CN112237628A

CN112237628A - EBV-targeted LMP2-mRNA nano vaccine

Info

Publication number: CN112237628A
Application number: CN202010687809.1A
Authority: CN
Inventors: 宋相容; 魏于全
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2019-07-17
Filing date: 2020-07-16
Publication date: 2021-01-19

Abstract

The invention belongs to the field of biological medicines, and particularly relates to LMP2 antigen-enhanced mRNA (messenger ribonucleic acid) and a design method and application thereof, a targeted nano preparation carrier and a preparation method and application thereof, and an LMP2mRNA liposome vaccine and a preparation method and application thereof. The design adopts MHC-I molecule intracellular sequences, and can obviously improve the presenting efficiency of the expressed antigen by MHC-I and MHC-II. The method has universality, the designed LMP2 antigen enhanced mRNA has stronger immunogenicity, and the prepared mRNA vaccine can be used for treating a series of cancers and tumors which are caused by the virus infection and represented by nasopharyngeal carcinoma.

Description

EBV-targeted LMP2-mRNA nano vaccine

Priority application

The present application claims priority of the "EBV targeted LMP2-mRNA nano vaccine" chinese patent application 201910648034.4 filed 2019, 7/17, which is incorporated by reference in its entirety.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to LMP2 antigen-enhanced mRNA (messenger ribonucleic acid) and a design method and application thereof, a targeted nano preparation carrier and a preparation method and application thereof, and an LMP2-mRNA liposome vaccine and a preparation method and application thereof.

Background

According to data of international cancer research institutions, in 2018, about 12.9 ten thousand new cases of nasopharyngeal carcinoma account for 0.7% of all cases diagnosed in 2018. However, its global distribution is highly unbalanced, especially prevalent in east and south east asia, accounting for over 70% of new cases. The incidence rate of nasopharyngeal carcinoma in men is higher than that in women, and the incidence rate of nasopharyngeal carcinoma in China in 2015 is about 2.5. Nasopharyngeal carcinoma is characterized by unique geographic distribution, and is different from other head and neck cancers in biology, epidemiology, histology, natural history, treatment response and the like, so the treatment of the nasopharyngeal carcinoma has specificity. These findings suggest that a combination of genetic, ethnic and environmental factors may influence the pathogenesis of nasopharyngeal carcinoma.

Radiotherapy is the main means of treating nasopharyngeal carcinoma at present, and is also the basic mode of treating the nasopharyngeal carcinoma without dissemination. Due to the special anatomy of the nasopharynx, the operation is not suitable for the initial treatment of nasopharyngeal carcinoma. But the non-specific damage of the body caused by radiotherapy is huge. Local recurrence and distant metastasis are two reasons for the failure of nasopharyngeal carcinoma treatment. Therefore, a new comprehensive treatment means is urgently needed to be found on the basis of the conventional treatment, and in recent decades, along with the research progress of molecular biology and tumor immunology, the molecular biology means is used for improving the cellular immunity of patients, preventing the recurrence and metastasis of nasopharyngeal carcinoma and serving as a supplement of the traditional treatment method, and more attention is paid to the treatment.

Viral antigens are the most immunogenic molecules in malignancies and the most significant tumor antigens in protective tumor immunity, and epstein barr virus, a member of the herpesviridae family, is the first virus found to be associated with human tumors. EB virus is now well established as one of the major causes of nasopharyngeal carcinoma, lymphoma and post-immunosuppressive lymphoma. The potential membrane proteins LMP1 and LMP2 expressed by EB virus play multiple oncogenic roles and can play roles by activating multiple signal transduction and regulating the expression of various oncogenic genes. In the aspect of therapeutic vaccines, EBV nuclear antigen EBNA1, latent membrane antigen LMP1 and LMP2 are the main therapeutic targets.

LMP2 is one of the virus proteins expressed continuously in the tissues of EB virus related tumor such as nasopharyngeal carcinoma, etc. and it does not cause B lymphocyte transformation, and in vitro and in vivo studies have proved that EB virus LMP2 gene can induce well to produce specific cellular immune response. Therefore, LMP2 becomes a good target antigen in inducing EB virus specific cellular immune response, and preventing and treating related tumors. To elicit a specific cellular immune response against LMP2, one must first select an LMP2 expression system suitable for therapeutic use.

At present, only polypeptide, DNA vaccine and CTL cell treatment of the vaccine aiming at LMP2 are in clinical research stage, and mRNA vaccine is not reported in literature.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a method for enhancing the immunogenicity of an EBV mRNA vaccine. The method has universality, and can be simply and conveniently expanded to the research and development of therapeutic mRNA vaccines of cancers caused by other EBVs.

In order to achieve the purpose, the invention adopts the following scheme:

a method for enhancing the immunogenicity of an EBV mRNA vaccine is characterized in that a transcription template DNA of mRNA is formed by sequentially connecting a promoter, a 5 'end untranslated region, a secretion signal of MHC-I type molecules, an antigen coding region, transmembrane and cytoplasmic domains, a 3' end untranslated region, A120 and a terminal sequence behind the A120.

Among them, the terminal sequence of A120 includes restriction sites which can be cut from the end of polyA, including but not limited to BspQI, SapI, EarI, etc.

Specifically, conventional mRNA transcription vectors are composed of only a promoter and a target protein coding region. The invention adds immunogenicity enhancing signal (IE) on the basis of traditional mRNA transcription vector design, namely: the two ends of the target antigen protein coding region are respectively additionally added with a secretion signal and transmembrane and cytoplasmic domains of MHC-I molecules so as to enhance the immunogenicity of the mRNA vaccine by enhancing the antigen presentation effect of the target antigen protein.

Wherein the sequence of the antigen coding region may be as set forth in SEQ ID NO: 7-10 and sequences with greater than 90% identity thereto.

The promoter is T7 or SP6 promoter.

Further, the nucleotide sequence of the 5' end untranslated region is shown in SEQ ID NO: 1 and synonymous codons thereof, and a sequence with more than 90% of identity with the sequence; the secretion signal of the MHC class I molecule includes but is not limited to various classes of MHC class I molecules from different species, such as: human MHC I molecule (HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-K, HLA-I), mouse MHC I molecule (H-2D, H-2K, H-2L), rat MHC I molecule, rhesus MHC I molecule.

Further, the nucleotide sequences of the transmembrane and cytoplasmic domains are shown in SEQ ID NO: 2 and synonymous codons thereof, and a sequence having more than 90% identity with the above sequence.

Further, the nucleotide sequence of the 3' end untranslated region is shown in SEQ ID NO: 3 and synonymous codons thereof, and a sequence having more than 90% identity with the above sequence.

Further, the nucleotide sequence of A120 is shown in SEQ ID NO: 4, the nucleotide sequence of the terminal sequence of A120 is shown as SEQ ID NO: 5, respectively.

Specifically, in the process of experimental research, mRNA can still be obtained when the amount of polyA in the nucleotide sequence of A120 is 50-500; when the amount is 80-200, a stable mRNA can be obtained, but when the amount is close to 120, the stability is the best.

Further, the object is a transcription template DNA of mRNA obtained by the method.

The other purpose of the invention is to provide an LMP2 immune-enhanced EBV mRNA vaccine, which is prepared from mRNA transcription template DNA. The vaccine can be used for preventing and treating a series of cancers represented by nasopharyngeal carcinoma and caused by the virus infection.

In order to achieve the purpose, the invention adopts the following scheme:

an immune-enhanced EBV mRNA vaccine of LMP2, wherein the mRNA transcription template DNA is sequentially connected by a promoter, a 5 'end untranslated region, a secretion signal of MHC-I type molecules, an antigen coding region, transmembrane and cytoplasmic domains, a 3' end untranslated region, A120 and a terminal sequence behind A120; the nucleotide sequence of the antigen coding region is shown as SEQ ID NO: 6 and sequences with identity over 90%.

Further, the nucleotide sequence of the mRNA is shown as SEQ ID NO: 7 and sequences with identity of more than 90%.

Furthermore, the targeting nano preparation carrier prepared by the mRNA vaccine.

Further, the targeted nano-preparation carrier is a liposome carrier.

Further, the targeting nano-preparation carrier is a core-shell nanoparticle.

Preferably, the liposome carrier is a cationic liposome carrier.

Specifically, mRNA is easily degraded by mRNA enzyme which is widely existed in vivo and in vitro, and proper carrier is selected to load mRNA, so that on one hand, mRNA can be protected from being degraded by mRNA enzyme, on the other hand, the mRNA can be enhanced to be taken by antigen presenting cells, even the encoded antigen protein can be continuously expressed, and the lasting immune activation effect can be exerted. Therefore, the invention firstly designs and constructs two carriers with better clinical transformation prospect, namely cationic liposome and core-shell nanoparticles; and taking GFP-mRNA as the model mRNA, and carrying out systematic optimization on the molding process parameters of the two preparations to determine the optimal preparation process and the prescription composition of the two carriers, so as to further carry out in-vitro cell evaluation on the two non-viral carrier preparations carrying the LMP 2-mRNA.

The cationic liposome has positive charges on the surface, can wrap mRNA molecules into the cationic liposome and phosphate radicals of nucleic acid through electrostatic interaction to form an mRNA-lipid complex, can also be adsorbed by cell membranes with negative charges on the surface, and then mRNA is transferred into cells through fusion of the membranes or endocytosis of the cells and occasionally through direct osmosis, and then is further expressed.

Further, the targeting nano-preparation carrier comprises a phospholipid material, an additive and an organic solvent; the phospholipid material comprises one or more of 1, 2-distearoyl-3-trimethylammonium propane, trimethyl ammonium chloride, octadecenoyloxy (ethyl-2-heptadecenyl-3-hydroxyethyl) imidazoline , didecyl adipate, 3- [ N- (N ', N' -dimethylaminoethane) carbamoyl ] cholesterol,

dimethylamino

1,2, 2-epoxypentyloxypropane and didecyl dimethyl ammonium bromide; the additive comprises one or more of total cholesterol, dioleoylphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylcholine; the organic solvent comprises one or more of absolute ethyl alcohol, methanol, ether, chloroform and methanol; the targeting nano-preparation carrier comprises the following components: 2.5mg/ml of 1, 2-distearoyl-3-trimethylammonium propane, 2.5mg/ml of total cholesterol, absolute ethanol;

or the following components: n- [ l- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium 3mg/ml, dioleoylphosphatidylethanolamine 2mg/ml, phosphatidylethanolamine 5mg/ml, methanol;

or the following components: octadecenoyloxy (ethyl-2-heptadecenyl-3-hydroxyethyl) imidazoline 4mg/ml, phosphatidylcholine 2.7mg/ml, diethyl ether;

or the following components: 8mg/ml of dimethyl dioctadecyl ammonium, 2mg/ml of dioleoyl phosphatidyl ethanolamine and chloroform;

or the following components: 3- [ N- (N ', N' -dimethylaminoethane) _ carbamoyl ] cholesterol 5mg/ml, 1, 2-distearoyl-3-trimethylammonium propane 2mg/ml, total cholesterol 2mg/ml, methanol;

or the following components: 7.5mg/ml 1, 2-distearoyl-3-trimethylammonium propane, 10mg/ml N- [ l- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium, 7.5mg/ml phosphatidylethanolamine, anhydrous ethanol;

or the following components: 4.2mg/ml of

dimethylamino

1,2, 2-epoxypentoxypropane, 4.2mg/ml of total cholesterol and methanol;

or the following components: 1.6mg/ml of didecyl dimethyl ammonium bromide, 2.4mg/ml of total cholesterol and chloroform.

