Lmp2a truncated mRNA related vaccine and preparation method and application thereof

Abstract

The application discloses an LMP2A truncated mRNA related vaccine and a preparation method and application thereof, the nucleotide sequence of the truncated mRNA is as shown in SEQ ID NO.1; the truncated body A has a better immune effect relative to a full length of LMP2A; and the mRNA vaccine is prepared based on the truncated body A, the sequence is as shown in SEQ ID NO.10, it is found that the mRNA vaccine can significantly inhibit tumor development, improve survival time, antibody response and immune response, and also has high safety. It is shown that the mRNA vaccine can provide good protection for mice, and can be used for developing a therapeutic vaccine for EBV related tumors.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to an LMP2A truncated mRNA-related vaccine, its preparation method, and its application. Background Technology

[0002] Epstein-Barr virus (EBV) was the first human tumor virus discovered and remains the only human pathogen capable of in vitro cell transformation. EBV infects over 90% of the world's population and causes approximately 2% of human malignancies. Since its discovery in Burkitt lymphoma 58 years ago, EBV has been found to be closely associated with various lymphomas and epithelial carcinomas, such as nasopharyngeal carcinoma (NPC), gastric cancer (GC), Burkitt lymphoma (BL), Hodgkin's lymphoma (HL), and post-transplant lymphoproliferative disorder (PTLD). Furthermore, EBV is a major cause of infectious mononucleosis and may play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis.

[0003] Nasopharyngeal carcinoma is primarily caused by EBV infection in areas where NPC is prevalent. LMP2A is an EBV-encoded late membrane protein that plays a crucial role in latent EBV infection. NPC is a squamous cell epithelial tumor with over 100,000 new cases worldwide each year. In areas where NPC is prevalent, EBV infection is considered a necessary and most important pathogenic factor for the development of NPC.

[0004] Over the past 20 years, with in-depth research into vaccine technology and a growing understanding of EBV immunology, an increasing number of EBV vaccine candidates have been developed. Various prophylactic / therapeutic vaccines targeting EBV and EBV-related tumors have been designed and studied, many of which have been shown to activate antigen-specific T-cell or B-cell responses. However, to date, no effective EBV vaccine has received clinical approval.

[0005] LMP2A is an EBV-encoded membrane protein expressed in various EBV-related malignancies and is crucial for establishing latent EBV infection. Furthermore, the widespread expression of LMP2A in EBV-related tumors makes it a potential therapeutic target. However, its relatively weak immunogenicity and natural membrane location make its use as an immune target a bottleneck in vaccine development. Summary of the Invention

[0006] The purpose of this invention is to provide a therapeutic vaccine for EBV-related tumors based on LMP2A truncated bodies.

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides an mRNA having a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.10.

[0009] A second aspect of the present invention provides a protein, the amino acid sequence of which is:

[0010] a) As shown in SEQ ID NO.2 or SEQ ID NO.11; or

[0011] b) The amino acid sequence shown in a) after substitution, deletion, or addition of one or more amino acids, and with the same or similar function; or

[0012] c) An amino acid sequence that has 99%, 95%, 90%, 85%, or 80% homology with the amino acid sequence defined in a) or b) and has the same function.

[0013] A third aspect of the present invention provides a DNA molecule that encodes the protein described in the second aspect of the present invention.

[0014] In some embodiments of the present invention, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO.3 or SEQ ID NO.12.

[0015] In a fourth aspect, the present invention provides an expression vector containing the DNA molecule described in the third aspect of the present invention.

[0016] In a fifth aspect, the present invention provides a recombinant cell containing the expression vector described in the fourth aspect of the present invention.

[0017] In a sixth aspect, the present invention provides a medicament comprising the mRNA described in the first aspect of the present invention, the protein described in the second aspect of the present invention, the DNA described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the recombinant cell described in the fifth aspect of the present invention.

