阅读说明：本技术 一种具有近红外性质的His-tag荧光探针及其制备和应用 (His-tag fluorescent probe with near-infrared property and preparation and application thereof ) 是由 胡琦 叶秀丽 于 2020-05-28 设计创作，主要内容包括：本发明提供一种具有近红外性质的His-tag荧光探针及其制备和应用,所述近红外性质的His-tag荧光探针可应用于His-tag富集蛋白的标记和纯化。所述的荧光检测原理为：荧光探针的亚氨基二乙酸(IDA)可以与镍离子进行螯合。螯合后的荧光探针可以与组氨酸标签结合,使标记后的蛋白呈现红色荧光,从而可以通过蛋白质凝胶电泳分离鉴定。本发明可以为蛋白的纯化和分离提供新的思路和方法。(The invention provides a His-tag fluorescent probe with near-infrared property, and preparation and application thereof. The fluorescence detection principle is as follows: iminodiacetic acid (IDA) of the fluorescent probe may chelate with nickel ions. The chelated fluorescent probe can be combined with a histidine tag, so that the labeled protein presents red fluorescence, and the protein can be separated and identified through protein gel electrophoresis. The invention can provide a new idea and method for the purification and separation of protein.)

1. A His-tag fluorescent probe with near infrared property is characterized in that the structure is as follows:

2. the His-tag fluorescent probe with near infrared properties according to claim 1, wherein the His-tag fluorescent probe has near infrared properties.

3. A His-tag fluorescent probe having near-infrared properties, characterized in that the His-tag fluorescent probe having near-infrared properties according to claim 1 and iminodiacetic acid (IDA) chelate nickel ion are formed.

4. Use of a His-tag fluorescent probe with near infrared properties, which is characterized in that the His-tag fluorescent probe with near infrared properties according to claim 3 is used for specifically binding histidine.

5. Use of a His-tag fluorescent probe with near infrared properties according to claim 4, for identifying and labeling proteins containing histidine enrichment.

6. Use of a His-tag fluorescent probe with near infrared properties according to claim 4, for detecting a labeled fluorescent protein by near infrared fluorescent gel scanning.

7. The preparation method of the His-tag fluorescent probe with the near-infrared property as claimed in claim 1, wherein the preparation method comprises the following steps:

Technical Field

The invention relates to a His-tag fluorescent probe with near-infrared property and preparation and application thereof.

Background

Protein purification uses the inherent similarity and difference between different proteins, uses the similarity between various proteins to remove contamination of non-protein substances, and uses the difference between proteins to purify a target protein from other proteins. The size, shape, charge, hydrophobicity, solubility and biological activity of each protein can vary, and the protein can be extracted from a mixture such as E.coli lysate to obtain recombinant protein.

Immobilized metal ion affinity chromatography (IMAC), abbreviated as metal chelating affinity chromatography, is a novel technology applied to prokaryotic protein purification. The method uses some special amino acids on the surface of protein to make them interact with metal ions so as to make affinity purification of protein. These effects include coordinate bond binding, electrostatic adsorption, covalent bond binding, etc., of which fusion Tag (His-Tag) combined with 6 histidine residues is most significantly used in prokaryotic protein expression.

Fluorescent probe technology is a relatively popular labeling and detection technology developed in recent years. Due to the sensitivity and stability of the compound, the compound attracts the wide attention of researchers. Among these fluorescent dyes, near infrared dyes are widely used for biological monitoring and applications because they can be used for less damage to organisms and have high penetration. Based on the assumption, the preparation and the application of the His-tag fluorescent probe with the near infrared property are designed.

Disclosure of Invention

The invention aims to provide a His-tag fluorescent probe with near infrared property and preparation and application thereof. The second purpose of the invention is to provide a method for protein purification and identification.

The invention adopts the following technical scheme for realizing the purpose:

the invention provides a compound shown as a formula (III), which is a fluorescent probe with near-infrared property:

a method for preparing a compound represented by the formula (III) according to the principle: iminodiacetic acid (IDA) of the fluorescent probe can be chelated with nickel ions, the chelated fluorescent probe can be combined with a histidine tag, so that the labeled protein presents red fluorescence, and can be separated and identified through protein gel electrophoresis, and the flow schematic diagram of the preparation method is shown in figure 1.

