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Virus-like protein cage particles as well as preparation method and application thereof

A virus and protein technology, applied in the field of virus-like protein cage particles, can solve problems such as failure of functional modification and incomplete protein molecular recombination, and achieve the effect of strong controllability, ensuring stability and activity, and mild conditions.

Active Publication Date: 2021-02-05
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This pH jump is only partially reversible, leading to possible incomplete protein recombination
Extreme assembly conditions bring challenges to subsequent functional modification or directly lead to failure of functional modification

Method used

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  • Virus-like protein cage particles as well as preparation method and application thereof
  • Virus-like protein cage particles as well as preparation method and application thereof
  • Virus-like protein cage particles as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 disassembles natural CCMV virus and purifies capsid protein

[0042] Collect 1mL of natural cowpea chlorotic mottle virus (CCMV virus) from fresh infected cowpea plants in virus buffer solution (0.1M sodium acetate, 1mM edetate disodium, 1mM sodium azide, pH 5.0), Quickly store in a refrigerator with a constant temperature of 4.0° C. to obtain a CCMV suspension. Prepare RNA buffer (50mM Tris, 500mM CaCl 2, 1 mM dithiothreitol, pH 7.5). The obtained CCMV suspension was first dialyzed in 200 mL RNA buffer for 2 hours, and then dialyzed in 800 mL buffer overnight at 4°C. When white flocs are observed, transfer to a centrifuge tube, centrifuge at 40,000rpm / min for 2h, extract the supernatant, and remove the white flocculent precipitate containing RNA. The supernatant (~1 mL) was dialyzed against 330 mL RNA buffer solution, and the buffer solution was changed every 3 hours for a total of 6 hours; followed by dialysis against 330 mL buffer solution overnight. ...

Embodiment 2

[0043] Embodiment 2: Preparation and performance characterization of nanobubbles

[0044] A self-made nano-bubble generating device is used to prepare bulk nano-bubbles by electrolyzing water. A 10 mM sodium chloride solution was prepared with deionized water (conductivity 18.4 MΩ·cm, 25° C.). Use a vacuum pump to flow the solution into the electrode tank that is electrolyzing water (electrolysis voltage 24V, current 3A) at a flow rate of 500mL / min. The electrolysis of water produces oxygen at the anode and hydrogen at the cathode, and the gas produced is dissolved in the sodium chloride solution at the same time, making the solution in a supersaturated state and generating bulk nanobubbles. The supersaturation rate of the solution is controlled by the amount of gas generated, and the surface potential of the nanobubbles is further controlled. Oxygen supersaturation measured by an oxygen measuring instrument (Fibox 3trace 3) was 110%-130%.

[0045] First filter the prepared...

Embodiment 3

[0047] Embodiment 3: Nanobubbles induce capsid protein self-assembly

[0048] At 4°C, 400 μL of nanobubble solution was quickly added to 200 μL of the purified CCMV capsid protein suspension. The mixed solution was mechanically shaken gently for 1 min, and then placed on a constant temperature shaker at 4°C overnight to induce self-assembly of the capsid protein to form a virus-like protein cage, and the formed virus-like protein cage was concentrated and collected by centrifugal filtration. The DLS, AFM and TEM test results of the prepared imitation virus protein cage particles can be found in figure 1 , figure 2 and image 3 .

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Abstract

The invention relates to virus-like protein cage particles as well as a preparation method and application thereof. Specifically, the invention provides a preparation method of virus-like protein cageparticles based on nanobubbles. The preparation method comprises the following steps: S1, preparing a bulk phase nanobubble solution; S2, disassembling viruses into oligomers, diluting the oligomerswith an assembly buffer solution, purifying the oligomers, and determining the concentration of the oligomers; and S3, adding the bulk phase nanobubble solution into the oligomers, carrying out uniform mixing to enable virus-like protein cages to be self-assembled, and then performing centrifugal purification to obtain the self-assembled virus-like protein cage particles. The preparation method ofthe virus-like protein cage particles has the advantages of strong adjustability, no invasion, mild preparation conditions and reversibility of a self-assembly process.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for preparing a nano-bubble-based imitation virus protein cage particle, and the imitation virus protein cage particle prepared therefrom and its application. Background technique [0002] Virus-like protein cage particles, referred to as protein nanocages for short, are hollow spherical complexes with a uniform shape and particle size formed by self-assembly of one or several protein subunits. The particle size is between 10-1000 nanometers, such as virus-like nanoparticles, encapsulin, ferritin, chaperonin, etc. These protein cage structures have the characteristics of wide distribution, many types, good biocompatibility, highly ordered structure, monodisperse, and the process of assembly and disassembly can be controlled artificially. The commonly used preparation methods are the template method that induces the self-assembly of protein molecules by negative...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C07K1/02A61K9/51A61K47/42
CPCC07K14/005A61K9/5169
Inventor 张敏敏水玲玲
Owner SOUTH CHINA NORMAL UNIVERSITY