CN113577258B

CN113577258B - Double-target mRNA vaccine and preparation method thereof

Info

Publication number: CN113577258B
Application number: CN202110876695.XA
Authority: CN
Inventors: 刘明录; 卢永灿; 冯建海; 强邦明; 王立新; 韩庆梅; 金海锋; 王亮; 许淼
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shandong Xinrui Biotechnology Co ltd
Priority date: 2021-07-31
Filing date: 2021-07-31
Publication date: 2024-04-16
Anticipated expiration: 2041-07-31
Also published as: CN113577258A

Abstract

The invention provides a double-target mRNA vaccine, which comprises a liposome mixture and double-target mRNA; the liposome mixture comprises DLin-MC3-DMA, DSPC, cholesterol and PEG200-DMG, wherein the molar ratio is 50:10:37.5:2.5; the double-target mRNA is transcribed from a linearized recombinant plasmid; the recombinant plasmid comprises a target gene fragment; the target gene fragment is obtained by sequentially connecting the following modules in series: 5'-UTR nucleic acid artificial sequence, P53 nucleic acid artificial sequence, linker nucleic acid artificial sequence, kras nucleic acid artificial sequence, MITD nucleic acid artificial sequence, 3' -UTR nucleic acid artificial sequence; the invention also provides a preparation method of the vaccine; the mRNA vaccine prepared by the invention can block the migration of mRNA, protect the mRNA from being degraded and improve the delivery efficiency of the mRNA.

Description

Double-target mRNA vaccine and preparation method thereof

Technical Field

The invention relates to a double-target mRNA vaccine and a preparation method thereof, belonging to the technical field of biological medicine.

Background

Tumor vaccine is a therapeutic tumor treatment method for activating the immune system of organism to generate specific anti-tumor cell immune response by using tumor cells, tumor cell lysate or tumor antigen, and comprises whole cell vaccine, polypeptide vaccine, RNA/DNA gene vaccine and dendritic cell vaccine. Among them, mRNA vaccine, which is a novel nucleic acid vaccine, can express any protein and can treat almost all protein-based diseases at the gene level. Compared with other traditional vaccine and DNA vaccine technologies, the synthesis of mRNA vaccine is simple in production process and low in cost. Meanwhile, the artificially designed nucleic acid does not induce immune response of human body, does not enter cell nucleus, does not have the risk of integrating into genome, and is relatively safer. However, mRNA has poor stability, is easily degraded by nuclease in vitro and in vivo, and has poor ability to penetrate cell membranes, which is a bottleneck for restricting development. Thus, mRNA vaccines require a suitable delivery vehicle to deliver them into the body for better immune response.

At present, tumor mRNA vaccines use nanometer preparation technology which is most widely based on cationic lipid and polymer, can protect nucleic acid drugs from degradation of nuclease, and successfully realize stable delivery of nucleic acid drugs including siRNA, mRNA, cas plasmid and the like in vivo. Cationic Lipid Nanoparticles (LNPs) are composed of cationic lipids, polyethylene glycol (PEG), cholesterol, etc., encapsulating nucleic acid drugs that are endocytosed by cell membranes into cells to form endosomal vesicles, which, along with a series of structural changes, promote mRNA to be released from the endosome and bound to the ribosome responsible for protein production, directing the synthesis of viral proteins. LNPs have good biocompatibility and physicochemical properties, exhibit high gene transfection effects in vivo and in vitro, and are proved to be the safest drug delivery vehicle. RNA drugs (Patisiran injection) have been approved by the FDA for the delivery of siRNA or other small oligonucleotides in vivo, but the materials used for mRNA vaccine delivery are very limited. Due to the susceptibility of mRNA to degradation and its own physicochemical characteristics, existing nanomaterials have limited efficiency in mRNA delivery, and the development and clinical use of mRNA-based tumor vaccines still face a number of problems. Therefore, there is a need to develop a new therapeutic approach to achieve efficient delivery of mRNA and efficient expression of the encoded antigen.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a double-target mRNA vaccine and a preparation method thereof, which realize the following aims:

(1) The mRNA vaccine prepared by the invention has high encapsulation efficiency;

(2) The LNP liposome prepared by the invention can block the migration of mRNA and protect the mRNA from degradation; the transfection efficiency of cells is improved;

(3) The antibody protein generated by the mRNA vaccine is higher than that of naked mRNA;

(4) The mRNA vaccine can effectively induce T cells to generate immune response;

(5) The mRNA vaccine has high killing rate on tumor cells.

In order to solve the technical problems, the invention adopts the following technical scheme:

a dual-target mRNA vaccine comprising a liposome mixture and a dual-target mRNA; the liposome mixture comprises DLin-MC3-DMA, DSPC, cholesterol and PEG200-DMG, wherein the molar ratio is 50:10:37.5:2.5;

the double-target mRNA is transcribed from a linearized recombinant plasmid; the recombinant plasmid comprises a target gene fragment; the target gene fragment is obtained by sequentially connecting the following modules in series: 5'-UTR nucleic acid artificial sequence, P53 nucleic acid artificial sequence, linker nucleic acid artificial sequence, kras nucleic acid artificial sequence, MITD nucleic acid artificial sequence, 3' -UTR nucleic acid artificial sequence; the 5' -UTR nucleic acid artificial sequence is shown as SEQ ID NO.1 in a sequence table; the artificial sequence of the P53 nucleic acid is shown as SEQ ID NO.2 in a sequence table; the Linker nucleic acid artificial sequence is shown as SEQ ID NO.3 in the sequence table; the Kras nucleic acid artificial sequence is shown as SEQ ID NO.4 in the sequence table; the MITD nucleic acid artificial sequence is shown as SEQ ID NO.5 in a sequence table; the 3' -UTR nucleic acid artificial sequence is shown as SEQ ID NO.6 in the sequence table.