The liposome consists of lipid (phospholipid) and additive. Phospholipids: including natural phospholipids and synthetic phospholipids. The structure of the phospholipid is characterized by a hydrophilic group consisting of a phosphate group and a quaternary ammonium salt group, and a lipophilic group consisting of two longer hydrocarbon groups; the natural phospholipid is mainly lecithin (phosphatidylcholine, PC), is derived from egg yolk and soybean, and is neutral; the synthetic phospholipid mainly comprises DPPC (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine) and the like, all belong to hydrogenated phospholipids, have the characteristics of stable property, strong oxidation resistance, stable finished product and the like, and are preferred auxiliary materials abroad. Cholesterol and phospholipids are the basic substances that together constitute cell membranes and liposomes. Cholesterol has the effect of regulating membrane fluidity and may be referred to as liposome a "fluidity buffer".

Liposomes generally have several characteristics: 1) targeting and lymph targeting; 2) sustained release effect; 3) the toxicity of the medicine is reduced; 4) the stability is improved.

Further, the cationic liposome carrier is prepared by a thin film hydration method. The method specifically comprises the following steps:

1) adding a proper amount of phospholipid material into an organic solvent for dissolving;

2) heating in water bath, and performing rotary evaporation under reduced pressure to remove the organic solvent to form a film from the phospholipid material;

3) adding hydration medium to hydrate the phospholipid membrane to obtain lipid colloid solution;

4) carrying out probe ultrasonic treatment on the lipid colloidal solution obtained in the step 3) under the water bath heat preservation condition or homogenizing the lipid colloidal solution by a homogenizer, a microjet and other equipment to obtain a liposome carrier;

5) adjusting the concentration of the solution obtained in the step 4) to a certain concentration, and then obtaining the cationic liposome carrier through a sterile water system filter membrane.

Further, the phospholipid material of step 1) is a cationic phospholipid material selected from the group consisting of DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC 14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and derivatives thereof, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, DOPE, and combinations thereof with an additive selected from the group consisting of one or more of Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), cholesterol (Chol), Dioleoylphosphatidylethanolamine (DOPE), and the like; the organic solvent is selected from ethanol, methanol, chloroform, diethyl ether, etc. capable of dissolving phospholipid material.

Further, the water bath heating temperature in the step 2) is 10-90 ℃, and the rotation speed of the reduced pressure rotary evaporation is 10-200 r/min.

Further, the time for hydrating the phospholipid membrane in the step 3) is 15-240 minutes, the temperature is 10-90 ℃, and the rotating speed is 10-200 revolutions per minute.

Further, the concentration in the step 5) is 1-100 mmol/L.

The invention also aims to provide a preparation method of the vaccine.

Specifically, the preparation method of the vaccine comprises the following steps: and mixing the prepared liposome carrier solution and the mRNA solution uniformly, and incubating to prepare the mRNA liposome vaccine.

Further, the liposome carrier is a cationic liposome.

Further, the N/P ratio of the phospholipid material to mRNA in the liposome vaccine is 0.5-10: 1.

Further, preferably, the N/P ratio of the phospholipid material and the mRNA in the liposome vaccine is 1-5: 1.

Further, the incubation temperature is 10-40 ℃, and the incubation time is 10-1000 minutes.

Further preferably, the incubation temperature is 20-30 ℃, and the incubation time is 30-240 minutes.

The fourth purpose of the invention is to provide the LMP2mRNA liposome vaccine for the application in resisting tumors, including but not limited to rhinitis tumors.

In addition to nasopharyngeal carcinoma, the mRNA in the present drug can also treat lymphoblastomas, Burkitt's lymphomas, Hodgkin's lymphomas, NK and T cell lymphomas, diffuse large B cell lymphomas, major effusion lymphomas, positive follicular lymphomas, multicenter Castleman disease, gastric Cancer, etc. caused by EB Virus, as described in a published article of interest (publication 1: Corey Smith, Rajiv Khanna. the Development of Prophylactic and Therapeutic EBV Vaccines [ J ]. Curr Top Microbiol Immunol 2015,391:455-

Further, the LMP2mRNA liposome vaccine promotes expression of the encoded antigen LMP2 and stimulates production of specific CTLs.

The fifth purpose of the invention is to provide the application of mRNA in enhancing the immunogenicity of the EBV vaccine.

In order to achieve the purpose, the invention adopts the following scheme:

the transcription template DNA of the mRNA is formed by connecting a promoter, a 5 'end untranslated region, a secretion signal of MHC-I type molecules, an antigen coding region, a transmembrane and cytoplasmic domain, a 3' end untranslated region, A120 and a terminal sequence behind A120 in sequence; the nucleotide sequence of the 5' end untranslated region is shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the 3' end untranslated region is shown in SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of A120 is shown as SEQ ID NO: 4, the nucleotide sequence of the terminal sequence of A120 is shown as SEQ ID NO: 5, respectively.

Further, the nucleotide sequence of the mRNA transcription template DNA is shown as SEQ ID NO: shown at 7.

The invention also provides a method for delaying the degradation of the mRNA vaccine, which is to uniformly mix the prepared liposome carrier solution and the mRNA vaccine solution and then incubate the mixture to prepare the mRNA liposome vaccine.

Further, the N/P ratio of the phospholipid material and the mRNA in the liposome vaccine is 0.5-10: 1.

Preferably, the N/P of the phospholipid material and the mRNA in the liposome vaccine is 1-5: 1.

Also provides the application of the mRNA vaccine, the LP-mRNA-LMP2 and the targeting nano-preparation carrier in the preparation of the T cell proliferation promoter.

Further, the application is in preparing CD8⁺Use of a promoter of T cell proliferation.

Further, the proliferation of said T cells is useful in the treatment or prevention of EB virus-induced diseases including lymphoblastoma, Burkitt's lymphoma, Hodgkin's lymphoma, NK cell and T cell lymphoma, diffuse large B cell lymphoma, major effusion lymphoma, positive follicular lymphoma, multicentric Castleman's disease, nasopharyngeal carcinoma, gastric carcinoma.

Further, the mRNA vaccine, the targeting nano-preparation carrier and the application of the LP-mRNA-LMP2 in preparing anti-tumor drugs are also provided.

Further, the application in the preparation of the promoter of the tumor killing cell factor.

Further, the tumor is lymphoblastoma, Burkitt's lymphoma, Hodgkin's lymphoma, NK cell and T cell lymphoma, diffuse large B cell lymphoma, major effusion lymphoma, positive follicular lymphoma, multicentric Castleman's disease, nasopharyngeal carcinoma, gastric carcinoma.

Further, the tumor is nasopharyngeal carcinoma.

The invention has the beneficial effects that:

1) the LMP2 antigen enhanced mRNA provided by the invention has stronger immunogenicity, and the prepared mRNA vaccine can be used for treating a series of cancers caused by the virus infection, which are represented by nasopharyngeal carcinoma;

2) the method for enhancing the immunogenicity of the EBV mRNA vaccine improves the structure of the traditional mRNA, so that the mRNA obtained by the method not only enhances the immunogenicity of the vaccine, but also can be simply and conveniently expanded to the design of other high-risk EBV mRNA, and has universality. (ii) a

3) The LP-mRNA-LMP2 vaccine provided by the invention has better storage stability, serum stability and transfection stability, can generate LMP2 specific T cells, stimulates the generation of specific CTL, and exerts high-efficiency anti-tumor activity; and is safe and has no toxic and side effects.

Drawings

FIG. 1 is an agarose gel electrophoresis of LMP2 antigen-enhanced mRNA template.

FIG. 2 shows denaturing agarose gel electrophoresis of LMP2 antigen-enhanced mRNA.

FIG. 3 shows the gel blocking results of the mRNA liposome vaccine.

FIG. 4 is a graph of the particle size of LMP2 mRNA.

FIG. 5 is a gel electrophoresis of LMP 2-mRNA.

FIG. 6 is a schematic representation of the LP-mRNA-LMP2 delivery system.

FIG. 7 is a transmission electron micrograph of LP-mRNA-LMP 2.

FIG. 8 shows the particle size and PDI changes of LP-mRNA-LMP2 and CS-mRNA-LMP2 before and after adsorption of serum proteins.

FIG. 9 shows Zeta potential changes of LP-mRNA-LMP2 and CS-mRNA-LMP2 before and after adsorption of serum proteins.

FIG. 10 is a graph showing the particle size, PDI, of LP-mRNA-LMP2 and CS-mRNA-LMP2 when stored at 4 ℃ for 0, 7, 14, and 21 days, respectively.

FIG. 11 shows the sum of Zeta potentials of LP-mRNA-LMP2 and CS-mRNA-LMP2 when stored at 4 ℃ for 0, 7, 14 and 21 days, respectively.

FIG. 12 is a gel electrophoresis of LP-mRNA-LMP2 and CS-mRNA-LMP2 when stored at 4 ℃ for days 0(A), 7(B), 14(C), and 21(D), respectively.

FIG. 13 shows the stability of LP-mRNA-LMP2 and CS-mRNA-LMP2 incubated with serum for different periods of time.

FIG. 14 is a review of the transfection efficiency of LP-mRNA-GFP (A/B) and CS-mRNA-GFP (C/D) in the absence/presence of serum; (E) is Lipo 2000-mRNA-GFP; (F) the transfection efficiency was counted (. p <0.05,. p <0.01,. p < 0.001).

FIG. 15 is a toxicity study on DC2.4 cells at various concentrations of LP-mRNA-LMP 2.

FIG. 16 is a toxicity study on DC2.4 cells at various concentrations of CS-mRNA-LMP 2.

FIG. 17 shows the uptake of LP-mRNA-Cy5 and CS-mRNA-Cy5 by incubation on DC2.4 cells for different periods of time.

FIG. 18 shows the maturation rate of BMDC cells.

FIG. 19 shows the uptake of LP-mRNA-Cy5 and CS-mRNA-Cy5 incubated on BMDCs for different periods of time.

FIG. 20 is an illustration of antigen presentation of LP-mRNA-OVA and CS-mRNA-OVA on BMDCs.

FIG. 21 is a graph of the detection of the intracellular LMP2 antigen in TC-1 and TC-1-GLUC-LMP 2.

FIG. 22 is a tumor-bearing mouse immunotherapy protocol.

