CN115232826A

CN115232826A - New coronavirus vaccine based on 1096 framework sequence

Info

Publication number: CN115232826A
Application number: CN202110434369.3A
Authority: CN
Inventors: 杨静; 王升启; 李蕾; 王鑫; 曹艺明; 龙晋蓉; 桑野; 任晋; 雷恩
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2022-10-25
Anticipated expiration: 2041-04-22
Also published as: CN115232826B

Abstract

The invention relates to a vaccine based on a 1096 framework sequence, which provides a novel vaccine for infection prevention and control of novel coronavirus and provides technical reference for research and development of other vaccines, antibodies, cytokines and the like based on mRNA. The active component of the vaccine is mRNA-1096, the mRNA-1096 comprises 5' -UTR8, mRNA of a target gene, 3' -UTR8 and Poly A which are sequentially connected, and the sequence of the 5' -UTR8 is obtained by replacing T in the 1 st to 50 th positions of the sequence 8 in a sequence table with U; the sequence of the 3' -UTR7 is obtained by replacing T in positions 822 to 956 of a sequence 8 in a sequence table with U; the sequence of the Poly A is obtained by replacing T in 957-1076 th positions of a sequence 8 in a sequence table with U.

Description

New coronavirus vaccine based on 1096 framework sequence

Technical Field

The invention relates to the field of nucleic acid vaccines, in particular to a novel coronavirus vaccine based on a 1096 framework sequence.

Background

The recently emerging mRNA vaccines are one of the novel nucleic acid vaccines. It can directly immunize target antigen gene coding script, and then the target antigen protein can be self-synthesized by body, and can quickly induce humoral immunity and cell immune response. mRNA vaccines are conceptually more advantageous than traditional inactivated vaccines, attenuated vaccines, novel subunit vaccines, viral vector vaccines and DNA vaccines: 1) The research and development speed of the mRNA vaccine technology is high, GMP production and quality control can be completed within three months, and the emergency prevention and control of the epidemic of the paroxysmal infectious diseases are facilitated; 2) mRNA vaccines are relatively safe, are not infectious by themselves, and do not require entry into the nucleus, without the risk of altering the gene. The mRNA vaccine is eliminated by enzymolysis after the translation of antigen protein is realized at the moment of short pause in vivo; 3) The mRNA vaccine has stronger immunogenicity, can induce stronger humoral immune response and cellular immune response, and can prevent virus from being outside cells and disinfect inside cells; 4) The mRNA vaccine research and development and preparation platform has the advantages of universality, low biological safety risk and strong industrialization capability, and can realize the standardized and safe production of vaccines. The large-scale production can be carried out only by changing the nucleotide sequence of mRNA, and other production equipment and condition items are not required to be changed.

The research and development of the novel coronavirus mRNA vaccine with independent intellectual property rights not only has important meaning to the current epidemic situation, but also can enrich the vaccine stock of normalized prevention and control. Successful development of a new corona mRNA vaccine depends largely on optimization of its own sequence. The sequence of an mRNA vaccine except for the coding region of the antigen gene can be considered as the backbone of an mRNA vaccine. The optimization of the mRNA vaccine framework sequence can improve the antigen expression efficiency, and can also realize the rapid research and development of different mRNA vaccines so as to deal with the outbreak of different infectious diseases and the variation of viruses.

Disclosure of Invention

The invention aims to provide mRNA based on a 1096 framework sequence and application thereof.

The invention provides an mRNA vaccine, wherein the mRNA is mRNA-1096, the mRNA-1096 comprises 5' -UTR8, mRNA of a target gene, 3' -UTR8 and Poly A which are sequentially connected, and the sequence of the 5' -UTR8 is obtained by replacing T in the 1 st to 50 th positions of the sequence 8 in a sequence table with U; the sequence of the 3' -UTR7 is obtained by replacing T in positions 822 to 956 of the sequence 8 in a sequence table with U; the sequence of the Poly A is obtained by replacing T in 957-1076 th positions of a sequence 8 in a sequence table with U.