[0018] In some embodiments of the present invention, the drug is a vaccine.

[0019] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable adjuvants, carriers, diluents, or excipients.

[0020] In some embodiments of the present invention, the carrier is a liposome.

[0021] In some embodiments of the present invention, the carrier is a cationic liposome, including cationic lipids and auxiliary lipids.

[0022] In some embodiments of the present invention, the cationic lipid includes at least one of DOTAP, DOTMA, DEOPC, DC-Chol, DDAB, DODMA, and DLinDMA.

[0023] In some embodiments of the present invention, the auxiliary lipid is a neutral auxiliary lipid.

[0024] The neutral auxiliary lipids include at least one of DSPC, DOPE, DOPC, DOPG, DOPS and cholesterol.

[0025] In some preferred embodiments of the present invention, the liposomes are: DOTMA:DOPA; the molar ratio is 1:10 to 10:1.

[0026] In some preferred embodiments of the present invention, the liposomes are: DOTMA:DOPA = 2:1.

[0027] In some embodiments of the present invention, the vaccine is prepared by mixing mRNA with liposomes, wherein the charge ratio of mRNA to liposomes is 5:1 to 1:10; and the mass ratio of mRNA to cationic lipids is 27:1 to 1:18.

[0028] In some embodiments of the invention, the drug is provided in a physiologically dosable form and is suitable for oral, intramuscular, intravenous, subcutaneous, or skin injection.

[0029] A seventh aspect of the present invention provides the use of a biomaterial in the preparation of a product, said biomaterial being any one of (a1) to (a6):

[0030] (a1) The mRNA described in the first aspect of the present invention;

[0031] (a2) The protein described in the second aspect of the present invention;

[0032] (a3) The DNA described in the third aspect of the present invention;

[0033] (a4) The expression vector described in the fourth aspect of the present invention;

[0034] (a5) The recombinant cells described in the fifth aspect of the present invention;

[0035] (a6) The medicine described in the sixth aspect of the present invention;

[0036] The product is preferably one of the functions of (b1) to (b6):

[0037] (b1) Prevention or treatment of EBV infection-related diseases;

[0038] (b2) Prevention or treatment of EBV infection-related tumors;

[0039] (b3) Inhibits tumor growth;

[0040] (b4) Increase survival time;

[0041] (b5) Triggers an immune response;

[0042] (b6) Preparation of EB virus antibodies.

[0043] In some embodiments of the present invention, the product is a drug.

[0044] In some embodiments of the present invention, the drug is preferably a vaccine.

[0045] The beneficial effects of this invention are:

[0046] This invention provides a truncated LMP2A mRNA and a related mRNA vaccine prepared based on the truncated mRNA. First, the applicant analyzed and compared the expression intensity of different truncated LMP2A sequences, finding that LMP2A (120-461aa) (truncated A) was strongly expressed and covered more epitopes. In vivo animal experiments showed that truncated A had better immunogenicity than the full-length LMP2A. Furthermore, an mRNA vaccine prepared based on truncated A was found to significantly inhibit tumor development, and compared to the full-length vaccine, it improved survival time, antibody response, and immune response, while also exhibiting higher safety. This indicates that the mRNA vaccine encoding truncated A can provide good protection in mice and can be used to develop therapeutic vaccines for EBV-related tumors. Attached Figure Description

[0047] Figure 1 The distribution of T cell epitopes in LMP2A.

[0048] Figure 2 The expression of different antigen truncated variants in 293T cells.

[0049] Figure 3 PCS particle size data for LMP2A-mRNA vaccine.

[0050] Figure 4 The zeta electronystrophic value of the LMP2A-mRNA vaccine.

[0051] Figure 5 A schematic diagram of the mRNA vaccine backbone sequence design.

[0052] Figure 6 To compare the efficacy of full-length mRNA vaccines encoding truncated variant A and LMP2A in eliciting cellular immune responses in mice using an enzyme-linked immunospot (ELISPOT) assay.