The following is a further improvement of the above technical scheme:

the method for preparing the double-target mRNA vaccine comprises the steps of constructing target gene fragments, constructing recombinant plasmids, linearly cutting and recovering the recombinant plasmids, transcribing to obtain mRNA and packaging the mRNA.

Constructing the recombinant plasmid, and cloning a target gene fragment to a PVAX1 vector to obtain the recombinant plasmid; the transcription yields mRNA which is transcribed into mRNA capped at the 5 'end and Poly (A) tail at the 3' end.

The packaging mRNA is to use liposome mixture to encapsulate mRNA.

The packaging mRNA specifically comprises: preparing ethanol solution of liposome mixture, preparing mRNA water solution, synthesizing LNP/mRNA vaccine particles, and preparing vaccine.

Preparation of the ethanol solution of the liposome mixture: dissolving the liposome mixture in ethanol to obtain an ethanol solution of the liposome mixture, wherein the mass volume ratio of the liposome mixture to the ethanol is 16.1-16.2mg:1mL;

preparation of the aqueous mRNA solution: dissolving the transcribed mRNA in a citric acid buffer solution, blowing and dispersing, adding PBS, and fully and uniformly mixing to obtain an mRNA aqueous solution; the mass volume ratio of the mRNA to the citric acid buffer solution to the PBS is as follows: 0.4-3.2 μg:1mL:2mL;

the LNP/mRNA vaccine particle synthesis method comprises the steps of synthesizing LNP/mRNA vaccine particles by using a nanoparticle synthesis system according to the volume ratio of an ethanol solution of a liposome mixture to an mRNA aqueous solution of 1:3;

the prepared vaccine is prepared by dialyzing LNP/mRNA vaccine particles in PBS solution for 24 hours, concentrating and filtering.

Cancer is a disease caused by somatic gene mutation, and mutations of P53 gene and K-ras gene exist in more than half of cancers and are overexpressed in tumors. After the P53 gene mutation is inactivated, the normal synthesis of P53 protein is affected, the function of the P53 protein is inhibited, the cell loses the monitoring of DNA damage, and mutation is easily accumulated to become cancer cells; the mutation of K-Ras gene can make cells continuously grow and inhibit autophagy, so that intracellular cell transduction is disordered, and many cancers such as lung cancer, pancreatic cancer and colorectal cancer are related to the K-Ras mutation.

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

(1) The mRNA vaccine prepared by the invention targets the P53 and Kras genes expressed by tumor cells, has strong specificity and is not easy to generate off-target effect.

(2) The particle size of the mRNA vaccine prepared by the invention is 92-96 nm, the encapsulation rate is above 98%, and the specific LNP liposome is selected to wrap the mRNA, so that the migration of the mRNA can be blocked, the mRNA is protected from being degraded, the delivery efficiency of the mRNA is improved, the mRNA is directly translated and expressed after entering cells, and the protein expression amount of the LNP/mRNA vaccine is 2.3 times that of naked mRNA.

(3) The mRNA vaccine prepared by the invention can effectively induce T cells to generate immune response, and improve secretion of cytokines IL-2 and IFN-gamma.

(4) The mRNA vaccine prepared by the invention has high killing effect on tumor cells and the killing rate on tumor target cells reaches 98.6 percent.

(5) The invention modifies the in vitro transcribed mRNA template, adds a cap at the 5 'end and a polyA tail at the 3' end, and protects mRNA from degradation.

(6) When the mRNA vaccine constructed by the invention is used for constructing a gene expression vector, the 5 'capping and 3' ploy (A) tail modification of the molecular structure of the mRNA vaccine can protect mRNA from degradation of nuclease in the transfection process, and the stability of the mRNA is improved.

Drawings

FIG. 1 is a schematic diagram of the structure of PVAX1-P53Kras carrier prepared in example 1.

FIG. 2 is a schematic diagram of the structure of PVAX1-EGFP vector prepared in example 1.

FIG. 3 is a block diagram of lipid nanoparticles.

FIG. 4 is a graph showing the fluorescent expression of LNP/EGFP transfected 293T cells at different concentrations according to example 7.

FIG. 5 is a graph showing IL-2 and IFN-gamma secretion from different LNP nanoparticle transfected cells of example 8.

Detailed Description

EXAMPLE 1 acquisition of the Gene of interest and construction of recombinant plasmid

The invention selects P53 and K-ras as target points and designs mRNA vaccine aiming at tumor.

