阅读说明：本技术 柚皮素抗原及其制备方法与应用 (Naringenin antigen and preparation method and application thereof ) 是由 崔永亮 焦必宁 赵静 赵其阳 张耀海 陈爱华 王成秋 何悦 于 2019-11-04 设计创作，主要内容包括：本发明公开了一种柚皮素抗原及其制备方法与应用,将柚皮素与同时含有氨基和羧基的化合物B进行反应得到化合物C；化合物C和N-羟基琥珀酰亚胺在二环己基碳二亚胺或1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐存在的条件下进行偶联反应得到化合物D；化合物D与载体蛋白经偶联反应得柚皮素抗原；柚皮素抗原在制备用于检测样品中柚皮素的酶联免疫试剂盒、柚皮素的发光免疫试剂盒或免疫亲和色谱柱中的应用。发明提供的制备柚皮素抗原的方法,能够方便、快捷地获得柚皮素抗原,合成步骤简洁明了、合成成本低,效果好。用本发明方法制备的柚皮素抗原进行免疫得到的抗体的特异性好、最低检测限值低。为柚皮素的快速免疫检测应用中将有广阔的前景。(The invention discloses a naringenin antigen and a preparation method and application thereof, wherein naringenin reacts with a compound B containing amino and carboxyl at the same time to obtain a compound C; carrying out coupling reaction on the compound C and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a compound D; coupling reaction is carried out on the compound D and carrier protein to obtain naringenin antigen; the naringenin antigen is applied to the preparation of an enzyme linked immunosorbent kit, a luminous immunoassay kit or an immunoaffinity chromatographic column for detecting naringenin in a sample. The method for preparing the naringenin antigen can conveniently and quickly obtain the naringenin antigen, and has the advantages of concise and clear synthesis steps, low synthesis cost and good effect. The antibody obtained by immunizing the naringenin antigen prepared by the method has good specificity and low minimum detection limit. Has wide prospect in the rapid immunodetection application of naringenin.)

1. A naringenin antigen characterized by: the structural general formula is shown as a formula A,

in the formula A, X is O, S, CH2 or NH, and n is an integer of 0-6; protein represents a carrier Protein selected from at least one of bovine serum albumin, ovalbumin and hemocyanin.

2. The method for preparing naringenin antigen of claim 1, comprising the steps of:

(1) reacting naringenin with a compound which is shown in a formula B and contains amino and carboxyl to obtain a compound shown in a formula C;

H₂N-X-(CH₂)n-COOH

structural formula B

In the formulas B and C, X is O, S, CH2 or NH, and n is an integer of 0-6;

(2) carrying out coupling reaction on the compound shown in the formula C and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a compound shown in a formula D;

in the formula D, X is O, S, CH2 or NH, and n is an integer of 0-6;

(3) coupling reaction is carried out on the compound shown in the formula D and carrier protein to obtain the naringenin antigen shown in the formula A; the carrier protein is at least one selected from bovine serum albumin, ovalbumin and hemocyanin.

3. The method of claim 2, wherein: in the step (1), the compound shown in the formula B is at least one selected from carboxymethoxylamine, hydrazinoacetic acid, aminoacetic acid, aminopropionic acid, aminobutyric acid, aminopentanoic acid and aminocaproic acid.

4. The method of claim 2, wherein: in the step (1), the feeding molar ratio of the compound shown in the formula B to naringenin is (0.1-10): 1;

the reaction temperature is 0-100 ℃, and the reaction time is 6-48 hours;

the solvent for the reaction is at least one selected from pyridine, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.

5. The method of claim 2, wherein: in the step (2), the molar part ratio of the compound shown in the formula C, N-hydroxysuccinimide and dicyclohexylcarbodiimide is 1 (1-5) to 1-5;

the molar ratio of the compound shown in the formula C to the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride is 1 (1-5) to 1-5;

the temperature of the coupling reaction is 0-50 ℃, and the time is 4-24 hours.

