阅读说明：本技术 一种抑霉唑半抗原ym-a、人工抗原、重链抗体及其制备方法和应用 (Imazalil hapten YM-A, artificial antigen, heavy chain antibody, and preparation method and application thereof ) 是由 沈玉栋 张咏仪 徐振林 王弘 杨金易 孙远明 肖治理 雷红涛 于 2020-04-28 设计创作，主要内容包括：本发明公开了一种抑霉唑半抗原YM-A、人工抗原、重链抗体及其制备方法和应用。本发明首先提供了一种抑霉唑半抗原YM-A,其结构式如式(I)所示：<Image he="345" wi="700" file="DDA0002471762610000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>该抑霉唑半抗原YM-A与待测物抑霉唑的骨架结构重叠度高,有效地提高了抑霉唑半抗原YM-A的免疫原性；进一步利用该抑霉唑半抗原YM-A与乳铁蛋白偶联得到的人工抗原作为免疫原免疫羊驼,将免疫效果好的羊驼血清交替使用Protein A和Protein G亲和层析柱进行分离,成功制备得到了抑霉唑重链抗体；并利用该重链抗体建立了一种基于重链抗体检测抑霉唑分析方法,对抑霉唑的最低检测限为12.39ng/mL,IC<Sub>50</Sub>为122.18ng/mL,线性范围为29.41～507.63ng/mL；且该抑霉唑半抗原YM-A、人工抗原和重链抗体的制备方法简单易行、成本低,应用前景广泛。(The invention discloses an imazalil hapten YM-A, an artificial antigen, a heavy chain antibody, a preparation method and application thereof. The invention firstly provides an imazalil hapten YM-A, the structural formula of which is shown in the formula (I): the skeleton structure overlapping degree of the imazalil hapten YM-A and the imazalil to be detected is high, and the method is effectiveThe immunogenicity of the imazalil hapten YM-A is improved; further using an artificial antigen obtained by coupling the imazalil hapten YM-A and lactoferrin as an immunogen to immunize alpaca, and separating alpaca serum with good immune effect by alternately using Protein A and Protein G affinity chromatography columns to successfully prepare an imazalil heavy chain antibody; and an analysis method for detecting imazalil based on the heavy chain antibody is established by using the heavy chain antibody, the minimum detection limit of the imazalil is 12.39ng/mL, IC 50 122.18ng/mL, and the linear range is 29.41-507.63 ng/mL; and the preparation methods of the imazalil hapten YM-A, the artificial antigen and the heavy chain antibody are simple and easy, low in cost and wide in application prospect.)

1. The imazalil hapten YM-A is characterized by having a structural formula shown in a formula (I):

2. the method for preparing imazalil hapten YM-A as claimed in claim 1, characterized in that sodium periodate and potassium permanganate are used to oxidize the allyl double bond of imazalil to aldehyde propyl, then oximation condensation derivatization reaction is carried out with carboxymethyl hydroxylamine hemihydrochloride, reflux and extraction are carried out, organic phase is taken for drying and concentration, and the concentrate is purified by silica gel column chromatography to obtain light yellow solid, namely the imazalil hapten YM-A.

3. Use of the imazalil hapten YM-A as defined in claim 1 for the preparation of an artificial imazalil antigen.

4. An imazalil artificial antigen is characterized by having a structural formula shown as a formula (II):

wherein the Protein is lactoferrin or ovalbumin.

5. A combination of an immunogen for detecting imazalil and a coating antigen, wherein the immunogen is a conjugate of imazalil hapten YM-A and lactoferrin as described in claim 1, and the coating antigen is a conjugate of imazalil hapten YM-A and ovalbumin as described in claim 1.

6. Use of the imazalil hapten YM-A as defined in claim 1, the artificial antigen of imazalil as defined in claim 4 or the combination of the immunogen and the coating antigen as defined in claim 5 for the detection of imazalil or for the preparation of an imazalil detection kit.

7. An imazalil heavy chain antibody is characterized in that the preparation method comprises the following steps: after the alpaca is immunized by the conjugate of the imazalil hapten YM-A and the lactoferrin in the claim 1, alpaca serum is separated by alternately using Protein A and Protein G affinity chromatography columns and eluted, and the imazalil heavy chain antibody is obtained.

8. A method for detecting imazalil, characterized in that a conjugate of imazalil hapten YM-A and ovalbumin as described in claim 1 is used as a coating antigen, and an imazalil heavy chain antibody as described in claim 7 is used as a detection antibody, and detection is performed by an indirect competitive ELISA method.

