the synthesis raw materials of the non-degradable lipoid molecule B are cyclodisinine andtrifluoromethyl 1, 2-epoxydodecane, and the principle of the synthesis reaction is as follows:

it should be noted that FX3 and cdK11F in the above two synthetic reaction principles are representative symbols of lipid molecule a and lipid molecule B, respectively, and are not the chemical names of lipid molecule a and lipid molecule B, and are not the abbreviations of the chemical names of lipid molecule a and lipid molecule B, and are used only for convenience of description.

The antigen coding mRNA is synthesized by using a kit for in vitro transcription, and firstly, a plasmid vector which can conveniently and rapidly load and stably express the epitope is established. In the mRNA antigen synthesis, antigens commonly used in the immunoassay, such as Ovalbumin (OVA) and TRP2 protein (expressed in mouse melanoma cells B16), were selected and used as the encoding antigens for the experiment. In the case of in vitro transcription of mRNA, if the objective is to synthesize the full-length sequence of the encoded protein, the transcription efficiency and stability are required to be high, and the expression of epitopes derived from a plurality of different proteins is not possible. Therefore, a better strategy is to select and design a partial sequence of mRNA encoding antigen short peptide for expression, such as OVA257-264 (the short peptide at position 257 and 264 of OVA protein contains recognizable antigen epitope and is commonly used in research) and TRP2180-188, wherein the two antigen peptide epitopes are proved mRNA which can be presented by MHC and synthesized by in vitro transcription, and are named as OVA mRNA and TRP2 mRNA respectively. Various sites of mRNA are modified, including 5 'end modification and 3' end plus more than 100 poly-a, as well as the use of linker sequences and signal peptides to link epitopes to enhance the stability of mRNA transcribed in vitro. The designed antigen epitope sequence can be replaced according to the requirement and can also be used for simultaneously placing a plurality of epitopes from different tumor antigens, thereby being suitable for the preparation of vaccines targeting single or a plurality of antigens.

The preparation process of the mRNA tumor vaccine comprises the following steps: firstly, according to the experiment, the dosage of the used nano material and mRNA is calculated, and the lipid molecules FX3 and cdK11F are mixed according to the ratio of 3: 7 to prepare syn3 solution (the concentration is 10mg/ml, the solvent is DMSO), and the solution is heated to 70 ℃ before use until the solution is completely dissolved to be transparent and clear; adding lipid molecule mixture syn3, mRNA and stabilizer DOPE, cholestrol and DSPE-PEG2000 (solvent DMSO, lipid molecule mixture syn3: DOPE: cholestrol: DSPE-PEG2000 is 20:10:5: 1) according to the mass ratio (lipid molecule mixture syn3: mRNA is 160: 1); after the reagent is added, uniformly mixing by using a Vortex oscillator; after mixing, adding the mixture into sterile water with a corresponding volume according to a proportion (DMSO: water is 1: 9), dropwise adding the mixed material into the water while Vortex the sterile water by using Vortex, and completely mixing the mixed material by using Vortex after all the liquid is dropwise added; standing for 10min after thoroughly mixing, performing ultrafiltration by using an ultrafiltration tube with the aperture of 100kDa to remove an organic reagent DMSO, washing nano vaccine particles by using sterile water during ultrafiltration, and obtaining the residual liquid after ultrafiltration, namely the mRNA tumor vaccine (syn3-mRNA nano vaccine) taking FX3 and cdK11F as delivery carriers. It should be noted that DOPE is dioleoylphosphatidylethanolamine, cholestrol is cholesterol, DSPE-PEG2000 is distearoylphosphatidylethanolamine-polyethylene glycol 2000, and DMSO is dimethyl sulfoxide.