[0053] Figure 7 This is a schematic diagram of an enzyme-linked immunospot test (ELISPOT).

[0054] Figure 8 A schematic diagram of the mouse tumor formation experiment design.

[0055] Figure 9 A diagram showing the detection of tumor cell lines expressing LMP2A.

[0056] Figure 10 Statistics on tumor development in mice injected with LMP2A-mRNA vaccine.

[0057] Figure 11 To detect tumor development in mice injected with the LMP2A-mRNA vaccine.

[0058] Figure 12 Survival time statistics for mice injected with LMP2A-mRNA vaccine.

[0059] Figure 13 The OD values ​​of serum gradient dilution antibody responses in mice injected with the LMP2A-mRNA vaccine.

[0060] Figure 14 The antibody response OD value of a mouse serum diluted tenfold after being injected with the LMP2A-mRNA vaccine.

[0061] Figure 15 The image shows the ELISPOT assay results of T cell responses in mice injected with the LMP2A-mRNA vaccine.

[0062] Figure 16 Changes in body weight in mice injected with the LMP2A-mRNA vaccine. Detailed Implementation

[0063] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0064] Example 1: Selection and Expression of Antigen Truncated Vertebrates

[0065] Based on the LMP2A sequence (GenBank: AGZ95187.1) of the prevalent EBV M81 strain in South China, this embodiment designs a coding region containing an LMP2A antigen-enriched fragment.

[0066] Because in LMP2A, T cell epitopes are thought to be more enriched primarily in the C-terminus ( Figure 1 Therefore, the applicant analyzed and compared the expression intensity of different truncated sequences and selected the fragment with stronger expression and richer T-cell epitopes for the next immunological control experiment.

[0067] A: LMP2A (120-461aa), RNA sequence as shown in SEQ ID NO.1; amino acid sequence as shown in SEQ ID NO.2; B: LMP2A (223-461aa), RNA sequence as shown in SEQ ID NO.3; amino acid sequence as shown in SEQ ID NO.4; C: LMP2A (293-434aa), RNA sequence as shown in SEQ ID NO.5; amino acid sequence as shown in SEQ ID NO.6; all C-termini are tagged with a Flag for detection (RNA sequence as shown in SEQ ID NO.7; amino acid sequence as shown in SEQ ID NO.8).

[0068] in:

[0069] SEQ ID NO.1: AUGAAUCCAGUAUGCCUGCCUGUAAUUGUUGCGCCCUACCUGUU。

[0070] SEQ ID NO.2:MNPVCLPVIVAPYLFWLAAIAASCFTASVSTVVSATGLALSLLLLAAVANSSAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSILFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGMMFLACLVVLIVDAVLQLSPLLGAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKVLLARLFLYALALLLLASALTAGGSILQTNFKSLSSTEFIPHLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMSLGGLLTMVAGAVWLTVMTNTLLSAWILTAGFLIFL。

[0071] SEQ ID NO.3:AUUUACGUUCUGGUGAUGCUUGUGCUCCUGAUACUAGCAUACAGAAGGAGAUGGCGCCGUUUGACUGUUUGUGGCGGCAUGAUGUUUUUGGCAUGUCUAGUUGUCCUCAUCGUCGACGCUGUUUUGCAGCUGAGUCCCCUCCUUGGAGCUGUAACUGUGGUUUCCAUGACGCUGCUGCUACUGGCUUUCGUCCUCUGGCUCUCUUCGCCAGGGGGCCUAGGUACUCUUGGUGCAGCCCUUUUAACAUUGGCAGCAGCUCUGGCACUGCUAGCGUCACUGAUUUUGGGCACACUUAACUUGACUACAAUGUUCCUUCUCAUGCUCCUAUGGACACUUGUGGUUCUCCUGAUUUGCUCUUCGUGCUCUUCAUGUCCACUGAGCAAGGUUCUUCUGGCACGACUGUUCCUAUAUGCUCUAGCACUCUUGUUGCUAGCCUCCGCGCUAACCGCCGGUGGCAGUAUUUUGCAAACAAACUUCAAGAGUUUAAGUAGCACUGAAUUUAUACCUCAUUUGUUCUGCAUGUUAUUACUGAUUGUCGCUGGCAUACUCUUUAUUCUUGCUAUCCUGACCGAAUGGGGAAGUGGAAAUAGAACAUACGGUCCCGUUUUUAUGUCCCUCGGCGGCCUGCUCACCAUGGUAGCCGGCGCUGUGUGGCUGACGGUGAUGACUAACACGCUUUUGUCUGCCUGGAUUCUUACAGCAGGAUUCCUGAUUUUCCUC。

