Construction method of multi-target mRNA (messenger Ribonucleic Acid) vaccine aiming at I-type and II-type dengue viruses

Abstract

The invention discloses a construction method of a multi-target mRNA vaccine aiming at type I and type II dengue viruses. The construction method comprises the following steps: S1, carrying out in-vitro transcription on linearized DNA: carrying out in-vitro transcription by using an all In One efficient mRNA synthesis kit; s2, carrying out mRNA purification, namely at the room temperature, carrying out purification by using an MEGA clear Transcription Clean-Up Kit kit, and carrying out centrifugation; s3, carrying out mRNA content determination and identification: S31, carrying out mRNA content determination: detecting the product concentration by using an ultramicro spectrophotometer and recording the product concentration; s32, carrying out agarose electrophoresis identification on the mRNA; and S33, judging whether the purified product meets the qualified standard or not. The mRNA vaccine obtained by the construction method disclosed by the invention is good in safety, strong in effectiveness and high in production efficiency, and has the potential of rapidness, low cost and extensible manufacturing.

Description

Technical Field

[0001] The present invention relates to the field of mRNA vaccine construction, and in particular to a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses. Background Art

[0002] Dengue virus belongs to the Flaviviridae family and the Flavivirus genus. It is a single-stranded positive-strand RNA virus, mainly transmitted by Aedes albopictus and Aedes aegypti, and mainly causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue virus is divided into different serotypes (DENV-Ⅰ, -Ⅱ, -Ⅲ and -Ⅳ) based on antigenicity. The virus has only one open reading frame (ORF), which is translated into a multimeric protein, which is cleaved into three structural proteins and seven non-structural proteins under the catalysis of host proteases and viral proteases, and plays their respective roles.

[0003] The sequence of the encoded protein is 5'-C-PrM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5-3'. Among them, the E protein is an envelope protein, which is mainly composed of three different domains (EDⅠ, EDⅡ and EDⅢ). Most of the neutralizing antibodies produced by the ED-Ⅰ and ED-Ⅱ regions have no neutralizing activity and have extensive cross-reactions with other flaviviruses. The ED-Ⅲ region is located at the C-terminus of the fusion E protein. This region is relatively conservative in structure and has an immunoglobulin-like β-barrel structure. It plays an important role in the recognition and fusion process between the virus and the host cell. It is also a key site for virus-induced neutralizing antibodies. Therefore, it is the main target for DENV vaccine and therapeutic antibody research.

[0004] Currently, vaccine development faces many difficulties due to the high heritability, variability, and antibody-dependent enhancement of infection after infection with DENV. In theory, vaccination is the most effective strategy to prevent dengue infection. An ideal dengue vaccine must have balanced protection against all four serotypes, sustained efficacy, and reliable safety. Currently, only The (TAK003) vaccine has been approved for clinical trials in some countries, but there is no dengue vaccine approved for use in my country.

[0005] The present invention designs a multi-target mRNA vaccine. In order to drive the target protein to the cell secretion pathway so that it can be quickly taken up and expressed in the cytoplasm, an mRNA modified with N1-methyl pseudouridine is designed, which encodes type 1 Cap (m7GPPPNm), tissue plasminogen activator (tPA) and 5' and 3' untranslated sequences and optimized target antigens (DENV-Ⅰ-EDⅢ+DENV-Ⅱ-EDⅢ), wherein tPA is used as an exogenous leader sequence and then encapsulated with lipid nanoparticles (LNP). After immunization, we evaluated the level of specific neutralizing antibodies and T cell immune responses induced by mRNA candidate vaccines. The results showed that this multi-target vaccine induced a strong antiviral immune response and produced balanced neutralizing antibodies against DENV-Ⅰ and DENV-Ⅱ. Subsequently, the protective effect of the vaccine was evaluated in gene-deficient mice to obtain the key parameters of the safety and effectiveness of the vaccine. The results showed that the gene-deficient mice vaccinated with the mRNA candidate vaccine were protected from the attack of lethal DENV. The research results of the present invention also emphasize that chimeric heterologous structural proteins can serve as effective targets for DENV vaccines and lay the foundation for further development of quadrivalent DENV vaccines based on mRNA technology. Summary of the invention

