Databa virus circular RNA and application thereof in preparation of vaccine

Abstract

The invention discloses a circular RNA (Ribose Nucleic Acid) of a Databan virus and an application of the circular RNA in preparation of vaccines, and belongs to the technical field of immunology and molecular virology. The sequence of the circular RNA of the large allofanda virus is formed by connecting a sequence shown in SEQ ID NO.2 end to end. After cells are transfected by the circular RNA of the Databu virus, virus-like particles of the Databu virus can be generated. After the circular RNA is encapsulated by the lipid nanoparticles, an immunized mouse can induce a high-level megabo virus neutralizing antibody and T cell immunity, and the immunized mouse serum can protect the mouse from being attacked by a lethal megabo virus. The invention provides a theoretical basis for further developing a safer, more efficient and quicker circular RNA vaccine for the GABA virus.

Description

Technical Field

[0001] The invention relates to the technical field of immunology and molecular virology, and in particular to a large Beaglebanda virus circular RNA and application thereof in vaccine preparation. Background Art

[0002] Dabie bandavirus (DBV), also known as severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), belongs to the Bunyaviridae family. Infection with this virus can cause severe fever with thrombocytopenia syndrome (SFTS). Severe patients will experience symptoms such as disseminated intravascular coagulation, multiple organ failure, persistent thrombocytopenia and high levels of inflammatory factors, and even death, with a mortality rate ranging from 12% to 30%. In 2017, the World Health Organization listed SFTS as one of the emerging infectious diseases that require priority research and intervention.

[0003] DBV is spherical, with a diameter of 80 to 120 nm, and the core contains three single-stranded negative-strand RNA segments. The L segment encodes RNA-dependent RNA polymerase; the M segment encodes a membrane glycoprotein polyprotein, which is cleaved by proteases to produce envelope proteins Gn and Gc; the S segment encodes nucleoprotein N and non-structural protein NS. Among them, Gn protein and Gc protein form heterodimers, wrapped on the surface of the virus, mediate receptor binding and virus entry into cells, and are the target proteins of neutralizing antibodies. So far, there is no specific treatment for DBV, and vaccination plays a vital role in the prevention and control of this infectious disease. In recent years, domestic research on DBV vaccines has mostly focused on the category of mRNA vaccines. Although mRNA vaccines have many advantages compared with traditional inactivated vaccines and live attenuated vaccines, due to the poor stability of mRNA itself, it is easily degraded by nucleases in the environment, and there is a risk of inducing side effects such as nonspecific immune responses. In actual production and application, mRNA vaccines still have many disadvantages.

[0004] "Circular RNA" (circRNA) is a type of RNA structure that forms a closed ring with a covalent bond. It is commonly found in viral and eukaryotic transcriptomes. Because it does not contain a 5' end cap and a 3' end poly (A) tail, it is not easily affected by RNA exonucleases and has higher stability. It can continuously express antigens in vivo and enhance immune effects, providing new ideas and methods for vaccine development. Therefore, taking advantage of circular RNA to develop a safer, more efficient, and faster Dabiebandha virus vaccine is of great significance for the prevention and control of fever with thrombocytopenia syndrome. Summary of the invention

[0005] The purpose of the present invention is to provide a Dabiebanda virus circular RNA and its application in the preparation of vaccines to solve the problems existing in the above-mentioned prior art. The circular RNA provided by the present invention can produce virus-like particles of Dabiebanda virus after transfecting cells. After the circular RNA is encapsulated by lipid nanoparticles, immunized mice can induce high levels of Dabiebanda virus neutralizing antibodies and T cell immunity, and the immune mouse serum can protect mice from lethal Dabiebanda virus attacks.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a DNA sequence for preparing a large Bepanda virus circular RNA, and the DNA sequence is shown as SEQ ID NO.1.

[0008] The invention also provides a recombinant vector containing the DNA sequence.

[0009] Furthermore, the recombinant vector uses pUC57 containing a T7 promoter as a backbone vector.

[0010] The present invention also provides a Dabiebandha virus circular RNA, wherein the sequence of the Dabiebandha virus circular RNA is composed of the sequence shown in SEQ ID NO.2 connected head to tail.

[0011] Furthermore, the recombinant vector is transcribed in vitro and self-cleaved to form the Dabiebandha virus circular RNA.

[0012] The present invention also provides the use of the DNA sequence, the recombinant vector or the Dabiebandha virus circular RNA in the preparation of a Dabiebandha virus circular RNA vaccine.

