Novel virus double-fluorescence labeling method based on nucleic acid and protein biosynthesis

A biosynthetic and fluorescent labeling technology, applied in the fields of chemistry and biomedicine, can solve the problems of inability to guarantee virus infectivity and virus activity, insufficient universality, cumbersome operation, etc., achieving small impact, good specificity, and easy operation. Effect

Inactive Publication Date: 2015-09-09
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to solve the problems that the existing virus labeling method is not only cumbersome to operate, but also cannot guarantee the infectivity and activity of the virus,

Method used

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  • Novel virus double-fluorescence labeling method based on nucleic acid and protein biosynthesis
  • Novel virus double-fluorescence labeling method based on nucleic acid and protein biosynthesis
  • Novel virus double-fluorescence labeling method based on nucleic acid and protein biosynthesis

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Effect test

Embodiment 1

[0025] A new method for dual fluorescent labeling of viruses based on nucleic acid and protein biosynthesis, the specific steps are as follows, as figure 1 Shown:

[0026] Step 1. Synthesizing methionine analogs --- azidohomoalanine (AHA) and deoxythymidine analogs --- ethylene deoxyuridine (5-Vinyl-2'-deoxyuridine, VdU);

[0027] The method of synthesizing azidomethionine is: dropwise to the NaN-containing 3 Distilled trifluoromethanesulfonic anhydride was added to the aqueous solution, and the reaction was stirred at room temperature for 2 h; the product was extracted twice with dichloromethane; at the same time, water and methanol were added to a round-bottomed flask equipped with a rotor, and then Boc-Dab (tert-butyl Oxycarbonyl-L-2,4-diaminobutyric acid), KCO 3 and catalyst CuSO 4 ·5H 2 O; add the extracted azide trifluoromethanesulfonic anhydride dropwise to the above reaction solution, and stir at room temperature overnight; rotate the reactant at 40°C to remove met...

Embodiment 2

[0043] A new method for dual fluorescent labeling of viruses based on nucleic acid and protein biosynthesis, the specific steps are as follows:

[0044] Step 1. Synthesizing methionine analogs --- azidohomoalanine (AHA) and deoxythymidine analogs --- ethylene deoxyuridine (5-Vinyl-2'-deoxyuridine, VdU);

[0045] Step 2, adding the virus into the culture medium containing the host cells for infection, and when the virus completely infects the host cells, that is, when the host cells become diseased, add azidomethionine and ethylene deoxyuridine to obtain cell A; Vinyl derivatization of viral nucleic acids and azido derivatization of proteins are achieved using the biosynthetic process of nucleic acids and proteins.

[0046] Step 3. Add fluorescent probe---tetrazine-Cy5 (Tetrazine-Cy5) to the cell A obtained in step 2, and obtain nucleic acid-labeled progeny virus by Diels-Alder reaction between tetrazine-alkene B; add a fluorescent probe --- cyclooctynylated Flour 525 (DBCO-Fl...

Embodiment 3

[0050] A new method for dual fluorescent labeling of viruses based on nucleic acid and protein biosynthesis, the specific steps are as follows:

[0051] Step 1. Synthesizing methionine analogs --- azidohomoalanine (AHA) and deoxythymidine analogs --- ethylene deoxyuridine (5-Vinyl-2'-deoxyuridine, VdU);

[0052] Step 2, adding the virus into the culture medium containing the host cells for infection, and when the virus completely infects the host cells, that is, when the host cells become diseased, add azidomethionine and ethylene deoxyuridine to obtain cell A; Vinyl derivatization of viral nucleic acids and azido derivatization of proteins are achieved using the biosynthetic process of nucleic acids and proteins.

[0053] Step 3. Add fluorescent probe---tetrazine-Cy5 (Tetrazine-Cy5) to the cell A obtained in step 2, and obtain nucleic acid-labeled progeny virus by Diels-Alder reaction between tetrazine-alkene B; add a fluorescent probe --- cyclooctynylated Flour 525 (DBCO-Fl...

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Abstract

The invention relates to a novel virus double-fluorescence labeling method based on nucleic acid and protein biosynthesis, and belongs to the field of chemistry and biomedicine. The method comprises specific steps as follows: a methionine analogue methionine azide and a deoxythymidine analogue ethylene deoxyuridine are synthesized; viruses are added to host cells for infection, methionine azide and ethylene deoxyuridine are added respectively, vinyl derivation of viral nucleic acid and azido derivation of protein are realized with biosynthesis processes of nucleic acid and protein; double-fluorescence labeling of viral nucleic acid and protein is naturally realized through two biological orthogonal reactions including a Diels-Alder reaction of tetrazine-oefin and a copper-free catalytic click chemical reaction of azido-cyclooctyne. The labeling method is simple, convenient and reliable, can be applied to all DNA (deoxyribonucleic acid) viruses including single-stranded DNA viruses, double-stranded DNA viruses, enveloped viruses and non-enveloped viruses, and is a universal virus fluorescence labeling method on an absolute basis.

Description

technical field [0001] The invention relates to a new virus double fluorescent labeling method based on nucleic acid and protein biosynthesis, which belongs to the fields of chemistry and biomedicine. Background technique [0002] Fluorescent labeling of viruses is crucial for the study of virus infection process and mechanism. Ideal virus labeling should realize the visualization of virus single particles under the premise of ensuring virus activity. The current methods mainly focus on labeling with fluorescent proteins or directly using dye molecules. However, these two types of methods have obvious deficiencies. For example, some organic dye labeling methods need to modify and separate and purify the virus, which will reduce the activity of the virus, and the labeling effect on the internal structure of the virus is poor, while those methods that directly embed organic dyes into the virus In the structure method, the signal is unstable because the dye molecule is easy to...

Claims

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Application Information

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IPC IPC(8): C12Q1/70
CPCC12Q1/02
Inventor 谢海燕黄利利朱厚舜
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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