[0006] In view of this, the present invention provides a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A method for constructing a multi-target mRNA vaccine against dengue virus type I and type II, comprising the following steps:

[0009] S1: Linearize DNA for in vitro transcription:

[0010] In vitro transcription was performed using the All In One High-Efficiency mRNA Synthesis Kit;

[0011] S2: Perform mRNA purification:

[0012] Purify using MEGA clear Transcription Clean-Up Kit and centrifuge at room temperature;

[0013] S3: mRNA content determination and identification:

[0014] S31: Determine the mRNA content: Use an ultra-micro spectrophotometer to detect and record the product concentration;

[0015] S32: mRNA agarose electrophoresis identification;

[0016] S33: Determine whether the purified product meets the qualified standards.

[0017] Preferably, the step S1 specifically includes the following steps:

[0018] S11: Preparation of reaction system: Add and prepare the IVT reaction system in the following order;

[0019]

[0020] S12: After mixing according to the above reaction system, centrifuge it instantly and place it in a PCR instrument for reaction at 37°C for 4 hours;

[0021] S13: Add 2 μL DNaseI to every 20 μL reaction system and react at 37°C for 15 min.

[0022] Preferably, the step S2 specifically includes the following steps:

[0023] S21: Fill the S1 reaction sample to 100 μL with Elution Buffer and mix gently with a pipette tip;

[0024] S22: Add 350 μL Binding Buffer and mix gently;

[0025] S23: Add 250 μL 100% ethanol and mix gently;

[0026] S24: Gently add the sample of S23 to the filter column, centrifuge at 14000g for 1 min, and discard the filtrate;

[0027] S25: Add 550 μL Washing Buffer, wash twice, centrifuge at 14000 g for 1 min each time, and discard the filtrate;

[0028] S26: centrifuge the empty RNA microadsorption column at 14000g for 2 min at room temperature;

[0029] S27: Add 50-100 μL RNase Free Water, let stand for 10 min, and centrifuge at 14000 g for 2 min;

[0030] S28: Transfer the dissolved mRNA to a new 1.5 mL centrifuge tube, mix thoroughly and mark it, take out 0.3 mL for content determination and identification, and store the rest at -20℃ for subsequent experiments.

[0031] Preferably, the step S32 specifically includes the following steps:

[0032] S321: Prepare electrophoresis buffer: Dissolve 50×TAE Buffer into 1×TAE Buffer using ultrapure water;

[0033] S322: Prepare the sample to be tested: take 100-300 ng of mRNA, add the same volume of Loading Buffer, and react at 70℃ for 5 min in a PCR instrument;

[0034] S323: mRNA was subjected to 1% agarose gel electrophoresis and then imaged, and markers were used to confirm whether the sample band size was correct.

[0035] Preferably, in step S33, Nanodrop detection is performed, and when the ratio of 260 / 280 is between 1.8-2.3 and 260 / 230≧2.0, the quality is qualified.

[0036] Preferably, in S323, the size of the agarose gel electrophoresis band is consistent with the size of the target band, and the band is clear without obvious diffusion.

[0037] Compared with the prior art, the present invention has achieved the following technical effects:

[0038] 1. The present invention is safe: there is no risk of "reversion to virulence" of attenuated live vaccines or induction of severe disease in some individuals. In addition, mRNA will be degraded by normal cellular processes, and its half-life in vivo can be adjusted by using various modifications and delivery methods;

[0039] 2. The invention is highly effective: various modifications make the mRNA more stable and efficient in translation, while avoiding anti-vector immune responses;

[0040] 3. The present invention has high production efficiency. Due to the high yield of its in vitro transcription reaction, mRNA has the potential for rapid, low-cost and scalable manufacturing. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The present invention is an mRNA agarose electrophoresis diagram of a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses.

