CN111377888B - Rhododendrin mollis toxin III hapten as well as preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a rhodojaponin III (Rhodojaponin III) hapten as well as a preparation method and application thereof. The rhodojaponin III hapten has the following structure:

Description

Rhododendrin mollis toxin III hapten as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of biochemical engineering, and particularly relates to a rhodojaponin III hapten as well as a preparation method and application thereof.

Background

In recent years, a plurality of poisoning accidents caused by eating honey are continuously outbreaked in mountainous areas of provinces and cities in the south of China, wherein the mountainous areas in the southwest are particularly severe. The honey poisoning is mainly caused by that people eat toxic honey by mistake, and the toxic honey is mainly related to toxic honey source plants. The honey source plants of the transection mountain in the southwest region are rich, and the honey source plants comprise a plurality of toxic honey source plants, such as rhododendron and tripterygium hypoglaucum, wherein the toxic rhododendron is rich in variety. In the bloom period of toxic rhododendron, honey is collected, phytotoxins in the honey migrate and are enriched in the honeycomb, and toxic honey is formed. Generally, the problem is that the honey contains high concentration of phytotoxin, once people eat the honey, the toxic reaction occurs, and serious people even die.

It is worth mentioning that turkish and nipaler also have a honey poisoning event all the year round, mainly caused by local toxic rhododendron honey-derived plants, causing the poisoning disease commonly called "crazy honey disease". Currently, the research on toxic honey leading to "mania honey" is very detailed, mainly honey containing high concentrations of arborinoxin iii (grayanotoxin iii). However, the rhododendron has a large variety, the composition of the rhododendron in different varieties and different production places is greatly different, and the variety and the content of the main toxic compounds of the rhododendron also have great difference. At present, whether the key phytotoxin in the toxic honey in China is consistent with that reported abroad and is also the lignocelluloses III is not clear at present.

The research team discovers that rhodojaponin III (Rhodojaponin III) has high content in toxic honey and high toxicity through systematic scientific research, and is one of key phytotoxins causing the toxic honey in China. Therefore, toxic honey can be identified by detecting whether the honey contains rhodojaponin III, so that the quality safety of the honey is guaranteed. At present, the detection method of rhodojaponin III in food is mainly based on technologies such as high performance liquid chromatography or liquid phase tandem mass spectrometry, and the methods mainly perform laboratory analysis, are long in time consumption, high in cost, high in requirement on professional knowledge of inspection personnel, and difficult to meet practical requirements of scenes such as on-site rapid detection.

Immunoassay is an important method for guaranteeing food safety at present, and is popular with food safety inspectors due to the advantages of high sensitivity, strong specificity, short time consumption, low cost and the like. In immunoassay, the acquisition of high-quality antibodies is the key to the successful development of immunoassay methods, and once high-quality antibodies are prepared, various immunoassay modes and methods can be developed. In the preparation process of the specific antibody of the small molecular compound, the design and synthesis of the hapten are the core, because only the proper hapten can effectively stimulate the body to obtain the high-quality antibody. However, no research on immunoassay methods of rhodojaponin III is available in the field.

Disclosure of Invention

Technical problem to be solved

The invention aims to design an appropriate rhodojaponin III hapten, and prepare the hapten by adopting a proper chemical synthesis means, lays a foundation for obtaining a subsequent rhodojaponin III high-quality antibody, and enables the rhodojaponin III antibody to be well applied to development of a detection kit and immunodetection of rhodojaponin III.

(II) technical scheme

The invention aims to provide a rhodojaponin III hapten which has the following structure and can effectively stimulate organisms to obtain high-quality rhodojaponin III antibodies.

。

The second purpose of the invention is to provide a preparation method of the rhodojaponin III hapten, wherein the rhodojaponin III reacts with succinic anhydride at 55-65 ℃ (preferably at 60 ℃) to obtain the rhodojaponin III hapten.

The invention discovers that under the conditions, the specific hydroxyl site of the rhodojaponin III can be introduced with the spacer arm succinic anhydride to obtain the rhodojaponin III hapten required by the invention.