The invention also claims an mRNA-1096 framework sequence, wherein the 1096 framework sequence comprises a5' -UTR8 connected to the 5' end of the mRNA of a target gene and a 3' -UTR8 and Poly A connected to the 3' end of the gene coding region of the target mRNA, and the sequence of the 5' -UTR8 is a sequence obtained by replacing T in the 1 st to 50 th positions of the sequence 8 in a sequence table with U; the sequence of the 3' -UTR7 is obtained by replacing T in positions 822 to 956 of a sequence 8 in a sequence table with U; the sequence of the Poly A is obtained by replacing T in 957-1076 th positions of a sequence 8 in a sequence table with U. And the application of the mRNA-1096 framework sequence in any one of the following parts is also within the protection scope of the invention:

1) Preparing an mRNA vaccine;

2) Preparing mRNA antibody;

3) Preparing mRNA cell factors;

4) Preparing related medicines used in mRNA therapy.

The mRNA-1096 skeleton sequence can be used for preparing mRNA vaccines, mRNA antibodies, mRNA cytokines or other applications, the mRNA of the target gene in the corresponding mRNA-1096 skeleton is different, and the mRNA can be applied to different fields according to the different properties of the mRNA of the target gene.

The invention also provides a vaccine, the active component of the vaccine is mRNA-1096 of claim 1, and the mRNA of the target gene is a sequence obtained by replacing T from 51 th to 821 th positions of a sequence 8 in a sequence table with U.

Wherein, the 5' end of the mRNA is connected with a Cap structure.

Wherein, the downstream of the polyA tail structure of the mRNA is connected with a recognition site sequence.

The application of the mRNA or the vaccine in the preparation of the novel coronary pneumonia vaccine product also belongs to the protection scope of the invention.

The invention also provides a biological material related to the vaccine, wherein the biological material is any one of B1) to B6):

b1 A nucleic acid molecule encoding the mRNA;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

b4 A recombinant plasmid containing the nucleic acid molecule according to B1) or a recombinant plasmid containing the expression cassette according to B2);

b5 A recombinant bacterium containing the nucleic acid molecule of B1), a recombinant bacterium containing the expression cassette of B2), or a recombinant bacterium containing the recombinant vector of B3);

b5 A transgenic cell line containing the nucleic acid molecule according to B1), or a transgenic cell line containing the expression cassette according to B2), or a transgenic cell line containing the recombinant vector according to B3).

Wherein, the recombinant vector is a recombinant vector which inserts a sequence shown in a sequence 8 in a sequence table into NheI-XhoI sites of a vector pcDNA3.1 (+) and keeps other sites of the vector unchanged.

Wherein, the recombinant bacterium is transformed into an Escherichia coli competent cell DH5 alpha of the recombinant plasmid.

The application of the above biological material in the preparation of vaccine products is also within the scope of the present invention, and further, the vaccine is a novel coronavirus vaccine.

The invention has the beneficial effects that: provides a novel vaccine for preventing and controlling the infection of the novel coronavirus and provides a technical reference for the research and development of other vaccines, antibodies, cytokines and the like based on mRNA.

Drawings

FIG. 1 is a schematic diagram of the structure of the vaccine of the present invention.

FIG. 2 is a schematic diagram of a recombinant vector structure in an embodiment of the present invention.

FIG. 3 is a schematic diagram of the structures of 13 vaccines in example 1 of the present invention.

Fig. 4 is a graph showing the results of mass analysis in example 1 of the present invention.

FIG. 5 is a graph showing the results of the expression test of cell-transfected proteins in example 1 of the present invention.

FIG. 6 is an evaluation of the in vivo efficacy of mice of the novel crown mRNA vaccine of example 2 of the present invention.

FIG. 7 shows neutralizing antibodies against the coronavirus of mice immunized with the vaccine sequences of mRNA-1096, mRNA-1710, mRNA-2568 and mRNA-4185 of example 2 of the present invention (NT) ₅₀ ) The situation is as follows.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 sequence design, preparation of novel coronavirus mRNA and detection of antigen expression by various cells in vitro

1. Sequence design of novel coronary mRNA vaccines

Sequence design of the novel coronary mRNA vaccine included the backbone code sequence of mRNA: 5'-Cap (5' -Cap); 5 '-untranslated region (5' -UTR); the 3 '-untranslated region (3' -UTR) and Poly A tail (Poly A) and the gene coding region (CDS) structural sequence of mRNA are shown in FIG. 1. CDS of new crown mRNA is composed of triplet codon from initiation codon AUG to termination codon, and determines amino acid sequence of different new crown antigen protein. In order to realize in vitro transcription of mRNA, a new crown mRNA sequence template is constructed between NheI-XhoI sites in a multiple cloning site region of pcDNA3.1 (+) to obtain a recombinant plasmid, and in vitro transcription is started under the action of T7 transcriptase by utilizing a T7 promoter sequence on a vector sequence, as shown in figure 2. Thus, 13 different new crown mRNA vaccine sequences were obtained by design optimization, as shown in fig. 3; the sequences of the 13 mRNAs are shown as the sequences in the sequence table:

1. sequence 1 (mRNA-1029):

5' -UTR1 from 1 to 49 at the 5' end, CDS (306-318 AA and RBD region encoding the novel crown S protein NTD), 3' -UTR1 from 821 to 889, and PolyA tail from 890 to 1009.