[0042] Figure 2 This is a graph showing the mRNA expression results of a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0043] Figure 3 This is a transmission electron microscopy observation diagram of mRNA-LNP of a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0044] Figure 4This is a diagram of the physicochemical properties of mRNA-LNP in a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0045] Figure 5 This is a specific antibody detection diagram of a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0046] Figure 6 This is a neutralizing antibody detection diagram of a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0047] Figure 7 This is a T lymphocyte subset detection diagram of a method for constructing a multi-target mRNA vaccine against type Ⅰ and type Ⅱ dengue viruses of the present invention;

[0048] Figure 8 This is a diagram of intracellular cytokine detection for a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0049] Fig. 9 The weight change of mice after virus attack according to the method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0050] Fig.10 This is a viremia detection diagram of a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention;

[0051] Fig.11 This is a brain tissue viral load detection diagram of a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses of the present invention. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The present invention discloses a method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses, comprising the following steps:

[0054] S1: Linearize DNA for in vitro transcription:

[0055] In vitro transcription was performed using the All In One High-Efficiency mRNA Synthesis Kit;

[0056] S11: Preparation of reaction system: Add and prepare the IVT reaction system in the following order;

[0057]

[0058] S12: After mixing according to the above reaction system, centrifuge it instantly and place it in a PCR instrument for reaction at 37°C for 4 hours;

[0059] S13: Add 2 μL DNaseI to each 20 μL reaction system and react at 37°C for 15 min;

[0060] S2: Perform mRNA purification:

[0061] Purify using MEGA clear Transcription Clean-Up Kit and centrifuge at room temperature;

[0062] The step S2 specifically includes the following steps:

[0063] S21: Fill the S1 reaction sample to 100 μL with Elution Buffer and mix gently with a pipette tip;

[0064] S22: Add 350 μL Binding Buffer and mix gently;

[0065] S23: Add 250 μL 100% ethanol and mix gently;

[0066] S24: Gently add the sample of S23 to the filter column, centrifuge at 14000g for 1 min, and discard the filtrate;

[0067] S25: Add 550 μL Washing Buffer, wash twice, centrifuge at 14000 g for 1 min each time, and discard the filtrate;

[0068] S26: centrifuge the empty RNA microadsorption column at 14000g for 2 min at room temperature;

[0069] S27: Add 50-100 μL RNase Free Water, let stand for 10 min, and centrifuge at 14000 g for 2 min;

[0070] S28: Transfer the dissolved mRNA to a new 1.5 mL centrifuge tube, mix thoroughly and mark it, take out 0.3 mL for content determination and identification, and store the rest at -20°C for subsequent experiments;

[0071] S3: mRNA content determination and identification:

[0072] S31: Determine the mRNA content: Use an ultra-micro spectrophotometer to detect and record the product concentration;

[0073] S32: mRNA agarose electrophoresis identification:

[0074] Preferably, the step S32 specifically includes the following steps:

[0075] S321: Prepare electrophoresis buffer: Dissolve 50×TAE Buffer into 1×TAE Buffer using ultrapure water;

[0076] S322: Prepare the sample to be tested: take 100-300 ng of mRNA, add the same volume of Loading Buffer, and react at 70℃ for 5 min in a PCR instrument;

[0077] S323: mRNA was subjected to 1% agarose gel electrophoresis and imaged, and markers were used to confirm whether the sample band size was correct;

[0078] Among them, the size of the agarose gel electrophoresis band is consistent with the size of the target band, and the band is clear without obvious diffusion;

[0079] S33: Determine whether the purified product meets the qualified standards:

[0080] Nanodrop test was performed, and when the ratio of 260 / 280 was between 1.8-2.3 and 260 / 230≧2.0, the quality was qualified.

[0081] The sequence of the mRNA vaccine is:

[0082] .

