III-HS-BSA-5 # can specifically recognize the veratryn III, and the cross reaction rate to other structural analogues is less than or equal to 40.7%.

TABLE 3 Cross-reactivity table of antiserum veratrum toxin III-HS-BSA-5 #

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. The quinotoxin III hapten is characterized in that the structure is as follows:

。

2. the method of producing the quinophthalone III hapten of claim 1 comprising: and (3) carrying out a light-resistant reaction on the chenopodium album toxin III and succinic anhydride at room temperature to obtain the veratrum album toxin III.

3. An artificial antigen of a quinoa toxin III, which is obtained by coupling a hapten of a quinoa toxin III according to claim 1 with a carrier protein;

wherein the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein, and human serum albumin.

4. The artificial antigen of quinugine toxin III according to claim 3, wherein the molar ratio of the artificial antigen of quinugine toxin III to the carrier protein is 23-25: 1.

5. The method of claim 3, wherein said step of coupling said carrier protein to said carboxyl carbon of said quinotoxin III hapten is performed by an activated ester method.

6. Use of the quinuproot toxin iii hapten of claim 1 or the artificial antigen of quinuproot toxin iii of claim 3 or 4 in any one of the following aspects:

the application of the quinoa toxin III specific antibody is prepared;

② the application in detecting the specific antibody of the veratryn III.

7. A polyclonal antibody specific to the wood veratryn III, which is prepared by immunizing an animal with the artificial antigen of the wood veratryn III according to claim 3 or 4 as an immunogen.

8. The use of the chenopodium album toxin iii specific polyclonal antibody of claim 7 in any one of the following aspects:

(1) the application in detecting the veratryn III;

(2) the application in the preparation of an immunochromatographic test strip of the quinugine toxin III;

(3) the application in preparing an enzyme-linked immunoassay detection kit of the quinugine toxin III.

9. A test reagent or test strip comprising the quinoxyrin III specific polyclonal antibody according to claim 7.

10. A kit comprising the chenopodium album toxin iii-specific polyclonal antibody according to claim 7.

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