9. An imazalil assay kit comprising the immunogen of claim 5 in combination with a coating source.

Technical Field

The invention belongs to the technical field of biotechnology. More particularly, relates to imazalil hapten YM-A, an artificial antigen, a heavy chain antibody, and a preparation method and application thereof.

Background

Imazalil, also known as imazalil, is a systemic fungicide, has good control effects on common penicilliosis, green mold and the like in fruits and vegetables, and is widely applied to corrosion prevention and fresh preservation of various fruits and vegetables after harvesting. The research shows that if a human body takes imazalil for a long time, endocrine system dysfunction is caused, and the nervous system and the liver are also affected. Due to abuse of imazalil, the requirement of China on the residual quantity of the imazalil in food is more and more strict, and the latest national standard GB 2763-2019 has more new 2016 new varieties for detection compared with the national standard GB 2763-.

The requirements for detecting imazalil are more and more demanding, and therefore, a rapid, sensitive and efficient detection method is needed to realize rapid detection of imazalil. Currently, methods for detecting pesticide residues of imazalil comprise gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, and other quantitative detection methods; the sample pretreatment and determination processes of the gas chromatography-mass spectrometry method, the liquid chromatography-mass spectrometry combined method and other instrument analysis methods are complicated, the cost is high, professional operation is required, and the method is not suitable for screening a large number of samples; the enzyme-linked immunosorbent assay method has the advantages of high sensitivity, strong specificity, low requirements on instruments and equipment, relatively simple pretreatment of samples and the like, and is a commonly used pesticide residue detection method in recent years. The enzyme-linked immunosorbent assay realizes qualitative and quantitative analysis of a target object in a detection sample based on specific recognition between an antigen and an antibody, and is characterized in that the antibody with high specificity and high sensitivity is prepared, and the primary condition for establishing the method is to design and synthesize effective hapten and complete antigen.

At present, sunglow et al disclose an imazalil hapten HIA, an artificial antigen of imazalil is successfully prepared, a rat polyclonal antibody with the titer of 1:6400 is obtained by immunizing animals, and IC of the antibody is measured by adopting an indirect competitive ELISA method₅₀The value was 1.76. mu.g/mL (synthesis and identification of the artificial antigen of the bactericide imazalil, 2010). Although the antibody can recognize imazalil, the recognition sensitivity is low; the hapten HIA is prepared by using hydroxyl substitution reaction of the imazalil analogue through two steps of substitution and hydrolysis, and the preparation process is complex and has complicated steps. Therefore, the method for detecting imazalil has the advantages of simple steps, high detection sensitivity and strong specificity and is of great significance.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing imazalil detection method and provides an imazalil hapten YM-A, an artificial antigen, a heavy chain antibody, a preparation method and application thereof. The synthesis method of the imazalil hapten YM-A prepared by the invention is simpler, the molecular structural formula of the imazalil is directly used for modification, carbon-carbon double bonds on the molecular structural formula are oxidized into aldehyde groups, then carboxyl with a section of carbon chain is connected, and the carboxyl is directly reacted in one step to form active group-carboxyl.

The first purpose of the invention is to provide imazalil hapten YM-A.

The second purpose of the invention is to provide a preparation method of the imazalil hapten YM-A.

The third purpose of the invention is to provide the application of the imazalil hapten YM-A in preparing the imazalil artificial antigen.

The fourth purpose of the invention is to provide an artificial antigen of imazalil.

The fifth purpose of the invention is to provide a combination of immunogen and coating antigen for detecting imazalil.

The sixth purpose of the invention is to provide the application of the imazalil hapten YM-A, the imazalil artificial antigen or the combination of the immunogen and the coating antigen in the detection of the imazalil or the preparation of an imazalil detection kit.

The seventh purpose of the invention is to provide the imazalil heavy chain antibody.

An eighth object of the present invention is to provide a method for detecting imazalil.

The ninth purpose of the invention is to provide an imazalil detection kit.

The above purpose of the invention is realized by the following technical scheme:

the invention provides an imazalil hapten YM-A, which has a structural formula shown in a formula (I):

the imazalil hapten YM-A and the imazalil molecular skeleton structure have high overlapping degree, are favorable for immune induction, and effectively improve the immunogenicity of the imazalil hapten YM-A.

The invention also provides a preparation method of the imazalil hapten YM-A, which is characterized in that after allylic double bonds of the imazalil are oxidized into aldehyde propyl by using sodium periodate and potassium permanganate, oximation condensation derivatization reaction is carried out on the allylic double bonds of the imazalil and carboxymethyl hydroxylamine hydrochloride, reflux and extraction are carried out, organic phase is taken for drying and concentration, and the concentrate is purified by silica gel column chromatography to obtain light yellow solid, namely the imazalil hapten YM-A.