[0083] Perform immunoblotting

[0084] The synthesized mRNA was stained with Lipofectamine TM MessengerMAX TM Transfection reagent was transfected into 293T cells. After 24 hours, cells were treated with RIPA lysis buffer and centrifuged at 14000 rpm for 10 minutes. The supernatant was collected and SDS-PAGE protein loading buffer (5×) was added. After mixing, the supernatant was boiled in boiling water for 8 minutes. SDS-PAGE gel electrophoresis was performed as follows:

[0085] 1) Electrophoresis: 120V 1h 30min;

[0086] 2) Transfer: Transfer the protein to a 0.45 μm nitrocellulose membrane at 15 V for 30 min.

[0087] 3) Blocking: Incubate in an incubator containing 5% skim milk for 2 h at room temperature;

[0088] 4) Washing: Wash 3 times with TBST;

[0089] 5) Primary antibody incubation: rabbit His-Tag antibody, incubate overnight at 4°C;

[0090] 6) Washing: repeat step 4);

[0091] 7) Secondary antibody incubation: HRP goat anti-rabbit IgG antibody, incubate at room temperature for 45 minutes;

[0092] 8) Washing: same as step 6);

[0093] 9) Development: Add ECL developer solution onto the membrane and place it into the instrument for imaging.

[0094] mRNA-LNP packaging:

[0095] The aqueous phase containing mRNA and the ethanol phase containing the lipid mixture are mixed by a T-type mixing device to prepare lipid nanoparticles (mRNA-LNP) loaded with mRNA. The specific steps are as follows:

[0096] ① Rinse: Select 5mL "HX" model syringes for both the aqueous phase and the ethanol phase, fill the syringes respectively, and drive out the bubbles in the syringes. After inserting them tightly into the chip inlet, call up the "wash" program, click Start, and complete a wash after receiving the corresponding waste liquid volume. Repeat the above process 3 times to complete the instrument cleaning work;

[0097] ② Liposome synthesis: mRNA was dissolved in citrate buffer (100 mM, pH 4.0), and the lipid mixture was dissolved in anhydrous ethanol. Then, ethanol and water were mixed in a T-junction device at a volume ratio of 3:1;

[0098] ③ Dialysis: Quickly put the product into a dialysis bag and dialyze the mRNA-LNP with 50 mL of 20 mM Tris-HCl solution (pH 7.2) for at least 6 hours. Then, sterilize the mRNA-LNP with a 0.22 μm filter membrane. Store the mRNA-LNP at 4°C for later use.

[0099] Transmission electron microscopy observation:

[0100] The sample was dropped onto a copper grid and observed under a transmission electron microscope.

[0101] Determination of mRNA-LNP particle size, PDI, potential and encapsulation efficiency:

[0102] The average diameter, polydispersity (PDI) and zeta potential of the prepared mRNA-LNPs were measured using NS-90Z (Malvern Panalytical).

[0103] Encapsulation efficiency: The concentration of unencapsulated mRNA was first determined using a fluorescence detection kit. Then, after the mRNA-LNP was cleaved with 0.4% Triton X-100, the total mRNA concentration was determined using a fluorescence detection kit. The encapsulation efficiency (EE) was calculated using the following equation:

[0104]

[0105] Mouse immunization grouping and plan:

[0106] SPF-grade AGB6 mice aged 5-6 weeks were selected as immunized subjects, and mRNA-LNP was immunized by intramuscular injection. The immunization procedure was to immunize the mice every two weeks for a total of 3 immunizations.

[0107] The challenge protection test was performed on the 42nd day after immunization. The mice were challenged with DENV-I and DENV-II by intraperitoneal injection. The mice were weighed and their mental state was tested for 14 consecutive days. The blood of the mice was collected on the 3rd day, and the brain tissue of the mice was collected on the 14th day, as shown in Table 1.

[0108] Table 1: Grouping of mouse immunization test for dengue virus mRNA candidate vaccine

[0109]

[0110] Specific antibody detection:

[0111] Enzyme-linked immunosorbent assay (ELISA) was used to detect specific antibodies.