On the basis, the preparation method is further optimized to obtain the following scheme:

in some preferred embodiments, the reaction is carried out in pyridine; preferably, the pyridine is anhydrous pyridine.

In some preferred embodiments, the molar ratio of the rhodojaponin III hapten to the succinic anhydride is 1 (0.8-1.2), more preferably 1: 1.

in some preferred embodiments, the reaction is carried out under exclusion of light.

In some preferred schemes, the reaction time is 7-9 h.

Conventionally, the reaction temperature in the present invention can be provided by some heat-insulating device or medium, for example, the reaction can be carried out under the condition of oil bath at 55-65 ℃.

Preferably, after the reaction, a purification process is further included, and the purification process specifically comprises: extracting the reaction product with ethyl acetate, washing the organic phase with dilute hydrochloric acid, drying and concentrating the organic phase, and purifying with a chromatographic column C₁₈Separating and purifying by chromatographic column to obtain rhodojaponin III hapten.

The above-described preferred embodiments can be combined by one skilled in the art to provide preferred embodiments of the present invention.

In a preferred embodiment, the preparation method of the rhodojaponin III hapten is as follows: dissolving rhodojaponin III (20 mg) and succinic anhydride (5.4 mg) with 5.0 mL of anhydrous pyridine respectively, mixing the two reaction solutions, and placing in an oil bath magnetic stirrer for reacting for 8 h in a dark place (60 h)^oC, 240 rpm) to obtain a reaction product. And adding water into the reaction system for quenching, extracting by using ethyl acetate, washing by using dilute hydrochloric acid, drying and concentrating the organic phase, and separating and purifying by using a preparative chromatograph to obtain the target compound rhodojaponin III hapten.

In a third aspect, the invention provides a rhodojaponin III artificial antigen, which is obtained by coupling a rhodojaponin III hapten with carrier protein, wherein the carrier protein is one or more selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein and human serum albumin; bovine serum albumin or ovalbumin is preferred.

Preferably, the molar ratio of the rhodojaponin III hapten to the carrier protein is (5.5-6): 1, and more preferably 5.8: 1.

The invention further provides a preparation method of the rhodojaponin III artificial antigen, which comprises the step of coupling the rhodojaponin III hapten and carrier protein by a carbodiimide method, an active ester method or a mixed anhydride method. The active ester method is preferred.

Preferably, the preparation method of the artificial antigen of rhodojaponin III comprises the following steps:

(1) the molar ratio of the raw materials is 1: (1.8-2.2): (1.8-2.2) weighing the rhodojaponin III hapten,N-hydroxysuccinimide and dicyclohexylcarbodiimide, withN,NDissolving dimethylformamide to obtain a reaction solution, reacting the reaction solution at 350-400 rpm at room temperature in a dark place for 5 h, centrifuging, and taking a supernatant to obtain a rhodojaponin III hapten activation solution;

dissolving the carrier protein in Phosphate Buffer Solution (PBS) to obtain a carrier protein solution with the final concentration of 4.5-5.5 mg/mL;

(2) dropping the rhodojaponin III hapten activated liquid into the carrier protein solution to enable the molar ratio of the rhodojaponin III hapten to the carrier protein to be (28-32): 1, 4^oC stirring overnight.

In the step (1), the preparation processes of the rhodojaponin III hapten activation solution and the carrier protein solution are not in sequence and can be adjusted according to actual conditions.

Preferably, after the step (2), the method further comprises:

(3) and (3) putting the reaction solution obtained in the step (2) into a dialysis bag, dialyzing for 3 days by using a phosphate buffer solution, and centrifuging to obtain a supernatant, thus obtaining the rhodojaponin III artificial antigen.

The artificial antigen is divided into immunogen and coating antigen, wherein the immunogen is rhodojaponin III-BSA, and the coating antigen is rhodojaponin III-OVA.

In a fourth aspect, the invention provides an application of the rhodojaponin III hapten or the rhodojaponin III artificial antigen in any one of the following aspects:

the application of the rhodojaponin III specific antibody is realized;

② the application in detecting anti-rhodojaponin III specific antibody.