[0072] SEQ ID NO.4:IYVLVMLVLLILAYRRRWRRLTVCGGMMFLACLVVLIVDAVLQLSPLL GAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKVLLARLFLYALALLLLASALTAGGSILQTNFKSLSSTEFIPHLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMSLGGLLTMVAGAVWLTVMTNTLLSAWILTAGFLIFL。

[0073] SEQ ID NO.5:

[0074] GGCCUAGGUACUCUUGGUGCAGCCCUUUUAACAUUGGCAGCAGCUCUGGCACUGCUAGCGUCACUGAUUUUGGGCACACUUAACUUGACUACAAUGUUCCUUCUCAUGCUCCUAUGGACACUUGUGGUUCUCCUGAUUUGCUCUUCGUGCUCUUCAUGUCCACUGAGCAAGGUUCUUCUGGCACGACUGUUCCUAUAUGCUCUAGCACUCUUGUUGCUAGCCUCCGCGCUAACCGCCGGUGGCAGUAUUUUGCAAACAAACUUCAAGAGUUUAAGUAGCACUGAAUUUAUACCUCAUUUGUUCUGCAUGUUAUUACUGAUUGUCGCUGGCAUACUCUUUAUUCUUGCUAUCCUGACCGAAUGGGGAAGUGGAAAUAGAACAUACGGUCCCGUUUUUAUGUCCCUCGGCGGCCUGCUCACCAUGGUA。

[0075] SEQ ID NO.6:GLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICS SCSSCPLSKVLLARLFLYALALLLLASALTAGGSILQTNFKSLSSTEFIPHLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMSLGGLLTMV。

[0076] SEQ ID NO.7:GACUACAAGGACGAUGAUGACAAG。

[0077] SEQ ID NO.8: DYKDDDDK.

[0078] The target antigen generation detection method (Western-Blot) is as follows:

[0079] 1) Digest 293T cells cultured for more than 24 hours and seed them into 12-well plates, controlling the cell density at 150,000 cells per well;

[0080] 2) Incubate the six-well plate at 37°C and 5% CO2 for 16-20 hours, then observe the cell status under a microscope. Once the cell confluence reaches 60% or more, transfection with the pcaggs plasmid encoding the antigen can be performed.

[0081] 3) Transfect the corresponding plasmid into 293 cells using PET (1 μg of plasmid per well, PEI: plasmid mass ratio of 3:1), and continue to culture at 37℃ and 5% CO2 for 24 hours.

[0082] 4) After the plasmid has been continuously expressed for 24 hours, remove the cell supernatant, add 100 μl / well of RIPA cell lysis buffer, react on ice for 10 min, and then transfer to a 1.5 ml EP tube.

[0083] 5) Add 25 μl of 5X SDS loading buffer (Genstar), mix well, denature at 95 degrees Celsius for 5 min, and then prepare for SDS-PAGE electrophoresis.

[0084] 6) After the protein samples have cooled to room temperature, load them into the 10.5% SDS-PAGE wells, with a loading volume of 35 μl per well. Perform electrophoresis at 80 V for 120 min, followed by membrane transfer.

