阅读说明：本技术 一种基于同步光源的x-射线多色遗传标记探针及其制备方法以及应用 (X-ray multicolor genetic marker probe based on synchronous light source and preparation method and application thereof ) 是由 樊春海 诸颖 孔华庭 张继超 闫庆龙 王丽华 胡钧 于 2019-11-11 设计创作，主要内容包括：本发明提供一种基于同步光源的X-射线多色遗传标记探针及其制备方法以及应用,包括：1)构建至少两种同时包括酶和目的蛋白的融合表达质粒,并将其同时转染进细胞；2)使用戊二醛固定液冰浴固定所述细胞；3)加入第一DAB-金属络合物,冰浴反应；4)去除反应液,加入封闭液；5)加入第二DAB-金属络合物,冰浴反应；6)若构建三种及三种以上融合表达质粒,则重复上述步骤4),5)；7)去除反应液,固定细胞；以及8)同步X射线成像观察,每种DAB-金属聚合物在X射线下具有特异性的荧光峰,即得。根据本发明,提供了一种能同时对细胞内多种生物分子进行高特异性识别和高分辨率成像的方法,具有良好的生物医学应用前景。(The invention provides an X-ray multicolor genetic marking probe based on a synchronous light source, a preparation method and application thereof, comprising the following steps: 1) constructing at least two fusion expression plasmids simultaneously comprising enzyme and target protein, and simultaneously transfecting the fusion expression plasmids into cells; 2) fixing the cells using an ice bath of glutaraldehyde fixing solution; 3) adding a first DAB-metal complex, and carrying out ice-bath reaction; 4) removing the reaction solution, and adding a sealing solution; 5) adding a second DAB-metal complex, and carrying out ice-bath reaction; 6) if three or more than three fusion expression plasmids are constructed, repeating the steps 4) and 5); 7) removing the reaction solution and fixing the cells; and 8) synchronous X-ray imaging observation, wherein each DAB-metal polymer has a specific fluorescence peak under X-rays, and the DAB-metal polymer is obtained. According to the invention, the method capable of simultaneously carrying out high-specificity identification and high-resolution imaging on multiple biomolecules in the cell is provided, and the method has a good biomedical application prospect.)

1. A preparation method of an X-ray multicolor genetic marker probe based on a synchronous light source is characterized by comprising the following steps:

1) constructing at least two fusion expression plasmids simultaneously comprising an enzyme and a protein of interest, and transfecting the at least two fusion expression plasmids simultaneously into a cell expressing at least two different fusion proteins;

2) fixing the cells using an ice bath of glutaraldehyde fixing solution;

3) adding a first DAB-metal complex aiming at the first fusion protein, carrying out ice-bath reaction, and polymerizing the first DAB-metal complex under the catalysis of the first fusion protein to generate a first DAB-metal polymer;

4) removing the reaction solution, and adding a sealing solution;

5) adding a second DAB-metal complex aiming at a second fusion protein, carrying out ice-bath reaction, and polymerizing the second DAB-metal complex under the catalysis of the second fusion protein to generate a second DAB-metal polymer;

6) if three or more than three fusion expression plasmids which simultaneously comprise enzyme and target protein are constructed, repeating the steps 4) and 5) until all the fusion proteins catalyze the corresponding DAB-metal complex to polymerize;

7) removing the reaction solution, and fixing the cells by using a fixing solution; and

8) dripping the cells in the step 7) on a synchronous imaging substrate, and observing synchronous X-ray imaging, wherein each DAB-metal polymer has a specific fluorescence peak under X-rays, thus obtaining the X-ray multicolor genetic marker probe based on a synchronous light source.

2. The method according to claim 1, wherein in step 1), the enzyme comprises: ascorbic acid peroxidase, mini singlet oxygen generating protein, tetra cysteine peptide, and horseradish peroxidase.

3. The method of claim 1, wherein in the steps 3) and 5), the metal of the DAB-metal complex is selected from the group consisting of: fe. Any one of the group consisting of Co, Ni, Cu, Zn, La, Sn, Cd, different kinds of DAB-metal complexes are selected for different fusion proteins.