FIG. 23 is a graph of the immune anti-tumor effect in TC-1-GLUC-LMP2 tumor-bearing mice.

FIG. 24 is the tissue distribution of the LP-mRNA-DID delivery system.

FIG. 25 shows that LMP2 antigen (n ═ 3) can be efficiently expressed in spleen (A) and lymph node (B) of TC-1-GLUC-LMP2 tumor-bearing mice after immunization with LP-mRNA-LMP 2.

FIG. 26 is a flow chart of LMP2 antigen expression in spleen and lymph nodes of TC-1-GLUC-LMP2 tumor-bearing mice immunized with LP-mRNA-LMP 2.

FIG. 27 is a determination of the maturity of DCs in the spleen. (. p <0.05,. p <0.01,. p < 0.001).

Fig. 28 shows mouse CD8+ T cell proliferation.

Fig. 29 is a graph of CD4+ T cells and CD8+ T cells detected in the spleen (n-3) (. sp < 0.05).

Figure 30 is detection of CD4+ T cells and CD8+ T cells in lymph nodes (./p < 0.05).

FIG. 31 shows the levels of IL-6(A) and IFN- γ (B) in mouse serum after intravenous injection of LP-mRNA-GFP and LP-mRNA-LMP 2.

Fig. 32 shows the body weight of mice before and after immunotherapy with LP-mRNA-LMP2 vaccine (mean ± sd, n-7).

FIG. 33 shows the biochemical results of the blood test of mice after immunotherapy with LP-mRNA-LMP 2.

FIG. 34 is a histopathological observation of the effect of the immune preparation on organ damage in TC-1-GLUC-LMP2 tumor-bearing mice (scale: 50 μm).

FIG. 35 is a schematic diagram of the structure of an mRNA transcription vector template.

Detailed Description

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1 construction of mRNA transcription vector template

Conventional mRNA transcription vectors are composed of only a promoter and a target protein coding region. The invention adds immunogenicity enhancing signal (IE) on the basis of traditional mRNA transcription vector design, namely: the two ends of the target antigen protein coding region are respectively additionally added with a secretion signal and transmembrane and cytoplasmic domains of MHC-I molecules so as to enhance the immunogenicity of the mRNA vaccine by enhancing the antigen presentation effect of the target antigen protein. In addition, the invention adds non-translated sequences at the 5 'end and the 3' end of the protein coding region so as to enhance the immunogenicity of the mRNA vaccine by enhancing the translation efficiency of the target antigen protein and the stability of mRNA.

The mRNA transcription template DNA is sequentially connected by a promoter, a 5 'end untranslated region, a secretion signal of MHC-I type molecules, an antigen coding region, a transmembrane and cytoplasmic domain, a 3' end untranslated region, A120 and a terminal sequence behind the A120.

The antigen coding region is connected with the corresponding position, and sequencing verification is carried out. The promoter is T7 promoter. The nucleotide sequence of the 5' end untranslated region is shown in SEQ ID NO: 1 is shown in the specification; the nucleotide sequences of the transmembrane and cytoplasmic domains are shown in SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the 3' end untranslated region is shown in SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of A120 is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the terminal sequence of A120 is shown as SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of the antigen coding region is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the mRNA transcription template DNA is shown as SEQ ID NO: 7 and sequences with identity greater than 90% are shown in detail in figure 35.

Example 2 in vitro transcription of mRNA

The template carrier of example 1 is linearized at the tail of the A120 element by Mfe I, and is extracted and purified by phenol chloroform isoamyl alcohol, so that the linearized template is obtained. Electrophoretic validation confirmed whether linearization was complete.

In vitro transcription reactions were performed as described in Table 1 and carried out at 37 ℃ for 30 min. The scale can be directly enlarged according to the demand:

TABLE 1

After the reaction was completed, the DNA template degradation reaction was carried out according to Table 2, and the reaction was carried out at 37 ℃ for 15 min. The scale can be directly enlarged according to the demand:

TABLE 2

After the reaction was complete, 179. mu.L of nuclease-free water was added, and then precooled ammonium acetate (5M) of equal volume was added and precipitated in an ice bath for 15min, followed by centrifugation at 17000g for 15min at 4 ℃. Washing with 70% nuclease-free ethanol twice, volatilizing ethanol, and redissolving with nuclease-free water. The concentration was measured with a spectrophotometer.

RNA denaturation was performed as in Table 3 and incubation at 65 ℃ for 10 min. The scale can be directly enlarged according to the demand:

TABLE 3

Precool in ice bath for 5 min. The capping reaction was carried out as in Table 4, and the reaction was carried out at 37 ℃ for 2 hr. The scale can be directly enlarged according to the demand:

TABLE 4

After the reaction is finished, 100 mu L of nuclease-free water is added, the equal volume of phenol, chloroform and isoamyl alcohol is added for extraction, 17000g of the mixture is centrifuged for 10min, the supernatant is taken, precooled equal volume of ammonium acetate (5M) is added for precipitation in an ice bath for 15min, and 17000g of the mixture is centrifuged for 15min at 4 ℃. Washing with 70% nuclease-free ethanol twice, and dissolving to obtain final product of mRNA. And finally, measuring the concentration by a spectrophotometer, and verifying the integrity by electrophoresis.

And (4) conclusion: lanes from left to right as shown in FIG. 2 are GFP mRNA (irrelevant mRNA), LMP2 mRNA. Denaturing agarose gel electrophoresis showed that LMP2mRNA was intact and not degraded.

EXAMPLE 3 preparation of cationic Liposomal vectors

Table 5: cationic liposome carrier preparation formula

The preparation method comprises the following steps: the method for preparing the cationic liposome delivery carrier by adopting the thin film hydration method comprises the following steps: respectively measuring the phospholipid material and the additive with corresponding prescription amount, adding the phospholipid material and the additive into a 250mL eggplant-shaped bottle, adding an organic solvent with corresponding volume, mixing and dissolving; carrying out water bath at 37 ℃, carrying out reduced pressure rotary evaporation under the condition of keeping out of the sun to remove the organic solvent to form a film, wherein the rotating speed is 50 r/min, and adding pure water with the corresponding volume for hydrating the phospholipid film for 45min under the condition of keeping out of the sun under the condition of 60 ℃ and 75rpm after 2 h; homogenizing the obtained lipid colloid solution with an ultrasonic/homogenizer or a high-pressure micro-jet device to obtain a liposome solution; taking liposome solution to a preset volume to make the lipid concentration be 6mM, and filtering with 0.22 μm sterile water system filter membrane to obtain the final product.

Example 4 particle size and potential measurement of cationic Liposome vectors

In this example, the particle size and the potential were measured in the same manner as in the other formulations, taking the sample prepared in formulation 1 of example 3 as an example. A volume of the liposome colloidal solution prepared in formulation 1 (lipid concentration of 6mM) was measured, diluted with purified water to a lipid concentration of 0.6mM, and the particle size, particle size distribution and potential of the liposomes were measured in a laser particle size analyzer (n ═ 3, i.e., 3 times per formulation), and the results of measurement of the particle size and potential of the liposomes were shown in table 6.

TABLE 6

Particle size	Electric potential
		70±3nm	50±3mV

And (4) conclusion: the particle size of the cationic liposome is about 70nm, and the potential is about 50 mV.

Example 5 preparation of LMP2mRNA liposome vaccine

Table 7: mRNA liposome vaccine preparation prescription and incubation parameter

Test examples	Cationic liposome carrier solution	mRNA solution	Incubation time (minutes)	Incubation temperature (. degree.C.)
					1	Prescription 1(1ml)	1ml	30	40
2	Prescription 1(1.5ml)	1ml	60	37
					3	Prescription 1(2ml)	1ml	45	35
4	Prescription 1(0.5ml)	1ml	90	30
					5	Prescription 5(3ml)	1ml	120	25
6	Prescription 5(6ml)	1ml	240	23
					7	Prescription 5(8ml)	1ml	480	20
8	Prescription 7(1.5ml)	1ml	720	16
					9	Prescription 7(0.8ml)	1ml	960	12
10	Prescription 7(3ml)	1ml	1000	10

Remarking: in the above table, the cationic liposome carrier solutions were all diluted in advance to a concentration of 6mmol/L, and the concentration of mRNA in the mRNA solution was 1 mg/mL.

The preparation method comprises the following steps: using the cationic liposome vector prepared in example 3, an mRNA liposome vaccine was prepared. The specific method comprises the following steps: taking the cationic liposome carrier solution and the mRNA solution according to the prescription amount in the table, fully mixing, and incubating at the temperature and time in the table to obtain the LM 2mRNA liposome vaccine.

Example 6 particle size and potential of LMP2mRNA Liposomal vaccine

In this example, the particle size and the potential were measured using the sample prepared in example 5 as an example, and the measurement method was similar to that of the other formulations. A certain volume of the mRNA liposome vaccine colloidal solution of experimental example 3 was measured, diluted with purified water to an mRNA concentration of 0.01mg/mL, and the particle size and potential of the mRNA liposome vaccine were measured in a laser particle size analyzer (n ═ 3, i.e., 3 times per prescription), and the results of the particle size and potential measurement of the mRNA liposome vaccine are shown in table 8.

TABLE 8

Particle size	Electric potential
		140±12nm	41±2mV

And (4) conclusion: the LMP2mRNA liposome vaccine prepared in example 5 has a particle size of about 140nm and a potential of about 40 mV. It was shown that the particle size of the liposomes increased to some extent after loading with mRNA and the potential decreased to some extent, presumably due to binding of mRNA to the liposome carrier surface.

Example 7 gel retardation experiment

1) DNA gel electrophoresis

Taking 0.15g of agarose and 15ml of water, placing the agarose and the water in a triangular flask, heating the agarose and the water in a microwave oven for 2min, cooling the agarose and the water to about 60 ℃, adding a plurality of drops of Gold View, uniformly mixing, pouring the mixture into a gel plate (controlling the thickness of the gel to be about 0.5 cm), inserting a comb, taking out the comb after solidification, and obtaining the agarose gel plate by placing the gel plate in an electrophoresis tank, adding 1 mul of 6 multiplied loading buffer into the gel plate, uniformly mixing and completely loading 6 mul. The loading of the DNA Marker was 5. mu.l. 120V, electrophoresis time 25 min. The results are shown in FIG. 1.

2) mRNA gel electrophoresis

Preparation of denatured agarose gel

Taking 36mL of RNase-free water and 0.4g of agarose, placing the mixture in a triangular flask, heating the mixture for 2min in a microwave oven, cooling the mixture to about 60 ℃, adding 4mL of 10 × MOPS for mixing, then adding 7.5mL of 37% formaldehyde, and mixing uniformly (the volume is about 40 mL). Pouring into gel plate (thickness of gel is controlled at about 0.5 cm), inserting comb, coagulating, taking out comb, placing gel plate into electrophoresis tank, adding pre-cooled 1 × MOPS electrophoresis buffer solution at 4 deg.C, and spreading gel.