2. Sequence 2 (mRNA-1060):

5' -UTR2 from 5' end 1 to 37, CDS from 38 to 808 (306-318A A and RBD region of NTD encoding novel crown S protein), 3' -UTR2 from 809 to 920, poly A tail from 921 to 1040.

3. Sequence 3 (mRNA-1069):

5' -UTR3 from 1 to 46 at the 5' end, CDS from 47 to 817 (306-318A A and RBD region of NTD encoding the novel crown S protein), 3' -UTR3 from 818 to 929, and Poly A tail from 930 to 1049.

4. Sequence 4 (mRNA-1069H):

5' -UTR4 from 1 to 46 from the 5' end, CDS from 47 to 817 (306-318A A and RBD region of NTD encoding the novel crown S protein), 3' -UTR4 from 818 to 929, poly A tail from 930 to 1049.

5. Sequence 5 (mRNA-1072):

5' -UTR5 from 1 to 49 of the 5' end, CDS from 50 to 820 (306-318A A and RBD region of NTD encoding the novel crown S protein), 3' -UTR5 from 821 to 932, and Poly A tail from 933 to 1052.

6. Sequence 6 (mRNA-1073):

5' -UTR6 from 1 to 50 at the 5' end, CDS (306-318A A and RBD region of NTD encoding novel crown S protein) from 51 to 821, 3' -UTR6 from 822 to 933, poly A tail from 934 to 1053.

7. Sequence 7 (mRNA-1083):

5' -UTR7 from 1 to 46 at the 5' end, CDS (306-318A A and RBD region of NTD encoding the novel crown S protein) from 47 to 817, 3' -UTR7 from 818 to 943, and Poly A tail from 944 to 1063.

8. Sequence 8 (mRNA-1096):

5' -UTR8 from 5' 1 to 50, CDS from 51 to 821 (306-318A A and RBD region of NTD encoding novel crown S protein), 3' -UTR8 from 822 to 956, and Poly A tails from 957 to 1076.

9. Sequence 9 (mRNA-1710):

5' -UTR7 from 5' 1 to 46, CDS from 47 to 1444 (306-318 AA and two segments of the receptor binding region RBD encoding NTD of the novel crown S protein), 3' -UTR7 from 1445 to 1570, and Poly A tail from 1571 to 1690.

10. Sequence 10 (mRNA-1941):

5' -UTR7 from 5' 1 to 46, CDS from 47 to 1675 (15-318A A and receptor binding region RBD encoding NTD of the novel crown S protein), 3' -UTR7 from 1676 to 1801, poly A tail from 1802 to 1921.

11. Sequence 11 (mRNA-2073):

5' -UTR7 from 1 to 46 from the 5' end, CDS from 47 to 1807 (15-318A A, receptor binding region RBD and HR2 encoding NTD of the novel crown S protein), 3' -UTR7 from 1808 to 1933, and Poly A tails from 1934 to 2053.

12. Sequence 12 (mRNA-2586):

5' -UTR7 from 1 to 46 of the 5' end, CDS from 47 to 2320 (15-318A A and two segments of the receptor binding region RBD encoding NTD of the novel crown S protein), 3' -UTR7 from 2321 to 2446, and Poly A tail from 2447 to 2566.

13. Sequence 13 (mRNA-4185):

5' -UTR7 from 1 to 46 from the 5' end, CDS (encoding a novel crown S protein) from 47 to 3913, 3' -UTR7 from 3920 to 4045, and Poly A tails from 4046 to 4165.