4. The method according to claim 1, wherein different enzyme reaction systems occur in steps 3) and 5) under different reaction conditions: generation of the ascorbate peroxidase reaction System requires H₂O₂(ii) a The generation of the mini singlet oxygen generating protein reaction system requires continuous O filling₂And 488nm illumination; the generation of the tetra-cysteine peptide reaction system requires the addition of ReAsH-EDT₂Continuous charging of O₂And 585nm illumination.

5. The method according to claim 4, wherein the order of occurrence of the enzymatic reaction system is controlled by controlling the reaction conditions.

6. The preparation method according to claim 1, wherein in the step 2), the concentration of the glutaraldehyde fixing solution is 1.5-3%, and the fixing time is 20-60 min.

7. The method according to claim 1, wherein in step 7), the fixative solution used comprises: paraformaldehyde, glutaraldehyde, ethanol, methanol, glacial acetic acid, acetone or formalin, and the fixing time is 10 min-2 h.

8. The method according to claim 1, wherein in step 8), different incident energies are selected according to different metal ions, the incident energy for Fe is 280-1500eV and 7112-15000eV, the incident energy for Co is 280-1700eV and 7709-16000eV, the incident energy for Ni is 280-1900eV and 8332-17000eV, the incident energy for Cu is 280-2000eV and 8979-18000eV, the incident energy for Zn is 280-2100eV and 9659-20000eV, the incident energy for La is 280-2500eV, 5483-12000eV and 38925-78000eV, the incident energy for Sn is 280-2000eV, 3929-11000eV and 29200-50000eV, and the incident energy for Cd is 280-1700eV, 3538-11000eV and 26711-50000 eV.

9. An X-ray multicolor genetic marker probe based on a synchronous light source prepared by the preparation method according to any one of claims 1 to 8.

10. Use of a simultaneous X-ray polychromatic genetic marking probe according to claim 9, for biomolecular recognition and imaging in cells, characterized in that it comprises: constructing a plurality of fusion expression plasmids simultaneously comprising enzyme and target protein, transfecting the fusion expression plasmids into cells simultaneously, expressing different fusion proteins of enzyme and protein simultaneously in the cells by the plasmids, catalyzing different DAB-metal complex polymerization sequentially by different enzyme reaction systems, generating DAB-metal polymers in situ at biomolecules in the cells, observing by using synchronous X-ray imaging, wherein each DAB-metal polymer has a specific fluorescence peak under X-ray, and thus realizing the characteristic recognition and positioning of the plurality of biomolecules in the cells.

Technical Field

The invention relates to the technical field of biochemistry, in particular to an X-ray multicolor genetic marker probe based on a synchronous light source and a preparation method and application thereof.

Background

Microscopic imaging techniques are one of the major drivers in the development of cell life sciences. Every physiological activity of a cell is a complex biological process involving interactions between multiple protein molecules and changes in their localization. This objectively requires the technology of cellular imaging research to acquire and image signals from multiple biomolecules simultaneously, thereby providing a complete description of the life process. Simultaneous X-ray based microscopy has unique advantages in the field of cell imaging. Since the wavelength of the X-ray is in the range of 0.1-10nm, the X-ray is a super-resolution microscopic imaging technology, and the resolution can reach several nanometers theoretically. In addition, X-rays are more penetrating into biological samples than electron beams, and thus do not require processing such as sectioning to image intact cells. More importantly, the X-ray microscopic imaging technology has unique X-ray fluorescence characteristic spectra for different elements and does not interfere with each other. Therefore, in combination with an X-ray sensitive imaging probe, high resolution identification and imaging of multiple biomolecules in a cell can be achieved simultaneously.

In our previous patent, an X-ray genetic marker probe based on a synchronized light source was developed for use in identifying and imaging specific biomolecules within cells. However, how to label multiple biomolecules of interest simultaneously is still a big bottleneck. Therefore, the development of synchronous X-ray multicolor imaging probes at the present stage has very important significance for realizing the accurate identification and positioning of various biomolecules in cells at the same time.