The nucleic acid sequences of each module of the PVAX1-P53Kras vector are as follows:

(1) 5' -UTR nucleic acid artificial sequence (SEQ ID NO. 1)

(2) P53 nucleic acid Artificial sequence (SEQ ID NO. 2)

(3) Linker nucleic acid Artificial sequence (SEQ ID NO. 3)

(4) Kras nucleic acid artificial sequence (SEQ ID NO. 4)

(5) MITD nucleic acid artificial sequence (SEQ ID NO. 5)

(6) 3' -UTR nucleic acid artificial sequence (SEQ ID NO. 6)

The sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 are sequentially connected, and the whole expression frame is synthesized by Nanjing Jinsrui biotechnology Co., ltd and inserted into a standard vector pUC57 vector, and the vector is named pUC-P53Kras. pUC-P53Kras vector and PVAX1 vector (SEQ ID NO. 7) were digested simultaneously with Fast Digest BamHI (from Thermo Fisher Co.) and Fast Digest NotI (from Thermo Fisher Co.), and the linearized DNA fragments were recovered by gel cutting, and ligated overnight at 16℃to form PVAX1-P53Kras expression vector. The PVAX1-P53Kras was transformed into E.coli (DH 5. Alpha.), and positive clones were picked up for PCR identification. Plasmids were extracted and sent to Nanjing Jinsri Biotechnology Co.Ltd for sequencing, sequencing identification was correct, and PVAX1-P53Kras was successfully constructed (see FIG. 1 for vector structure). PVAX1-P53Kras plasmid was extracted from positive clones, diluted to 2. Mu.g/. Mu.L and stored at-80℃until use.

By adopting the same method, the EGFP gene fragment (SEQ ID NO. 8) is cloned on a PVAX1 vector, and is named PVAX1-EGFP (the vector structure is shown in figure 2), sequencing identification is correct, and PVAX1-EGFP is successfully constructed. PVAX1-EGFP plasmid is extracted from positive clone, diluted to 2 mug/mu L and stored at-80 ℃ for standby.

EXAMPLE 2 linearization cleavage and recovery of recombinant plasmid

The template for in vitro transcription requires an RNA polymerase promoter and requires that plasmid DNA must be linearized by restriction enzymes, since circular DNA will produce a long heterogeneous RNA template, and restriction enzyme sites must be downstream of the gene of interest, cleavage must be complete and complete, not I restriction enzyme is selected to cleave the plasmid singly (PVAX 1-P53Kras and PVAX 1-EGFP), respectively, and the optimized cleavage system (total 50. Mu.L) is as follows:

10xBuffer:3 μL; notI enzyme: 3 μL; PVAX1-P53Kras plasmid: 1. Mu.L (2. Mu.g/. Mu.L); water: 43 μl.

10xBuffer:3 μL; notI enzyme: 3 μL; PVAX1-EGFP plasmid: 1. Mu.L (2. Mu.g/. Mu.L); water: 43 μl.

The two plasmids are respectively subjected to single enzyme digestion reaction;

the mixture is prepared in a 150 mu L centrifuge tube according to the system, and is subjected to enzyme digestion for 1h in a water bath at 37 ℃, quickly transferred into the water bath at 65 ℃ for 5min, and the enzyme digestion reaction is terminated. The fragment size obtained was expected by electrophoresis on a 1% agarose gel. The target fragment is excised and recovered by agarose gel DNA recovery kit.

The concentration of the recovered DNA was measured by a spectrophotometer. The concentration of the fragment after cleavage of PVAX1-P53Kras was 1.1. Mu.g/. Mu.L, A260/280=1.83; the concentration of the fragment after cleavage of PVAX1-EGFP was 1.2. Mu.g/. Mu.L, A260/280=1.82, and the result was consistent with the DNA measurement standard (A260/280=1.8-2.1). Storing at-20deg.C for use.

Example 3 in vitro transcription mRNA (IVT mRNA)

Using the linearized DNA of example 2 as a template, 5' capped mRNA was synthesized by in vitro transcription using a cap mMESSAGE mMACHINE T kit (Ambion Corp.) as follows:

TABLE 1 transcription reaction system

1. According to the instructions, the reaction system (see Table 1) was prepared on ice, and after thoroughly mixing, centrifuged to the bottom of the PCR tube and incubated overnight at 37 ℃.

2. 1. Mu.L of Dnase I was added, mixed well, incubated at 37℃for 20min, and the DNA was removed from the product.

3. mRNA was purified by LiCl precipitation:

(1) 25. Mu.L DEPC water was added to the 50. Mu.L system;

(2) Adding 25 mu L of LiCl solution, and uniformly mixing;

(3) Incubating in a refrigerator at-20deg.C for 30min;

(4) Centrifuging at 4deg.C for 15min at 12000g, and removing supernatant;

(5) Adding 500 mu L70% ice ethanol into the precipitate, 12000g, centrifuging at 4 ℃ for 15min, and removing the supernatant;

(6) Repeating step (5);

(7) mRNA was resuspended in an appropriate amount of DEPC water and quantified using a micro-spectrophotometer to give a concentration of 2. Mu.g/. Mu.L.

4. Poly (A) tails were added to the 3' end of the mRNA and the reaction system was placed on ice (see Table 2).

Table 2 3' end Poly (A) tail reaction system

5. Well mixed, centrifuged to the bottom of the tube and incubated for 3h at 37 ℃.

6. The mRNA was purified again by LiCl precipitation, in the same manner as in step 3. The purified mRNA was resuspended in an appropriate amount of DEPC water to give an mRNA solution, and the mRNA was quantified using a differential spectrophotometer to give a concentration of 2. Mu.g/. Mu.L mRNA, and the mRNA product was stored in a refrigerator at-80℃with 50. Mu.L of mRNA per tube.

