阅读说明：本技术 S-腺苷同型半胱氨酸人工完全抗原、制备方法及其应用 (S-adenosyl homocysteine artificial complete antigen, preparation method and application thereof ) 是由 马岚 刘丹 吴峰 岑瑜 于 2020-05-09 设计创作，主要内容包括：本发明提供了一种S-腺苷同型半胱氨酸人工完全抗原的制备方法,包括以下步骤：将S-腺苷同型半胱氨酸与二碳酸二叔丁基酯混合,得到一第一中间体,所述二碳酸二叔丁基酯用于保护S-腺苷同型半胱氨酸中的抗原表位；将载体蛋白加入到所述第一中间体中,反应后得到一第二中间体；以及去除所述第二中间体中的所述二碳酸二叔丁基酯,从而得到所述S-腺苷同型半胱氨酸人工完全抗原。该方法制备出的S-腺苷同型半胱氨酸人工完全抗原具有较高特异性、选择性和免疫原性。本发明还提供一种该方法制备的S-腺苷同型半胱氨酸人工完全抗原以及该S-腺苷同型半胱氨酸人工完全抗原在抗体制备上的应用。(The invention provides a preparation method of an artificial complete antigen of S-adenosyl homocysteine, which comprises the following steps: mixing S-adenosyl homocysteine with di-tert-butyl dicarbonate to obtain a first intermediate, wherein the di-tert-butyl dicarbonate is used for protecting an epitope in the S-adenosyl homocysteine; adding carrier protein into the first intermediate, and reacting to obtain a second intermediate; and removing the di-tert-butyl dicarbonate in the second intermediate, thereby obtaining the S-adenosylhomocysteine artificial complete antigen. The S-adenosyl homocysteine artificial complete antigen prepared by the method has higher specificity, selectivity and immunogenicity. The invention also provides the S-adenosyl homocysteine artificial complete antigen prepared by the method and the application of the S-adenosyl homocysteine artificial complete antigen in antibody preparation.)

1. A preparation method of an artificial complete antigen of S-adenosyl homocysteine is characterized by comprising the following steps:

mixing S-adenosyl homocysteine with di-tert-butyl dicarbonate to obtain a first intermediate, wherein the di-tert-butyl dicarbonate is used for protecting an epitope in the S-adenosyl homocysteine;

adding carrier protein into the first intermediate, and reacting to obtain a second intermediate; and

removing the di-tert-butyl dicarbonate in the second intermediate to obtain the S-adenosylhomocysteine artificial complete antigen.

2. The method for preparing an artificial complete antigen of S-adenosylhomocysteine according to claim 1, characterized in that the epitope comprises an alpha-amino group.

3. The method of preparing an artificial complete antigen of S-adenosylhomocysteine according to claim 1, wherein the carrier protein has amino groups on its surface, and after adding the carrier protein to the first intermediate, further comprising:

adding a cross-linking agent to the first intermediate;

wherein the first intermediate and the carrier protein are subjected to a cross-linking reaction through the cross-linking agent, so that the second intermediate is obtained.

4. The method of claim 3, wherein the cross-linking agent comprises glutaraldehyde.

5. The method of claim 1, wherein the carrier protein comprises at least one of bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, and human serum albumin.

6. The method for preparing an artificial complete antigen of S-adenosylhomocysteine as claimed in claim 1 wherein the reagent used to remove said di-tert-butyl dicarbonate comprises trifluoroacetic acid.

7. The method of claim 6, wherein the reagent for removing di-tert-butyl dicarbonate further comprises silica gel.

8. The method of claim 1, wherein the first intermediate is located on the surface of the carrier protein.

9. An artificial complete antigen of S-adenosyl homocysteine prepared by the method of preparing an artificial complete antigen of S-adenosyl homocysteine according to any of claims 1 to 8.

10. Use of an artificial complete antigen of S-adenosylhomocysteine according to claim 9 in the preparation of antibodies.

Technical Field

The invention relates to the technical field of organic chemistry and immunology, in particular to an artificial complete antigen of S-adenosyl homocysteine, a preparation method and application thereof.

Background

In an organism, the activated methide S-adenosylmethionine (SAM) transfers methyl groups to various protein or nucleic acid molecules through enzyme catalysis, and thus forms corresponding methylation products, which are called methylation of the organism. The SAM with the methyl removed generates S-Adenosyl-L-homocysteine (SAH), and the SAM with the methyl removed generates homocysteine (Hcy) after further hydrolysis, and the process is the only way for generating the Hcy by the currently known vertebrates.