[0013] The present invention also provides a method for preparing a Dabie Bandha virus circular RNA vaccine, comprising constructing the recombinant vector, wherein the recombinant vector is transcribed in vitro and self-cleaved and cyclized to form the Dabie Bandha virus circular RNA, and the Dabie Bandha virus circular RNA is encapsulated by lipid nanoparticles to obtain the Dabie Bandha virus circular RNA vaccine.

[0014] The present invention also provides a Dabiebandha virus circular RNA vaccine obtained by the preparation method.

[0015] The present invention discloses the following technical effects:

[0016] The present invention designs a DNA sequence, which comprises 3'Intron, exon 2, Internal Ribosome Entry Site (IRES), signal peptide (SP), a gene encoding Dabiebanda virus Gn / Gc protein, exon 1 and 5'Intron sequence from 5' end to 3' end. After the DNA sequence is cloned into a plasmid containing a T7 promoter, a linearized recombinant plasmid obtained by single enzyme digestion can generate a circular RNA during in vitro transcription, wherein the two ends of the circular RNA are SP, IRES, Exon 2 and Exon 1 sequences. After the circular RNA is transfected into cells, virus-like particles of Dabiebanda virus can be generated. After the circular RNA is encapsulated by lipid nanoparticles, high-level Dabiebanda virus neutralizing antibodies and T cell immunity can be induced in immunized mice, and the immune mouse serum can protect the mice from the attack of lethal Dabiebanda virus.

[0017] The present invention provides a theoretical basis for further developing a safer, more efficient and faster Dabiebandha virus circular RNA vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram for preparing circRNA by in vitro transcription and circularization;

[0019] Figure 2 Agarose gel electrophoresis of DBV-Gn / Gc circRNA prepared by in vitro transcription for 6 h + GTP catalysis for 15 min and in vitro cyclization;

[0020] Figure 3 High-performance liquid chromatography (HPLC) analysis results of DBV-Gn / Gc circRNA prepared at different times of in vitro transcription and circularization;

[0021] Figure 4 Agarose gel electrophoresis of DBV-Gn / Gc circRNA prepared by in vitro transcription for 6 hours after RNase R digestion;

[0022] Figure 5 The analysis results of DBV-Gn / Gc circRNA transcribed and circularized in vitro were collected by high performance liquid chromatography, where A is the sample collected from two parts of the second half of the peak of the in vitro transcription circularization product (transcription for 6 hours + GTP circularization for 15 minutes) on HPLC, and B is the comparison of the two parts of the sample collected by HPLC with the peak of the in vitro transcription circularization product on HPLC;

[0023] Figure 6 This is the agarose gel electrophoresis of dephosphorylated DBV-Gn / Gc circRNA;

[0024] Figure 7 This is the agarose gel electrophoresis of dephosphorylated DBV-Gn / Gc circRNA after RNase R treatment;

[0025] Figure 8 is the resistance of DBV-Gn / Gc circRNA to environmental temperature, wherein A is the gel electrophoresis of DBV-Gn / Gc circRNA after being placed in 28℃ and 37℃ for different time periods; B is the remaining amount of DBV-Gn / Gc circRNA after being placed in 28℃ for different time periods; C is the remaining amount of DBV-Gn / Gc circRNA after being placed in 37℃ for different time periods;

[0026] Fig. 9 The large benda virus-like particles produced in 293T cells transfected with DBV-Gn / Gc circRNA were observed by transmission electron microscopy;

[0027] Fig.10 is the particle size and polymer dispersibility index of DBV-Gn / Gc circRNA-LNP, where A is the observation of DBV-Gn / Gc circRNA-LNP particles using a transmission electron microscope; B is the analysis of the size dispersion of DBV-Gn / Gc circRNA-LNP particles using a particle size analyzer;

[0028] Fig.11 The LNPs made of different doses of DBV-Gn / Gc circRNA were used to immunize mice to induce Gn and Gc reactive IgG. A is the ELISA determination of Gn reactive IgG in the serum of 6-week-old C57BL / 6 mice immunized with 0.5, 5, and 50 μg of DBV-Gn / Gc circRNA; B is the OD of Gn reactive IgG in the serum of mice immunized with 0.5, 5, and 50 μg of circRNA. 450 (AUC) statistical analysis; C is the ELISA determination of Gc-reactive IgG in the serum of 6-week-old C57BL / 6 mice immunized with 0.5, 5, and 50 μg of DBV-Gn / Gc circRNA; D is the OD of Gc-reactive IgG in the serum of mice immunized with 0.5, 5, and 50 μg of circRNA 450 (AUC) Statistical analysis;