2. In vitro synthesis of novel coronary mRNA vaccines

1. The 13 new crown mRNA vaccine sequences are subjected to DNA template synthesis and verified by sequencing.

2. Amplifying the DNA template synthesized in the step 1:

the DNA templates of the 13 mRNAs are respectively connected between NheI-XhoI sites of a vector pcDNA3.1 (+), so as to obtain 13 recombinant plasmids of pcDNA3.1-1029, pcDNA3.1-1060, pcDNA3.1-1069H, pcDNA3.1-1072, pcDNA3.1-1073, pcDNA3.1-1083, pcDNA3.1-1096, pcDNA3.1-1710, pcDNA3.1-1941, pcDNA3.1-2073, pcDNA3.1-2586 and pcDNA3.1-4185. The 13 recombinant plasmids were transformed into competent cells DH 5. Alpha. Respectively, and cultured in host E.coli to obtain a large amount of amplified cells. Then, recombinant plasmids amplified in the amplified bacteria were extracted with the aid of endotoxin-free plasmid macroextraction kits (Tiangen Biotech, beijing, ltd., DP 117), respectively. The amplified recombinant plasmids were linearized separately: utilizing XbaI and SapI to carry out enzyme digestion linearization on the extracted recombinant plasmid, obtaining a template for mRNA in vitro synthesis after purification, and adopting the Qubit ^TM Quantification was performed using the dsDNA BR Assay Kit (Invitrogen, Q32850).

3. Taking the DNA amplified in the step 2 as an in vitro synthesis template of mRNA, preparing a reaction system shown in the table 1, incubating at 37 ℃ for 1h for in vitro transcription to obtain a large amount of in vitro transcription RNA, and adopting Qubit ^TM Quantification was performed using the dsDNA BR Assay Kit (Invitrogen, Q32850).

TABLE 1 in vitro transcription reaction Using T7-FlashScribe ^TM Transcription kit (Cellscript, C-ASF 3507)

4. Purifying the in vitro transcription RNA product in the step 3: adding 1 μ l RNase-Free DNase I into the transcription reaction system, and incubating for 15min at 37 ℃ to remove the DNA template in the in vitro transcription product system to obtain the transcription product. And purifying the obtained transcription product by the following method:

(1) Addition of RNase-Free H to the transcript ₂ O make up volume 200. Mu.l;

(2) Add 200. Mu.l of mixture A (water-saturated phenol: chloroform: isoamyl alcohol, v: v: v,25: 24), vortex for 10s,4 ℃, centrifuge for 5min at 13800 Xg, then move the upper aqueous phase in the tube to a new tube;

(3) Adding an equal volume of the mixed solution B (chloroform: isoamyl alcohol, v: v,24: 1) into the new tube, vortexing for 10s, centrifuging at 13800 Xg for 5min, and then transferring the upper aqueous phase in the tube into the new tube;

(4) Adding 5M ammonium acetate solution with the same volume into a new tube, vortex, mixing uniformly, placing on ice for 15min, centrifuging at 4 ℃ and 13800 Xg for 15min, and removing supernatant.

(5) Adding 70% glacial ethanol to clean RNA, and discarding 70% ethanol; adding an appropriate amount of RNase-Free water (Solarbio, R1600) for resuspension, and adopting a Qubit ^TM RNA BR Assay Kit (Invitrogen, Q10211) Kit for quantification.

5. And (5) performing mRNA capping reaction on the RNA transcription purified product obtained in the step (4), wherein the specific steps are as follows:

(1) RNA denaturation: 60. Mu.g of the purified product of the transcription was taken, incubated at 65 ℃ for 15min for denaturation and then transferred to ice.

(2) And (3) mRNA capping reaction, namely adding the RNA denatured product, then preparing a reaction system according to the specification shown in the table 2, and incubating at 37 ℃ for 0.5h to obtain a capped product of which the mRNA has Cap 0.

TABLE 2 Cap 0 reaction System for mRNA preparation Using scriptCap ^TM Cap 1 trapping System kit (Cellscript, C-SCCS 1710).

mRNA capping product purification: the same as step 4.

Mass analysis of mRNA

The quality of the synthesized mRNA was analyzed using Agilent 2100 bioanalyzer and RNA Nano 6000Assay Kit (Aglient, 5067-1511) as follows:

(1) mRNA denaturation: mRNA and mRNA Ladder were denatured at 70 ℃ for 2min and then immediately ice-washed.

(2) Preparing a gel: RNA gel matrix was added to the filter tube as indicated, centrifuged at 1500 Xg for 10min at room temperature, and stored at 4 ℃ until use.