[0085] 7) First, activate the PVDF membrane (0.22μm, 5.5*8.5cm) with anhydrous methanol (1min), then immerse it in the transfer solution. Use the Bio-Rad standard wet transfer apparatus with a transfer current of 250mA and a transfer time of 120min.

[0086] 8) After the transfer is complete, immediately place the protein membrane into a Western immersion container pre-filled with PBST (0.1% Tween 20), rinse for 1-2 minutes, and discard the washing buffer. Add an appropriate amount of 5% skim milk (dissolved in PBST), shake slowly on a shaker, and block at room temperature for 45 minutes.

[0087] 9) Primary antibody incubation: Add primary antibody (anti-Flag, Sigma, or anti-beta-actin, CST) diluted 1:1000 with 5% BSA, and incubate overnight at 4°C with gentle shaking. After removing the primary antibody, add an appropriate amount of PBST (0.1% Tween 20) and wash on a side-shaking rack for 10 minutes each time; wash 3 times.

[0088] 10) Secondary antibody incubation: Add secondary antibody (anti-mouse, CST, or anti-rabbit, CST) diluted 1:3000 with 5% BSA, and incubate overnight at 4°C with gentle shaking. After removing the primary antibody, add an appropriate amount of PBST (0.1% Tween 20) and wash on a side-shaking rack for 10 minutes each time; wash 3 times.

[0089] 11) Protein detection: Mix equal volumes of chromogenic solutions A and B, immerse the PVDF membrane in the chromogenic solution, and after 1 minute, place it in a chemiluminescence analyzer (Biorad) for imaging; results are shown below. Figure 2 The truncated variant A (LMP2A 120-461aa, 36kDa) showed good in vitro expression efficacy and contained a large number of T cell epitopes. The truncated variant C (LMP2A 293-434aa, 15kDa) also showed good in vitro expression efficacy, but contained fewer T cell epitopes. Therefore, truncated variant A was used as the candidate vaccine in subsequent animal experiments.

[0090] Example 2: Detection of Cellular Immunological Response

[0091] To compare whether the truncated LMP2A (120-461aa), which showed the best in vitro expression effect, could elicit a more effective cellular immune response in mice, the applicant prepared mRNA encoding a 5'-UTR element, a truncated LMP2A (120-461aa), a 3-UTR element, and a polyA tail element. The DNA sequence is shown in SEQ ID NO. 9, the RNA sequence in SEQ ID NO. 10, and the amino acid sequence in SEQ ID NO. 11. A mixture of mRNA and liposomes was also prepared, along with mRNA encoding a 5'-UTR element, full-length LMP2A, a 3-UTR element, and a polyA tail element (DNA sequence in SEQ ID NO. 12, RNA sequence in SEQ ID NO. 13, and amino acid sequence in SEQ ID NO. 14). The PCS particle size data of the mRNA-liposome mixture are shown in [link to mRNA-liposome mixture]. Figure 3 It can be seen that the particle size before packaging is approximately 180 nm; the particle size of mRNA encoding full-length or truncated LMP2A after packaging is approximately 350 nm (n=3); the zeta potential of the mRNA-liposome mixture is shown in [reference needed]. Figure 4 .

[0092] in:

[0093]

[0094]

[0095] SEQ ID NO.11:MNPVCLPVIVAPYLFWLAAIAASCFTASVSTVVSATGLALSLLLLAAVANSSAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSILFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGMMFLACLVVLIVDAVLQLSPLLGAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKVLLARLFLYALALLLLASALTAGGSILQTNFKSLSSTEFIPHLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMSLGGLLTMVAGAVWLTVMTNTLLSAWILTAGFLIFL。

[0096]

[0097]

[0098] SEQ ID NO.14: MGSLEMVPMGAGPPSPGGDPDGDDGGNNSQYPSASGSSGNTPTPPN DEERESNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAASCFTASVSTVVSATGLAL SLLLLAAVANSSAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSILFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGMMFLACLVVLIVDAVLQLSPLLGAV TVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKVLLARLFLYALALLLLASALTAGGSILQTNFKSLSSTEFIPHLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMSLGGLLTMVAGAVWLTVMTNTLLSAWILTAGFLIFLIGFALFGVIRCYYCLTLESEERPPTPYRNTV.