[0029] Fig.12Figure 2 shows the high-efficiency neutralization of Dabiebanda virus by sera of mice immunized with DBV-Gn / Gc circRNA at different doses. A shows the neutralization of DBV by sera of mice immunized with 0.5, 5, and 50 μg circRNA at different dilutions. B shows the NT value of DBV neutralized by sera of mice immunized with 0.5, 5, and 50 μg circRNA in vitro. 50 Comparison of values;

[0030] Fig.13 Different doses of DBV-Gn / Gc circRNA immunized mice to induce a broad range of CD8 + T cell response, where A is the CD8 induced by 0.5 μg circRNA / mouse + T cell responses to 35 epitope peptides from Gn and Gc proteins; B is the CD8 + T cell responses to 35 epitope peptides from Gn and Gc proteins; C is the CD8 + T cell responses to 35 epitope peptides from Gn and Gc proteins; * indicates a positive response;

[0031] Fig.14 The protective effect of serum from mice immunized with DBV-Gn / Gc circRNA-LNP on one-day-old suckling mice against lethal Dabiebanda virus attack. DETAILED DESCRIPTION

[0032] Example 1 Preparation of Dabiebandha virus circular RNA

[0033] 1. Gene synthesis and recombinant plasmid construction

[0034] In order to prepare the circRNA of Dabiebandha virus in vitro, a DNA sequence was designed and commissioned to be synthesized by Jinweizhi Company. The 5′ to 3′ ends of the DNA sequence are 3′Intron, exon 2, Internal ribosome entry site (internal ribosome entry site, IRES), signal peptide (signal peptide, SP), gene encoding Dabiebandha virus Gn / Gc protein (DBV-Gn / Gc), exon 1 and 5′Intron sequence, as shown in SEQ ID NO.1. The DNA sequence was cloned into the pUC57 plasmid containing the T7 promoter to obtain the recombinant plasmid pUC57-DBV-Gn / Gc circRNA. The recombinant plasmid was then transformed into the Escherichia coli strain EPI400. The linear RNA precursor produced by the in vitro transcription of the recombinant plasmid pUC57-DBV-Gn / Gc circRNA can be self-cleaved and circularized into a circular RNA (circRNA), namely DBV-Gn / Gc circRNA, such as Figure 1 As shown, the sequence of DBV-Gn / Gc circRNA is composed of the sequence shown in SEQ ID NO.2 connected end to end.

[0035] SEQ ID NO.1:

[0036]

[0037]

[0038]

[0039] SEQ ID NO.2:

[0040]

[0041]

[0042] In SEQ ID NO.1 and SEQ ID NO.2, the “” part represents the 3′Intron. The marked part represents exon 2. The marked part represents IRES, and the italic part represents SP. The marked part represents DBV-Gn / Gc, The marked part represents exon1. The marked part represents the 5' Intron.

[0043] 2. Extraction of recombinant plasmid

[0044] (1) Take 10 mL of LB medium and add it to a sterile 50 mL centrifuge tube, then add 10 μL of AMP (1000×) and 10 μL of Escherichia coli strain EPI400 containing the recombinant plasmid pUC57-DBV-Gn / Gc circRNA, and culture at 37°C / 200 rpm until the logarithmic growth phase.

[0045] (2) Add 10 mL of bacterial solution to 500 mL of LB medium and add 500 μL of AMP (1000×), and culture overnight at 37°C / 200 rpm.

[0046] (3) Plasmid was extracted using a plasmid extraction kit (Tiangen). 2.5 mL of equilibration solution BL was added to the adsorption column CP6 of the plasmid extraction kit (the adsorption column was placed in a 50 mL collection tube), centrifuged at 8000 rpm (~8228 × g) for 2 min, the waste liquid in the collection tube was discarded, and the adsorption column was placed back in the collection tube.

[0047] (4) Take 150 mL of overnight cultured bacterial solution and add it to a 50 mL collection tube. Centrifuge at 8000 rpm (~8228 × g) for 8 min to collect the bacteria. Use a 1 mL pipette to remove the supernatant as much as possible.

[0048] (5) Add 12 mL of solution P1 (to which RNase A has been added) to the centrifuge tube containing the bacterial cell pellet and use a pipette or vortex oscillator to thoroughly suspend the bacterial cell pellet.

[0049] (6) Add 12 mL of solution P2 to the centrifuge tube and immediately and gently invert the tube 6-8 times to fully lyse the bacteria. Leave the tube at room temperature for 5 min.

