X-ray multicolor genetic marker probe based on synchronous light source as well as preparation method and application thereof

A technology of genetic markers and synchronous light sources, applied in the field of biochemistry, can solve problems such as the inability to identify and locate multiple biomolecules, and achieve good biomedical application prospects

Active Publication Date: 2020-02-21
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide an X-ray multicolor genetic labeling probe based on a synchronous light source and its preparation method and application, so as to solve the problem that the existing X-ray microscopy technology cannot simultaneously identify and detect multiple biomolecules in cells. positioning problem

Method used

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  • X-ray multicolor genetic marker probe based on synchronous light source as well as preparation method and application thereof
  • X-ray multicolor genetic marker probe based on synchronous light source as well as preparation method and application thereof
  • X-ray multicolor genetic marker probe based on synchronous light source as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of simultaneous X-ray two-color genetically labeled probe and its application in nucleus + mitochondria two-color imaging.

[0042] Construction of pcDNA3-NLS-APEX2 plasmid. The construction process of the plasmid was carried out by conventional molecular biology methods, as follows: First, according to the human NLS-APEX2 protein sequence, its DNA sequence was optimized using human-biased codons and the complete sequence was synthesized. The complete sequence was provided by Shanghai Synthesized by Quanyang Biotechnology Co., Ltd. The NLS-APEX2 sequence was then cloned into the backbone of the pcDNA3 mammalian expression vector to construct the pcDNA3-NLS-APEX2 plasmid. The plasmid sequence was sequenced and verified, and the plasmid sequence is shown in SEQ ID NO:1. Among them, pcDNA3 is a commercial mammalian expression vector backbone (purchased from Invitrogen TM ). After the sequence of the NLS-APEX2 fusion protein is cloned into the pcDN...

Embodiment 2

[0050] Example 2 Preparation of simultaneous X-ray two-color genetically labeled probe and its application in microfilament + mitochondria two-color imaging.

[0051] The source of the pEGFP-mito-MiniSOG plasmid is the same as in Example 1. pEGFP-APEX2-Actin plasmid (Addgeneplasmid #66172), the sequence is shown in SEQ ID NO:3. The source and culture method of HeLa cells are the same as in Example 1.

[0052] The pEGFP-APEX2-Actin and pEGFP-mito-MiniSOG plasmids were simultaneously transfected into HeLa cells. Use liposome Lipo3000 method for transfection, add 1.5μL Lipo3000, 500ng pEGFP-APEX2-Actin plasmid, 500ng pEGFP-mito-MiniSOG plasmid and 2μL P3000 to each well. After 24h, remove the medium and fix with 2% glutaraldehyde in ice bath . Add containing 0.03% H 2 o 2 The 3,3'-diaminobenzidine (DAB-Ni) reaction solution was reacted in an ice bath for 20 minutes, and the reaction solution in the well was removed. Add 10 mM acetic anhydride solution to block, and after 10...

Embodiment 3

[0055] Example 3 Comparison of the application of simultaneous X-ray two-color genetic labeling probes based on APEX2, MiniSOG, and Tetracysteine ​​in two-color imaging of cells.

[0056]The construction method of pcDNA3-NLS-APEX2 plasmid is the same as that in Example 1. The source of the pEGFP-mito-MiniSOG plasmid is the same as in Example 1. The pcDNA3-mito-Tetracysteine ​​and pcDNA3-NLS-MiniSOG plasmids were constructed respectively. First, according to the human mito-Tetracysteine ​​and NLS-MiniSOG protein sequences, the human biased codons were used to optimize the DNA sequence and synthesize the full sequence, which was synthesized by Shanghai Quanyang Biotechnology Co., Ltd. Then the mito-Tetracysteine ​​and NLS-MiniSOG sequences were cloned into the backbone of the pcDNA3 mammalian expression vector, thereby constructing the pcDNA3-mito-Tetracysteine ​​and pcDNA3-NLS-MiniSOG plasmids respectively, sequenced and verified the plasmid sequences, and the plasmid sequence...

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Abstract

The invention provides an X-ray multicolor genetic marker probe based on a synchronous light source as well as a preparation method and an application thereof. The preparation method comprises the following steps: 1) constructing at least two fusion expression plasmids simultaneously comprising enzymes and target proteins, and simultaneously transfecting the fusion expression plasmids into cells;2) fixing the cells in an ice bath by using glutaraldehyde fixing liquid; 3) adding a first DAB-metal complex, and carrying out an ice bath reaction; 4) removing the reaction liquid, and adding a confining liquid; 5) adding a second DAB-metal complex, and carrying out the 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 liquid and fixing the cells; and (8) performing synchronous X-ray imaging observation, wherein each DAB-metal polymer has a specific fluorescence peak under X-rays, and obtaining the X-ray multicolor genetic marker probe. According to the invention, a method capable of carrying out high-specificity recognition and high-resolution imaging on various biomolecules in cells atthe same time is provided, and the method has a good biomedical application prospect.

Description

technical field [0001] The invention relates to the technical field of biochemistry, and more specifically relates to an X-ray multicolor genetic marker probe based on a synchronous light source, a preparation method and application thereof. Background technique [0002] Microscopic imaging technology is one of the main driving forces for the development of cell life science. Every physiological activity of cells is a complex biological process, involving the interaction between various protein molecules and their localization changes. This objectively requires that the technology of cell imaging can simultaneously acquire and image the signals from multiple biomolecules, so as to make a complete elaboration of the life process. Microscopy based on synchrotron X-rays has unique advantages in the field of cell imaging. Since the wavelength of X-rays is in the range of 0.1-10nm, it is naturally a super-resolution microscopic imaging technology, and the resolution can theoret...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62G01N23/223G01N1/30
CPCC12N15/62C12N15/85C12N2800/107C12N2800/22G01N1/30G01N23/223G01N2001/305G01N2223/076G01N2223/1016G01N2223/612
Inventor 樊春海诸颖孔华庭张继超闫庆龙王丽华胡钧
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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