The method adopts the combined action of sodium periodate and potassium permanganate to carry out oxidation reaction on the imazalil, and the allyl double bond of the imazalil is oxidized into the aldehyde propyl group.

Preferably, the temperature of the oxidation reaction is 20 ℃ to 30 ℃.

More preferably, the temperature of the oxidation reaction is 20 ℃.

Preferably, the temperature of the oximation condensation derivatization reaction is 58 ℃ to 62 ℃.

More preferably, the temperature of the oximation condensation derivatization reaction is 60 ℃.

Preferably, the extraction agent for the extraction is ethyl acetate and saturated NaCl.

The application of the imazalil hapten YM-A in preparing the artificial imazalil antigen also belongs to the protection scope of the invention.

The invention also provides an imazalil artificial antigen, the structural formula of which is shown as the formula (II):

wherein the Protein is lactoferrin or ovalbumin.

When the imazalil hapten YM-A is coupled with carrier Protein (Protein), the specific structure of the imazalil hapten YM-A protrudes out of the surface of the carrier Protein, and the antigen epitope serving as a carrier is exposed to an animal immune system, so that a foundation is laid for obtaining a high-specificity and high-quality nano antibody.

The invention also provides a combination of an immunogen and a coating antigen for detecting imazalil, wherein the immunogen is a conjugate of the imazalil hapten YM-A and lactoferrin, and the coating antigen is a conjugate of the imazalil hapten YM-A and ovalbumin.

Preferably, the mole ratio of coupling of imazalil hapten YM-A and lactoferrin is 1: 58 to 62.

More preferably, the mole ratio of coupling of imazalil hapten YM-A and lactoferrin is 1: 60.

preferably, the mole ratio of coupling of imazalil hapten YM-A and ovalbumin is 1: 78-82.

More preferably, the mole ratio of the imazalil hapten YM-A to the egg white albumin is 1: 80.

the application of the imazalil hapten YM-A, the imazalil artificial antigen or the combination of the immunogen and the coating antigen in the detection of the imazalil or the preparation of an imazalil detection kit also belongs to the protection scope of the invention.

The invention also provides an imazalil heavy chain antibody, and the preparation method comprises the following steps: and (3) immunizing alpaca by using the conjugate of the imazalil hapten YM-A and lactoferrin, separating alpaca serum by alternately using Protein A and Protein G affinity chromatography columns, and eluting to obtain the imazalil heavy chain antibody.

The invention also provides a method for detecting imazalil, which takes the conjugate of the imazalil hapten YM-A and ovalbumin as a coating antigen, takes the imazalil heavy chain antibody as a detection antibody and adopts an indirect competitive ELISA method for detection.

The invention also provides an imazalil detection kit which comprises the immunogen and the coating antigen.

The invention has the following beneficial effects:

the invention firstly provides the imazalil hapten YM-A, the overlap degree of the skeleton structure of the imazalil hapten YM-A and the imazalil to be detected is high, and the immunogenicity of the imazalil hapten YM-A is effectively improved; further using an artificial antigen obtained by coupling the imazalil hapten YM-A and lactoferrin as an immunogen to immunize alpaca, and obtaining a camel source heavy chain antibody capable of specifically recognizing imazalil through serum separation;

the preparation method of the imazalil hapten YM-A, the artificial antigen and the heavy chain antibody provided by the invention is simple and easy to implement, the prepared imazalil hapten YM-A and the imazalil complete antigen can effectively stimulate immunized animals to generate heavy chain antibodies with high sensitivity and strong specificity, the minimum detection limit of the established method for detecting the imazalil is 12.39ng/mL, and IC₅₀122.18ng/mL, linear range of 29.41-507.63 ng/mL, simple and rapid process, strong specificity and high sensitivity,has good application prospect and wide development space in the rapid and effective detection of the imazalil.

Drawings

FIG. 1 is a graph of the results of UV full-wavelength scan of imazalil hapten YM-A, lactoferrin LF and imazalil artificial antigen.

FIG. 2 is a graph of the results of UV full-wavelength scans of imazalil hapten YM-A, ovalbumin OVA and imazalil artificial antigen.

FIG. 3 is a graph showing the results of SDS-PAGE identifying three subtypes in alpaca serum.

FIG. 4 is a graph of the standard indirect competition ELISA established based on heavy chain antibody.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.