[0050] (7) Add 12 mL of solution P4 to the centrifuge tube, and immediately gently invert the tube 6-8 times to mix thoroughly until white dispersed flocculent precipitates appear in the solution. Then place it at room temperature for 10 minutes. Centrifuge at 8000 rpm (~8228×g) for 20 minutes to allow the white precipitate to settle to the bottom of the tube. Aspirate the supernatant in the front into the filter CS1, push the push handle to filter, and collect the filtrate in a clean 50 mL tube.

[0051] (8) Add 0.3 times the volume of isopropanol to the filtrate, mix by inversion, and transfer to adsorption column CP6.

[0052] (9) Centrifuge at 8000 rpm (~8228×g) for 2 min, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0053] (10) Add 10 mL of rinse solution PW to the adsorption column CP6, centrifuge at 8000 rpm (~8228 × g) for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0054] (11) Repeat the previous step.

[0055] (12) Add 3 mL of anhydrous ethanol to the adsorption column CP6, centrifuge at 8000 rpm (~8228 × g) at room temperature for 2 min, and discard the waste liquid.

[0056] (13) Place the adsorption column CP6 back into the collection tube and centrifuge at 8000 rpm (~8228 × g) for 5 min to remove the remaining rinse solution in the adsorption column.

[0057] (14) Place the adsorption column CP6 in a new 50 mL collection tube, add 1 mL of RNase-free water (preheated at 65 °C) to the middle of the adsorption membrane, let it stand at room temperature for 5 min, and then centrifuge it at 8000 rpm (~8228 × g) for 2 min. Transfer all the eluate in the 50 mL centrifuge tube to a sterile 1.5 mL centrifuge tube and store it at -20 °C.

[0058] (15) Take 25 μL of the eluate and measure the plasmid concentration using a K5500 PLUS ultra-micro spectrophotometer.

[0059] 3. Linearization of recombinant plasmid

[0060] Add the reagents in Table 1 to a sterile, RNase-free EP tube. Place in a 37°C water bath for 30 min. Heat at 65°C for 20 min to inactivate the enzyme, and use agarose gel electrophoresis to identify the size of the enzyme-cut band.

[0061] Table 1 Recombinant plasmid single restriction enzyme digestion reaction system

[0062]

[0063] 4. Recover the linearized plasmid

[0064] (1) Briefly centrifuge the enzymatic mixture, measure its volume with a pipette, and transfer it to a 1.5 mL centrifuge tube without enzyme.

[0065] (2) Add an equal volume of Buffer GDP and mix by inversion.

[0066] (3) Place the DNA column in a collection tube, transfer the mixed solution to the DNA column, and centrifuge at 12,000 rpm for 1 min.

[0067] (4) Discard the filtrate, put the column back into a 2 mL centrifuge tube, add 600 μL Buffer DW2 (diluted with anhydrous ethanol) to the column, and centrifuge at 12,000 rpm for 1 min.

[0068] (5) Repeat step (4) once.

[0069] (6) Discard the filtrate, put the column back into a 2 mL centrifuge tube, and centrifuge at 12,000 rpm for 2 min.

[0070] (7) Open the column lid and air dry for 5 min to completely remove the ethanol.

[0071] (8) Place the column in a 1.5 mL centrifuge tube and add 30 μL of DEPC water to the center of the column membrane. Allow to stand for 5 minutes and centrifuge at 12,000 rpm for 2 minutes. The resulting liquid is high-purity DNA.

[0072] (9) Detect the DNA concentration and perform agarose gel electrophoresis.

[0073] 5. In vitro transcription

[0074] (1) Mix all components of the T7 RNA Transcription Kit (Sangon) except T7 RNA Polymerase Mix (Table 2) by vortexing, centrifuge briefly, and place on ice.

[0075] (2) Add the components in Table 2 at room temperature (DEPC water, buffer and NTP first, and linearized recombinant plasmid and enzyme last).

[0076] Table 2 In vitro transcription reaction system

[0077]

[0078] The components were mixed, collected by brief centrifugation, and incubated at 37°C for 6 h.

[0079] 6. Digestion of linear DNA template

[0080] (1) Add 1 μL of DNase I (Sangon) to the above 20 μL reaction system.

[0081] (2) Mix thoroughly and centrifuge to allow the solution to settle to the bottom of the centrifuge tube.

[0082] (3) 37°C water bath for 15 min.

[0083] 7. RNA Circularization

[0084] (1) Add GTP (Sangon) to the above reaction system to make the final concentration 2 mM.

[0085] (2) 55°C water bath for 15 min.

[0086] 8. RNA Purification

[0087] (1) Add 30 μL of Lithium Chloride Precipitation Solution (novo protein) and 30 μL of DEPC water (the final concentration of Lithium Chloride should be maintained at 2.5-2.8 M) to the above 20 μL transcription product system;

[0088] (2) After mixing, place at -20℃ for 30 min.