[0112] The specific operation method is as follows: a certain dose of DI-EDⅢ+DI-EDⅢ, DI-EDⅢ and DI-EDⅢ proteins were coated onto the ELISA plate, 1% BSA was added to each well after coating at 4°C overnight, and the plates were blocked at 37°C for 2h; the plates were washed with PBST and the serum to be tested (2-fold dilution) was added, and the plates were incubated at 37°C for 2h; the plates were washed with PBST for 3 times, HRP secondary antibody was added, and the plates were incubated at 37°C for 45min; the plates were washed with PBST for 5 times, 100μL TMB colorimetric solution was added to each well, and the plates were incubated at room temperature for 15min; after adding the stop solution, the plates were immediately placed in an ELISA reader for reading.

[0113] Neutralizing antibody detection:

[0114] 1) Inactivate the serum in a 56°C water bath for 30 min and store at 4°C for later use;

[0115] 2) Serum dilution: DMEM medium was used for 2-fold dilution (initial dilution factor was 1:8), with a total of 9 dilution gradients;

[0116] 3) Virus solution preparation: The virus dose to be added to each well is 100 TCID 50 ;

[0117] 4) Take 60 μL of the serum dilution in step 2) and an equal volume of the virus solution in step 3) and mix them thoroughly in a 96-well cell plate, incubate at 37° C., 5% CO2 for 1 h, and set up negative serum control, DENV virus control and cell control wells at the same time;

[0118] 5) Prepare cells and observe results: Adjust the Vero cell concentration to 1×10 6 / mL, then add the virus-serum mixture in step 4), culture in an incubator for 5 days, observe and record the cytopathic effect every day.

[0119] T lymphocyte subset flow cytometry detection:

[0120] 1) After isolating the spleen lymphocytes of mice and counting the cells, 1×10 6 Cells were fluorescently stained;

[0121] 2) Fluorescent antibody staining: According to the antibody instructions, 1×10 6 PE / Cy7 anti-mouse CD3ε (2.5μL), FITC anti-mouse CD4 Antibody (0.5μL), and APC anti-mouse CD8aAntibody (1.25μL) were added to each lymphocyte, and a blank cell group, CD3ε, CD4, and CD8a single staining groups were set up at the same time, and the cells were stained at 4°C in the dark for 45 minutes.

[0122] 3) Washing: Wash twice with cell staining buffer and centrifuge at 2500 rpm for 5 min;

[0123] 4) Detection on the flow cytometer: Add 300 μL cell staining buffer to each well and resuspend, then transfer to a flow cytometer to detect CD3 in 10,000 cells. + CD4 + , CD3 + CD8 + The number of positive cells.

[0124] Detection of intracellular cytokines IL-4, IFN-γ and TNF-α:

[0125] 1) Absorb 3×10 6 / mL of spleen lymphocytes were transferred to a 12-well plate, 1mL / well, and a control group was set up at the same time;

[0126] 2) Add 10 μg / mL protein to each well, add the same stimulus to the control group, and place in a 37°C 5% CO2 incubator for 1 hour;

[0127] 3) Add protease transport inhibitor, 1 μL / well, and culture in the incubator for 16 h;

[0128] 4) Staining: Collect cells and wash, add 6 μL TruStain FcX to each tube TM (anti-mouse CD16 / 32)Antibody, 4℃, dark for 5 min; each 1×10 6 Each spleen lymphocyte was added with 2.5 μL of CD3ε, 0.5 μL of CD4, and 1.25 μL of CD8, respectively, and stained at 4°C in the dark for 40 min;

[0129] 5) Cell fixation and permeabilization: After washing the cells twice, add 300 μL of Fixation and Permeabilization Solution to each tube and incubate at 4°C in the dark for 20 min.

[0130] 6) Washing: After washing with 1× BD buffer, the cells were divided into 3 tubes, with 1×10 cells in each tube. 6 indivual;

[0131] 7) Incubation with IFN-γ, IL-4 and TNF-α antibodies: Add 1.25 μL of PE-IFN-γ, PE-IL-4 and PE-TNF-α antibodies to each tube of cells and stain for 40 min at 4°C in the dark;

[0132] 8) Detection after washing: After washing the cells, use a flow cytometer to detect the number of positive cells.