7. Verification of in vitro transcribed mRNA length:

placing mRNA in a water bath kettle at 70 ℃ for 10min, placing on ice for 3min, performing electrophoresis in urea-acrylamide gel, performing 100v constant-pressure electrophoresis for 30min, photographing by a gel imager, and verifying the size and integrity of mRNA.

As a result analysis shows, P53Kras mRNA and EGFP mRNA transcribed from the linearized PVAX1-P53Kras and PVAX1-EGFP in vitro are subjected to electrophoresis in urea acrylamide gel, and the obtained mRNA fragments are 1681bp and 817bp respectively, and the obtained mRNA accords with the expectation.

Example 4 preparation of LNP for packaging mRNA and characterization thereof

The LNP component includes: ionizable cationic lipids (DLin-MC 3-DMA), positively charged, interact electrostatically with negatively charged mRNA, assemble into virus-sized particles, which facilitate intracellular delivery and release of mRNA to the cytoplasm; cholesterol can enhance the fluidity of the liposome and stabilize the nanoparticles; phospholipids (DSPC) forming a lipid bilayer structure; PEG-lipid (PEG 200-DMG), can improve the stability of nanostructure, and prolong the metabolism time of nanometer medicine in blood. The mRNA is wrapped in the cationic liposome through electrostatic action, the hydrophobic end of the PEG-lipid is combined with the hydrophobic end of the cationic lipid, the hydrophilic end of the PEG-lipid (connected with PEG) outwards forms the shell of the nucleic acid lipid nanoparticle, and in order to enhance the stability of the nucleic acid lipid nanoparticle, a proper amount of cholesterol and other components are added, so that a finished product of the nucleic acid lipid nanoparticle is finally obtained (see figure 3).

The specific operation steps are as follows:

the preparation method of the P53Kras mRNA vaccine comprises the following steps:

1. preparation of ethanol solution

10.4mg of DLin-MC3-DMA, 2.7mg of DSPC, 4.9mg of cholesterol and 2.2mg of PEG200-DMG were weighed out as solids (molar ratio: 50:10:37.5:2.5), placed in a 15mL RNase-free tube, 1.25mL of ethanol was added thereto, and the mixture was dissolved by stirring sufficiently to obtain an ethanol solution of a liposome mixture.

2. Preparation of aqueous solutions

mu.L of the mRNA solution prepared in example 3 (mRNA mass: 2. Mu.g) was dissolved in 1.25mL of 10mM citrate buffer (pH=4) and blown up and down 50 times; 2.5mL of PBS was added, and the mixture was blown with force 50 times and thoroughly mixed to obtain an aqueous mRNA solution.

3. Synthesis of LNP/mRNA vaccine

LNP particles were synthesized by mixing an ethanol solution of the liposome mixture with an aqueous mRNA solution in a ratio of 1:3 (volume ratio) using a nanoparticle synthesis system (Ignite);

dialyzing the obtained LNP in PBS solution (pH 7.4) for 24h, concentrating to 1mL by using an Amicon ultracentrifuge filter, filtering with a 0.22um filter membrane for 2 times, and storing at-20deg.C for use to obtain P53Kras mRNA vaccine, wherein the concentration of mRNA is 2 μg/mL as expressed by LNP/P53 Kras;

the concentration is the mass to volume ratio of the initially added mRNA to the mass of the final vaccine.

Preparation of EGFP mRNA vaccine:

EGFP mRNA vaccine, expressed as LNP/EGFP, was prepared in the same manner;

PVAX1-P53Kras (DNA) vaccine:

based on the preparation method of the P53Kras mRNA vaccine, the following changes are:

when the aqueous solution is prepared, the equal amount of PVAX1-P53Kras plasmid of the example 1 is used for replacing mRNA solution, and the PVAX1-P53Kras (DNA) vaccine is expressed by LNP/PVAX1-P53 Kras;

blank control:

when the aqueous solution is prepared, equal amount of DEPC water is used for replacing the mRNA solution, the rest preparation methods are the same as those for preparing the P53Kras mRNA vaccine, and blank control is expressed by LNP/blank.

Example 5 LNP/mRNA nanoparticle characterization analysis

The size, the carried potential, the polydispersity and the encapsulation efficiency of the nanoparticles were determined for each group of vaccines prepared in example 4:

1. size, carried potential and polydispersity determination of nanoparticles

The size, carried potential and Polydispersity (PDI) of the prepared nanoparticles were analyzed using a Malvern Zetasizer Nano ZS 90.90 nm particle size potentiometric analyzer. The specific method comprises the following steps:

preparing 100 mu L of three groups of samples of LNP/blank, LNP/P53Kras and LNP/EGFP respectively, adding 10mM citric acid buffer solution to 1mL, and dripping into a cuvette; and (5) placing the mixture into an instrument to measure the particle size, the potential and the PDI.

The results show (see Table 3) that LNP/blank, LNP/P53Kras, LNP/EGFP particle diameters are 108nm, 96nm, 92nm, respectively, since LNP and mRNA are attracted to each other by electrostatic interaction, the diameter of the nanoparticle is slightly reduced, and related studies show that cationic liposomes with small size are more efficient than cationic liposomes with large size; the PDI of the three components is smaller than 0.3, and the dispersity is good; LNP/blank, LNP/P53Kras, LNP/EGFP potentials were 32.3mV, 20.2mV and 21.2mV in this order, and the potential was reduced after binding to liposomes due to the negative charge of the nucleic acid, which potential helped the particles to cross the cell membrane.