Disclosure of Invention

The invention aims to provide an X-ray multicolor genetic marker probe based on a synchronous light source, a preparation method and application thereof, so as to solve the problem that the existing X-ray microscopic technology cannot simultaneously identify and locate various biomolecules in cells.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention, there is provided a method for preparing an X-ray multicolor genetic marker probe based on a synchronous light source, the method comprising the steps of: 1) constructing at least two fusion expression plasmids simultaneously comprising an enzyme and a protein of interest, and transfecting the at least two fusion expression plasmids simultaneously into a cell expressing at least two different fusion proteins; 2) fixing the cells using an ice bath of glutaraldehyde fixing solution; 3) adding a first DAB-metal complex aiming at the first fusion protein, and carrying out ice-bath reaction to ensure that the first fusion protein catalyzes the first DAB-metal complex to polymerize; 4) removing the reaction solution, and adding a sealing solution; 5) adding a second DAB-metal complex aiming at a second fusion protein, and carrying out ice-bath reaction to ensure that the second fusion protein catalyzes the second DAB-metal complex to polymerize; 6) if three or more than three fusion expression plasmids which simultaneously comprise enzyme and target protein are constructed, repeating the steps 4) and 5) until all the fusion proteins catalyze the corresponding DAB-metal complex to polymerize; 7) removing the reaction solution, and fixing the cells by using a fixing solution; and 8) dropwise adding the cells on a synchronous imaging substrate, and synchronously carrying out X-ray imaging observation, wherein each DAB-metal polymer has a specific fluorescence peak under X-rays, so that the X-ray multicolor genetic marker probe based on the synchronous light source is obtained.

According to the preparation method provided by the invention, the working principle is as follows: the plasmid expresses fusion proteins of different enzymes and proteins in a cell simultaneously, constructs a plurality of fusion expression plasmids simultaneously comprising the enzymes and target proteins, transfects the fusion expression plasmids into the cell simultaneously, expresses the fusion proteins of different enzymes and proteins in the cell simultaneously by the plasmid, can catalyze different DAB-metal polymerization by different enzyme reaction systems sequentially, and generates DAB-metal polymers in situ at biomolecules in the cell. And each DAB-metal polymer has a specific fluorescence peak under X-rays by using synchronous X-ray imaging observation, so that the characteristic identification and positioning of various biomolecules in cells are realized.

In step 1), the enzyme comprises: ascorbic acid peroxidase (APEX, APEX2), mini singlet oxygen generating protein (MiniSOG), tetra cysteine peptide (tetracystein), and horseradish peroxidase (HRP), and the like. Most preferred is APEX 2. Among them, APEX2, MiniSOG, tetracystein are suitable for any intracellular protein, and HRP is most suitable for endoplasmic reticulum-associated proteins.

It is to be understood that for multicolor imaging purposes in the present invention, at least two fusion expression plasmids are required. In the present method, the kind of fusion expression depends on the kind of enzyme having catalytic function, and at present, there are mainly the above four enzymes suitable for the present method: ascorbic acid peroxidase, mini singlet oxygen generating protein, tetra-cysteine peptide and horseradish peroxidase, however, theoretically, the method does not limit the kind of fusion expression plasmid, and after more and more enzymes are discovered and developed in the future, the method can be applied to more fusion expression plasmids of enzymes and target proteins.

The Transfection Reagent used in step 1) is Lipofectamine 3000Transfection Reagent, Lipofectamine 2000CD Transfection Reagent, Lipofectamine LTX Reagent or jetPRIME Transfection Reagent.

The transfection method is a conventional method, and the ratio of the transfection reagent to the plasmid is 3: 1-1: 1, and the optimal ratio is 1.5: 1. The transfection time is 12-48 h, preferably 24 h.

The cell is a cell strain of conventional passage or primary culture, and in the practical application process, a DNA sequence for expressing an enzyme with catalytic activity and a target protein is designed when a fusion expression plasmid is constructed according to the species source of the cell. Preferably, the DNA sequence is designed and optimized for better expression in the cell based on the codon usage preferences of different species.