[0089] (3) Centrifuge at 12000 rpm for 15 min, discard the supernatant, and collect the precipitate.

[0090] (4) Wash three times with 70% ethanol precooled at -20°C, each time for 5 min, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and collect the precipitate.

[0091] (5) Add 100 μL of DEPC water to reconstitute and dilute, then measure the RNA concentration.

[0092] 9. RNase R enzyme digestion experiment

[0093] (1) Add RNA to a sterile, RNase-free EP tube and heat at 65°C for 3 min. Place on ice for 5 min.

[0094] (2) Add the components in Table 3 in sequence.

[0095] Table 3 RNase R enzyme digestion reaction system

[0096]

[0097] Mix well and centrifuge to the bottom of the tube, and bathe in 37℃ water for 30min.

[0098] 10. Agarose gel electrophoresis

[0099] (1) Add 0.6 g agarose and 30 mL DEPC water to a conical flask and heat in a microwave oven to completely dissolve the agarose. Cool to 55°C and quickly add 4 mL 10×MOPS Electrophoresis Buffer and 6.8 mL Denaturant in a clean bench and shake quickly.

[0100] (2) 0.5 μg of RNA was mixed with 2× RNA loading buffer (Thermo Scientific RNAmaker SM1821) at a volume ratio of 1:1, heated at 70°C for 10 min, and cooled on ice.

[0101] (3) Transfer the agarose gel into an electrophoresis tank, add 1×MOPS Electrophoresis Buffer (covering the gel), and perform electrophoresis at 100 V.

[0102] (4) Observe and take photos on a gel imager.

[0103] Results: Figure 2-Figure 4 As shown in Figure 2, in general, the longer the transcription time, the fewer linear RNA precursors and the more circRNAs. There is almost no linear RNA precursor in the product obtained after 6 h of transcription plus 15 min of guanosine triphosphate (GTP) catalysis ( Figure 2 ). The in vitro transcription products were further analyzed by high performance liquid chromatography (HPLC), and the results showed that the linear RNA precursor and circRNA appeared as two closely connected peaks in the HPLC graph. As the transcription time increased, the linear RNA precursor peak decreased and the circRNA peak increased. Especially after 6 hours of transcription, the addition of GTP for 15 minutes could almost cyclize all linear RNA precursors into circRNA ( Figure 3 When the product of transcription for 6 hours was subjected to agarose gel electrophoresis, linear RNA precursor bands and circRNA bands were visible. After the product of transcription for 6 hours was treated with RNase R, the linear RNA precursor band was almost invisible, while the circRNA band remained unchanged ( Figure 4 ).

[0104] Example 2 Purification of DBV-Gn / Gc circRNA

[0105] 1. HPLC analysis of in vitro transcription products and circRNA recovery

[0106] (1) Experimental consumables and instruments: High performance liquid chromatograph (Shimadzu), SEC purification column (Yuxu).

[0107] (2) Experimental reagents: TE buffer (Thermo), gel recovery RNA purification kit (Magen).

[0108] (3) Dilute the total RNA produced by transcription to 0.5 μg / μL, take 100 μL and analyze it on a high performance liquid chromatograph to observe the peak sample and waveform.

[0109] (4) After the waveform is determined, the RNA at different peak times is recovered and tested on a machine to observe the waveform at the selected time and then collect a large amount of RNA at that time.

[0110] (5) The RNA purification method is the same as that in Part 8 of Example 1.

[0111] 2. Dephosphorylation of DBV-Gn / Gc circRNA

[0112] The reaction system was prepared according to Table 4.

[0113] Table 4 CircRNA dephosphorylation reaction system

[0114]

[0115] Incubate at 37°C for 15 min.

[0116] The dephosphorylated DBV-Gn / Gc circRNA was then purified by lithium chloride and subjected to agarose gel electrophoresis. Figure 5-Figure 7 shown. Figure 5 It shows that the second half of the peak of the sample (in vitro transcription 6h + GTP cyclization 15min) in the high performance liquid chromatography (enriched in DBV-Gn / Gc circRNA) is divided into two parts for collection, that is, the time period indicated by the red arrow is collection part 1 and the time period indicated by the cyan arrow is collection part 2. Figure 6 The results showed that there were no visible linear RNA precursors in the two collected fractions by agarose gel electrophoresis analysis. Figure 7 It was shown that these collected fractions were highly resistant to RNase R digestion, indicating that the two collected fractions were rich in DBV-Gn / Gc circRNA and contained almost no linear RNA precursors.