Preparation of electrophoresis sample

0.3 mu L of mRNA (0.3 mu g) and 5.7 mu L of RNase-free water are mixed uniformly, 5 mu L of formaldehyde loading buffer solution containing ethidium bromide is added, heating is carried out at 70 ℃ for 5min, then instantaneous centrifugation is carried out at 4 ℃ to obtain an mRNA sample, and the LPX sample prepared in the embodiment 5 is taken to obtain the LPX sample by the same method.

③ gel electrophoresis

Samples were added to gel wells (sample wells near the black end of the electrophoresis chamber), each sample was added in a volume of 10. mu.L, voltage was set at 200V (current 300mA, power 60W), electrophoresis was started, electrophoresis was stopped until the indicator front reached 1/2 (about 25min) at the gel, the gel was removed and placed in a gel imager for observation, and the results are shown in FIG. 3.

And (4) conclusion: in the first diagram, a lane 1 is a linearized plasmid, and a lane 2 is an mRNA Marker;

lanes

1 and 2 in FIG. 2 are LMP 2-mRNA;

lanes

4 and 5 are LPX. It can be seen that when N/P is 3, no band appears in the mRNA-loaded preparation at the site of free mRNA, indicating that under these conditions the liposomes fully ensure mRNA integrity and no degradation, consistent with experimental expectations.

Example 8 preparation of LMP2-mRNA

1. IVT mRNA was prepared synthetically using the Cellscript C-MSC11610S in vitro transcription kit.

2. The relevant components were added to a 0.6mL centrifuge tube in the order and dosage shown in Table 9, gently tapped with a pipette gun to mix them well, and the reaction was collected at the bottom of the tube after simple centrifugation. Reacting at 37 ℃ for 2h to obtain the product.

TABLE 9 LMP2-mRNA reaction System

3. Adding 1.0 mu L of DNase I into the reacted system, slightly blowing and beating the DNase I, uniformly mixing the DNase I and the DNase I, and incubating the DNase I and the DNase I at the temperature of 37 ℃ for 15min to remove the DNA template in the reacted system.

mRNA purification: (1) adding 179 mu L of RNase-free water into the system after the reaction in the step 3), then adding an equal volume of PCIAA (phenol/chloroform/isoamylol) solution, and vortexing for 10 s;

(2) centrifuging at 15000rpm for 5min, and separating two phases;

(3) transferring the supernatant to a new EP tube, adding 5M ammonium acetate solution with the same volume, mixing, and standing at-20 deg.C for 20 min;

(4) centrifuging at 15000rpm and 4 deg.C for 2 min;

(5) discarding the supernatant, adding 500 μ L70% ethanol, washing and precipitating;

(6) discarding the supernatant, and volatilizing at room temperature for 5 min;

(7) adding 75 μ L RNase-free water to redissolve to obtain LMP2 IVT mRNA.

mRNA capping: (1) incubating 75 μ L of the obtained LMP2 IVT mRNA at 65 deg.C for 10min, performing thermal denaturation, and cooling on ice;

(2) the components were added in the order and dosage shown in Table 10, mixed well and centrifuged briefly to collect the reaction solution at the bottom of the tube. Then, the reaction was carried out at 37 ℃ for 2 hours, and the reaction did not require tailing because the template contained the polyA fragment.

TABLE 10 mRNA capping reaction System

6. Capped mRNA purification

(1) Adding 100 μ L of RNase-free water into the reaction system under the condition of '2.3.4 mRNA capping', then adding an equal volume of PCIAA (phenol/chloroform/isoamyl alcohol) solution, and vortexing for 10 s;

(2) centrifuging the solution system obtained in the step (1) at 15000rpm for 5min, and separating two phases;

(3) taking the supernatant obtained in the step (2), transferring the supernatant into a new EP tube, adding an equal volume of 5M ammonium acetate solution, lightly blowing the solution by using a liquid transfer gun to uniformly mix the solution, and standing the solution for 20min at the temperature of minus 20 ℃;

(4) centrifuging at 15000rpm at 4 deg.C for 2 min;

(5) discarding the supernatant, and adding 500 μ L70% ethanol solution to wash the precipitate;

(6) discarding the supernatant, standing at room temperature for 5min, and volatilizing to remove residual ethanol solution;

(7) and (4) adding 75 mu L of RNase-free water into the precipitate in the step (6), and re-dissolving to obtain LMP 2-mRNA.

Example 9 concentration determination of LMP2-mRNA

The mRNA solution obtained in example 8 was vortexed and mixed, and after simple centrifugation, the plasmid solution was collected at the bottom of the tube, and 1. mu.L of the mixture was measured for concentration and purity in the Nanodrop, and the results are shown in Table 10:

TABLE 10 plasmid concentration and purity measurement results

Example 10 gel electrophoresis for structural confirmation of LMP2-mRNA

A1% agarose gel (indicator Gold View) was prepared, and the test sample was loaded with an RNA marker as a reference, and the results are shown in FIG. 5: the LMP2-mRNA has only a single band, which indicates that the LMP2-mRNA has higher purity. The length of the resulting LMP2-mRNA should be 2533nt according to design, and FIG. 5 shows that the band of LMP2-mRNA is located around 2500nt, indicating that the band is correct.

Example 11 preparation of LMP2-mRNA non-viral Immunity preparation/cationic Liposome-LMP 2-mRNA Complex (LP-mRNA-LMP2)

The present invention prepares an N/P-3 LMP2-LPS delivery system by co-incubating LP with LMP2-mRNA with or without an immunogenicity enhancing signal (IE). An optimized blank LP 10. mu.L was placed in an EP tube, and 40. mu.L of RNase-free water was added to make the total volume 50. mu.L. A certain amount of LMP2-mRNA solution was added to 10. mu.L of RNase-free water containing 1.5mM NaCl (for osmotic pressure adjustment), and mixed well, and a certain amount of RNase-free water was added thereto to make the total volume 50. mu.L. And adding the LMP2-mRNA solution into the blank LP solution, blowing and stirring the solution up and down by using a pipette gun, and incubating the solution at room temperature for 10min to obtain the LP-mRNA-LMP 2. A schematic of the structure of LP-mRNA-LMP2 is shown in FIG. 6. Preparation of core-shell nanoparticle-mRNA Complex (CS-mRNA-LAMP 2): weighing a proper amount of PLGA, and dissolving the PLGA in ethyl acetate to obtain an oil phase (O); an appropriate amount of PVA was then weighed and dissolved in RNase-free water as an aqueous phase (W). And (3) quickly injecting the oil phase (O) into the water phase (W) by using an injector, and carrying out ultrasonic emulsification by using an ultrasonic cell disruptor under the ice bath condition. The emulsified solution was quickly transferred to an eggplant-type flask and rotary evaporated in a water bath at 37 ℃ and 75rpm to volatilize ethyl acetate. And (5) obtaining the blank PLGA nanoparticles after the organic solvent is removed. And then, coating the cationic lipid shell on the surface of the blank PLGA nanoparticle by a thin film hydration-ultrasonic homogenization method. The specific operation is as follows: adding a proper amount of DOTAP and Chol into an eggplant-shaped bottle according to the molar ratio of 1:1, and performing rotary evaporation in a water bath at 37 ℃ under the condition of 50rpm to obtain a uniform DOTAP/Chol lipid film; and adding the obtained blank PLGA nanoparticle solution into the lipid membrane for hydration, transferring the solution into a 10mL EP tube after the lipid membrane is completely hydrated, and ultrasonically homogenizing by using a probe to obtain blank CS. Blank CS 5. mu.L was put into an EP tube, and 45. mu.L of RNase-free water was added to make the total volume 50. mu.L. A certain amount of mRNA solution was added to 10. mu.L of 1.5mM NaCl (osmotic pressure-adjusted) RNase-free water, and mixed well, and a certain amount of RNase-free water was added thereto to make the total volume 50. mu.L. And adding the mRNA solution into the blank CS solution, blowing and beating the gun tip up and down, mixing uniformly, and incubating at room temperature for 10min to obtain the CS-mRNA-LMP2 with the N/P being 3, wherein the final concentration of the mRNA is 0.01 mg/mL.

Example 12 particle size and Zeta potential of LP-mRNA-LMP2

Blank LP according to particle size and particle size distribution and Zeta potential measurement method_DOTAPThe particle size, PDI and Zeta potential of LP-mRNA-LMP2 were measured, and the results are shown in Table 11.

TABLE 11 LP_DOTAPParticle size, PDI and Zeta potential of LP-mRNA-LMP2

From the results, it was found that the particle size of LP-mRNA-LMP2 was increased and the Zeta potential was decreased after LMP2-mRNA was incubated as compared with that before the incubation.

Example 13 morphological Observation of LP-mRNA-LMP2

The morphological and structural characteristics of LP-mRNA-LMP2 were characterized by Transmission Electron Microscopy (TEM). The specific operation is as follows: the prepared LP-mRNA-LMP2 solution was dropped on a copper mesh covered with a carbon support film, left to stand for 1min, most of the solution was sucked off with filter paper, left to dry at room temperature, 1% (w/v) phosphotungstic acid was dropped to carry out negative staining for 5min, excess staining solution was removed, naturally dried at room temperature, and then placed in a TEM to observe and photograph, and the results are shown in FIG. 7. The TEM image shows that LP-mRNA-LMP2 is in a sphere-like, multi-compartment liposome structure.

Example 14 serum protein adsorption

LP-mRNA-LMP2 and CS-mRNA-LMP2 (core-shell nanoparticle Cy5-mRNA complex) were mixed with equal volume of 20% FBS or normal saline injection, and incubated at 37 deg.C for 2 h. After the incubation time was reached, the mixture was centrifuged at 4 ℃ for 60min at 10000rpm, and the supernatant was carefully discarded. Adding 1.5mL of normal saline injection again, mixing, centrifuging at 4 deg.C for 2 times, discarding supernatant after final centrifugation, resuspending with normal saline injection, and determining particle size and potential. The results of the particle size and potential changes after adsorption of the serum proteins LP-mRNA-LMP2 and CS-mRNA-LMP2 are shown in FIGS. 8-9.

Comparing the particle size and PDI of LP-mRNA-LMP2 and CS-mRNA-LMP2 before and after adsorption of serum protein, the particle size of LP-mRNA-LMP2 and CS-mRNA-LMP2 without serum treatment was 136.10 + -3.03 nm and 191.07 + -3.30 nm, respectively. After serum treatment, the particle sizes of LP-mRNA-LMP2 and CS-mRNA-LMP2 were increased to 426.93 + -26.58 nm and 404.47 + -12.89 nm, respectively, and the particle size distribution was increased. As can be seen from FIG. 9, the surfaces of LP-mRNA-LMP2 and CS-mRNA-LMP2 particles were both positively charged before serum treatment at 39.40. + -. 1.15mV and 18.77. + -. 0.40mV, respectively. After the serum treatment, the particle surface is negatively charged, and the potential is between-2 mV to-5 mV.