Furthermore, the invention provides an anti-rhodojaponin III antibody, which is prepared by immunizing animals with the rhodojaponin III artificial antigen as an immunogen; the antibodies include polyclonal antibodies and monoclonal antibodies.

The animal can be Balb/c mouse, New Zealand white rabbit, sheep, etc., preferably Balb/c mouse.

Preferably, when the rhodojaponin III artificial antigen is obtained by coupling the hapten and bovine serum albumin, the immune effect is better.

In a fifth aspect, the invention provides the use of the anti-rhodojaponin III antibody in any one of the following aspects:

(1) application in detecting rhodojaponin III;

(2) the application in the preparation of the immunochromatographic test strip of rhodojaponin III;

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

Furthermore, the invention provides a detection reagent or detection test paper, which contains the anti-rhodojaponin III antibody.

(III) advantageous effects

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

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

(2) The rhodojaponin III artificial antigen provided by the invention can keep the chemical structure of rhodojaponin III unchanged, and is exposed on the surface of protein to serve as an antigenic determinant, thereby laying a foundation for preparing a high-sensitivity anti-rhodojaponin III antibody.

(3) The rhodojaponin III antibody (especially polyclonal antibody) prepared by using the rhodojaponin III artificial antigen provided by the invention has the advantages of simple and economic preparation process, highest antibody titer of 32000, detection sensitivity of 5.2 ng/mL, high practical value and good application prospect in public health safety detection.

Drawings

FIG. 1 is a scheme showing the synthesis of the rhodotoxin III hapten of example 1.

FIG. 2 is a mass spectrum of rhodojaponin III hapten as in example 1.

FIG. 3 is a MALDI-TOF-MS chart of rhodojaponin III-BSA in example 2.

FIG. 4 is a standard graph of antiserum # 2 detected rhodojaponin III in example 3.

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. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 preparation and characterization of Rhododendrin III hapten

(1) Dissolving rhodojaponin III (20 mg) and succinic anhydride (5.4 mg) with 6.0 mL of anhydrous pyridine, mixing the two solutions, and placing the mixture in an oil bath magnetic stirrer to react for 9 h in a dark place (60 h)^oC，240 rpm) to obtain a reaction product. The synthetic route is shown in figure 1.

(2) The reaction was quenched with water, extracted with ethyl acetate, washed with 0.1M dilute hydrochloric acid and the organic phase was concentrated by drying.

(3) Separating by column chromatography to obtain white target compound rhodojaponin III hapten.

And identifying the rhodojaponin III and the purified hapten by adopting liquid phase tandem high-resolution mass spectrometry, wherein according to the synthetic route, if succinic anhydride is connected in the chemical structure of the target object, the accurate molecular weight of the hapten is increased by 100 Da compared with that of the rhodojaponin III. Rhododendrin III in ESI negative modem/zIs 367.21261 ([ M-H)]^-) The element composition of which is C₂₀H₃₁O₆ ^-(ii) a Rhododendrin mollis toxin III haptenm/zIs 467.22811 ([ M-H)]^-) (FIG. 2) having an element composition of C₂₄H₃₅O₉ ^-. The precise molecular weight of the hapten is increased by 100 Da (C) compared with that of rhodojaponin III₄H₄O₃) The result shows that succinic anhydride is connected to the chemical structure of the target object, which indicates that the rhodojaponin III hapten is successfully synthesized and can be used for preparing antibodies.

Example 2 preparation of Rhododendrin III Artificial antigen

(1) The rhodotoxin III hapten (10 mg), N-hydroxysuccinimide (4.3 mg) and dicyclohexylcarbodiimide (8.6 mg) prepared in example 1 were weighed in a glass reaction flask, and 1 mL of DMF was added thereto. The glass bottle containing the reaction solution was placed on a magnetic stirrer and reacted at 400 rpm for 5 hours at room temperature in the dark.

(2) 60 mg BSA (OVA) was dissolved in 12 mL PBS containing 10% (volume percent) DMF to obtain a protein solution.

(3) And (3) dropwise adding the liquid phase which completes the step (1) into the protein solution prepared in the step (2), placing the reaction solution into a magnetic stirrer, and reacting for 5 hours at 4 ℃.