2. Encapsulation efficiency determination

The encapsulation efficiency of the nano particles is measured by adopting an ultracentrifugation method, and the specific operation steps are as follows:

three groups of LNP/blank, LNP/P53Kras, LNP/EGFP were prepared 1mL each, the concentration was noted as C0 (2. Mu.g/mL), and the three groups were placed in DEPC-treated ultrafilters, respectively, and centrifuged at 4℃and 5000rpm for 1 hour to separate unpacked mRNA from liposomes, and the supernatant after centrifugation was unpacked mRNA, and the concentration was measured as C. The total mRNA amount (C0) was calculated from the sample before centrifugation, and the supernatant after centrifugation was the free mRNA amount (C), and the encapsulation efficiency was calculated.

Encapsulation (%) = C0-C/C0x100%.

The results show (see Table 3) that the encapsulation efficiency of the LNP/P53Kras and LNP/EGFP is as high as 98% or more, and the mRNA vaccine is substantially completely encapsulated.

Table 3 nanoparticle characterization

EXAMPLE 6 gel blocking experiment

To evaluate LNP binding to mRNA, it was verified by gel blocking experiments. The prepared nanoparticle LNP/blank and LNP/P53Kras in example 4 were selected, and the mRNA solution (naked mRNA) obtained in step 6 of example 3 was subjected to agarose gel electrophoresis at 150v for 15min, and then observed under a chemiluminescent gel imager.

The results indicate that the control LNP/blank did not emit light; the naked mRNA is in a dispersion state due to partial degradation; LNP/P53Kras stays in the gel well and can completely block mRNA migration. Thus, LNP liposomes prepared according to the present invention can block mRNA migration and protect mRNA from degradation.

Example 7 evaluation of in vitro cell transfection Effect

1. To verify the dose of LNP/mRNA required and the effect of transfection on cells, EGFP mRNA was selected for ease of observation and detection as a model.

293T cells were seeded onto 24-well plates at a density of 5X10 ⁵ Cells/well, cells were cultured by adding 1ml of LDMEM medium (containing 10% FBS) to each well, and when the cell density reached 80%, cells were transfected by adding 1ml of LNP/EGFP at different concentrations, and six experiments were set up, 3 replicates per group, and the groups were as follows:

bare mRNA (2. Mu.g/mL), blank (LNP/blank), LNP/EGFP (0.5. Mu.g/mL), LNP/EGFP (1. Mu.g/mL), LNP/EGFP (2. Mu.g/mL), LNP/EGFP (4. Mu.g/mL), and 5% CO at 37deg.C ₂ After 48h of co-incubation under the conditions, cell suspensions were obtained and the percentage of EGFP positive cells was detected by flow cytometry.

Naked mRNA group: example 3 mRNA solution obtained in step 6.

The amounts of mRNA added were changed in the LNP/EGFP (0.5. Mu.g/mL), LNP/EGFP (1. Mu.g/mL), LNP/EGFP (4. Mu.g/mL) and the LNP/EGFP (2. Mu.g/mL) groups, with reference to the preparation method of the LNP/EGFP (2. Mu.g/mL) groups.

The results show (see FIG. 4) that with increasing LNP/EGFP concentration, EGFP positive cell expression rate increases, and EGFP expression level is highest, up to more than 95% when LNP/EGFP concentration is 2 μg/mL, indicating that EGFP encoded mRNA is translated into fluorescent protein. The green fluorescence expression of the naked mRNA group is lower, only 31%, and the naked mRNA is degraded in the transfection process, so that the expression of the protein is affected. Therefore, the lipid prepared by the invention can protect mRNA from being degraded, and improve the transfection efficiency of cells.

2. ELISA method detection

293T cells were seeded onto 24-well plates at a density of 5X10 ⁵ Cells/well, cells were cultured by adding 1mL of DMEM medium (containing 10% FBS) to each well, and when the cell density reached 80%, cells were transfected by adding 1mL of LNP/P53Kras (2. Mu.g/mL).

Three sets of experiments were set up, grouped as follows: naked mRNA group (mRNA solution obtained in step 6 of example 3), blank group (equal volume of DMEM medium (containing 10% FBS)), experimental group LNP/P53Kras (2. Mu.g/mL), after a total incubation of 48h, 293T cells were centrifuged at 1500rpm for 5min, and the supernatant was collected and cell pellet was discarded. Cell supernatants were directly diluted 10-100-fold with coating solution and subjected to ELISA detection. The ELISA detection comprises the following specific operation steps:

(1) The dilutions were added to 96-well ELISA plates, 100. Mu.L/well, and placed in a refrigerator at 4℃overnight.

(2) The plates were washed 5 times with pre-configured PBST, 200. Mu.L/well each time, at least 1min, and the plates were spun dry.

(3) 5% BSA blocking solution, 200. Mu.L/well, was added and the plates were incubated in an incubator at 37℃for 2h. After the incubation, the blocking solution was thrown off, and the plates were washed again with PBST 5 times, as in the washing plate above.