Example 15 storage stability of LMP2-mRNA Nanolipid Carrier Complex

The obtained LP-mRNA-LMP2 and CS-mRNA-LMP2 were stored at 4 ℃ and sampled at 0, 7, 14 and 21 days, respectively, to determine particle size and potential, and gel retardation test was carried out, and the results are shown in FIGS. 10 to 12.

As can be seen from FIGS. 10 to 11, the particle size and potential of LP-mRNA-LMP2 and CS-mRNA-LMP2 did not change significantly when stored at 4 ℃ for 21 days, indicating that LP-mRNA-LMP2 and CS-mRNA-LMP2 have better stability at 4 ℃. Furthermore, it can be seen from the gel electrophoresis test (FIG. 12) that the free LMP2-mRNA was not degraded when stored for 21 days, indicating that LMP2-mRNA had better stability at 4 ℃. LP-mRNA-LMP2 and CS-mRNA-LMP2 had no bands at the corresponding positions, indicating that there was no leakage of mRNA and the structure remained intact for both delivery systems.

Taken together, LP-mRNA-LMP2 and CS-mRNA-LMP2 can be stored at 4 ℃ for at least 21 days with good stability.

Example 16 serum stability

(1) Gel retardation study

According to the reference scheme, LP-mRNA-LMP2 and CS-mRNA-LMP2 are incubated with equal volume of FBS (FBS final concentration is 50%) for 2min, 5min, 10min, 30min, 1h and 2h, corresponding mass of free LMP2-mRNA is taken for the same treatment, and gel electrophoresis is carried out to examine the serum stability of LP-mRNA-LMP2 and CS-mRNA-LMP 2. The results are shown in FIG. 13.

As can be seen in FIG. 13, the serum is a diffuse band, and the free LMP2-mRNA is a complete single band. At 2min of incubation, the serum-treated free LMP2-mRNA lanes had both diffuse bands of serum and bands of LMP2-mRNA after degradation. Indicating that the free LMP2-mRNA is unstable in serum and is easily degraded. Furthermore, after serum treatment, no band of degraded mRNA was present in the lane LP-mRNA-LMP2, while a band of degraded mRNA was present in the lane CS-mRNA-LMP2, indicating that CS-mRNA-LMP2 did not effectively protect LMP2-mRNA from degradation. When the incubation time exceeded 30min, the LMP2-mRNA degradation band substantially disappeared, indicating that LMP2-mRNA was completely degraded. Degradation bands appear in the LP-mRNA-LMP2 lane, and the brightness of the degradation bands gradually becomes lighter as the incubation time is prolonged; similarly, the degradation band of CS-mRNA-LMP2 became gradually lighter, and the degradation band substantially disappeared when incubated for 2 h. In contrast, LP-mRNA-LMP2 can delay the degradation of LMP2-mRNA and can protect LMP2-mRNA from degradation in a short time.

Example 17 transfection stability

In order to further investigate the influence of the serum on the transfection efficiency of the two immune preparations, the GFP-mRNA is used as the model mRNA, and an optimal preparation process is adopted to prepare LP-mRNA-GFP and CS-mRNA-GFP; and transfection experiments were performed in the presence and absence of serum, respectively, with Lipo2000-mRNA-GFP as a positive control. The specific scheme is as follows:

transfection under serum-free conditions: the culture medium of DC2.4 cells cultured overnight was discarded, the cell surface was gently washed with PBS, 1mL of serum-free and antibiotic-free RPMI-1640 basic culture medium was added, and after incubation in an incubator for 30min, a certain amount of LP-mRNA-GFP and CS-mRNA-GFP were added, respectively, at a final concentration of 1. mu.g/well GFP-mRNA. Incubate at 37 ℃ for 6h, after incubation is complete, plates are supplemented with an equal volume of antibiotic-free medium containing 20% FBS and incubation is continued for 24 h.

Transfection with serum conditions: the culture medium of DC2.4 cells cultured overnight was discarded, the cell surface was gently washed with PBS, and 2mL of RPMI-1640 medium containing 10% FBS was added. Then, a certain amount of LP-mRNA-GFP and CS-mRNA-GFP were added, respectively, to a final concentration of GFP-mRNA of 1. mu.g/well. Incubate at 37 ℃ for 24 h.

After the culture is finished, the culture solution is gently aspirated by a pipette gun, the cells in a 6-well plate are collected and washed 3 times (1200rpm, 5min) with PBS, 300 mu L of PBS solution is added into the cells washed for the last time, the resuspended cells are gently blown and blown, and the transfection efficiency and the transfection fluorescence intensity are detected by a flow cytometer. The results are shown in FIG. 14: under the serum-free condition, the transfection efficiencies of the LP-mRNA-GFP and the CS-mRNA-GFP are similar and are about 70 percent; in the serum (10% FBS), the transfection efficiencies of LP-mRNA-GFP and CS-mRNA-GFP are respectively 62.05 +/-1.22% (p <0.05) and 21.86 +/-3.93% (p <0.001), which are remarkably reduced, and the serum influences the transfection efficiency of mRNA. The effect of serum on the transfection efficiency of CS-mRNA-GFP was greater than that of LP-mRNA-GFP, suggesting that LP-mRNA-GFP may be more advantageous when both immune agents are administered intravenously.

EXAMPLE 18 cytotoxicity Studies of LMP2-mRNA non-viral vector formulations

The preparation LP-mRNA-LMP2 and CS-mRNA-LMP2 were diluted with serum-free medium to total lipid concentrations of 0.0001. mu.M, 0.001. mu.M, 0.01. mu.M, 0.1. mu.M and 1. mu.M, respectively, for use.

DC2.4 cells with a confluency of 80% were digested, counted, diluted with medium and added at 1X 10⁴The density of each well is inoculated in a 96-well plate, and the plate is placed in an incubator to be cultured for 24 h. After the culture was completed, the old medium was discarded, and media containing different concentrations of LP-mRNA-LMP2 and CS-mRNA-LMP2 were added to a 96-well plate at 100. mu.L per well in parallel with 6 wells per concentration. Meanwhile, the control group used fresh medium without serum. After incubating 96-well plates in an incubator for 24 hours, 20. mu.L of MTT solution (5mg/mL) was added to each well, and then incubated in an incubator for 4 hours, the supernatant was discarded, 150. mu.L of DMSO was added to each well, and the mixture was shaken on a shaker to dissolve blue-violet formazan crystals. And detecting the absorbance value of each hole by using a multifunctional microplate reader, wherein the detection wavelength is 492 nm. After completion of the assay, cell viability (cell viability) was calculated using the following formula:

Cell viability＝Abs_test/Abs_control*100％

wherein, Abs_test: absorbance values measured by culturing cells in a culture solution containing an immune preparation; abs_control: absorbance values measured by culturing cells in medium. The results are shown in FIGS. 15-16: after the LP-mRNA-LMP2 and CS-mRNA-LMP2 are incubated with DC2.4 cells, the cell survival rates are all above 90%, which indicates that the LP-mRNA-LMP2 and CS-mRNA-LMP2 have lower cytotoxicity and better safety.

Example 19 DC2.4 cellular uptake

Reference LP-mRNAThe preparation process of the-LMP 2 comprises the steps of replacing LMP2-mRNA with Cy5-mRNA in a mode, preparing a cationic liposome Cy5-mRNA complex (LP-mRNA-Cy5) and a core-shell nanoparticle Cy5-mRNA complex (CS-mRNA-Cy5), and enabling the content of Cy5-mRNA to be 0.01 mg/mL. Culturing DC2.4 cells, collecting DC2.4 cells when the cell confluency reaches 80%, counting, diluting the cell suspension with the culture medium, and inoculating the cells in 12-well plate at a density of 1.5 × 10⁵One/well, volume 1mL, cultured for 24 h. Prior to the assay, the old medium was gently aspirated, the cell surface was gently washed 2 times with PBS, and then 1mL of serum-free RPMI-1640 basal medium was added. After incubating the cells in an incubator at 37 ℃ for 30min, LP-mRNA-Cy5 and CS-mRNA-Cy5 (Cy 5-mRNA 1. mu.g per well) were administered, respectively, and then the plates were placed in an incubator for 0.5h, 1h, 2h, 4h and 6h, respectively. After the incubation was completed, the supernatant was quickly aspirated, and uptake was stopped by adding 4 ℃ PBS, and the cell surface was gently washed 3 times with PBS. After the last wash was complete, pancreatin was added for digestion, the cells were collected and transferred to a flow tube and washed 2 times with PBS. After the final centrifugation, 300 μ L PBS was added to each flow tube to resuspend the cells. The fluorescence intensity of Cy5 in the cells was measured by flow cytometry, and the results are shown in fig. 17: the mean fluorescence intensity MFI of Cy5 in the cells, measured by flow cytometry, represents the absolute amount of uptake of the nanopreparative in the cells; thus, it was found that the uptake of LP-mRNA-Cy5 and CS-mRNA-Cy5 on DC2.4 cells gradually increased with the increase of the incubation time. When the incubation time exceeded 1h, the cellular uptake of LP-mRNA-Cy5 increased significantly compared to CS-mRNA-Cy 5. The uptake of LP-mRNA-Cy5 and CS-mRNA-Cy5 was maximal at 4h of incubation. In contrast, the uptake of LP-mRNA-Cy5 decreased when incubated for 6h, whereas the uptake of CS-mRNA-Cy5 did not differ significantly from that of 4 h.

Example 20 BMDC culture

1) RPMI-1640450 mL, heat-inactivated FBS 50mL, 100 XPicillin/streptomycin 5mL and 1000 XP 2-mercaptoethanol 500 μ L, mixing well to obtain complete culture solution (CM). Mixing 49mL of CM and 1 mu g/mL of GM-CSF 1.0mL uniformly to obtain BMDCs culture solution;

2) mice were sacrificed, tibia and femur were separated, and syringes were usedSucking the culture solution to wash the tibia and the femur to obtain a bone marrow suspension, filtering by using a sterile nylon filter screen with the diameter of 70 mu m, and collecting the bone marrow single cell suspension. Centrifuging the cell suspension, discarding supernatant, adding precooled erythrocyte lysate to lyse erythrocyte, adding 20mL RPMI1640, centrifuging, discarding supernatant, adding 3mL CM, resuspending cells, and counting (2.0-2.5 × 10)⁷cells)；

3) Adding 10mL of BMDCs culture solution into a cell culture dish, and adding the BMDCs culture solution to a culture dish containing 4-5 multiplied by 10⁶The cell suspension was dropped on different positions of a petri dish, gently mixed back and forth, left and right, and cultured at 37 ℃ under 5% CO2, which was recorded as Day 0.

4) Day 3 was supplemented with 10mL BMDCs per dish and continued until Day 8.