[0099] The RNA-liposome complex was prepared as follows: mRNA was diluted to 200 μl with PBS, and liposome suspension was added. The mixture was then stirred and allowed to stand for 10 minutes. The RNA-liposome complex formed with an RNA to liposome charge ratio of 2:1.3 (17.6 μl of liposome suspension was added for every 40 μg mRNA solution) had a particle zeta potential of approximately -30 mV (negatively charged) and a size of approximately 300-400 nm.

[0100] The liposome preparation method is as follows: DOTMA and DOPE are dissolved in anhydrous ethanol at a molar ratio of 2:1 (100mg DOTMA and 40.92mg DOPE are dissolved in 680μl of anhydrous ethanol). The ethanol solution is then slowly added dropwise to 34ml of distilled water. The mixture is stirred continuously at 200rpm for 1 hour and then filtered through a 0.45μm filter membrane and stored at 4 degrees Celsius.

[0101] A schematic diagram of the mRNA vaccine backbone sequence design can be found in [link / reference]. Figure 5 .

[0102] C57 mice were immunized via tail vein injection with LMP2A (120-461aa) and the full-length LMP2A mRNA vaccine (20 μg each time, administered 4 times on days 3, 6, 10, and 15, n=7). Spleens were harvested 7 days after the last injection to compare the immune response.

[0103] The ELISPOT assay method is as follows:

[0104] 1) Seven days after the last immunization, the spleens of mice in the experimental group and the control group were taken, ground and the cell suspension was collected. After centrifugation, the suspension was washed once with 1640 (Sigma) medium.

[0105] 2) Lysis of red blood cells: Spleen cell samples from each mouse were lysed with 2 ml of erythrocyte lysis buffer (BioSharp) for 2 minutes, then the reaction was terminated with 10 ml of PBS, centrifuged, and resuspended in an appropriate amount of 1640 containing 10% FBS before cell counting.

[0106] 3) Add sterile DPBS (Sigma) to a pre-coated IFN-r ELISPOT plate (Mabtech), 200 μl / well, blot clean, repeat 4 times. Then add 200 μl of 1640 containing 10% FBS to each well and block at room temperature for 30 minutes, blot clean.

[0107] 4) Add 2*10 to each hole 5 Spleen cells were cultured at 37°C and 5% CO2 for 16-20 hours before spot detection. The LMP2A peptide group was treated with LMP2A peptide (final concentration 10 μg / ml), the blank control group was treated with 1640 containing 2% FBS, and the positive control group was treated with PMA (final concentration 50 ng / ml) and Ionomycin (final concentration 1 μg / ml).

[0108] 5) Discard the cell contents and perform spot analysis according to the instructions provided by the Mabtech kit manufacturer.

[0109] The results are as follows Figure 6 and Figure 7 As can be seen from the above, the immune effect of LMP2A (120-461aa) is significantly better than that of the full-length LMP2A.

[0110] Example 3: Detection of Tumor Formation Effect in Mice

[0111] To investigate whether the mRNA vaccine encoding LMP2A (120-461aa) (referred to as 2A-RNP) has a therapeutic / alleviating effect on tumorigenesis, the applicant established a stable monoclonal cell line of B16-luc expressing the full length of LMP2A, and then injected 2*10 cells via tail vein. 5A tumor-bearing mouse model was established using tumor cells. A schematic diagram of the mouse tumorigenesis experimental design is shown below. Figure 8 The expression of LMP2A protein in the cell line was detected, and the results are shown in [the table below]. Figure 9 It can be seen that the plant successfully established a stable strain.

