阅读说明：本技术 一种用于核酸疫苗增强剂的纳米材料 (Nano material for nucleic acid vaccine enhancer ) 是由 吴延恒 于 2020-05-06 设计创作，主要内容包括：本发明提供了一种用于核酸疫苗增强剂的纳米材料,其特征在于,所述纳米材料通过以下方法制备获得：(1)制备A溶液：将CaC<Sub>l2</Sub>与Tris buffer溶于水中；(2)制备B溶液：将HEPES缓冲液与水混合；(3)将B溶液滴加到A溶液中,搅拌,制得CP纳米颗粒沉淀；(4)将帕米膦酸钠滴加到步骤(3)的AB混合液中,滴加人血丙种球蛋白,获得本发明的纳米材料。本发明的纳米材料显著提高核酸疫苗对靶细胞的转染效率,增加抗原表达,促进抗原提呈细胞成熟从而建立有效的免疫反应,实现核酸疫苗的增强或增效功能。(The invention provides a nano material for a nucleic acid vaccine enhancer, which is characterized by being prepared by the following method: (1) preparation of solution A: mixing CaC l2 Dissolving with Tris buffer in water; (2) preparing a solution B: mixing HEPES buffer solution with water; (3) dropwise adding the solution B into the solution A, and stirring to obtain CP nanoparticle precipitate; (4) and (4) dropwise adding sodium pamidronate into the AB mixed solution obtained in the step (3), and dropwise adding human gamma globulin to obtain the nano material. The nanometer material of the invention obviously improves the transfection efficiency of the nucleic acid vaccine to target cells, increases antigen expression, promotes the maturation of antigen presenting cells so as to establish effective immune response and realize the enhancement or synergy function of the nucleic acid vaccine.)

1. A nanomaterial for a nucleic acid vaccine enhancer, characterized in that the nanomaterial is prepared by the following method:

(1) preparation of solution A: adding CaCl₂Dissolving with Tris buffer in water;

(2) preparing a solution B: mixing HEPES buffer solution with water;

(3) dropwise adding the solution B into the solution A, and stirring to obtain CP nanoparticle precipitate;

(4) and (4) dropwise adding sodium pamidronate into the AB mixed solution obtained in the step (3), and dropwise adding human gamma globulin to obtain the nano material.

2. The nanomaterial of claim 1, wherein the CaCl is₂The concentration of (A) is 1.0-2.0M; the concentration in the Trisbuffer is 10mM, and the pH value is 10.

3. The nanomaterial of claim 1, wherein the HEPES buffer comprises the following components: 200 to 280mM NaCl, 10 to 15mM Na₂HPO₄And 24 to 50mM HEPES.

4. The nanomaterial of claim 1, wherein the concentration of the pamidronate sodium is from 0.1 to 5 mg/mL.

5. The nanomaterial of claim 1, wherein the concentration of human gamma globulin is from 0.04 to 0.8 ug/mL.

Technical Field

The invention belongs to the technical field of nano materials, and particularly relates to a nano material for a nucleic acid vaccine enhancer.

Background

The nucleic acid vaccine is prepared through introducing exogenous gene (DNA or RNA) encoding certain antigen protein into animal or human body cell, synthesizing antigen protein via the expression system of host cell, and inducing the host to produce immune response to the antigen protein for preventing and treating diseases. The nucleic acid vaccine has many advantages compared with the traditional vaccine, and the existing nucleic acid vaccine enters the clinical stage and is applied to the market at present. However, how to make the nucleic acid vaccine reach the target cell smoothly after entering the body and express and present the antigen protein effectively is a bottleneck that hinders the rapid development and accelerated application of the nucleic acid vaccine.

At present, only a very small amount of nucleic acid vaccines (2%) can freely enter cells after entering an organism, but fewer vaccines which can reach target cells (antigen presenting cells) exist, so that the nucleic acid vaccines are large in immune dosage, large in side effect, high in cost and not ideal in immune effect. After the nucleic acid vaccine immunizes an organism, the antigen protein must be effectively expressed in the antigen presenting cells, the antigen is processed and presented, and the cell maturation immune reaction is completed, but if the nucleic acid vaccine enters the immature antigen presenting cells, the expression of the antigen protein still can not be realized, so that the low immune efficiency is caused. Many developed nucleic acid vaccines cannot be finally used in clinic, but because the vaccine is designed, a nucleic acid vaccine enhancer is not provided to improve the transfection efficiency of the nucleic acid vaccine enhancer on effective cells, an effective immune response cannot be established, and finally, further development and popularization and application of the vaccine are abandoned carelessly.

