Influenza virosome coated bionic nano vaccine and preparation method thereof

A kind of influenza virus, bionic nano technology, applied in biochemical equipment and methods, virus/bacteriophage, vaccine, etc., can solve the problem of low pDNA expression efficiency, and achieve the effect suitable for large-scale production, low production cost and good repeatability

Active Publication Date: 2020-02-11
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression efficiency of pDNA modified with NLS is not high. In addition, the charge ...

Method used

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  • Influenza virosome coated bionic nano vaccine and preparation method thereof
  • Influenza virosome coated bionic nano vaccine and preparation method thereof
  • Influenza virosome coated bionic nano vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] (1) Preparation of VI: Influenza virus PR8 was ultracentrifuged, and the bottom precipitate was collected. For every 5 mg of precipitated virus, 375 μL of 200 mM 1,2-dihexanoylphosphatidylcholine was added for 30 minutes on ice. Collect the supernatant by ultracentrifugation, dialyze the supernatant in HBS buffer for 24 hours, remove 1,2-dicaproyl lecithin, and obtain recombinant VI, wherein the ultracentrifugation conditions are 4°C, 100,000×g, 1.5h;

[0060] (2) Synthesis of fluorinated cationic polymer: react according to the molar ratio of amino group, heptafluorobutyric anhydride and triethylamine in the cationic polymer is 1:3:1.2, the reaction medium is methanol, and stirred at room temperature for 48 hours. After the reaction, the reaction product was dialyzed in deionized water with a pH of 3 to 4 for 2 days, and freeze-dried to obtain a fluorinated modified cationic polymer. The cationic polymer used in this example was 25kDa PEI;

[0061] (3) FAu preparation:...

Embodiment 2

[0065] Low pH conditions in cellular endosomes / lysosomes can induce conformational changes in influenza virus hemagglutinin (HA), causing fusion of the viral envelope with endosome / lysosome membranes. Therefore, under acidic conditions, when the conformation of HA adsorbed on red blood cells changes, it will cause the red blood cell membrane to rupture, thereby releasing hemoglobin and causing hemolysis. Thus, the fusion process of the membrane can be simulated by using the hemolysis experiment. Such as image 3 As shown in A, ARV and VI prepared in Example 1 were incubated with red blood cells respectively, OD 540 UV absorption increased with the decrease of pH, both ARV and Ⅵ had hemolytic effect, which indicated that membrane fusion had occurred. Further, anionic liposomes are used to simulate the membrane structure of cells and carry fluorescence resonance energy transfer (FRET) compounds, which are composed of DOPC, cholesterol and FRET reagent pair NBD-PE and Rho-PE, w...

Embodiment 3

[0067] The ARV prepared in Example 1 was immunized into mice (C57BL / 6, 20±2 g, male) by subcutaneous injection at the base of the tail at the first week and the third week, and each dose was 40 μg of ARV protein. Isolate the spleen at the 4th week, prepare a single cell suspension, collect 2 million cells into a 1.5mL centrifuge tube, centrifuge at 1600rpm for 5min, remove the supernatant, wash gently with 1mL PBS twice, and finally resuspend in 100μl PBS, wash with CD3, CD8 Antibody and CD3, CD4 antibody labeled cells, incubated at 4°C in the dark for 30min. Subsequently, it was fixed with 4% paraformaldehyde for flow detection. Such as Figure 4 As shown in A-4B, VI, pFAu and ARV can effectively induce CD8 + T and CD4 + The content of T cells increased, among which ARV had the strongest induction ability. Serum samples of immunized mice were prepared in the 4th week, and the IgG titer in the serum was determined, such as Figure 4 As shown in C, the prepared ARV has a...

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Abstract

The invention discloses an influenza virosome (VI) coated bionic nano vaccine and a preparation method thereof. The bionic nano vaccine comprises VI, small-particle-size fluorinated particles and DNAvaccines, the bionic nano vaccine is a core-shell structure nano system, the VI is lipid vesicles containing influenza virus envelope protein, inner cores of the nano system are small-particle-size fluorinated particles loaded with the DNA vaccines, and the VI wraps the surfaces of the inner cores. The VI provided by the invention has receptor binding activity, lysosome membrane fusion activity and antigen activity of influenza virus; and the small-particle-size fluorinated inner cores can deliver DNA into cores to promote protein expression. The nano system can realize the common loading of protein vaccines and the DNA vaccines and the site-specific delivery of each component, and finally a synergistic effect is achieved to improve the immune effect. The invention belongs to the field ofpharmaceutical preparations and biological medicines. Influenza virus infection can be effectively prevented, and very high clinical application value is achieved.

Description

technical field [0001] The invention relates to the field of pharmaceutical preparations and the field of biomedicine technology, in particular to a biomimetic nano-vaccine coated with influenza virus bodies and a preparation method thereof. Background technique [0002] The spread of seasonal influenza virus poses a huge threat to global public health security and seriously affects human health and economic development. Vaccination is still the main means of preventing and dealing with influenza virus. The currently approved influenza vaccines are mainly inactivated vaccines (such as split vaccines) and live attenuated vaccines. However, there are still many safety hazards in the use of influenza vaccines based on modified and inactivated virus strains, mainly including reverse mutations of pathogens, allergy and autoimmunity and other adverse reactions. In comparison, DNA vaccines and protein vaccines have the advantages of good safety, high purity, and strong specificity...

Claims

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Application Information

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IPC IPC(8): A61K39/125A61K9/14A61K47/02A61P31/16
CPCA61K39/12A61K9/143A61P31/16C12N2760/16134A61K2039/53
Inventor 陈刚周昕李真真
Owner YANGZHOU UNIV
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