(3) Preparation of the gel-dye mixture: the RNA dye was equilibrated for 30min in the dark, then vortexed for several seconds, and after transient centrifugation a gel dye mixture was prepared at a ratio of 65.

(4) Loading the gel-dye mixture: prior to use of the RNA nano-chip, the chip-maker clamp was adjusted to the uppermost position. Put RNA chip into chip groove, add 9. Mu.l gel-dye mixture (without air bubbles) into the hole marked black G; closing the glue injector when the injector piston is at the position of 1ml, pressing the injector, fixing for 30s by using a clamp, then loosening, and pulling the piston back to the position of 1ml after 5 s; the injector was opened and 9. Mu.l of the gel-dye mixture was added to the other wells marked with white G.

(5) Loading a Marker: add 6. Mu.l of RNA Marker to all sample wells and ladder wells.

(6) Loading Ladder with mRNA: add 1. Mu.l ladder to the ladder pattern labeled wells, add mRNA to the remaining 12 wells (unused wells can be replaced by RNase-free water), place the chip on a chip vortex shaker, shake at 2400r/min for 1min, then place the chip in an Agilent 2100 instrument for detection within 5 min.

The results are shown in FIG. 4. The results showed that the bands of mRNA-1029 (FIG. 4-1), mRNA-1060 (FIG. 4-2), mRNA-1083 (FIG. 4-3), mRNA-1096 (FIG. 4-4), mRNA-1710 (FIG. 4-5), mRNA-1941 (FIG. 4-6), mRNA-2073 (FIG. 4-7), mRNA-2568 (FIG. 4-8) and mRNA-4185 (FIG. 4-9) were synthesized and all coincided with the target band. Sequencing shows that each product obtained is consistent with the mRNA sequence designed in the step one.

3. mRNA cell transfection protein expression assay

1. Inoculating cells: 293T cells (ATCC) were seeded in 6-well plates at 1X 10 per well, respectively ⁶ Individual cell, 37 ℃,5% CO ₂ Transfection was performed in the incubator until cells reached 80-90% confluency.

2. Preparing a transfection complex: transfection complexes of 13 mRNAs were prepared according to the following formulation.

By using

mRNA Transfection kit (Mirus, MIR 2250): mu.l of Opti-MEM + 2.5. Mu.g of mRNA + 5. Mu.l of mRNA boost reagent + 5. Mu.l of TransIT-mRNA reagent, mixed and left to stand at room temperature for 2-5min to form a transfection complex.

3. Transfecting cells: 13 respective drops of the transfection complexes into the cells and shaking up and down and left and right to evenly distribute the transfection complexes, 37 ℃,5% CO ₂ And (4) after incubation for 18h, collecting cells, and before and after transfection, replacing a cell culture solution.

4. Extracting total cell protein: after the cells were washed twice with PBS, the cells were vortexed using cell lysate RIPA (Jin Pulai, P06M 11) + proteinase inhibitor 100 × (Jin Pulai, P01C 01) to lyse the cells sufficiently. After ice-cooling for 30min, centrifuging at 4 deg.C for 13800 Xg for 15min, and collecting supernatant.

5. Quantification of total cellular protein: total protein in cell lysis supernatants was quantified using BCA protein quantification kit (Jin Pulai, P06M 16). Mixing the cell lysis supernatant with BCA working solution, incubating at 37 deg.C for 45min ₅₆₂ _nm The absorbance was measured, and the total cell protein concentration was calculated.

Western Blotting (WB) detection of target protein expression: prefabricated gel Bolt by protein electrophoresis ^TM Total Protein (30. Mu.g) was separated by 4 to 12%, bis-Tris,1.0mm, mini Protein Gel (Invitrogen, NW04120 BOX) electrophoresis (200V, 22 min). The separated proteins on the gel were transferred to iBlot 2Transfer stacks, PVDF (Invitrogen, IB 24001) membrane under gradient voltage (20V, 1min; 23V,4min;25V, 2min), incubated at room temperature in 1 XTBST containing 5% skimmed milk powder, 20r/min, and blocked for 1h. Anti-adoptive new coronavirus spike protein receptorRBD Rabbit polyclonal dilution (1. The membrane was washed with 1 XTBST at 60r/min, incubated at room temperature for 10min and repeated 3 times to completely remove the primary antibody residues. The Secondary Antibody was prepared from Goat Anti-Rabbit IgG Secondary Antibody diluent (1. The membrane was washed with 1 XTBST at 60r/min, incubated at room temperature for 10min and repeated 3 times to completely remove the secondary antibody residues. The HRP-labeled antibody-bound antigen was detected in a chemiluminescence apparatus using ECL chemiluminescence super-sensitivity chromogenic kit (assist in san Francisco, 36208ES 60) incubated with membrane chamber Wen Biguang for 3 min. Visualization of bands and protein marker by exposure, pageRuler ^TM The expression of the target antigen Protein was verified by alignment of the Prestained Protein Ladder (Invitrogen, 26617) bands. The antibody on the membrane is removed by using membrane regeneration liquid (Solebao, SW 3020), the membrane is sealed again, and then the internal reference antibody is incubated to detect the consistency of the amount of the protein loaded. Primary antibodies were diluted with β -actin Rabbit monoclonal antibodies (1.