The invention also provides a synthesis method of the near-infrared His-Tag fluorescent probe, which comprises the following steps:

the compound (1-1) and N-hydroxysuccinimide (NHS) generate a compound (1-2) under the action of Dicyclohexylcarbodiimide (DCC);

wherein the compound (1-1) has the following formula:

wherein the compound (1-2) has the following formula:

the preparation method comprises the following steps: 1.0mmol of rhodamine 6G and 1.2mmol of N-hydroxysuccinimide (NHS) were dissolved in 10ml of a N, N-dimethylformamide solution, and 1.0mmol of Dicyclohexylcarbodiimide (DCC) was added thereto to carry out a reaction at room temperature for 12 hours. Adding 30mL of saturated sodium chloride solution into the reaction solution, extracting with ethyl acetate (30mL) for three times, collecting an organic phase, washing with 30mL of saturated sodium chloride solution for three times, drying the organic layer with anhydrous magnesium sulfate, filtering, and rotationally evaporating at normal temperature to remove the solvent to obtain a crude product; and (3) performing silica gel column chromatography on the crude product, taking a solution of dichloromethane and methanol in a volume ratio of 1:30 as a mobile phase, tracking and collecting an eluent with the Rf value of 0.3-0.5 by TLC, decompressing and removing the solvent from the collected eluent, and drying to obtain the compound 1-2.

A preparation method of a novel near-infrared fluorescent probe comprises the following steps:

in DMF solution, compound (1-2) and iminodiacetic acid (IDA) generate compound His-1; the preparation method comprises the following steps: 1.0mmol of the compound 1-2, 1.2mmol of iminodiacetic acid (IDA) was dissolved in 10ml of N, N-dimethylformamide solution and reacted at room temperature for 12 hours. After the reaction is finished, adding 30mL of saturated sodium chloride solution into the reaction solution, extracting with dichloromethane (30mL) for three times, collecting an organic phase, washing with 30mL of saturated sodium chloride solution for three times, drying the organic layer with anhydrous magnesium sulfate, filtering, and rotationally evaporating the solvent at normal temperature to obtain a crude product; performing silica gel column chromatography on the crude product, taking a solution with a volume ratio of dichloromethane to methanol of 1:15 as a mobile phase, tracking and collecting an eluent with an Rf value of 0.3-0.5 by TLC, decompressing and removing a solvent from the collected eluent, and drying to obtain a compound fluorescent probe (III);

the reaction route is as follows:

the invention provides application of detecting that the fluorescent probe (III) has near infrared fluorescence characteristics. The near-infrared characteristics of the fluorescent probe are as follows: the compound (III) has good solubility in aqueous solution, and under the action of excitation light at 540nm, the compound (III) can show a remarkable near-infrared fluorescence emission wavelength at 625 nm.

Further, the specific method for detecting the fluorescence property of the fluorescent probe is that the compound (III) is respectively added into ultrapure water, and the fluorescence intensity of the probe at the emission wavelength of 620nm when the excitation is 540nm is measured; thus indicating that the probe has good near infrared characteristics.

The compound (III) can be used as a fluorescent probe and can be applied to marking and purifying His-tag enriched protein. The fluorescence detection principle is as follows: iminodiacetic acid (IDA) of the fluorescent probe may chelate with nickel ions. The chelated fluorescent probe can be combined with a histidine tag, so that the labeled protein presents red fluorescence.

Further, the labeled protein may be separated by a gel protein.

Further, the separated gel exhibited a red fluorescence band under the gel scanner.

Drawings

FIG. 1 is a schematic flow chart of a method for preparing a fluorescent probe having near infrared properties.

FIG. 2 is a nuclear magnetic hydrogen spectrum of the compound (III) prepared in example 2 of the present invention.

FIG. 3 shows fluorescence emission spectra of compound (III) of example 2 in the present invention, wherein the excitation wavelength of the fluorescent probe is 540 nm.

FIG. 4 shows compound (III) prepared in example 2 of the present invention, and the emission wavelength of the fluorescent probe is 620nm.

FIG. 5 Coomassie brilliant blue staining of fluorescent probes binding to histidine-tagged proteins.

FIG. 6 is a fluorescent coagulum of fluorescent probes bound to histidine-tagged proteins. The fluorescent signal is only displayed in the electrophoretic lane to which the fluorescent probe is added.

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 that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.