Although there are some rare studies to enhance immune response by using nanomaterial adjuvant nucleic acid vaccines, they are called vaccine adjuvants and present only a certain link to antigen. The nucleic acid vaccine reinforcing agent is different from an adjuvant, and comprises the steps of protecting a nucleic acid vaccine, promoting the nucleic acid vaccine to enter antigen presenting target cells, improving the maturity of the antigen presenting target cells, enhancing the transcription expression and the antigen processing and presentation of protein, and comprehensively improving the efficiency of the nucleic acid vaccine.

There is no report of nucleic acid vaccine enhancing (efficacy) agent at present. The method makes up the blank, can remarkably improve the transfection efficiency of the nucleic acid vaccine to target cells, increases antigen expression, promotes the maturation of antigen presenting cells so as to establish effective immune response, and realizes the enhancement or synergy function of the nucleic acid vaccine.

Disclosure of Invention

The present invention aims at overcoming the demerits of available technology, and provides one kind of nanometer material for nucleic acid vaccine intensifier, which can send nucleic acid vaccine into target cell and raise the expression and presentation of antigen protein effectively to raise immune reaction in several steps.

In order to achieve the purpose, the invention adopts the technical scheme that: a nanometer material for nucleic acid vaccine enhancers is prepared by the following steps:

(1) preparation of solution A: adding CaCl₂Dissolving with Tris buffer in water;

(2) preparing a solution B: mixing HEPES buffer solution with water;

(3) dropwise adding the solution B into the solution A, and stirring to obtain CP nanoparticle precipitate;

(4) and (4) dropwise adding sodium pamidronate into the AB mixed solution obtained in the step (3), and dropwise adding human gamma globulin to obtain the nano material.

Preferably, CaCl₂The concentration of (A) is 1.0-2.0M; the concentration of the Tris buffer is 10mM, and the pH value is 10.

Preferably, the HEPES buffer comprises the following components: 200 to 280mM NaCl, 10 to 15mM Na₂HPO₄And 24 to 50mM HEPES. The HEPES is 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid.

Preferably, the concentration of the Pamidronate sodium (Pamidronate) is 0.1-5 mg/mL.

Preferably, the concentration of the human gamma globulin is 0.04-0.8 ug/mL.

Compared with the prior art, the invention has the following beneficial effects:

1. the nucleic acid vaccine nano enhancer has cell selectivity, can be specifically combined with antigen presenting cells, and has the cell targeting property (antigen presenting cells) of nucleic acid vaccine delivery.

2. The cell membrane is a phospholipid bilayer, and an electronic dense band with the thickness of about 2.5nm is respectively arranged at the inner side and the outer side of the membrane, so that the characteristics of the cell membrane are not beneficial to most of nucleic acid fragments or plasmids to enter cells, and the probability of nucleic acid entering the cells is greatly reduced. The nucleic acid vaccine nano reinforcing agent is a nano particle with good physical and chemical compatibility, can effectively establish bridge connection between nucleic acid and cell membranes, greatly improve the contact probability of the nucleic acid and the cell membranes and the efficiency of entering cytoplasm and cell nucleus, and can stimulate the phagocytic function and cell maturation of antigen presenting cells and promote the immune reaction of the nucleic acid vaccine.

3. After the nanoparticles carrying the nucleic acid vaccine are phagocytized by antigen presenting cells, the pH value in a phagocytosis lysosome can be changed, and then the nucleic acid is protected from being degraded and destroyed by nuclease in the phagocytosis lysosome.

4. The DNA can be transcribed and translated only after entering the cell nucleus, and finally the antigen protein is expressed. The nucleic acid vaccine nano enhancer disclosed by the invention has the capability of improving the high-efficiency entry of the nucleic acid vaccine into cell nucleus.

5. Immature antigen-presenting cells, even after taking up nucleic acid material and entering the nucleus, are unable to present the relevant expressed antigen, and only after these cells are mature, can achieve efficient presentation of the antigen to T or B cells, inducing an immune response. The nucleic acid vaccine nano enhancer disclosed by the invention can obviously promote the conversion of immature antigen presenting cells to mature antigen presenting cells, so that the immune efficacy of the nucleic acid vaccine is effectively improved.

Drawings

FIG. 1 is a transmission electron microscope image of the MCP nanoparticles of the present invention.

FIG. 2 is a graph showing the efficiency of uptake of 4-hour macrophage RAW264.7 to CP-Cy5, Liposome-Cy5, MCP-Cy 5.

FIG. 3 is a diagram showing the in vitro delivery efficiency of MCP nanoparticles after pGFP transfection to macrophages, wherein A is the percentage of positive cells and B is the result of flow cytometry.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the present invention will be further described in detail with reference to specific embodiments.