The results are shown in FIG. 5, which shows: HEK-293t cells (shown in figure 5-1) were transfected by mRNA-1029, mRNA-1060, mRNA-1069, H, mRNA-1072, mRNA-1073, mRNA-1083 and mRNA-1096, and WB all detected the expression of the target antigen; after HEK-293t cells were transfected with mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568 (FIG. 5-2) and mRNA-4185 (FIG. 5-3) designed based on the mRNA-1083 backbone code sequence, WB detected the expression of the antigen of interest. The screened mRNA-1083, mRNA-1096, mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568 and mRNA-4185 are used as candidate sequences of the new crown mRNA vaccine to evaluate the effectiveness in vivo.

Example 2 conditions of antibody production following immunization of mice with the New crown mRNA vaccine

1. Preparation of high-efficiency delivery preparation of novel coronary mRNA vaccine

Preparation of the preparation and Shanghai microbial company cooperate to prepare a new crown mRNA-LPPs lipid polymer vaccine.

2. In vivo efficacy evaluation of novel coronary mRNA vaccines

Experimental animals BALB/c mice (female, 6-8 weeks, 16-18g, beijing Bei Fu) were randomly assigned as shown in Table 3 (5 mice per group), and mice were immunized by intramuscular injection. Adequate mouse sera were obtained by orbital bleeds on day 10 after each immunization. Detecting the generation and titer of RBD specific antibodies in the serum of the immunized mouse by an ELISA method; and detecting the generation and dripping degree of a pseudovirus neutralizing antibody in the serum of the immunized mouse by using the new corona S protein pseudovirus, and evaluating the in vivo immunization efficacy of the new corona mRNA vaccine.

The ELISA method is used for detecting the condition of the RBD specific antibody in the serum of the immunized mouse, and the specific detection method comprises the following steps:

(1) Coating: RBD protein (40592-VNAH, yinqiao, proteus) was diluted to 1 ng/. Mu.l in carbonate buffer (50mM, pH 9.6, 0.22 μm filter). Adding 100 mu g of RBD protein diluent into each hole of a 96-hole plate, sealing, and standing at 4 ℃ overnight;

(2) Washing the plate: after overnight coating, pour 96-well plate to remove protein coating solution, add 200 μ L of washing solution (1 × TBS containing 0.2% Tween-20) per well, shake gently with hand for 30s, pat dry on paper, repeat 6 times;

(3) And (3) sealing: add 200. Mu.l of blocking solution (1 × TBS containing 2% BSA) to each well, incubate at 37 ℃ for 2h;

(4) Washing the plate: pour 96-well plate to remove blocking solution, add 200. Mu.l of washing solution (1 × TBS containing 0.2% Tween-20) per well, shake gently with hand for 30s, pat dry on paper, repeat 6 times;

(5) Incubating the primary antibody: diluting immune mouse serum with antibody diluent (containing 0.5% BSA washing solution) 10 times to obtain 10 times of diluted solution ^-1 To 10 ^-6 Adding 100 mul serum diluent into each hole of serum with different dilutions, and incubating for 2h at 37 ℃ in an incubator;

(6) Washing the plate: pouring a 96-well plate to remove serum diluent, adding 200 mu L of washing solution (1 xTBS containing 0.2% Tween-20) into each well, slightly shaking by hand for 30s, then patting dry on paper, and repeating for 6 times;