Example 21 BMDC transfection

1) Collecting the BMDCs of Day 8, resuspending in serum-free and antibiotic-free basal medium, counting, and adjusting the cell density to 10 × 10⁵one/mL. 0.5mL of the basal medium was added to each well of the 6-well plate, followed by 0.5mL of the cell suspension such that the cell density was 5X 10⁵each/mL/hole, gently shaking and mixing all the materials, and culturing the materials in an incubator at 37 ℃ for about 2 hours for later use;

2) to each well was added 0.5 μ g of LMP2-mRNA loaded with IE and without IE. Incubating for 4h in a cell incubator;

3) continuously culturing 0.5ml of antibiotic-free culture solution supplemented with 20% FBS per well for 24h, discarding the culture medium, washing with cold PBS for 1 time, collecting cells in a 6-well plate, washing the cells with PBS for 3 times (1200rpm, 5min), and resuspending the cells in 100 μ L of PBS solution;

4) adding 1 mu L of CD16/32 into each flow tube, uniformly mixing, incubating for 10min at 4 ℃, adding 1 mu of LPE-anti-mouse CD11c and APC-anti-mouse CD86 flow antibodies into the corresponding flow tubes, and incubating for 40min at 4 ℃ in a dark place; after washing 2 times with 2mL of PBS and adding about 300. mu.L of PBS per flow tube, the cells were activated by flow cytometry, and the results are shown in FIG. 18, where LMP2-mRNA containing antigen-presenting signal was more able to stimulate the maturation of BMDC cells than LMP2-mRNA without antigen-presenting signal.

Example 22 uptake of BMDCs

LP-mRNA-LMP2 and CS-mRNA-LMP2 were prepared by replacing LMP2-mRNA with Cy5-mRNA in the model, and LP-mRNA-Cy5 and CS-mRNA-Cy5 were prepared so that Cy5-mRNA content was 0.01 mg/mL. BMDCs were cultured and immature BMDCs with a purity of greater than 80% were used for cell uptake assays. Collecting BMDCs, centrifuging at 1000rpm for 3min, discarding supernatant, re-suspending cells in serum-free RPMI-1640 medium, inoculating in 12-well plate at 1.5 × 10⁵Cell uptake assays were performed in 1mL volumes per well by placing plates in a 37 ℃ incubator for 2 h. LP-mRNA-Cy5 and CS-mRNA-Cy5 (Cy 5-mRNA 1. mu.g per well) were administered separately to each well and the plates were placed in incubators for 0.5h, 1h, 2h, 4h and 6h, respectively. After incubation, BMDCs were collected in a flow tube and the cell surface was washed 3 times with PBS. After washing, 300. mu.L of PBS was added to each flow tube to resuspend the cells. The fluorescence intensity of Cy5 in the cells was measured by flow cytometry, and the results are shown in fig. 19: with increasing incubation time, the uptake of LP-mRNA-Cy5 on BMDCs increased and then decreased, and the cellular uptake reached a maximum when incubated for 1 h. The cell uptake amount is not significantly different from 1h when the culture medium is incubated for 2h, but gradually decreases when the culture medium is incubated for more than 2 h. The uptake of CS-mRNA-Cy5 was significantly lower than LP-mRNA-Cy5 at different times and did not change much with incubation time.

Combining the uptake of both non-viral vectors on DCs, it was found that LP-mRNA-Cy5 was more readily taken up by DCs than CS-mRNA-Cy 5.

Example 23 antigen presentation of BMDCs

Cationic liposome-OVA-mRNA-complex (LP-mRNA-OVA) and core-shell nanoparticle-OVA-mRNA-complex (CS-mRNA-OVA) were prepared using OVA-mRNA as model mRNA, replacing LMP2-mRNA with model OVA-mRNA, in reference to the preparation processes of LP-mRNA-LMP2 and CS-mRNA-LMP2, to achieve an OVA-mRNA content of 0.01 mg/mL. BMDCs were cultured and immature BMDCs with a purity of greater than 80% were used for antigen presentation assays. Collecting cultured BMDCs, centrifuging at 1000rpm for 3min, discarding supernatant, suspending cells in serum-free RPMI-1640 medium, adding medium to dilute cells to obtain cell density of 1.510⁶BMDCs were seeded into 24-well plates at 250. mu.L per well per mL. The mixture was gently blown to disperse the mixture evenly, and the plate was incubated at 37 ℃ for 2 hours in an incubator to carry out the experiment. LP-mRNA-OVA and CS-mRNA-OVA were added to each well at a final mRNA concentration of 1. mu.g/well, respectively. After 6h incubation, 250 μ L of RPMI-1640 medium with 20% FBS was added to each well and incubation continued for 24 h. After the incubation was completed, the medium was discarded, and 300. mu.L of PBS solution was added to each well. Cells in the 24-well plate were collected with a cell scraper and transferred to a flow tube, washed 1 time with 4 ℃ PBS. Add 100. mu.L PBS to resuspend the cells. 0.5. mu.L of anti-mouse CD16/32 was added to each flow tube, and incubated at 4 ℃ for 15min, and then 0.5. mu.L of PE-anti-mouse CD11c and 0.5. mu.L of LAPC-anti-OVA (257-264)/mouse H-2Kb 25-D1.16 flow antibody were added, and incubated at 4 ℃ for 40 min. After the incubation was completed, the cells were washed 1 time with PBS, and then the percentage of H-2 Kb-positive cells among CD11 c-positive cells was measured by flow cytometry.

The results are shown in FIG. 20, and it can be seen that LP-mRNA-OVA and CS-mRNA-OVA both have better antigen-presenting effects, and that the antigen-presenting effect of LP-mRNA-OVA (10.1 + -0.5%) is significantly higher than that of CS-mRNA-OVA (8.1 + -0.4%).

Example 24 detection of LMP2 antigen in TC-1-GLUC-LMP2 tumor cells

Count 5X 10⁵Collecting TC-1-GLUC-LMP2 cells, placing in a flow tube, centrifuging at 1000rpm for 5min, discarding supernatant, washing with sterile PBS twice, adding 1 μ LPE-EBV-LMP2A monoclonal antibody, and incubating at room temperature in dark for 30 min. After incubation was complete, the cells were washed twice with PBS (5 min centrifugation at 1000 rpm), and finally 300. mu.L of PBS resuspended cells were added to the flow tube and detected on the flow cytometer.

The results are shown in FIG. 21: TC-1-GLUC-LMP2 cell highly expresses LMP2 antigen, and the suggestion is that the LMP2 antigen is used for establishing a nasopharyngeal carcinoma mouse model and has EBV-related LMP2 antigen expression characteristics; when LP-mRNA-LMP2 immunotherapy is given, the generated LMP2 specific T cells can be used for identifying the cells in vivo, effectively inhibiting the proliferation of the cells and inducing the apoptosis of the cells, thereby promoting the tumor to be faded.

EXAMPLE 25 pharmacodynamic evaluation-tumor growth Curve

Collecting TC-1-GLUC-LMP2 cells in logarithmic growth phase, washing with sterile PBS, centrifuging to remove supernatant, adding sterile PBS to resuspend cells, and adjusting cell density to 8 × 10⁶one/mL. 25 mice were inoculated subcutaneously in the right flank, 100. mu.L cells per mouse. 3 days after inoculation, the mice developed macroscopically visible nodules; after 7 days of inoculation, the mice form obvious nodules subcutaneously, and the tumor forming rate is as high as 100%.

7 days after tumor inoculation, 21 mice with similar tumor sizes are selected and randomly divided into a Control group (Saline), an irrelevant mRNA group (LP-mRNA-GFP) and an LP-mRNA-LMP2 group, and the weight of each mouse is weighed and the tumor volume is measured; control groups were injected intravenously with 300. mu.L of Saline per mouse, and the remaining two groups were injected intravenously with LP-mRNA-GFP and LP-mRNA-LMP2 in an amount of 30. mu.g of mRNA (300. mu.L) per mouse, respectively, and the mouse immunotherapy protocol is shown in FIG. 22.

During the treatment period, the physiological changes of tumor mice are counted, and from the first immunization administration, the longest diameter (a) of the tumor and the shortest diameter (b) perpendicular to the longest diameter are measured by a vernier caliper every 2 days according to the following formula:

tumor volume was calculated and mouse mean tumor volume growth curves were plotted, with the results shown in figure 23: according to the tumor volume growth curve of the salt group in FIG. 23, it can be seen that TC-1-GLUC-LMP2 cells grow rapidly and have better tumorigenicity after being inoculated under the skin of a mouse; but grows to 1000mm in tumor volume³In time, tumor growth gradually stagnated, and there was also a large variation among mice. The TC-1-GLUC-LMP2 cell is a self-constructed cell, so that the subsequent test further optimizes the tumor model by methods of increasing the inoculation number of tumor cells and the like, and lays a foundation for the stable research model for further pharmacodynamic evaluation of the nasopharyngeal carcinoma immunotherapy candidate drug. GFP-mRNA was chosen as a control for the LMP2-mRNA unrelated sequence to clarify that after loading of LMP2-mRNA into LP, it was LMP2-mRNA itself that exerted an immunotherapeutic effect, rather than the load usedTumor regression induced by adjuvant effects of body or mRNA sequences. LP-mRNA-LMP2 vaccine is injected intravenously, which can obviously inhibit tumor growth and exert high-efficiency anti-tumor activity.

Example 26 tissue distribution study

In vivo distribution studies were performed using 6-7 week old male C57BL6/C mice. DID is taken as a tracer dye, the DID is mixed with a lipid material to prepare a blank liposome, LMP2-mRNA is incubated to prepare LP-mRNA-DID, and in-vivo distribution research is carried out. Mice were intravenously administered LP-mRNA-DID, and the Control group was administered the same amount of physiological saline solution, and after the administration, mice had free access to water. Mice were sacrificed 6h after administration, and organs of tissues such as heart, liver, spleen, lung, kidney, brain, lymph node, etc. were removed and imaged using a small animal in vivo imager.

As shown in FIG. 24, no fluorescence was observed in the major tissues and organs such as heart, liver, spleen, lung, kidney, brain, and lymph node of Control mice, suggesting that the tissue distribution method has good specificity and no interference of tissue background, and can be used for distribution detection and evaluation of LP-mRNA-DID. LP-mRNA-DID is mainly distributed in the liver, spleen and lung after intravenous injection, and the suggestion is that the immune preparation is mainly taken by DCs of the major organs, thereby realizing antigen presentation and playing a role in high-efficiency immunotherapy activity.

Example 27 LMP2 antigen expression in spleen and lymph nodes

Based on the distribution results of example 26, it was preliminarily concluded that LP-mRNA-DID should be expressed efficiently in the spleen rich in DCs, and that there is also a possibility that LMP2 pro-expression detection assay, antigen expression, could be performed in the lymph nodes rich in DCs. Therefore, TC-1-GLUC-LMP2 tumor-bearing mice are selected, LMP2 is designed to preliminarily explore the anti-cancer action mechanism of LP-mRNA-LMP2, and the test steps are as follows:

(1) tumor-bearing mice were constructed, 6 mice with similar tumor sizes were selected 7 days after tumor inoculation, randomly divided into 2 groups of 3 mice each, and immunized with Saline and LP-mRNA-LMP2 through tail vein, respectively. Immunizations were performed every 5 days, 24h after the 2 nd immunization, mice were sacrificed;

(2) the spleen and inguinal lymph node of the mouse were removed and ground, and the cell suspensions were collected in 15mL centrifuge tubes, washed 2 times with PBS, 2mL each time.