[0117] Example 3 DBV-Gn / Gc RNA Environmental Stability Experiment

[0118] The DBV-Gn / Gc RNA purified by high performance liquid chromatography was placed at 28°C and 37°C for 0 days (0d), 2d, 5d, 14d and 28d respectively. The samples were then subjected to agarose gel electrophoresis. The results are shown in Figure 2. Figure 8 As shown, although the amount of DBV-Gn / GccircRNA gradually decreased over time, more than 50% of the circRNA remained after exposure to 28°C or 37°C for 5 days, and a small amount remained after exposure to both temperatures for 28 days, suggesting that the circRNA has a high ambient temperature stability.

[0119] Example 4 DBV-Gn / Gc circRNA transfected cells and electron microscopic observation

[0120] 1. Subculture 293T cells into a cell dish (10 cm in diameter) and culture in a 37°C / 5% CO2 incubator for 24 h;

[0121] 2. Discard the supernatant of 293T cells, add 8 mL of serum-free MEM culture medium, and culture in a 37°C / 5% CO2 incubator for 4 hours;

[0122] 3. Mix 10 μg DBV-Gn / Gc circRNA and 20 μg polyetherimide (PEI) transfection reagent and incubate at room temperature for 20 min. Add the transfection reagent to 293T cells and culture in a 37°C / 5% CO2 incubator for 4 h;

[0123] 4. Discard the transfection reagent, add MEM complete culture medium, and culture in a 37℃ / 5% CO2 incubator for 24h;

[0124] 5. Discard the cell supernatant, fix the cells with glutaraldehyde, and observe the virus-like particles (VLPs) in the cells using a transmission electron microscope.

[0125] Observation results such as Fig. 9 As shown, there are a large number of VLPs of Bepbanda virus in 293T cells transfected with DBV-Gn / Gc circRNA, with an average diameter of about 100 nm, which is consistent with the diameter of complete virus particles appearing in DBV-infected Vero cells.

[0126] Example 5 Preparation of DBV-Gn / Gc RNA lipid nanoparticles

[0127] 1. Prepare the aqueous phase: dilute DBV-Gn / Gc circRNA to 170 μg / mL with citric acid buffer (pH 4, 50 mM) and filter with a 0.22 μm filter membrane.

[0128] 2. Prepare the organic phase: dissolve 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA), distearoyl phosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000 in anhydrous ethanol to concentrations of 6.25 mM, 1.25 mM, 4.8125 mM, and 0.1875 mM, respectively, and filter with a 0.22 μm filter membrane.

[0129] 3. Preparation of lipid nanoparticles (LNP): The aqueous phase and the organic phase were added simultaneously to the Microflow S microfluidic nanodrug preparation instrument (model MT-MF-S) at a volume ratio of 3:1 and a flow rate of 12 mL / min to prepare DBV-Gn / GccircRNA-LNP.

[0130] 4. Immediately dilute the synthesized sample 40 times with PBS buffer (pH 7.4, 0.01M), take 2 mL of the solution after dilution and use a particle size analyzer to measure the particle size, polymer dispersibility index and Zeta potential of DBV-Gn / Gc circRNA-LNP. Concentrate the remaining sample with a 100 kDa ultrafiltration tube, collect the concentrate, which is the DBV-Gn / Gc circRNA-LNP solution, and store it at 4 °C for later use.

[0131] The results are as follows Fig.10 As shown, the average particle size of DBV-Gn / Gc circRNA-LNP was 134.1 nm, the polymer dispersity index was 0.024, and the Zeta potential was -4.7 mV.

[0132] Example 6 Immune protection effect of DBV-Gn / Gc RNA-LNP

[0133] 1. DBV-Gn / Gc RNA-LNP immunization of mice

[0134] Different doses (0.5, 5 or 50 μg circRNA / mouse) of DBV-Gn / Gc circRNA-LNP were injected subcutaneously into the back of 6-week-old C57BL / 6 mice. Control mice were given only the same volume of PBS (called naive mice). Two weeks later, the mice were boosted once with the same dose of DBV-Gn / Gc circRNA-LNP. Two weeks after the booster immunization, the mice were killed and the mouse serum was collected for enzyme-linked immunosorbent assay (ELISA) and viral plaque reduction neutralization test (PRNT). Splenocyte suspension was prepared and CD8 + T cells used in ELISpot experiments.