(7) Incubation of secondary antibody: diluting goat anti-mouse IgG (H + L) (Biyunyan, A0216) labeled with horseradish peroxidase 250 times with antibody diluent (containing 0.5% BSA washing solution) to obtain secondary antibody diluent, adding 100 μ L of secondary antibody diluent into each well, and incubating at 37 deg.C in incubator for 1H;

(8) Washing the plate: pouring a 96-well plate to remove the secondary antibody diluent, adding 200 μ l of washing solution (1 × TBS containing 0.2% Tween-20) into each well, shaking gently by hand for 30s, patting dry on paper, and repeating for 6 times;

(9) Color development: adding 100 μ l TMB substrate (Tiangen) into each well, incubating at room temperature in dark for 20min;

(10) And (3) stopping color development: add 50. Mu.L of 2M H per well ₂ SO ₄ Detection of A on a microplate reader ₄₅₀ OD value of (1);

(11) And (3) judging the serum antibody titer: the OD of a certain diluted serum/OD of a negative control is more than or equal to 2.1, and the OD/OD of the negative control is less than 2.1, wherein the dilution is the corresponding antibody titer of the serum sample (if the OD of the negative control is less than 0.05, the calculation is carried out according to 0.05).

Table 3: mouse intramuscular injection vaccine sequence and immune condition

The results are shown in FIG. 6: RBD-specific IgG antibodies were detected in the sera of immunized mice mRNA-1096, mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568 and mRNA-4185. Wherein the titer of the primary immune serum of the mRNA-1096 and mRNA-1710 immunized mice reaches about 1:10000.

the new crown S protein pseudovirus is used for detecting the condition of a pseudovirus neutralizing antibody in the serum of an immunized mouse, and the specific detection method comprises the following steps:

(1) Cell plating: one day prior to the experiment, HEK293T-ACE2 cells to be infected (Yeasen Biotech, HB 200710) were seeded in 96-well cell culture plates at an inoculum size of about 1X 10 ⁴ One cell/well, next day, pseudoviral infection was performed with cell density around 30%. HEK293T-ACE2 cell culture fluid is 90% DMEM +10% FBS +1% diabody +0.75 mug/mL puromycin;

(2) Immune sera inhibited pseudovirus infection: diluting immune mouse serum with cell culture solution by 10 times gradient to obtain 10 ^-1 To 10 ^-6 Serum at different dilutions. The cryopreserved pseudovirus was removed (titer 1X 10) ⁶ TU/mL, 0.5. Mu.l) (Yeasen Biotech, HB 200707) was thawed on ice, mixed with 100. Mu.l of the diluted serum, incubated at 37 ℃ for 1h in a incubator, and then the cultured cells were added. Replacing a fresh culture medium for continuous culture after 6 hours of virus infection;

(3) Infection detection: 48h after the cells are infected with pseudovirus and the solution is changed, the expression of green fluorescent protein is observed by a fluorescence microscope and the activity of luciferase is detected by a firefly luciferase reporter gene detection kit (Yeasen Biotech, HB 181218).

(4) Serum pseudovirus neutralizing antibody titers (NT) ₅₀ ) And (3) detection: NT (NT) ₅₀ Defined as the serum dilution fold at which the pseudoviral control group produced 50% reduction in fluorescent light units (RLUs). NT ₅₀ Determined by GraphPad Prism 6.0 software nonlinear regression log (inhibitor) v.s. normalized response (Variable slope).

The results are shown in FIG. 7: the serum antibodies of the immunized mice of mRNA-1096, mRNA-1710, mRNA-2568 and mRNA-4185 all have the neutralizing activity of the new crown S protein pseudovirus. Primary and secondary serum neutralizing antibodies (NT) against the mRNA vaccine against the new crown S protein pseudovirus ₅₀ ) The titers of the neutralizing antibodies against the vims-derived serum-clear coronavirus proteins of immunized mice mRNA-1096, mRNA-1710 and mRNA-2568 shown in Table 4 (NT) ₅₀ ) The titer of the serum pseudovirus NT of the hyperimmune mouse is better than that of the serum pseudovirus NT of the RBD-mRNA-LNPs vaccine of the New York blood center under the same dose ₅₀ The titer (1. Wherein mRNA-1096 priming mouse serum pseudovirus NT ₅₀ The titers all exceed 1.