(4) Transferring the protein activating solution obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 7 KDa, and then putting the dialysis bag into PBS buffer for dialysis for 3 days at 4 ℃ (changing every 12 h).

(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 rhodojaponin III-BSA solution.

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

The immunogen rhodojaponin III-BSA is identified by MALDI-TOF-MS (shown in figure 3), the coupling molar ratio of the obtained carrier protein BSA to the rhodojaponin III hapten is 1:5.8, and the result proves that the immunogen is successfully synthesized and can be used for immunizing mice and preparing monoclonal antibodies.

Example 3 preparation and characterization of Rhododendrin III polyclonal antibodies

Preparation of polyclonal antibody of rhodojaponin III

The rhodojaponin III-BSA prepared in example 2 was used as immunogen to immunize mice, and rhodojaponin III-OVA was used as coating antigen to detect mouse antiserum. The concentration of complete antigen was measured by the Bradford method and both immunogen and coating antigen were measured to be 4.5 mg/mL.

Diluting the immunogen to 1 mg/mL (diluting with PBS (0.01M and pH 7.4)) during the first immunization, taking the diluted immunogen, mixing the diluted immunogen with Freund's complete adjuvant in equal volume, fully emulsifying, and inoculating Balb/c mice (5 mice are immunized) with 6-8 weeks old subcutaneously at multiple points on the back of the neck, wherein the dose of the inoculated immunogen is 100 mu g/mouse, and the injection dose is 0.2 mL/mouse. Boosts were 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 3.

Second, detection of mouse polyclonal antibody

Blood is collected from the eyeballs of the mice one week after the 3 rd boosting immunization, 5 antiserum (namely, polyclonal antibody) are obtained through centrifugation at 3000 rpm, and the antiserum is named according to the numbers of 1# -5 #. 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 is detected by an indirect ELISA method, and the specific steps are as follows:

(1) coating: the coated rhodojaponin III-OVA was diluted to 0.3. mu.g/mL with carbonate buffer (0.05M, pH 9.6), added to a 96-well clear plate (100. mu.L/well), incubated at 37 ℃ for 2 h in an incubator, and the plate was washed 3 times with PBST buffer (0.05% Tween-20 PBS, pH 7.4).

(2) And (3) sealing: add blocking solution (5% skim milk) 150. mu.L/well, incubate at 37 ℃ for 1 h, discard blocking solution, wash with PBST buffer 1 time, pat dry.

(3) Adding the antibody to be detected: mu.L of 0.01M PBS (pH 7.4) was added to each well, and 50. mu.L of the diluted anti-rhodotoxin III polyclonal antibody was added, starting from 1:2000, diluted with 0.01M PBS in a 2-fold gradient, for a total of 8 gradients. The sample addition was 50. mu.L/well, 37^oC incubator reaction for 30 min, PBST buffer washing 3 times, pat dry.

At the same time, the non-immunized mouse antiserum was set as a negative control.

(4) Adding an enzyme-labeled secondary antibody: adding HRP-labeled goat anti-mouse IgG antibody diluted by enzyme-labeled secondary antibody diluent according to the volume ratio of 1:5000, reacting at the temperature of 37 ℃ in an incubator for 30 min, washing with PBST buffer solution for 3 times, and patting to dry.

(5) Color development: mixing horseradish peroxidase substrate 3,3 ', 5, 5' -tetramethylbenzidine solution and 30% hydrogen peroxide by mass according to a volume ratio of 1:1, adding into a microplate (100 muL/hole), and developing at 37 ℃ for 15 min.

(6) And (4) terminating: 50 μ L of 2 mol/L concentrated sulfuric acid was added to each well.

(7) Reading: by OD₄₅₀Wavelength measurement of each well OD value. The negative OD value is less than or equal to 0.15, and the corresponding antibody dilution with the maximum OD value between 1.5 and 1.8 is taken as the antibody titer. The optimal dilution of antiserum is shown in table 1, and the data in the table show that the dilution of all antiserum is 1:8000 or more, which indicates that the mouse immunized by the synthetic hapten-conjugated carrier protein can obtain better immunization effect. The titer of antiserum No. 2 was the highest among them, and was1:32000。

2. Polyclonal antibody IC₅₀Measurement of (2)

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

(2) Adding standard substances and antibodies: add 50. mu.L of rhodojaponin III standard solution and 50. mu.L of diluted antibody solution (diluted according to the antibody titers in Table 1) to each well, incubate for 30 min at 37 ℃ and then wash 3 times with PBST solution and pat dry. The solvent of the standard solution was PBS buffer, and the concentrations of the standard were 0.25, 0.75, 2.25, 6.75,20.25, 60.75 and 182.25 ng/mL solutions, three for each concentration.