[0135] 2. ELISA experiment

[0136] Coating ELISA plate: Gn expressed by E. coli 60-446 and Gc 628-1024The protein (amino acid sequence is from the HB154 / China / 2011 strain of DBV, and the specific sequence is shown in SEQ ID NO.3 and SEQ ID NO.4) was diluted to 50 μg / mL with coating buffer and added to the ELISA plate (100 μL / well), and incubated at 4°C overnight. The liquid in each well was discarded, and the plate was washed twice with PBS buffer. 100 μL of 5% skim milk powder was added to each well and incubated at 4°C overnight. The liquid in each well was discarded, and the plate was washed twice with PBS buffer. The mouse serum was serially diluted 3 times and added to each well (50 μL / well), and incubated at 37°C for 1.5h. Discard the liquid in each well, wash the plate 6 times with PBST (PBS buffer containing 0.5% Tween-20), add 50 μL of freshly prepared HRP-labeled goat anti-mouse IgG (1:5000, Invitrogen, Cat. No#A16066) to each well, and incubate at 37°C for 1.5 h. Discard the liquid in each well, wash the plate 6 times with PBST, add 40 μL of freshly prepared TMB (3,3',5,5'-tetramethylbenzidine) (Sigma, Cat. No#T0440) substrate to each well, and react at room temperature for 10 min. Measure the OD of each well using an enzyme reader 450nm The area under the curve (AUC) was used to compare the differences between groups.

[0137] Gn 60-446 Amino acid sequence (SEQ ID NO.3):

[0138] WLRNHSQFQGYVGQRGGRSQVSYYPAENSYSRWSGLLSPCDADWLGMLVVKKAKESDMIVPGPSYKGKVFFERPTFDGYVGWGCGSGKSRTESGELC SSDSGTSSGLLPSDRVLWIGDVACQPMTPIPEETFLELKSFSQSEFPDICKIDGIVFNQCEGESLPQPFDVAWMDVGHSHKIIMREHKTKWVQESSS KDFVCYKEGTGPCSESEEKACKTSGSCRGDMQFCKVAGCEHGEEASEAKCRCSLVHKPGEVVVSYGGMRVRPKCYGFSRMMATLEVNPPEQRIGQCTGCHLECINGGVRLITLTSELRSATVCASHFCSSATSGKKSTEIHFHSGSLVGKTAIHVKGALVDGTEFTFEGSCMFPDGCDAVDCTFCREFLKNPQ.

[0139] G 628-1024 Amino acid sequence (SEQ ID NO.4):

[0140] KCKKSSSYFVPDARSRCTSVRRCRWAGDCQSGCPPHFTSNSFSDDWAGKMDRAGLGFSGCSDGCGGAACGCFNAAPSCIFWRKWVENPHGIIWKVSPCAAWVPSAVIELTMPSGEVRTFHPMSGIPTQVFKGVSVTYLGSDMEVSGLTDLCEIEELKSKKLALAPCNQAGMGVVGKVGEIQCSSEESARTIKKDGCIWN ADLVGIELRVDDAVCYSKITSVEAVANYSAIPTTIGGLRFERSHDSQGKISGSPLDITAIRGSFSVNYRGLRLSLSEITATCTGEVTNVSGCYSCMTGAKVSIKLHSSKNSTAHVRCKGDETAFSVLEGVHSYIVSLSFDHAVVDEQCQLNCGGHESQVTLKGNLIFLDVPKFVDGSYMQTYHSTVPTGANIPSPTDW.

[0141] The results are as follows Fig.11 As shown, 0.5 μg of DBV-Gn / Gc circRNA also induced a high level of antibody response in 6-week-old adult mice, with 5 μg of circRNA induced the highest level of antibody response.

[0142] 3. PRNT experiment

[0143] Vero cells were plated in 24-well plates (1×10 5 / well) and cultured for 24 h.

[0144] The mouse serum to be tested was diluted 3-fold in a 96-well plate (starting from 1:30), DBV [50 viral plaque-forming units (PFU) / well] was added to each well, and incubated at 37°C for 1 h.

[0145] The Vero cell supernatant was discarded, and the virus / mouse serum mixture was added to the cells and infected at 37°C for 1.5 h.

[0146] The virus solution was discarded, and 1% methylcellulose culture medium was added to each well to cover the cells and cultured for 3-6 days.

[0147] Add 4% paraformaldehyde to each well for fixation at room temperature for 1 hour, and then wash the plate.

[0148] Add 0.5% crystal violet staining solution to each well for 10 min at room temperature and wash the plate.

[0149] The number of virus plaques in each well was counted, and the neutralization titer (half inhibitory concentration, NT) of mouse sera at different dilutions against DBV was calculated. 50 value).