The fixative used in step 2) is only glutaraldehyde, because glutaraldehyde can maximally maintain the catalytic activity of the enzyme. The concentration of the glutaraldehyde stationary liquid is 1.5-3%. Among them, 2% is preferable. The fixing time is 20-60 min, preferably 40 min.

In the steps 3) and 5), the metal in the DAB-metal complex is selected from the following group: fe. Any one of the group consisting of Co, Ni, Cu, Zn, La, Sn, Cd, different kinds of DAB-metal complexes are selected for different fusion proteins. It should be understood that there is no fixed one-to-one correspondence between the DAB-metal complex and the fusion expression plasmid, and the two can be arbitrarily paired according to actual needs.

In steps 3) and 5), different enzyme reaction systems need different reaction conditions for generation: generation of the ascorbate peroxidase reaction System requires H₂O₂(ii) a The generation of the mini singlet oxygen generating protein reaction system requires continuous O filling₂And 488nm illumination; the generation of the Tetracysteine peptide reaction system requires the addition of ReAsH-EDT2, a continuous O-charging₂And 585nm illumination.

That is, the substrate molecule reaction solution has different components for different enzymes. For example, APEX and APEX2 can catalyze H₂O₂Generating¹O₂，¹O₂The catalyst can further catalyze the polymerization of substrate molecules, so that the substrate molecule reaction liquid must contain hydrogen peroxide for APEX and APEX 2. Hydrogen peroxide is not an essential component for MiniSOG and Tetracysteine.

The substrate molecule reaction conditions are different for different enzymes. For example, for MiniSOG and Tetracysteine, fluorescence irradiation and O are required₂Is continuously supplied. Whereas for APEX and APEX2, fluorescence irradiation and O₂And are not required ingredients.

In the step 3) and the step 5), the ice-bath reaction time is 30 s-2 h. The optimal ice-bath reaction time is selected according to different target proteins.

In the step 4), the used sealing solution is an acetyl imidazole solution or an acetic anhydride solution. The concentration is 1 to 100 mM.

The fixing solution used in step 7) is optionally paraformaldehyde, glutaraldehyde, ethanol, methanol, glacial acetic acid, acetone or formalin, etc. Among these, paraformaldehyde is most preferred, particularly 4% paraformaldehyde fixing fluid. The paraformaldehyde fixing liquid enables proteins in cells to be fixed, maintains the structure of the cells, and is beneficial to further dehydration and observation.

It is to be understood that the fixative used in step 2) is only glutaraldehyde, while the fixative used in step 7) is not only glutaraldehyde, but may be other types of fixatives, but is most preferably paraformaldehyde.

The fixing time of the fixing solution in the step 7) is 10 min-2 h. The preferred time is 15 min.

In step 8), different incident energies are selected according to different metal ions, wherein the incident energies for Fe are 280-1500eV and 7112-15000eV, the incident energies for Co are 280-1700eV and 7709-16000eV, the incident energies for Ni are 280-1900eV and 8332-17000eV, the incident energies for Cu are 280-2000eV and 8979-18000eV, the incident energies for Zn are 280-2100eV and 9659-20000eV, the incident energies for La are 280-2500eV, 5483-12000eV and 38925-78000eV, the incident energies for Sn are 280-2000eV, 3929-11000eV and 29200-50000eV, and the incident energies for Cd are 280-1700eV, 3538-11000eV and 26711-50000 eV.

The selected X-ray incident energy range can image corresponding metal elements, but the optimal incident energy is selected, for example, Co, Ni, Cu and Zn, and the incident energy is selected to be 10000 eV.

According to a second aspect of the present invention, there is provided an X-ray multicolor genetic marker probe based on a synchronized light source prepared according to the above-mentioned preparation method.