The syn3-mRNA nano vaccine prepared is identified to be in a form that mRNA is wrapped in the center by cationic lipid polymer to form nano particles. The nanoparticles have a particle size of about 100 nm and have been found to have good stability when monitored over a period of time (7 days). After nanoparticle formation, the mRNA is centrally encapsulated by cationic lipid polymers, thereby protecting the mRNA from nuclease degradation. The used stabilizer DAPE-PEG2000 can make the nanoparticles more stable and not easily influenced by physiological environment, thereby prolonging the circulation time, reducing the phagocytosis of cells to materials and making the nanoparticles have better biocompatibility. As shown in FIG. 1, it is the morphology and size of syn3-mRNA nano vaccine particles under a transmission electron microscope.

OVA mRNA (mRNA coding OVA257-264) synthesized by in vitro transcription is used as a delivery vector by using 15 different biological materials to prepare the OVA mRNA nano vaccine. The 15 nano vaccines were incubated with mouse dendritic cell DC2.4, and then B3Z cells (OVA-specific T cells, which can recognize MHC presented OVA epitopes and are activated) were stimulated to secrete cytokine IL-2. The secretion amount of cytokine IL-2 was measured by ELISA, and the results in FIG. 2B show that syn-3, as the delivery vehicle for mRNA, showed the highest degree of T cell activation, and stimulated secretion of cytokine IL-2 at higher levels than the other 6 different ratios of the mixture of two lipid molecules (0: 10, 1:9, 2: 8, 4: 6, 6: 4, 10: 0) as the vehicle. In this experiment, OVA257-264 protein short peptide-stimulated DC2.4 was used as a positive control, and the ratio of 7 different biomaterials used as delivery vehicles is shown in the following table:

in FIG. 2, A is a schematic diagram of an in vitro antigen presentation experiment. The mRNA encoding the antigen is taken up by the dendritic cells, the encoded antigen is expressed translationally and processed and presented to T cells, and the activated T cells secrete the cytokine IL-2. B shows that ELISA results show that 7 proportions of biological materials are respectively used as mRNA vaccine carriers in-vitro antigen presentation experiments, wherein the highest level of inducing T cells to activate and secrete IL-2 is achieved when OVA mRNA is loaded on the lipid molecule mixture syn-3. C shows that ELISA results show that the optimal mass ratio of syn-3 to OVA mRNA in vaccine preparation is 160: 1. the vaccine with syn-3 as the delivery vehicle was compared to Lip2000(Lip2000, i.e., Lipfectamine 2000, a liposome reagent commonly used in biological experiments for nucleic acid transfection). The secretion amount of the cytokine IL-2 was measured by ELISA, and D results showed that syn-3 as a delivery vector for mRNA showed higher levels of secretion of the cytokines IL-2 and IFN-gamma than that of liposome lipofectamine 2000.

In order to verify the effect of syn-3-OVA mRNA (coding OVA257-264) nano vaccine on stimulating antigen presentation and activating specific T cells in mice, the effect of the vaccine is verified by animal experiments. The test is divided into three groups (a control group, an OVA + Alum control group and a syn-3-OVA mRNA nano vaccine group), wherein Alum in the OVA + Alum group is an immunologic adjuvant aluminum hydroxide which is approved to be used in clinic at present, and the immune effect of an organism on OVA protein can be effectively improved. Mice were immunized 2 times with 7 day intervals by subcutaneous injection into the groin, draining the regional lymph nodes 7 days later, and the proportion of antigen-specific T cells (CD8+ OVA-Tetramer +) was analyzed by flow assay, as shown in FIG. 3A. As shown in FIG. 3B, a significant increase in the proportion of antigen-specific T cells in lymph nodes of the syn-3-OVA mRNA nano-vaccine immunized group can be seen. Therefore, the syn-3-mRNA nano vaccine can activate the presentation of the antigen and promote the activation of the antigen-specific T cells.