6. The method of claim 2, wherein: in the step (3), the molar part ratio of the compound shown in the formula D to the carrier protein is (5-100): 1;

the temperature of the coupling reaction is 0-50 ℃, and the time is 8-36 hours; the coupling reaction is carried out under the condition that the pH value is 4-11;

and (2) carrying out coupling reaction on the compound shown in the formula D in a carrier protein solution, wherein the carrier protein solution is obtained by adding the carrier protein into a buffer solution, the buffer solution is at least one selected from carbonate buffer, phosphate buffer, borate buffer and 4-hydroxyethyl piperazine ethanesulfonic acid buffer, and the pH value of the buffer solution is 7.4.

7. The method of claim 2, wherein: the method further comprises a step of dialyzing the reaction system of the coupling reaction; in the dialysis step, the used dialysate is phosphate buffer solution with the pH value of 4-10 and the concentration of 0.01-0.2 mol/L.

8. The method of claim 2, wherein: the naringenin antigen is a conjugate formed by connecting naringenin and carrier protein through an amido bond; the amido bond is formed by carboxyl on the formula C and amino on the carrier protein through active ester.

9. Use of the naringenin antigen of claims 1-8 wherein the use comprises: the application of the naringenin in detecting samples, the application of the naringenin in preparing enzyme linked immunosorbent assay kits for detecting the naringenin in the samples, and the application of the naringenin in preparing luminescent immunoassay kits or immunoaffinity chromatographic columns for detecting the naringenin in the samples.

10. The method of application according to claim 9, characterized in that: the detection sample is water, medicine, food or soil.

Technical Field

The invention belongs to the technical field of biology, and relates to a preparation method of a naringenin antigen, the naringenin antigen prepared by the preparation method and application thereof.

Background

Naringenin is aglycone of naringin, widely exists in rutaceae plants and is commonly known as Naringenin; the chemical name is as follows: 4', 5, 7-trihydroxyflavone; the trade name is: naringin, naringenin; CAS registry number 480-41-1; the molecular formula is: c₁₅H₁₂O₅(ii) a The relative molecular weight is: 272.25. the chemical structural formula is as follows:

naringenin (Naringin) has estrogen-like activity, and thus has the effect of promoting endothelial cells to secrete NO; it can reduce oxidative damage of cells; it can effectively reduce the damage of retina function and tissue structure, improve visual function; naringenin also has obvious effects of relieving cough, eliminating phlegm and relieving asthma; naringenin can directly inhibit activation of T lymphocyte, thereby preventing secretion of various inflammatory factors and further having protective effect on immunological acute liver injury.

At present, naringenin extraction methods mainly comprise a reflux method, an organic solvent method, a solid phase extraction method and an ultrasonic extraction method. The naringenin content analysis method comprises a spectrophotometric method, a High Performance Liquid Chromatography (HPLC), an Ultra Performance Liquid Chromatography (UPLC), an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), a reversed phase high performance liquid chromatography-diode array detection method (HPLC-DAD), a capillary zone electrophoresis method, an RP-HPLC method and the like, and has high sensitivity and good accuracy. Compared with an instrumental analysis method, the immunoassay method has the advantages of rapidness, simplicity, convenience, real-time performance, easiness in field detection, simplicity in sample pretreatment, high sensitivity, strong selectivity, suitability for high-throughput analysis and the like, and can also greatly reduce the detection cost.

Based on the analysis, a naringenin antigen which can rapidly, simply and conveniently carry out on-site detection on naringenin in real time, has high sensitivity and strong selectivity and is suitable for high-throughput analysis is urgently needed in the industry at present.

Disclosure of Invention

In view of the defects, the invention provides a method for preparing a naringenin antigen which has high sensitivity and strong selectivity and is suitable for high-throughput analysis, the naringenin antigen prepared by the method and application thereof.

The invention is realized by the following means:

the naringenin antigen provided by the invention has a structural general formula shown in formula A,

in the formula A, X is O, S, CH2 or NH, and n is an integer of 0-6; protein represents a carrier Protein selected from at least one of bovine serum albumin, ovalbumin and hemocyanin.