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

The measured data are plotted on the abscissa as the-log 10 (competitor) value and on the OD₄₅₀The value is the ordinate, the four parameter equation in origin8.0 software is used for fitting, a standard curve is established to obtain IC₅₀The value is obtained. IC of 5 polyclonal antibodies₅₀The values are shown in Table 1, and the data in the table show the IC of the 2# antiserum₅₀And the lowest. The standard curve chart of the detection of rhodojaponin III by the 2# antiserum is shown in FIG. 4, and the linear detection range (IC) of rhodojaponin III by the 2# antiserum can be seen from the graph₂₀-IC₈₀) 1.08-29.2 ng/mL, IC₅₀It was 5.2 ng/mL. The result shows that the hapten structure designed in the invention is used as an antigenic determinant, which can stimulate mice to generate high-sensitivity antibodies.

TABLE 1 characterization of antiserum Properties

Note: the detection standard when IC50 is determined is rhodojaponin III.

3. Detection of polyclonal antibody specificity

Selection of antiserum IC₅₀The lowest 2# antiserum was tested for specificity. The detection method is the same as above. The curve concentration of rhodojaponin III structural analogs (rhodojaponin I, quinoa toxin III, quinoa toxin I) is adjusted to 0, 1, 3, 9, 27,81. 243, 729 ng/mL, 3 replicates were set, and the specificity of the antibody was determined. The specificity of the antibody is expressed by cross-reactivity (CR) and is calculated by the following formula: CR (%) = IC₅₀(Rhododendrin III)/IC₅₀(structural analogue) × 100%

The results are shown in Table 2, 2# antiserum can specifically recognize rhodojaponin III, and the cross reaction rate of the rhodojaponin III to other structural analogues is less than 40.3%.

TABLE 2 Cross-reactivity Table of antiserum No. 2

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. A rhodojaponin III hapten, which has the following structure:

。

2. the method for preparing the rhodojaponin III hapten as claimed in claim 1, wherein the rhodojaponin III hapten is obtained by reacting the rhodojaponin III with succinic anhydride at 55-65 ℃.

3. The preparation method according to claim 2, wherein the molar ratio of the rhodojaponin III to the succinic anhydride is 1 (0.8-1.2);

and/or, the reaction is carried out under the condition of keeping out light;

and/or the reaction time is 7-9 h.

4. An artificial antigen of rhodojaponin III, which is obtained by coupling the hapten of rhodojaponin III as claimed in claim 1 with a carrier protein;

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

5. The artificial antigen of rhodojaponin III according to claim 4, wherein the molar ratio of the rhodojaponin III hapten to the carrier protein is (5.5-6): 1.

6. The use of the artificial antigen of rhodojaponin III according to claim 4 or 5 in any one of the following aspects:

the application of the rhodojaponin III specific antibody is realized;

② the application in detecting anti-rhodojaponin III specific antibody.

7. An anti-rhodojaponin III antibody, which is prepared by immunizing animals with the rhodojaponin III artificial antigen of claim 4 or 5 as an immunogen; the anti-rhodojaponin III antibody is a polyclonal antibody.

8. The anti-rhodojaponin III antibody of claim 7, wherein the rhodojaponin III artificial antigen is derived from the hapten of claim 1 coupled to bovine serum albumin.

9. The anti-rhodojaponin III antibody of claim 7 or 8 for use in any one of:

(1) application in detecting rhodojaponin III;

(2) the application in the preparation of the immunochromatographic test strip of rhodojaponin III;

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

10. A detection reagent or test strip comprising the anti-rhodojaponin III antibody according to claim 7 or 8.

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