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Rhodojaponin III hapten as well as preparation method and application thereof

A technology of halotoxin and hapten, applied in chemical instruments and methods, animal/human protein, serum albumin, etc.

Inactive Publication Date: 2020-07-07
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on the immunoassay method of romantoxin III in this field

Method used

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  • Rhodojaponin III hapten as well as preparation method and application thereof
  • Rhodojaponin III hapten as well as preparation method and application thereof
  • Rhodojaponin III hapten as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation and Identification of Hapten of Aminotoxin III

[0056] (1) Dissolve merinotoxin III (20 mg) and succinic anhydride (5.4 mg) in 6.0 mL of anhydrous pyridine, mix the two solutions, and place them in an oil-bath magnetic stirrer for 9 h in the dark (60 o C, 240 rpm), to obtain the reaction product. Synthetic route see figure 1 .

[0057] (2) The reaction system was quenched with water, extracted with ethyl acetate, washed with 0.1M dilute hydrochloric acid, and the organic phase was dried and concentrated.

[0058] (3) Column chromatographic separation to obtain the white target compound haptenin III hapten.

[0059] The hapten toxin III and the purified hapten were identified by liquid phase tandem high-resolution mass spectrometry. According to its synthetic route, if the chemical structure of the target compound is connected with a carbonyl imidazole group, the hapten and the hapten The exact molecular weight is increased by 100 Da compared t...

Embodiment 2

[0060] Example 2 The preparation of the artificial antigen of merinotoxin III

[0061] (1) Weigh the hapten III hapten (10 mg), N-hydroxysuccinimide (4.3 mg) and dicyclohexylcarbodiimide (8.6 mg) prepared in Example 1, respectively, and react with the glass In the bottle, add 1 mL of DMF. The glass bottle containing the reaction solution was placed on a magnetic stirrer, and reacted at room temperature for 5 h at 400 rpm in the dark.

[0062] (2) Dissolve 60 mg of BSA (OVA) in 12 mL of PBS buffer containing 10% (volume percent) DMF to obtain a protein solution.

[0063] (3) Add the liquid phase completed in step (1) dropwise to the protein solution prepared in step (2), place the reaction solution on a magnetic stirrer, and react at 4°C for 5 h.

[0064] (4) The protein activation solution in step (3) was transferred to a dialysis bag with a molecular weight cut-off of 7 KDa, and then the dialysis bag was placed in PBS buffer and dialyzed at 4 °C for 3 days (the medium was c...

Embodiment 3

[0068] Example 3 Preparation and Identification of Polyclonal Antibody to Aminotoxin III

[0069] 1. Preparation of Polyclonal Antibody to Aminotoxin III

[0070] The aminotoxin III-BSA prepared in Example 2 was used as an immunogen to immunize mice, and the aminotoxin III-OVA was used as a coating source to detect the mouse antiserum. The concentration of the complete antigen was determined by the Bradford method, and the concentrations of the immunogen and the coating agent were both 4.5 mg / mL.

[0071] For the first immunization, the immunogen was diluted to 1 mg / mL (diluted with 0.01 M PBS, pH 7.4), and the diluted immunogen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified. Spot inoculate 6-8 week old Balb / c mice (5 mice were immunized), the dose of immunogen was 100 μg / mouse, and the injection dose was 0.2 mL / mouse. Immunization was boosted every 4 weeks, and the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant...

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Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a rhodojaponin III hapten as well as a preparation method and an application thereof. The rhodojaponin III hapten has the following structure shown in the specification, by utilizing the conjugate of the rhodojaponin III hapten and the carrier protein provided by the invention, an anti-rhodojaponinIII specific antibody can be obtained by immunizing a mouse, and the antibody preparation process is simple, economic, high in sensitivity and high in practical value. The product can be used for establishing an enzyme-linked immunosorbent assay method so as to realize rapid detection of rhodojaponin III. The hapten of the rhodojaponin III disclosed by the invention has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of biochemical industry, in particular to a halotoxin III hapten and its preparation method and application. Background technique [0002] In recent years, there have been many cases of poisoning incidents caused by eating honey in the mountainous areas of some provinces and cities in southern my country, especially in the southwestern mountainous areas. Honey poisoning is mainly caused by people eating poisonous honey by mistake, and poisonous honey is mainly related to poisonous nectar plants. The Hengduan Mountains in Southwest China are very rich in nectar plants, including many poisonous nectar plants, such as poisonous rhododendron and Kunming mountain crabapple, among which the species of poisonous rhododendron is particularly rich. During the blooming period of the poisonous rhododendron, honey will collect its nectar, transfer and enrich the phytotoxins in the hive, and form poisonous honey. Usual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D303/17C07D301/00C07K14/765C07K14/795C07K14/47C07K16/44G01N33/53
CPCC07D301/00C07D303/17C07K14/47C07K14/765C07K14/77C07K14/795C07K16/44G01N33/5308
Inventor 杨术鹏李熠傅怡周金慧张金震杨宇晖黄京平金玥赵文王鹏
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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