The invention provides a method for preparing naringenin antigen (formula A), which comprises the following steps:

(1) reacting naringenin with a compound which is shown in a formula B and contains amino and carboxyl to obtain a compound shown in a formula C;

H₂N-X-(CH₂)n-COOH

structural formula B

In the formulas B and C, X is O, S, CH2 or NH, and n is an integer of 0-6;

(2) carrying out coupling reaction on the compound shown in the formula C and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a compound shown in a formula D;

in the formula D, X is O, S, CH2 or NH, and n is an integer of 0-6;

(3) coupling reaction is carried out on the compound shown in the formula D and carrier protein to obtain the naringenin antigen shown in the formula A; the carrier protein is at least one selected from bovine serum albumin, ovalbumin and hemocyanin.

Further, in the step (1), the compound shown in the formula B is at least one selected from carboxymethoxyamine, hydrazinoacetic acid, aminoacetic acid, aminopropionic acid, aminobutyric acid, aminopentanoic acid and aminocaproic acid.

Further, in the step (1), the feeding molar ratio of the compound shown in the formula B to naringenin is (0.1-10): 1

The reaction temperature is 0-100 ℃, and the reaction time is 6-48 hours;

the solvent for the reaction is at least one selected from pyridine, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.

Further, in the step (2), the molar part ratio of the compound shown in the formula C, the N-hydroxysuccinimide and the dicyclohexylcarbodiimide is 1: (1-5): (1-5);

the molar part ratio of the compound shown as the formula C to the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride is 1: (1-5): (1-5);

the temperature of the coupling reaction is 0-50 ℃, and the time is 4-24 hours.

Further, in the step (3), the molar part ratio of the compound shown in the formula D to the carrier protein is (5-100): 1;

the temperature of the coupling reaction is 0-50 ℃, and the time is 8-36 hours; the coupling reaction is carried out under the condition that the pH value is 4-11;

carrying out coupling reaction on the compound shown in the formula D in a carrier protein solution, wherein the carrier protein solution is obtained by adding the carrier protein into a buffer solution, the buffer solution is selected from at least one of carbonate buffer solution, phosphate buffer solution, borate buffer solution and 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, and the pH value of the buffer solution is 7.4;

further, the method further comprises the step of dialyzing the reaction system of the coupling reaction; in the dialysis step, the used dialysate is phosphate buffer solution with the pH value of 4-10 and the concentration of 0.01-0.2 mol/L.

The naringenin antigen prepared by the method also belongs to the protection scope of the invention. The naringenin antigen is a conjugate formed by connecting naringenin and carrier protein through amido bond; the amido bond is formed by carboxyl on the formula C and amino on the carrier protein through active ester.

The antibody obtained from the naringenin antigen provided by the invention, the application of the antigen and/or the antibody in detecting naringenin in a sample, the application in preparing an enzyme linked immunosorbent kit for detecting naringenin in the sample, a chemiluminescence immunoassay kit for naringenin or an immunoaffinity chromatographic column belong to the protection scope of the invention. Wherein the detection sample is water, medicine, food or soil.

The invention has the beneficial effects that:

the method for preparing the naringenin antigen can conveniently and quickly obtain the naringenin antigen, and has the advantages of concise and clear synthesis steps, low synthesis cost and good effect. The antibody obtained by immunizing the naringenin antigen prepared by the method has good specificity and low minimum detection limit. The method for preparing the naringenin antigen and the naringenin antigen obtained by the method have wide prospects in the application of rapid immunodetection of naringenin.

Drawings

FIG. 1 is a scheme of the synthesis of naringenin antigen;

FIG. 2 is a standard curve of the established naringenin indirect ELISA method.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

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

Dicyclohexylcarbodiimide (DCC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) Freund's complete adjuvant, Freund's incomplete adjuvant, bovine serum albumin and ovalbumin were purchased from Sigma, goat anti-mouse IgG-HRP from Jackson, o-phenylenediamine (OPD), carboxymethoxylamine hemihydrochloride, naringenin and other conventional reagents were purchased from Shanghai Arlatin Biotechnology Ltd.