[0150] The results are as follows Fig.12 As shown in Figure 2, the sera of mice immunized with three doses of DBV-Gn / Gc circRNA-LNP all had a neutralizing effect on DBV, with NT 50 The values ​​were 1:2511, 1:7943, and 1:5011, respectively. 5 μg of circRNA induced the highest level of DBV neutralizing antibodies.

[0151] 4.ELISPOT assay

[0152] The above mouse spleen cell-derived CD8 + T cells (1×10 5 cells / well) and LPS-Blast (1×10 5 / well) were plated into each well of a mouse IFNγ ELISpot plate (MabTech, pre-coated) and incubated in a 37°C / 5% CO2 incubator for 24 h.

[0153] The cells and liquid in each well were discarded, and after washing the plate, biotinylated anti-IFNγ monoclonal antibody was added to each well and incubated at 37°C for 1 hour.

[0154] The liquid in each well was discarded, and HRP-labeled streptavidin was added to each well, and incubated at 37°C for 1 h.

[0155] The liquid in each well was discarded, and after washing the plate, AEC substrate was added to each well and reacted at 37°C for 10 min.

[0156] Discard the liquid in each well, wash the plate and count the spot forming cells (SFC) in each well. The results are expressed as IFNγSFC / 10 6 CD8 + T cells. When the experimental well result is >20SFCs / 10 6 CD8 + If the number of T cells is more than 1 times greater than that of the wells without peptide stimulation, the reaction is considered positive.

[0157] Results: The information of 35 T cell epitope peptides in Dabiebandha virus is shown in Table 5. Fig.13 As shown, peptide-specific CD8 + The frequency and breadth of T cells were proportional to the amount of circRNA used. 0.5 μg of circRNA did not induce significant peptide-specific CD8 in adult mice. +T cell immune response, and 50 μg of circRNA induced higher levels of CD8 than 5 μg of DBV-Gn / Gc circRNA + T cell immunity. CD8 isolated from mice immunized with 50 μg of DBV-Gn / Gc circRNA-LNP + T cells responded to each of the 35 peptide stimuli.

[0158] Table 5 H-2 from Dabiebandha virus b Restricted CD8 + T cell epitope peptide

[0159]

[0160]

[0161] 5. Passive transfer experiment

[0162] Different doses (0.5, 3, and 15 μL) of naive or DBV-Gn / Gc circRNA-LNP-immunized mouse serum were injected subcutaneously into the back of one-day-old suckling mice. Two hours later, 1000 PFU of DBV was injected subcutaneously into the back of each mouse. Three days later, the mice were killed, and the mouse serum was collected. The virus titer in the mouse serum was determined by the virus plaque formation assay, and the results were expressed as PFU / mL.

[0163] The results are as follows Fig.14 As shown, the DBV titer in the serum of mice infused with 0.5 μL of DBV-Gn / Gc circRNA-LNP immune serum was significantly lower than that in the serum of mice given naive serum (468 PFU / mL vs 71935 PFU / mL, P=0.0047). More importantly, the DBV virus titer was not detected in the serum of mice infused with 3 or 15 μL of DBV-Gn / Gc circRNA-LNP immune serum.

[0164] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A DNA sequence for preparing a Dabiebandha virus circular RNA, characterized in that: The DNA sequence is shown as SEQ ID NO.

1.

2. A recombinant vector containing the DNA sequence according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The recombinant vector uses pUC57 containing T7 promoter as the backbone vector.

4. A Dabiebandha virus circular RNA, characterized in that The sequence of the Dabiebandha virus circular RNA is composed of the sequence shown in SEQ ID NO.2 connected end to end.

5. The Dabiebandha virus circular RNA according to claim 4, characterized in that The recombinant vector according to claim 2 or 3 is transcribed in vitro and self-cleaved and circularized to form the Dabiebandha virus circular RNA.

6. Use of the DNA sequence according to claim 1, the recombinant vector according to claim 2 or 3, or the Dabiebandha virus circular RNA according to claim 4 or 5 in the preparation of a Dabiebandha virus circular RNA vaccine.

7. A method for preparing a Dabiebandha virus circular RNA vaccine, characterized in that: The method comprises constructing the recombinant vector as described in claim 2 or 3, wherein the recombinant vector is transcribed in vitro and self-cleaved and circularized to form the Dabie Bandha virus circular RNA as described in claim 4 or 5, and the Dabie Bandha virus circular RNA is encapsulated by lipid nanoparticles to obtain the Dabie Bandha virus circular RNA vaccine.

8. The Dabiebandha virus circular RNA vaccine obtained by the preparation method according to claim 7.

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