[0133] Fluorescence quantitative PCR detection

[0134] The viral load of RNA extracted from blood and brain tissue was detected by probe method. The primers and probes are shown in Table 2, the reaction system is shown in Table 3, and the Bio-Rad fluorescent quantitative PCR instrument was used with the following program: reverse transcription at 55°C for 15 min; pre-denaturation at 95°C for 30 sec; 95°C for 10 sec, 60°C for 40 sec for 45 cycles.

[0135] Table 2: Primers and probes for dengue virus detection

[0136]

[0137] Table 3: Fluorescence quantitative PCR reaction system

[0138]

[0139] result:

[0140] 1) mRNA quality detection

[0141] Depend on Figure 1 It can be seen that the size of the agarose gel electrophoresis band is 990bp, which is consistent with the size of the target band. The band is clear without obvious diffusion and the quality is qualified.

[0142] 2) Verification of mRNA expression in vitro:

[0143] like Figure 2 As shown, an obvious target band can be seen at 28 kDa, and the band size is consistent with expectations, indicating that mRNA is successfully expressed.

[0144] 3) Observation of mRNA-LNP morphology:

[0145] like Figure 3 As shown, the coated mRNA-LNPs are nearly spherical.

[0146] 4) Determination of particle size, PDI, potential and encapsulation efficiency:

[0147] like Figure 4 As shown, the average particle size of the prepared mRNA-LNP was 158.4 nm, the PDI was 0.1345, the Zeta potential was -0.35 mV, the encapsulation efficiency was 96.2%, and the physical and chemical properties met the requirements of subsequent experiments.

[0148] 5) Specific antibody detection:

[0149] like Figure 5 As shown in Figure 2, the average specific IgG antibody titers against DI-EDⅢ+DI-EDⅢ, DI-EDⅢ and DI-EDⅢ proteins on day 42 after immunization were all 10 6 about.

[0150] 6) Neutralizing antibody detection

[0151] like Figure 6 As shown, the mRNA vaccine has good immunogenicity, and vaccination can induce AGB6 mice to produce neutralizing antibodies. The average neutralizing antibody titers against DENV-Ⅰ and DENV-Ⅱ were 1:179.2 and 1:121.6, respectively.

[0152] 7) T lymphocyte subset detection

[0153] like Figure 7As shown, immune group CD3 + CD4 + The proportion of positive T cells was 20.66%, 1.17 times that of the control group; + CD8 + The proportion of positive T cells was 13.12%, 1.7 times that of the control group. mRNA vaccine can induce CD4 + and CD8 + The secretion of T lymphocytes enhances the humoral immune response, the ability to specifically kill target cells and resist viral infection.

[0154] 8) Detection of intracellular cytokines IL-4, IFN-γ and TNF-α:

[0155] like Figure 8 As shown, among the cytokines secreted by Th1 cells, the immune group secretes CD4 + IFN-γ + 、CD4 + TNF-α + 、CD8 + IFN-γ + and CD8 + TNF-α + The proportions were 0.66%, 0.37%, 0.27% and 0.22% respectively, which were significantly higher than those in the control group. Among the cytokines secreted by Th2 cells, the immune group secreted CD4 + IL-4 + and CD8 + IL-4 + The proportions were 0.27% and 0.19% respectively, both significantly higher than the control group. mRNA candidate vaccines can induce Th1 and Th2 cellular immune responses.

[0156] 9) Body weight changes after challenge:

[0157] like Fig. 9 As shown, the weight of mice infected with DENV-Ⅰ decreased in both the immunized group and the control group before 7 days, but the weight of mice in the immunized group gradually recovered after 7 days, while the weight of mice in the control group decreased and died; the weight of mice infected with DENV-Ⅱ decreased briefly on the 4th day, while the weight of mice in the control group continued to decrease, and they developed paralysis symptoms and then died.