(3) Erythrocytes were lysed in a centrifuge tube using 1mL of a 4 ℃ pre-cooled erythrocyte lysate, followed by dilution of the lysate with 20mL of RPMI-1640, centrifugation, and discarding the supernatant.

(4) Washing 2 times with 2mL PBS, resuspending the cells, preparing 300. mu.L of each tissue cell, counting, and diluting the cell suspension to 4X 10⁶each/mL, corresponding to the 3 flow tube split.

(5)1mL of 4% paraformaldehyde was added to each flow tube, the cells were fixed for 15min, washed 2 times with 2mL of PBS, the supernatant was discarded, the membranes were disrupted with 200. mu.L of 1% Triton-X100 solution for 15min, washed 2 times with 2mL of PBS, and the supernatant was discarded.

(6) 5% BSA blocked for 30min, washed 2 times with PBS, 2mL each time. Add 100 u L PBS heavy suspension cells, to the flow tube respectively add 1 u L PE-EBV-LMP2A monoclonal antibody, lightproof incubation for 30 min.

(7) After the incubation time was reached, the cells were washed 2 times with 2mL portions of PBS. About 300. mu.L of PBS was added to each flow tube, and the expression of the target antigen LMP2 in lymphocytes was detected by flow cytometry.

Data processing was performed to plot LMP2 antigen expression in mouse spleen and lymph nodes as shown in fig. 25, and representative flow results are shown in fig. 26.

As can be seen from FIG. 25, after two immunizations with LP-mRNA-LMP2 vaccine, significant LMP2 antigen expression could be detected in spleen and lymph node of mice enriched with DCs compared to Control group; the suggestion is that the LP-mRNA-LMP2 can effectively deliver main drug LMP2-mRNA to DCs and effectively promote the expression of coding antigen LMP2 in tumor-bearing mice, thereby presenting the DCs to T cells, stimulating the generation of specific CTL and exerting high-efficiency anti-tumor activity.

Example 28 maturation of DCs in spleen

In order to further explore the immunological mechanism of the LP-mRNA-LMP2 vaccine, the maturity of DCs in the spleen of mice after immunotherapy with LP-mRNA-LMP2 was examined, i.e., the mice at the pharmacodynamic end point were sacrificed, spleen tissues were isolated, and single cell suspensions were prepared. The specific operation is as follows: mouse spleen was submerged in syringe and passed through a 70 μm cell strainer to obtain individual splenocytes. Centrifuging the single cell suspension of the spleen at 4 ℃ and 2000rpm for 5min, removing the supernatant, adding 1mL of erythrocyte lysate for cracking for 10min, removing erythrocytes, adding PBS (phosphate buffer solution) for uniformly mixing, centrifuging at 4 ℃ and 2000rpm for 5min, removing the supernatant, repeating PBS washing once, and adding PBS for later use. Adding a proper amount of single cell suspension into flow tubes, then adding 1 mu L of anti-mouse CD16/32 into each flow tube, uniformly mixing, incubating at 4 ℃ for 15min, after the incubation is finished, respectively adding 1 mu L of PE-anti-mouse CD11c, 1 mu L of FITC-anti-mouse CD80 and 1 mu L of APC-anti-mouse CD86 into the corresponding flow tubes, incubating at 4 ℃ in a dark place for 40min, after the incubation is finished, washing for 2 times by PBS, and detecting by a flow cytometer, wherein the result is shown in FIG. 27.

As can be seen from A and B in FIG. 27, DCs were increased in the spleen of mice after immunization with LP-mRNA-LMP2 (CD11c +), and the CD11c + cells were significantly higher in the LP-mRNA-LMP2 group than in the Saline group and the LP-mRNA-GFP group. In addition, as can be seen from C and D, the MFI of CD80+ and CD86+ DCs in the mouse spleen was significantly increased compared to the Saline group and the LP-mRNA-GFP group (p < 0.001). It was suggested that LP-mRNA-LMP2 resulted in stronger maturation activation of DCs in the spleen compared to LP-mRNA-GFP, probably related to efficient expression of the encoded antigen LMP2 and efficient formation of pMHC.

Example 29 mouse CD8+ T cell proliferation assay

Preparation of lymphocytes

C57BL/6 mice were sacrificed, the spleen of the mice was removed from the capsule, mechanically ground with an injector head, and passed through a cell strainer with a pore size of 70 μm. Cells were harvested and washed 2 times with PBS (1200rpm, 3 min). Then, the cells were resuspended in FBS-free RPMI1640 medium and the cell density was adjusted to 1X 10⁵Per mL; spleen cells were seeded in 24-well plates at 0.5ml per well and cultured acclimatically in a cell culture incubator for 30 min.

Spleen cell transfection

1) Separately, 0.5. mu.g of IE-containing LMP2-mRNA liposome and IE-free LMP2-mRNA liposome were added to each well; and LMP2-mRNA liposomes without antigen presentation. After culturing for 4 hours in a cell culture box, supplementing 0.5ml of RPMI1640 medium containing 20% FBS to each hole, and continuously culturing for 48 hours in the cell culture box;

2) collecting cells cultured for 48h, adding FITC-labeled anti-mouse CD3, PE-labeled anti-mouse CD4 and APC-labeled anti-mouse CD8, and dyeing for 30min in dark place; after staining for 30min, the cells were washed twice with PBS and then tested for proliferation of mouse T cells by flow cytometry, and the results are shown in FIG. 28, where LMP2-mRNA containing an antigen-presenting signal is more able to promote proliferation of mouse spleen CD8+ T cells than LMP2-mRNA without an antigen-presenting signal.

Example 30 detection of CD4+ T cells and CD8+ T cells in spleen and lymph nodes

The key of the mRNA vaccine for immunotherapy of tumors is the induction of antigen-specific CTL, so that after LP-mRNA-LMP2 immunotherapy of mice, the generation of LMP 2-specific CTL is detected, which is helpful for explaining the relevant mechanism of high-efficiency therapeutic activity of nasopharyngeal carcinoma. Antigen-specific CTLs are often detected using MHC tetramers, which are composed of four monomeric molecules bound by major histocompatibility complexes and antigen peptides, and labeled with fluorescence, which can label antigen-specific T cells at the single cell level. The research related to nasopharyngeal carcinoma immunotherapy belongs to the emerging field, and at present, the commercially available MHC tetramer is only used for human CTL detection, and no commercially available reagent for mouse detection is available. For this reason, CD4 was administered directly first to spleen and lymph nodes⁺T cells and CD8⁺T cells were detected. Specifically, mice at the pharmacodynamic end point were sacrificed, spleen tissue was isolated, and single cell suspensions were prepared. The specific operation is as follows: mouse spleen was submerged in syringe and passed through a 70 μm cell strainer to obtain individual splenocytes. Taking lymph nodes, cutting into pieces, placing into an EP tube, adding preheated collagenase solution, incubating at 37 ℃ for 2h, transferring to a cell filter screen after digestion, grinding, and filtering to obtain single lymphocyte. And finally, centrifuging the single cell suspension of the spleen and the lymph nodes for 5min at 4 ℃ and 2000rpm, discarding the supernatant, adding 1mL of erythrocyte lysate for lysis for 10min, removing erythrocytes, adding PBS (phosphate buffer solution) for uniformly mixing, centrifuging for 5min at 4 ℃ and 2000rpm, discarding the supernatant, repeating PBS washing once, and adding PBS for later use. In the flowAdding a proper amount of single cell suspension into a formula tube, then adding 1 mu L of anti-mouse CD16/32 into each flow tube, uniformly mixing, incubating at 4 ℃ for 15min, after the incubation is finished, respectively adding 1 mu L of FITC-anti-mouse CD3, 1 mu L of APC-anti-mouse CD8a and 1 mu L of PE-anti-mouse CD4 into the corresponding flow tube, incubating at 4 ℃ in a dark place for 40min, after the incubation is finished, washing for 2 times by PBS, and detecting by a flow cytometer, wherein the results are shown in FIGS. 29 and 30.

As can be seen from FIG. 29, the proportion of T cells in the spleen was increased after immunization with LP-mRNA-LMP2 (FITC), and the CD8+ T cells in the LP-mRNA-LMP2 group were higher than that in the Saline group. From this, it was concluded that the administration of LP-mRNA-LMP2 activates a specific T cell response in mice. As can be seen in FIG. 30, the T cell proportion in lymph nodes increased after immunization with LP-mRNA-LMP2 (FITC), and both CD4+ T cells and CD8+ T cells were significantly higher in the LP-mRNA-LMP2 group than in the Saline group (p <0.05) and GFP group, suggesting that specific T cell responses may be activated in mice after administration of LP-mRNA-LMP 2.

Example 31 detection of IL-6 and IFN-. gamma.in serum

Male C57BL6/C mice 6-7 weeks old were randomly divided into 3 groups of 3 mice each, and Saline, LP-mRNA-GFP and LP-mRNA-LMP2 were administered by intravenous injection, respectively, at an mRNA dose of 30. mu.g/mouse. After 24h, blood was drawn from the orbit and centrifuged at 2000rpm for 10min at 4 ℃ and the supernatant was collected and assayed. The standard curve and the sample content were determined by using corresponding ELISA kits for IL-6 and IFN-gamma, respectively, according to the instructions, and the results are shown in FIG. 31.

As shown in FIG. 31, both intravenous injection of LP-mRNA-GFP and LP-mRNA-LMP2 induced more IL-6 and IFN- γ production in mice compared to the Saline group, and the LP-mRNA-LMP2 group was significantly elevated compared to the LP-mRNA-GFP group; it is concluded that LP-mRNA-LMP2 induces LMP2 specific CTL in vivo, thereby generating larger amount of tumor killing cytokine and exerting high anti-tumor activity.

Example 32 evaluation of safety-physiological status and body weight of mice

The physiological status of the mice was monitored as day 0 from the day of the 1 st immunization of the mice. The body weight of each group of mice was measured and recorded every 2 days, and the mean body weight of each immunotherapy group of mice was calculated. The results are shown in FIG. 32.

The result shows that the LP-mRNA-GFP and LP-mRNA-LMP2 vaccines immunize mice through a venous approach, the weight of the mice is not obviously influenced, the activity state of the mice is active before and after immunization, the hairs are black and bright, and the feces are normal; and the physiological conditions of the mice in the LP-mRNA-LMP2 group are similar to those of the salt group, which indicates that the LP-mRNA-LMP2 has no obvious toxicity in vivo.