(4) Anti-p53 Rabbit Monoclonal Antibody (diluted 1:600) was added and the plates were incubated in an incubator at 37℃for 2h at 100. Mu.L/well. After incubation, the plates were washed again 5 times with PBST and the plate washing procedure was followed as described above.

(5) Secondary antibodies GoatAnti-MouseigG, HRP Conjugated (diluted 1:5000) were added and the plates incubated in a 37℃incubator for 2h. After incubation, the plates were washed again 5 times with PBST and the plate washing procedure was followed as described above.

(6) Adding 100 mu L/hole of TMB color development liquid, reacting for 20min at room temperature in dark, adding 100 mu L/Kong Zhongzhi liquid, and stopping the reaction. The absorbance at 450nm was read by a multifunctional microplate reader.

The results are shown in Table 4, the exposed mRNA group and the LNP/P53Kras both produce antigen proteins which can be specifically combined with the P53 antibody protein, and the ELISA detection result shows that the antibody protein produced by the LNP/P53Kras nano vaccine of the experimental group is 2.3 times higher than that of the exposed mRNA group. The experimental result further shows that the LNP nano delivery system provided by the invention can protect the degradation of P53Kras mRNA, successfully enter cells and express P53 protein.

TABLE 4 expression of antigenic proteins in 293T cells

Example 8 cytokine secretion assay

IL-2 and IFN-gamma are key indicators for assessing whether T cells produce a strong immune response, and the cytokine secretion of T cells is detected by the ELSIA method.

1. Cell culture

(1) Effector cells

Activated T cells were seeded onto 24-well plates at a density of 5X10 ⁵ 1mL DMEM medium (containing 10% FBS) was added to each well to culture the cells to 80% cell density, 1mL LNP/P53Kras (2. Mu.g/mL), 1mL LNP/PVAX1-P53Kras (2. Mu.g/mL), 1mL naked mRNA (2. Mu.g/mL) were separately added to transfect the cells, the cells were incubated for 48 hours, collected, centrifuged at 1500rpm for 5min, and the supernatant was discarded, and the cells obtained were three effector cells,

the control group was not added with vaccine or naked mRNA, and the rest was the same.

(2) Target cells

Human hepatoma cells Hep3B (purchased from the upper sea biomass) were used as target cells and cultured in DMEM medium (containing 10% FBS) to logarithmic phase.

(3) Co-culture of cells

Effector cells were conditioned with DMEM medium containing 10% FBS (1×10 ⁵ Well) and target cells (1X 10 ⁵ Per well) and inoculated in 96-well plates, 100 μl/well, divided into four groups, specifically grouped as follows:

experiment group a: t cells (LNP/P53 Kras transfection) and Hep3B cells were co-cultured;

experimental group B: t cells (LNP/PVAX 1-P53Kras transfection) and Hep3B cells were co-cultured;

experiment group C: t cells (naked mRNA transfection) and Hep3B cells were co-cultured;

control group: t cells (untransfected) and Hep3B cells were co-cultured;

three replicates of each group were placed at 37℃with 5% CO ₂ The incubator was co-cultured for 72 hours, and the culture supernatant was collected for detection of cytokines IL-2 and IFN-gamma.

2. Detection of IFN-gamma and IL-2 in cell culture supernatants

(1) IFN-gamma and IL-2 kits were removed and left to stand at room temperature, 5 standard concentration gradients of IFN-gamma and IL-2 each were prepared according to instructions. 100. Mu.L of standard was added to the antibody pre-coated wells.

(2) Sample preparation, 100 μl of cell culture supernatant was added directly to the well plate, while a blank control group was set.

(3) Adding 50 mu L of biotin-labeled primary antibody diluted in proportion into each hole, and incubating for 90min at 37 ℃; after the incubation is finished, the supernatant is discarded, and the washing liquid is washed for 5 times for 1min each time; adding 100 mu L of enzyme-binding secondary antibody diluted in proportion, and incubating for 30min at 37 ℃; after the incubation, the supernatant was discarded, and the wash was performed 5 times for 1min each.

(4) 100 mu L of substrate TMB (3, 3', 5' -tetramethyl benzidine) is added, the mixture is incubated at 37 ℃ for 5-15min for color development, 100 mu L of stop solution is added after the color development is obvious, and the mixture is placed in an enzyme-labeling instrument for OD450 nm reading.

ELISA results showed (see FIG. 5 and Table 5): the concentration of IL-2 and IFN-r was higher in the LNP/P53Kras group, the DNA group and the naked mRNA group than in the control group, and the concentration of IL-2 and IFN-r was the highest in the LNP/P53Kras group. Therefore, the LNP/mRNA vaccine constructed by the invention can effectively induce T cells to generate immune response.

TABLE 5 cytokine secretion (pg/mL)

Example 9 LNP/mRNA vaccine in vitro killing experiments

1. T cell preparation

50mL of peripheral blood was collected and separated with TBD sample density separation solution (from Tianjin, hua Kochiaceae) to obtain PMBC. After induction culture with DMEM (available from Corning Inc., 88-551-CM) medium containing 1000IU/mL recombinant interferon alpha 2a (available from Shenyang Sansheng Co., ltd.) for 24 hours, induction culture was continued with the addition of 1000IU/mL recombinant IL-2 (available from Shenyang Sansheng Co., ltd.), 50ng/mL OKT-3 and 5% autologous plasma of the patient for 24 hours. Adding liquid at two-time ratio every two days, culturing until 14 th day, and detecting CD3 in T cells by flow cytometry ⁺ 、CD56 ⁺ The positive expression rate of (CD 3-FITC, CD16/CD56-PE antibody was purchased from BECKMAN, inc., A07735). CD3 ⁺ Positive rate>90%，CD3 ⁺ CD56 ⁺ Double positive rate>20%, T cell induction was considered successful.