[0112] Then, on days 3, 6, 10, and 15, mice were injected with either 2A-RNP or a blank liposome control (n=14), and tumor growth was observed. It can be seen that tumor development in mice injected with LMP2A-RNP was significantly inhibited compared to the control group. Figure 10 & Figure 11 Furthermore, the survival time of mice injected with LMP2A-RNP ( Figure 12 ), serum antibody titer against LMP2A ( Figure 13 & Figure 14 ) and T-cell immune response ( Figure 15 The results were significantly better than those of the control group.

[0113] The antibody titer against LMP2A in serum was detected by an ELISA assay, and the detection steps are as follows:

[0114] 1) The immunosorbent assay plate was coated overnight at 4°C with LMP2A peptide (100 ng / well, 100 μL PBS).

[0115] 2) On the second day, the plate was blocked with 3% bovine serum albumin (BSA) diluted with 0.1% PBST (PBS containing 0.1% Tween-20) at 37°C for 1 hour and washed 3 times.

[0116] 3) Serially dilute mouse plasma samples in 3% BSA and incubate at 37°C for 1 hour.

[0117] 4) Wash the plate 5 times with 0.1% PBST and incubate it with goat anti-mouse IgG-HRP (ab6789) (1:10000 diluted in blocking buffer) at 37°C for 30 minutes.

[0118] 5) After washing the plate 5 times with 0.1% PBST, add 100 μL of 3,3',5,5'-tetramethylbenzidine substrate (TMB) (Tiangen Cat#PA107-02) to each well and store in the dark. After 5 minutes, terminate the reaction with 1M hydrochloric acid (HCl) and measure the OD450 using a BioTek Epoch microplate.

[0119] T-cell immune responses were detected by ELISPOT, using the same method as before.

[0120] Furthermore, the body weight of mice injected with LMP2A-RNP was not significantly lower than that of mice injected with blank liposomes. However, due to tumor growth leading to a decline in the mice's condition and body weight, the body weight of mice in the blank control group was lower than that of the LMP2A-RNP experimental group in the later stages. Figure 16 This indicates that LMP2A-RNP has a high level of security.

[0121] In summary, it can be seen that the mRNA vaccine prepared by LMP2A (120-461aa) has good tumor suppression and immune effects.

[0122] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. An mRNA, characterized in that, The nucleotide sequence of the mRNA is shown in SEQ ID NO. 1 or SEQ ID NO.

10.

2. A protein, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A DNA molecule encoding the protein of claim 2.

4. An expression vector comprising the DNA molecule of claim 3.

5. A recombinant cell comprising the expression vector of claim 4.

6. A medicament comprising the mRNA of claim 1, the protein of claim 2, the DNA of claim 3, the expression vector of claim 4, or the recombinant cell of claim 5.

7. The medicament according to claim 6, characterized in that, The medicament is a vaccine.

8. The medicament according to claim 6, characterized in that, The medicament further comprises a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient.

9. The medicament according to claim 8, characterized in that, The carrier is a liposome.

10. The medicament according to claim 6, characterized in that, The medicament is provided in a physiologically administrable form and is suitable for oral, intramuscular, intravenous, or subcutaneous injection.

11. Use of a biological material in the manufacture of a product, the biological material being any one of (a1) to (a6); (a1) the mRNA of claim 1; (a2) the protein of claim 2; (a3) the DNA of claim 3; (a4) the expression vector of claim 4; (a5) the recombinant cell of claim 5; (a6) the medicament of any one of claims 6 to 10; The product functions as any one of (b1) to (b2): (b1) prevention or treatment of a tumor; (b2) preparation of an EB virus antibody; The tumor is a melanoma.

12. Use according to claim 11, characterized in that, The product is a medicament.

13. Use according to claim 12, characterized in that, The medicament is a vaccine.

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