Example 33 evaluation of safety-measurement of blood Biochemical indicators

At the end point of pharmacodynamic test, collecting venous blood of mice, centrifuging at 3500rpm for 10min at 4 ℃, taking serum, and analyzing the blood composition condition of each mouse by using a blood biochemical automatic analyzer. And (4) performing quality control on the biochemical analyzer according to a standard operation flow, and displaying the normal use of the instrument according to a quality control detection result. And (3) placing the prepared plasma samples in a total blood biochemical analyzer, and respectively using corresponding detection kits to determine the content of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Total Protein (TP), Uric Acid (UA) and urinary anhydride (UREAL) in each plasma sample. The results are shown in FIG. 33.

As a result, after the mice are immunized by the LP-mRNA-LMP2, all indexes in the blood of the mice are similar to those of mice in a salt group. The result shows that various biochemical indexes in the plasma of mice are not affected after mice are immunized by the LP-mRNA-LMP2, and the safety of LP-mRNA-LMP2 is further suggested.

Example 34 histopathological evaluation

After the treatment, one mouse was randomly selected from each group, and pathological tissue sections of each organ in the body were prepared and stained with H & E. And (4) placing the stained tissue in a pathological section scanner for observation and photographing, and inspecting pathological change conditions of important tissues and organs such as the liver, the kidney, the spleen, the lung and the like of the mouse. The results are shown in FIG. 34.

After treatment, one mouse was randomly selected from each group, and the mice, heart, liver, spleen, lung, and kidney were removed and sectioned and H & E stained according to standard procedures. The stained tissues are observed by a pathological section scanner and photographed, and the result shows that the LP-mRNA-GFP and the LP-mRNA-LMP2 have no obvious organ toxicity.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Sequence listing

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accgtcatgt ctaacactct cctgagtgcc tggattctta cagctggctt tcttatattc 1380

ctgatcggct ttgcgctctt cggggtgatc cgctgctgca gatattgttg ttactactgc 1440

cttacgcttg aaagcgaaga aaggcctcca acaccatata ggaatactgt t 1491

<210> 10

<211> 1491

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgggatcac tggaaatggt acctatgggc gctggtccac catctccagg tggagatcct 60

gatggatatg atggaggtaa caactcacag tacccaagcg cttctgggtc tagcgggaat 120

actcctacac ctcccaatga cgaagagagg gagtctaacg aagaacctcc cccaccatac 180

gaggaccctt actggggcaa cggagacaga cactccgact accagcccct gggaacacag 240

gaccagtccc tgtatttggg ccttcagcat gatgggaatg atggtttgcc cccaccccct 300

tactcaccta gggacgattc ctctcagcat atctacgaag aggctggacg aggctctatg 360

aaccccgtct gcctccctgt catcgtggca ccatatctct tctggctcgc tgccattgca 420

gctagctgtt tcaccgcctc agtgtccacg gtagtcactg ctaccggcct ggcactgagt 480

ctcctcctcc tggcagcagt cgcctcatct tatgccgccg cccaaaggaa attgctgacg 540

ccagtgactg tgctgacagc agtggtaaca ttctttgcca tctgcctgac gtggaggata 600

gaggaccctc cctttaacag cctgctgttc gctcttctgg ccgctgccgg tggacttcaa 660

gggatttacg tcctggttat gctcgtactg cttatcctcg cttacaggag acgatggaga 720

agacttacag tttgcggggg aattatgttc ctcgcttgtg tgttggtctt gatcgtggac 780

gctgtattgc agctttcacc tctgctggga gccgtcacgg tcgtatccat gaccttgctg 840

ctccttgcct tcgttttgtg gctgagtagc ccgggaggat tgggtacttt gggagccgct 900

cttttgacac ttgcagccgc actggctctt ctggcttctc ttatcctggg cactctgaat 960

ctcacaacca tgttcctcct catgttgctg tggaccctcg tggtcctcct gatctgttcc 1020

agttgctcta gttgtccgct ctcaaaaatc ctgcttgcca gattgtttct gtatgctctg 1080

gctctgcttc tcctcgcttc agcactcatt gctggcggct ccattttgca gaccaacttt 1140

aagagcctga gctccacaga atttattccg aacttgttct gtatgcttct tcttatcgtc 1200

gcaggcatcc tcttcatcct cgccatactt acagagtggg ggtccggtaa caggacttat 1260

gggccagtat ttatgtgtct cggcggactt ctcacaatgg tggctggtgc cgtttggctc 1320

actgtaatga gtaacaccct tctgtcagca tggatactga ccgccgggtt tctgattttc 1380

ttgatcggct tcgcactgtt cggggtgatc cggtgttgca gatactgttg ctattattgt 1440

cttactctgg agtctgaaga acgccctccg acaccctata gaaatactgt g 1491

Claims

1. A method for enhancing the immunogenicity of an EBV mRNA vaccine is disclosed, wherein a template DNA is formed by sequentially connecting a promoter, a 5 'end untranslated region, a secretion signal of MHC-I type molecules, an antigen coding region, transmembrane and cytoplasmic domains, a 3' end untranslated region, A120 and a terminal sequence behind A120.

2. The method of enhancing the immunogenicity of an EBV mRNA vaccine according to claim 1, wherein said promoter is the T7 or SP6 promoter.

3. The method for enhancing the immunogenicity of an EBV mRNA vaccine according to claim 1, wherein the nucleotide sequence of the 5' untranslated region is as set forth in SEQ ID NO: 1 and synonymous codons thereof, and a sequence with more than 90% of identity with the sequence; the secretion signal of the MHC class I molecule includes but is not limited to various classes of MHC class I molecules from different species, such as: human MHC I molecule (HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-K, HLA-I), mouse MHC I molecule (H-2D, H-2K, H-2L), rat MHC I molecule, rhesus MHC I molecule.

4. The method of enhancing the immunogenicity of an EBV mRNA vaccine according to claim 1, wherein the nucleotide sequences of the transmembrane and cytoplasmic domains are as set forth in SEQ ID NO: 2 and synonymous codons thereof, and a sequence having more than 90% identity with the above sequence.

5. The method for enhancing the immunogenicity of an EBV mRNA vaccine according to claim 1, wherein the nucleotide sequence of the 3' untranslated region is as set forth in SEQ ID NO: 3 and synonymous codons thereof, and a sequence having more than 90% identity with the above sequence.

6. The method for enhancing the immunogenicity of an EBV mRNA vaccine according to claim 1, wherein the nucleotide sequence of a120 is as set forth in SEQ ID NO: 4, the nucleotide sequence of the terminal sequence of A120 is shown as SEQ ID NO: 5, respectively.

7. A template DNA transcribed from mRNA obtained by the method of any one of claims 1 to 6.

8. An mRNA vaccine prepared by transcribing the template DNA with the mRNA according to claim 7.

9. The mRNA vaccine of claim 8 wherein the antigen coding region has the nucleotide sequence set forth in SEQ ID NO: 6 and sequences with identity over 90%.

10. The mRNA vaccine of claim 8, wherein the mRNA transcription template DNA has a nucleotide sequence as set forth in SEQ ID NO: 7 and sequences with identity of more than 90%.

11. A targeted nanoformulation carrier prepared from the mRNA vaccine of claim 8.

12. The targeted nanoformulation carrier of claim 11, wherein the targeted nanoformulation carrier is a liposome carrier.

13. The targeted nanoformulation carrier of claim 11, wherein the targeted nanoformulation carrier is a cationic liposome carrier.

14. The targeted nanoformulation carrier of claim 11, wherein the targeted nanoformulation carrier is a core-shell nanoparticle.

15. The targeted nanoformulation vector of claim 11, wherein the liposome vector is prepared using conventional liposome preparation methods including, but not limited to, thin film hydration, organic solvent injection, reverse evaporation, and freeze-thaw.

16. The targeted nanoformulation carrier of claim 11, comprising a phospholipid material, an additive, an organic solvent; the phospholipid material comprises one or more of 1, 2-distearoyl-3-trimethylammonium propane, trimethyl ammonium chloride, octadecenoyloxy (ethyl-2-heptadecenyl-3-hydroxyethyl) imidazoline , didecyl adipate, 3- [ N- (N ', N' -dimethylaminoethane) carbamoyl ] cholesterol, dimethylamino 1,2, 2-epoxypentyloxypropane and didecyl dimethyl ammonium bromide; the additive comprises one or more of total cholesterol, dioleoylphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylcholine; the organic solvent comprises one or more of absolute ethyl alcohol, methanol, ether, chloroform and methanol; the targeting nano-preparation carrier comprises the following components: 2.5mg/ml of 1, 2-distearoyl-3-trimethylammonium propane, 2.5mg/ml of total cholesterol, absolute ethanol;

or the following components: 4.2mg/ml of dimethylamino 1,2, 2-epoxypentoxypropane, 4.2mg/ml of total cholesterol and methanol;

17. The method for delaying the degradation of the mRNA vaccine of claim 8, wherein the mRNA liposome vaccine is prepared by mixing the prepared liposome carrier solution and the mRNA vaccine solution, and incubating.

18. The method of claim 17, wherein the N/P of the phospholipid material and mRNA in the liposomal vaccine is 0.5-10: 1.

19. The method of claim 17, wherein the N/P ratio of the phospholipid material and the mRNA in the liposomal vaccine is 1-5: 1.

20. The mRNA liposome vaccine of claim 15, LP-mRNA-LMP 2.

21. Use of the mRNA vaccine of claim 8, the targeted nanoformulation vector of claim 11, and the LP-mRNA-LMP2 of claim 20 in the preparation of a promoter of T cell proliferation.

22. Use according to claim 21, characterized in thatIn that the application is in the preparation of CD8⁺Use of a promoter of T cell proliferation.

23. Use according to claim 21, wherein the proliferation of T cells is in the treatment or prevention of epstein barr virus (EB) induced lymphoblastomas including lymphoblastomas, Burkitt's lymphomas, Hodgkin's lymphomas, NK cell and T cell lymphomas, diffuse large B cell lymphomas, primary effusion lymphomas, positive follicular lymphomas, multicentric Castleman's disease, nasopharyngeal carcinomas, gastric carcinomas.

24. The use of the mRNA vaccine of claim 8, the targeted nanoformulation vector of claim 11, and the LP-mRNA-LMP2 of claim 20 in the preparation of an anti-tumor medicament.

25. The use according to claim 24, for the preparation of a promoter for a tumor killing cytokine.

26. The use according to claim 25, wherein the tumor is lymphoblastoma, Burkitt's lymphoma, Hodgkin's lymphoma, NK cell and T cell lymphoma, diffuse large B cell lymphoma, major effusion lymphoma, positive follicular lymphoma, multicentric Castleman's disease, nasopharyngeal cancer, gastric cancer.

27. The use of claim 26, wherein the tumor is nasopharyngeal carcinoma.

28. The use according to claim 24, wherein the LMP2mRNA liposome vaccine promotes expression of the encoded antigen LMP2 and stimulates production of specific CTLs.