Methylation is an important biochemical reaction ubiquitous in organisms, is closely related to the regulation of genes, organism metabolism, aging, diseases, hormones and other life processes, and quantitative indexes of the methylation are also important physiological indexes of various diseases such as Alzheimer's Disease (AD), arthritis, Parkinson's Disease (PD), systemic lupus erythematosus, tumors, even depression, schizophrenia and the like. Meanwhile, the high blood Hcy content is of great significance in clinical medicine as an independent risk factor of cardiovascular and cerebrovascular diseases. Therefore, the SAH and Hcy can be detected quickly, conveniently and accurately, and the method has important scientific value and wide market prospect. The traditional detection methods mainly comprise a High Performance Liquid Chromatography (HPLC), a mass spectrometry method, an electrochemical method, a fluorescence polarization method (FPIA), a capillary electrophoresis method and the like, and the methods are time-consuming, labor-consuming, high in cost and seriously dependent on various large-scale equipment and operators with professional skills. Although the cyclic enzyme method is relatively simple, the reaction is complicated, the procedure is complex, instantaneous direct detection of target molecules cannot be realized, and the development of a rapid detection reagent is more difficult.

However, SAH is a small molecule substance belonging to a hapten, has no immunogenicity, cannot directly immunize an animal to obtain a corresponding antibody, and needs to be coupled with a suitable carrier protein to prepare a complete antigen having both immunogenicity and immunoreactivity. In theory, haptens are monovalent antigens, i.e.: there is only one epitope on the molecule. Thus, to obtain a highly effective and specific antibody, it is necessary to protect the critical epitopes when they are attached to the carrier protein and to expose them completely to the surface of the carrier, only to allow recognition and sufficient stimulation of the BCR receptor of B cells. When the small molecule has a carboxyl group, it is usually attached to the surface of the carrier protein by condensation with an amidation agent and an amino group on the surface of the carrier. Although this method has few by-products and high efficiency, the prerequisite is that the carboxyl group used for coupling is not an epitope of the antigen. The SAH molecule can be regarded as a Hcy molecule combined with adenosine through-S-C-bond, wherein only one carboxyl is positioned in a homocysteine part, and the carboxyl is a key group of an epitope, for example, if an antibody capable of simultaneously responding to the SAH molecule and the Hcy molecule is to be obtained, any group at the Hcy end is intact. If the carrier is directly used for connecting, the immune specificity of the synthetic antigen and the specificity of the subsequent generated antibody are certainly greatly influenced. However, the key problem is ignored in the existing preparation research of only a few SAH and Hcy monoclonal antibodies, the optimized and reasonable antigen preparation process based on the SAH molecular structure is lacked, and the SAH antigen and antibody with high quality and high efficiency cannot be stably produced.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing an artificial complete antigen of S-adenosyl homocysteine, which can effectively prevent antigen epitopes from being damaged by reaction in the subsequent preparation process.

In addition, the artificial complete antigen of S-adenosyl homocysteine prepared by the preparation method of the artificial complete antigen of S-adenosyl homocysteine is also needed to be provided.

In addition, the application of the S-adenosylhomocysteine artificial complete antigen in antibody preparation is also needed to be provided.

The invention provides a preparation method of an artificial complete antigen of S-adenosyl homocysteine, which comprises the following steps:

mixing S-adenosyl homocysteine with di-tert-butyl dicarbonate to obtain a first intermediate, wherein the di-tert-butyl dicarbonate is used for protecting an epitope in the S-adenosyl homocysteine;

adding carrier protein into the first intermediate, and reacting to obtain a second intermediate; and

removing the di-tert-butyl dicarbonate in the second intermediate to obtain the S-adenosylhomocysteine artificial complete antigen.

The invention also provides the S-adenosyl homocysteine artificial complete antigen prepared by the preparation method of the S-adenosyl homocysteine artificial complete antigen.

The invention also provides an application of the S-adenosyl homocysteine artificial complete antigen in antibody preparation.

The method comprises the steps of firstly protecting key alpha-amino on an S-adenosyl homocysteine SAH molecule by a di-tert-butyl dicarbonate (Boc) reagent so as to shield key antigenic sites of the SAH molecule, thereby preparing a first Boc-SAH intermediate. The first intermediate can effectively prevent the key epitope of the antigen from being damaged by reaction in the subsequent process of preparing the complete antigen, and simultaneously can selectively enable the primary amino group at the adenosine end of the SAH molecule to become a reaction site coupled with the carrier protein. The Boc-SAH-BSA second intermediate was then prepared by coupling the amino group at the adenosine terminus of the SAH molecule with a carrier protein (BSA) via glutaraldehyde. And finally, removing protection from the alpha-amino protected by the Boc, and repairing a key antigen epitope of the alpha-amino protected by the Boc, thereby preparing the S-adenosylhomocysteine artificial complete antigen with higher specificity, selectivity and immunogenicity.

Drawings

FIG. 1 is a flow chart of the preparation of the artificial complete antigen of S-adenosyl-homocysteine according to the preferred embodiment of the present invention.

FIG. 2 shows the molecular formula of the first intermediate according to the preferred embodiment of the present invention.

FIG. 3 shows the result of ELISA competition assay of SAH complete antigen and SAH standard prepared by the preferred embodiment of the present invention.

FIG. 4 shows the result of ELISA competition assay of SAH complete antigen and Hcy standard prepared by the preferred embodiment of the present invention.

