A kind of virus-like protein cage particle and its preparation method and application
A protein and virus 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.
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Embodiment 1
[0041] Example 1 Disassembly of native CCMV virus and purification of capsid protein
[0042] 1 mL of native cowpea chlorotic mottle virus (CCMV virus) was collected from freshly infected cowpea plants in virus buffer solution (0.1 M sodium acetate, 1 mM disodium EDTA, 1 mM sodium azide, pH 5.0), It was quickly stored in a refrigerator with a constant temperature of 4.0°C to obtain a CCMV suspension. Prepare RNA buffer (50 mM Tris, 500 mM CaCl 2, 1 mM dithiothreitol, pH 7.5). The obtained CCMV suspension was first dialyzed in 200 mL RNA buffer for 2 h, and then dialyzed in 800 mL buffer overnight at 4°C. When white floccules are observed, transfer to a centrifuge tube, centrifuge at 40,000 rpm / min for 2 h, extract the supernatant, and remove the white flocculent precipitate containing RNA. The supernatant (~1 mL) was dialyzed against 330 mL of RNA buffer solution, and the buffer solution was changed every 3 hours for a total of 6 h; followed by dialysis against 330 mL of bu...
Embodiment 2
[0043] Example 2: Preparation and performance characterization of nanobubbles
[0044] A self-made nanobubble generator was used to prepare bulk nanobubbles by electrolyzing water. A 10 mM sodium chloride solution was prepared with deionized water (conductivity 18.4 MΩ·cm, 25° C.). The solution was flowed into the electrode tank where water was being electrolyzed (electrolysis voltage 24V, current 3A) with a vacuum pump at a flow rate of 500 mL / min. The electrolyzed water generates oxygen at the anode and hydrogen at the cathode, and the generated gas is dissolved in the sodium chloride solution at the same time, so that the solution is in a supersaturated state and bulk nanobubbles are generated. The supersaturation rate of the solution is controlled by the amount of gas generated, while the surface potential of the nanobubbles is further controlled. The oxygen supersaturation was 110%-130% measured with an oxygen meter (Fibox 3trace 3).
[0045] The prepared nanobubble so...
Embodiment 3
[0047] Example 3: Nanobubbles induce self-assembly of capsid proteins
[0048] To 200 μL of the purified CCMV capsid protein suspension, 400 μL of nanobubble solution was rapidly added at 4°C. The mixed solution was gently mechanically shaken for 1 min, and then placed on a constant temperature shaker at 4°C overnight to induce the self-assembly of capsid proteins to form virus-like protein cages. The formed virus-like protein cages were concentrated and collected by centrifugal filtration. The DLS, AFM and TEM test results of the prepared virus-like protein cage particles are shown in figure 1 , figure 2 and image 3 .
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