According to a third aspect of the present invention there is provided a use of a simultaneous X-ray polychromatic genetic marker probe in biomolecular recognition and imaging in a cell, comprising: constructing a plurality of fusion expression plasmids simultaneously comprising enzyme and target protein, transfecting the fusion expression plasmids into cells simultaneously, expressing different fusion proteins of enzyme and protein simultaneously in the cells by the plasmids, catalyzing different DAB-metal complex polymerization sequentially by different enzyme reaction systems, generating DAB-metal polymers in situ at biomolecules in the cells, observing by using synchronous X-ray imaging, wherein each DAB-metal polymer has a specific fluorescence peak under X-ray, and thus realizing the characteristic recognition and positioning of the plurality of biomolecules in the cells.

According to a preferred embodiment of the invention, the nuclear and mitochondrial marking is achieved by selecting for fusion proteins and enzymes that localize to the nucleus and mitochondria. For example, the nuclear marker may be selected from NLS, a protein tag that localizes within the nucleus, and the mitochondria may be selected from COX8 protein, a protein that localizes within the mitochondria; after the constructed fusion protein expression plasmid is transfected into a cell, the cell can express the fusion protein of the enzyme and the target protein and is positioned at the target protein. Similarly, labeling of other regions in the cell can also be achieved, provided that fusion proteins of the enzyme and the protein localized to other regions in the cell are designed.

The positive progress effects of the invention are as follows: the synchronous X-ray microscopic imaging technology and various molecular probes suitable for the synchronous X-ray microscopic imaging technology are of great significance for understanding cell life processes. However, the X-ray microscopy is currently used for imaging a single biological target in a cell, and the types of molecular probes suitable for the technique to specifically recognize important biological targets in a cell are limited. The invention utilizes the characteristic that the X-ray microscopic imaging technology has unique X-ray fluorescence characteristic spectrums for different elements and does not interfere with each other, plasmids expressing fusion proteins of different enzymes and proteins are transfected into cells simultaneously, and the plasmids express the fusion proteins of different enzymes and proteins in the cells simultaneously. Different enzyme reaction systems can catalyze different DAB-metal polymerization in sequence, and DAB-metal polymers are generated in situ at biological molecules in cells. And (3) using synchronous X-ray imaging observation, each DAB-metal polymer has a specific fluorescence peak under X-rays, and the characteristic identification and positioning of various biomolecules in cells are realized.

In conclusion, the invention provides a method capable of simultaneously carrying out high-specificity identification and high-resolution imaging on various biomolecules in cells, and the method has a good biomedical application prospect.

Drawings

FIG. 1A is an image of the nuclear and mitochondrial marking images of simultaneous X-ray bi-color genetic probes (white area, nucleus on left, mitochondria on right);

FIG. 1B is a representative X-ray fluorescence spectrum of a simultaneous X-ray two-color genetic probe labeling the nuclear and mitochondrial regions;

FIG. 1C is an overlay of the two left and right panels of FIG. 1A (gray sections indicate nuclei, white sections indicate mitochondria; gray, white are both pseudo-colors);

FIG. 2A is an image of the markers of the cell microfilament and mitochondria for a simultaneous X-ray bicolor genetic probe (white area, left cell microfilament, right mitochondria);

FIG. 2B is a representative X-ray fluorescence spectrum of a simultaneous X-ray two-color genetic probe labeling of cellular microfilaments and mitochondrial regions;

FIG. 2C is an overlay of the left and right panels of FIG. 2A (gray portions indicate cellular microfilaments, white portions indicate mitochondria, gray, white both in false color);

FIG. 3A is a labeled image of the nucleus and mitochondria of three simultaneous X-ray bicolor genetic probes in sequence from top to bottom;

FIG. 3B is a representative X-ray fluorescence spectrum of the nucleus and mitochondrial regions after labeling with the corresponding bi-color probe combination;

fig. 3C is an overlay of the left and right panels of each of the groups of fig. 3A.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

The invention selects human HeLa cells, a double-color genetic marker system, enzyme with catalytic activity mainly takes APEX2+ MiniSOG, biological target selects cell nucleus + mitochondrial protein as a representative, substrate molecules select DAB-Co and DAB-Ni as a representative, and an X-ray multi-color genetic marker probe is constructed and applied to the imaging research of the cells.