FIG. 5 is a Fourier infrared identification map of the SAH complete antigen prepared in the preferred embodiment of the present invention.

FIG. 6 is a concentration-absorbance standard curve of SAH fitted.

FIG. 7 is a concentration-absorbance standard curve of SAH fitted.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Referring to fig. 1, a preferred embodiment of the present invention provides a method for preparing an artificial complete antigen of S-adenosyl homocysteine, comprising the following steps:

and step S11, mixing the S-adenosyl homocysteine with di-tert-butyl dicarbonate to obtain a first intermediate, wherein the di-tert-butyl dicarbonate is used for protecting the epitope in the S-adenosyl homocysteine.

Wherein an epitope in S-adenosylhomocysteine (SAH) is protected by di-tert-butyl dicarbonate (Boc). As shown in FIG. 2, the SAH molecule is homocysteine (Hcy) molecule and adenosine are combined through-S-C-bond, and only one carboxyl group in the SAH molecule is positioned in homocysteine part and forms the characteristic epitope together with the adjacent alpha-amino group. The α -amino group must not react during subsequent antigen preparation. Thus, the α -amino group of the cysteine moiety of the SAH molecule is selectively protected by Boc. Wherein, the first intermediate can be represented as Boc-SAH.

And step S12, adding the carrier protein into the first intermediate, and obtaining a second intermediate after reaction.

Wherein the surface of the carrier protein has amino groups. The carrier protein comprises at least one of Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA) and Human Serum Albumin (HSA). In this embodiment, the carrier protein is BSA. I.e., the second intermediate may be denoted Boc-SAH-BSA.

Specifically, a cross-linking agent may also be added to the first intermediate, and the cross-linking agent may be glutaraldehyde. Wherein primary amino groups in the SAH molecule of the first intermediate at adenosine sites react with a substantial amount of free amino groups on the surface of the carrier protein via the cross-linking agent. Wherein the aldehyde group at the end of the glutaraldehyde molecule forms schiff bases with the amino group of the carrier protein surface and the amino group of SAH, respectively, thereby linking the first intermediate to the surface of the carrier protein.

The invention can retain the carboxyl of cysteine position in SAH molecule by glutaraldehyde coupling method. That is, the coupling method can avoid the destruction of the carboxyl group of SAH.

In this embodiment, the first intermediate and BSA may undergo nonspecific carrier protein autogenous crosslinking during glutaraldehyde crosslinking, and the aggregated protein may be removed by a concentration centrifugal tube having a molecular weight of 10000, so as to maintain the prepared Boc-SAH-BSA complete antigen precursor having a uniform molecular weight and a good solubility and dispersibility.

Step S13, removing the di-tert-butyl dicarbonate in the second intermediate, thereby obtaining the S-adenosylhomocysteine artificial complete antigen.

In this embodiment, the protecting group Boc in the second intermediate is removed by trifluoroacetic acid (TFA), thereby reducing the a-amino group in the SAH molecule and repairing the chemical groups affecting the epitope. And a silica gel catalysis method can be used for repairing the epitope, the silica gel catalysis method can reduce the use concentration of TFA, and the carrier protein is prevented from being hydrolyzed under the action of TFA to the maximum extent.

In this embodiment, the S-adenosylhomocysteine artificial complete antigen may be represented as SAH-BSA.

The invention also provides the S-adenosyl homocysteine artificial complete antigen prepared by the preparation method of the S-adenosyl homocysteine artificial complete antigen.

The invention also provides an application of the S-adenosyl homocysteine artificial complete antigen in antibody preparation.

The present invention will be specifically described below with reference to examples.

The Phosphate (PBS) buffer at pH 7.4 and 0.02M in the following examples was prepared as follows: weighing 2.3g of Na₂HPO₄、0.524g NaH₂PO₄.H₂O and 8.77g NaCl are dissolved in pure water, the volume is adjusted to 1L by deionized water, the pH is adjusted to 7.4, and PBS buffer solution with the pH of 7.4 and the concentration of 0.02M is obtained.

The dialysate in the following examples was prepared as follows: the prepared PBS with pH of 7.4 and concentration of 0.02M is diluted 2 times to obtain dialysate.

The PBST in the following examples was formulated as follows: taking the prepared PBS solution with the pH of 7.4 and the concentration of 0.02M, adding Tween20 according to the proportion of 0.05 percent, dissolving and mixing uniformly to obtain the PBST solution.

The blocking solutions in the following examples were prepared as follows: and (3) adding BSA (bovine serum albumin) into the prepared PBST solution according to the proportion of 1% to dissolve the PBST solution uniformly to obtain a blocking solution.

The pH of the following examples was 9.6 and a 0.5M Carbonate (CBS) flush was prepared as follows: 1.59g of Na was weighed₂CO₃、2.93g NaHCO₃Dissolving in deionized water to 100ml, adjusting pH to 9.6 to obtain CBS buffer solution with pH of 9.5 and concentration of 0.5M.