Subsequently, in order to verify that syn-3-mRNA nano vaccine can exert a preventive anti-tumor effect against tumor specific antigen and has a better effect than that of the traditional method (Alum aluminum hydroxide adjuvant is approved as a vaccine adjuvant to be clinically used in Europe, and Protamine Protamine is used as an mRNA carrier to be clinically tested), the function of inhibiting tumor growth is verified through experiments. First, experiments were performed using the constructed pancreatic cancer BxPC3 as a mouse tumor model, tumor cell inoculation (dorsal subcutaneous inoculation of pancreatic cancer BxPC3 cells, 106 cells per mouse). The experimental groups are: a control group, an OVA + Alum group, a Protamine-OVA mRNA group, and a syn-3-OVA mRNA group; protamines have been demonstrated to act as vehicles for delivery of mRNA and are therefore set as pairsGroup control; OVA + Alum was the commonly used immunostimulant in the experiment. The tumor volume was about 50mm 14 days after inoculation of the cell solution³And injecting the immune preparation intravenously, and observing the growth of the tumor. Experimental results show that syn-3-OVA mRNA nano vaccine can effectively inhibit the growth of tumor as a preventive tumor vaccine (figure 4B), and OVA + Alum group and Protamine-OVA mRNA group do not show good tumor inhibition effect. Fig. 4B shows the tumor growth curve of each mouse in each group. Therefore, syn-3-OVA mRNA can effectively inhibit tumor growth as a prophylactic tumor vaccine in a pancreatic cancer mouse tumor model.

The above description is only a part of or preferred embodiments of the present invention, and neither the text nor the drawings should be construed as limiting the scope of the present invention, and all equivalent structural changes, which are made by using the contents of the present specification and the drawings, or any other related technical fields, are included in the scope of the present invention.

Claims

1. An mRNA vaccine delivery vehicle comprising a lipid molecule a and a lipid molecule B, wherein the lipid molecule a is synthesized by reacting bromononene and trifluoromethyloct-2-en-1-ol, and the lipid molecule B is synthesized by reacting cyclodisinine and trifluoromethyl 1,2 epoxydodecane.

2. The mRNA vaccine delivery vehicle according to claim 1, wherein the molar ratio of bromononene and trifluoromethyloct-2-en-1-ol is 1:2 and the molar ratio of cyclodisinine and trifluoromethyl 1,2 epoxydodecane is 1: 6.

3. A method for preparing an mRNA vaccine delivery vector, which is characterized by comprising the following steps:

(2) mixing the cyclolysyl and the trifluoromethyl 1, 2-epoxydodecane according to a molar ratio of 1:6, and adding excessive methanol; heating the mixed solution in an oil bath, and controlling the reaction temperature to be 80 ℃ and the reaction time to be 48 h; after the reaction is finished, carrying out rotary evaporation on a product obtained by the reaction to obtain an oily mixture, wherein the oily mixture is the lipoid molecule B;

(3) and mixing the lipoid molecule A, the lipoid molecule B and the stabilizer to obtain the mRNA vaccine delivery carrier.

4. An mRNA vaccine comprising mRNA encoding a specific antigen and the mRNA vaccine delivery vector of claim 1 or 2.

5. The mRNA vaccine of claim 4, wherein the stabilizing agent comprises dioleoylphosphatidylethanolamine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.

6. The mRNA vaccine of claim 5, wherein the mass ratio of the total mass of lipid molecule A and lipid molecule B in the mRNA vaccine delivery vehicle to the dioleoylphosphatidylethanolamine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 20:10:5: 1.

7. The mRNA vaccine of claim 6, wherein the ratio of the mass of lipid molecule A and lipid molecule B to the mass of mRNA in the mRNA vaccine delivery vehicle is 48:112: 1.

8. The mRNA vaccine of claim 4, wherein the mRNA encodes an influenza antigen.

9. A method for preparing mRNA vaccine is characterized by comprising the following steps:

10. The method for preparing an mRNA vaccine according to claim 9, wherein the ultrafiltration is performed using an ultrafiltration tube having a pore size of 100 kDa.