[0158] 10) Detection of viremia after challenge:

[0159] like Fig.10 As shown in Figure 2, after infection with DENV-Ⅰ, the viral loads of the immunized group and the control group were 1672 copies / mL and 1.19×10 5The viral loads in the immune group and the control group were 72 times lower than those in the control group. After infection with DENV-Ⅱ, the viral loads in the immune group and the control group were 728 copies / mL and 9.15×10 4 copies / mL, which was 125 times lower than that of the control group.

[0160] 11) Detection of viral load in brain tissue after challenge:

[0161] like Fig.11 As shown, in AGB6 mice inoculated with DENV-Ⅰ, the RNA copy numbers detected in the brain tissue homogenates of the immunized group and the control group were 1091 copies / mL and 1.64×10 5 In AGB6 mice inoculated with DENV-Ⅱ, the RNA copy numbers detected in the brain tissue homogenates of the immunized group and the control group were 1.37×10 4 copies / mL and 1.19×10 5 copies / mL.

[0162] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for constructing a multi-target mRNA vaccine against type Ⅰ and type Ⅱ dengue viruses, characterized in that: The following steps are involved: S1: Linearize DNA for in vitro transcription: In vitro transcription was performed using the All In One High-Efficiency mRNA Synthesis Kit; S2: Perform mRNA purification: Purify using MEGA clear Transcription Clean-Up Kit and centrifuge at room temperature; S3: mRNA content determination and identification: S31: Determine the mRNA content: Use an ultra-micro spectrophotometer to detect and record the product concentration; S32: mRNA agarose electrophoresis identification; S33: Determine whether the purified product meets the qualified standards.

2. The method for constructing a multi-target mRNA vaccine against dengue virus type I and type II according to claim 1, characterized in that: The step S1 specifically includes the following steps: S11: Preparation of reaction system: Add and prepare the IVT reaction system in the following order; S12: After mixing according to the above reaction system, centrifuge it instantly and place it in a PCR instrument for reaction at 37°C for 4 hours; S13: Add 2 μL DNaseI to every 20 μL reaction system and react at 37°C for 15 min.

3. The method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses according to claim 1, characterized in that: The step S2 specifically includes the following steps: S21: Fill the S1 reaction sample to 100 μL with Elution Buffer and mix gently with a pipette tip; S22: Add 350 μL Binding Buffer and mix gently; S23: Add 250 μL 100% ethanol and mix gently; S24: Gently add the sample of S23 to the filter column, centrifuge at 14000g for 1 min, and discard the filtrate; S25: Add 550 μL Washing Buffer, wash twice, centrifuge at 14000 g for 1 min each time, and discard the filtrate; S26: centrifuge the empty RNA microadsorption column at 14000g for 2 min at room temperature; S27: Add 50-100 μL RNase Free Water, let stand for 10 min, and centrifuge at 14000 g for 2 min; S28: Transfer the dissolved mRNA to a new 1.5 mL centrifuge tube, mix thoroughly and mark it, take out 0.3 mL for content determination and identification, and store the rest at -20℃ for subsequent experiments.

4. The method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses according to claim 1, characterized in that: The step S32 specifically includes the following steps: S321: Prepare electrophoresis buffer: Dissolve 50×TAE Buffer into 1×TAE Buffer using ultrapure water; S322: Prepare the sample to be tested: take 100-300 ng of mRNA, add the same volume of Loading Buffer, and react at 70℃ for 5 min in a PCR instrument; S323: mRNA was subjected to 1% agarose gel electrophoresis and then imaged, and markers were used to confirm whether the sample band size was correct.

5. The method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses according to claim 1, characterized in that: In step S33, Nanodrop detection is performed, and when the ratio of 260 / 280 is between 1.8-2.3 and 260 / 230≧2.0, the quality is qualified.

6. The method for constructing a multi-target mRNA vaccine against type I and type II dengue viruses according to claim 4, characterized in that: In the S323, the size of the agarose gel electrophoresis band is consistent with the size of the target band, and the band is clear without obvious diffusion.

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