2. Cell culture

(1) Effector cells

The activated T cells are inoculated on a 24-well plate with an inoculation density of 5x10 ⁵ When 1mL of DMEM medium (containing 10% FBS) was added to each well to culture cells to 80% cell density, 1mL of LNP/P53Kras (2. Mu.g/mL), 1mL of LNP/PVAX1-P53Kras (2. Mu.g/mL) and 1mL of naked mRNA (2. Mu.g/mL) were respectively added to transfect the cells, and the cells were incubated for 48 hours, collected, centrifuged at 1500rpm for 5min, and the supernatant was discarded, and the cells were three effector cells.

(2) Target cells

Human hepatoma cells Hep3B (purchased from the upper sea afforest organisms) were used as target cells and cultured to logarithmic growth phase using DMEM medium and 10% FBS.

(3) Co-culture of cells

Effector cells were conditioned with DMEM medium containing 10% FBS (1×10 ⁵ Well) and target cells (1X 10 ⁵ Per well) and inoculated in 96-well plates, 100 μl/well, divided into three groups, specifically grouped as follows:

LNP/P53Kras group: t cells (LNP/P53 Kras transfection) and Hep3B cells were co-cultured;

LNP/PVAX1-P53Kras group: t cells (LNP/PVAX 1-P53Kras transfection) and Hep3B cells were co-cultured;

naked mRNA group: t cells (naked mRNA transfection) and Hep3B cells were co-cultured;

control group: t cells (untransfected) and Hep3B cells were co-cultured;

culturing in an incubator with 5% CO2 at 37 ℃, adding 20mL CCK-8 into each hole after 24 and h, continuously incubating for 2 hours, detecting the wavelength of 450nm by using an enzyme-labeling instrument, and reading the OD value. Calculating the cell killing rate: killing rate/% = [1- (blank OD value-effector OD value)/blank OD value ] ×100%.

The results show that the killing rates of the LNP/P53Kras group, the LNP/PVAX1-P53Kras group and the naked mRNA group are 98.6%, 62.1% and 41.3%, respectively, the killing rate of T cells transfected by the LNP/P53Kras group is highest and is obviously higher than that of the LNP/PVAX1-P53Kras group and the naked mRNA group, and the killing rates of the LNP/P53Kras group transfected by the LNP/PVAX1-P53Kras group and the mRNA group are 1.6 times and 2.4 times respectively. Therefore, the LNP/P53Kras constructed by the invention can protect mRNA from degradation during transfection, induce cells to generate stronger immune response, and has remarkable effect of killing tumor cells.

In conclusion, the invention successfully constructs an effective mRNA vaccine delivery system, which not only can protect mRNA from degradation in the transfection process, but also can enhance the cellular immune response of organisms.

Sequence listing

<110> Shandong Xinrui biotechnology Co., ltd

<120> a double-target mRNA vaccine and method for preparing the same

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 89

<212> DNA

<213> race (Homo sapiens)

<400> 1

tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60

gaagagtaag aagaaatata agagccacc 89

<210> 2

<211> 810

<212> DNA

<213> race (Homo sapiens)

<400> 2

accaccatcc actacaacta catgtgtaac agttcctgca tgggcggcat gaaccaaagg 60

cccatcctca ccatcatcac actggaagac tccagtggta atctactggg aggatctgga 120

ggtggaggtt caggaggaac caccatccac tacaactaca tgtgtaacag ttcctgcatg 180

ggcggcatga actggaggcc catcctcacc atcatcacac tggaagactc cagtggtaat 240

ctactgggag gatctggagg tggaggttca ggaggatctg actgtaccac catccactac 300

aactacatgt gtaacagttc ctgcatgggc tctatgaacc ggaggcccat cctcaccatc 360

atcacactgg aagactccag tggtaatgga ggatctggag gtggaggttc aggaggaatc 420

acactggaag actccagtgg taatctactg ggacggaaca gctttgaggt gtgtgtttgt 480

gcctgtcctg ggagagaccg gcgcacagag gaagagaatc tccgcaaggg aggatctgga 540

ggtggaggtt caggaggagt ccgcgccatg gccatctaca agcagtcaca gcacatgacg 600

gaggttgtga ggcattgccc ccaccatgag cgctgctcag atagcgatgg tctggcccct 660

cctcagcatg gaggatctgg aggtggaggt tcaggaggac tactgggacg gaacagcttt 720

gaggtgcgtg tttgtgcctg tcctgggaga gactggcgca cagaggaaga gaatctccgc 780

aagaaagggg agcctcacca cgagctgccc 810

<210> 3

<211> 30

<212> DNA

<213> race (Homo sapiens)

<400> 3

ggaggatctg gaggtggagg ttcaggagga 30

<210> 4

<211> 570

<212> DNA

<213> race (Homo sapiens)

<400> 4

actgaatata aacttgtggt agttggagct gttggcgtag gcaagagtgc cttgacgata 60

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 120

actgaatata aacttgtggt agttggagct gatggcgtag gcaagagtgc cttgacgata 180

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 240

actgaatata aacttgtggt agttggagct tgtggcgtag gcaagagtgc cttgacgata 300

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 360

actgaatata aacttgtggt agttggagct agaggcgtag gcaagagtgc cttgacgata 420

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 480

actgaatata aacttgtggt agttggagct gctggcgtag gcaagagtgc cttgacgata 540

cagctaattc agaatcactt tgtggatgaa 570

<210> 5

<211> 171

<212> DNA

<213> race (Homo sapiens)

<400> 5

gtgggcatca ttgctggcct ggttctcctt ggagctgtga tcactggagc tgtggtcgct 60

gccgtgatgt ggaggaggaa gagctcagat agaaaaggag ggagttacac tcaggctgca 120

agcagtgaca gtgcccaggg ctctgatgtg tccctcacag cttgtaaagt g 171

<210> 6

<211> 119

<212> DNA

<213> race (Homo sapiens)

<400> 6

tgataatagg ctggagcctc ggtggccatg cttcttgccc cttgggcctc cccccagccc 60

ctcctcccct tcctgcaccc gtacccccgt ggtctttgaa taaagtctga gtgggcggc 119

<210> 7

<211> 2999

<212> DNA

<213> race (Homo sapiens)

<400> 7

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc 780

ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 840

gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 900

ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 960

attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 1020

gcaggcatgc tggggatgcg gtgggctcta tggcttctac tgggcggttt tatggacagc 1080

aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt 1140

aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca 1200

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 1260

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 1320

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 1380

cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac 1440

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 1500

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 1560

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 1620

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 1680

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 1740

caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 1800

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 1860

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 1920

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 1980

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt 2040

cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacaggtggc 2100

acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 2160

atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca cgtgctaaaa 2220

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 2280

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 2340

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2400

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2460

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2520

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2580

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2640

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2700

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2760

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2820

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2880

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2940

agcaacgcgg cctttttacg gttcctgggc ttttgctggc cttttgctca catgttctt 2999

<210> 8

<211> 717

<212> DNA

<213> race (Homo sapiens)

<400> 8

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

Claims

1. A double-target mRNA vaccine, characterized in that: the double-target mRNA vaccine comprises a liposome mixture and double-target mRNA; the liposome mixture comprises DLin-MC3-DMA, DSPC, cholesterol and PEG200-DMG, wherein the molar ratio of the liposome mixture is 50:10:37.5:2.5;

the double-target mRNA is transcribed from a linearized recombinant plasmid; the recombinant plasmid comprises a target gene fragment; the target gene fragment is obtained by sequentially connecting the following modules in series: 5'-UTR nucleic acid artificial sequence, P53 nucleic acid artificial sequence, linker nucleic acid artificial sequence, kras nucleic acid artificial sequence, MITD nucleic acid artificial sequence, 3' -UTR nucleic acid artificial sequence; the 5' -UTR nucleic acid artificial sequence is shown as SEQ ID NO.1 in a sequence table; the artificial sequence of the P53 nucleic acid is shown as SEQ ID NO.2 in a sequence table; the Linker nucleic acid artificial sequence is shown as SEQ ID NO.3 in the sequence table; the Kras nucleic acid artificial sequence is shown as SEQ ID NO.4 in the sequence table; the MITD nucleic acid artificial sequence is shown as SEQ ID NO.5 in a sequence table; the 3' -UTR nucleic acid artificial sequence is shown as SEQ ID NO.6 in the sequence table; and constructing the recombinant plasmid, and cloning the target gene fragment to a PVAX1 vector to obtain the recombinant plasmid.

2. A method of preparing the double-target mRNA vaccine of claim 1, characterized in that: the method comprises the steps of constructing a target gene fragment, constructing a recombinant plasmid, carrying out linear digestion and recovery on the recombinant plasmid, carrying out transcription to obtain mRNA, and packaging the mRNA.

3. The method according to claim 2, characterized in that: the mRNA obtained by transcription is transcribed into mRNA with 5 'capping and 3' end Poly (A) tail.

4. The method according to claim 2, characterized in that: the packaging mRNA is to use the liposome mixture to encapsulate mRNA.

5. The method according to claim 4, wherein: the packaging mRNA specifically comprises: preparing an ethanol solution of the liposome mixture, preparing an mRNA aqueous solution, synthesizing LNP/mRNA vaccine particles, and preparing the vaccine.

6. The method according to claim 5, wherein:

preparation of the aqueous mRNA solution: dissolving the transcribed mRNA in a citric acid buffer solution, blowing and dispersing, adding PBS, and fully and uniformly mixing to obtain an mRNA aqueous solution; the mass volume ratio of the mRNA to the citric acid buffer solution to the PBS is 0.4-3.2 mug: 1mL:2mL;

the LNP/mRNA vaccine particle synthesis method comprises the steps of synthesizing LNP/mRNA vaccine particles by using a nanoparticle synthesis system, wherein the volume ratio of an ethanol solution to an mRNA aqueous solution of the liposome mixture is 1:3;

the prepared vaccine is obtained by dialyzing LNP/mRNA vaccine particles in PBS solution for 24h, concentrating and filtering.