TABLE 4 neutralizing antibodies against serum Neocoronaviruses (NT) from mice immunized with neocoronaviruses mRNA-1096, mRNA-1710, mRNA-2568 and mRNA-4185 vaccine sequences ₅₀ ) The titer.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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<400> 1

cacaggctct ttttttgcag aagcttaaaa taaacgctca gctttgacca tggtgttcgt 60

gttcctggtg ctgctgcccc tggtgagcag cttcaccgtg gagaagggca tctaccagac 120

cagcaacttc cgcgtgcagc ccaccgagag catcgtgcgc ttccccaaca tcaccaacct 180

gtgccccttc ggcgaggtgt tcaacgccac ccgcttcgcc agcgtgtacg cctggaaccg 240

caagcgcatc agcaactgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag 300

caccttcaag tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt 360

gtacgccgac agcttcgtga tccgcggcga cgaggtgcgc cagatcgccc ccggccagac 420

cggcaagatc gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc 480

ctggaacagc aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtaccgcct 540

gttccgcaag agcaacctga agcccttcga gcgcgacatc agcaccgaga tctaccaggc 600

cggcagcacc ccctgcaacg gcgtggaggg cttcaactgc tacttccccc tgcagagcta 660

cggcttccag cccaccaacg gcgtgggcta ccagccctac cgcgtggtgg tgctgagctt 720

cgagctgctg cacgcccccg ccaccgtgtg cggccccaag aagtccacca acctggtgaa 780

gaacaagtgc gtgaacttcc accaccacca ccaccactaa accaacctca agaacatgtg 840

actggagtct cttagctaca cagaaacaaa atctcgtttt ttttcaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gaagagc 1017

<210> 2

<211> 1048

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

actcttctgg tccccacaga ctcagagaga acccaccatg gtgttcgtgt tcctggtgct 60

gctgcccctg gtgagcagct tcaccgtgga gaagggcatc taccagacca gcaacttccg 120

cgtgcagccc accgagagca tcgtgcgctt ccccaacatc accaacctgt gccccttcgg 180

cgaggtgttc aacgccaccc gcttcgccag cgtgtacgcc tggaaccgca agcgcatcag 240

caactgcgtg gccgactaca gcgtgctgta caacagcgcc agcttcagca ccttcaagtg 300

ctacggcgtg agccccacca agctgaacga cctgtgcttc accaacgtgt acgccgacag 360

cttcgtgatc cgcggcgacg aggtgcgcca gatcgccccc ggccagaccg gcaagatcgc 420

cgactacaac tacaagctgc ccgacgactt caccggctgc gtgatcgcct ggaacagcaa 480

caacctggac agcaaggtgg gcggcaacta caactacctg taccgcctgt tccgcaagag 540

caacctgaag cccttcgagc gcgacatcag caccgagatc taccaggccg gcagcacccc 600

ctgcaacggc gtggagggct tcaactgcta cttccccctg cagagctacg gcttccagcc 660

caccaacggc gtgggctacc agccctaccg cgtggtggtg ctgagcttcg agctgctgca 720

cgcccccgcc accgtgtgcg gccccaagaa gtccaccaac ctggtgaaga acaagtgcgt 780

gaacttccac caccaccacc accactaagc tggagcctcg gtggccatgc ttcttgcccc 840

ttgggcctcc ccccagcccc tcctcccctt cctgcacccg tacccccgtg gtctttgaat 900

aaagtctgag tgggcggcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa agaagagc 1048

<210> 3

<211> 1057

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccatgg tgttcgtgtt 60

cctggtgctg ctgcccctgg tgagcagctt caccgtggag aagggcatct accagaccag 120

caacttccgc gtgcagccca ccgagagcat cgtgcgcttc cccaacatca ccaacctgtg 180

ccccttcggc gaggtgttca acgccacccg cttcgccagc gtgtacgcct ggaaccgcaa 240

gcgcatcagc aactgcgtgg ccgactacag cgtgctgtac aacagcgcca gcttcagcac 300

cttcaagtgc tacggcgtga gccccaccaa gctgaacgac ctgtgcttca ccaacgtgta 360

cgccgacagc ttcgtgatcc gcggcgacga ggtgcgccag atcgcccccg gccagaccgg 420

caagatcgcc gactacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg 480

gaacagcaac aacctggaca gcaaggtggg cggcaactac aactacctgt accgcctgtt 540

ccgcaagagc aacctgaagc ccttcgagcg cgacatcagc accgagatct accaggccgg 600

cagcaccccc tgcaacggcg tggagggctt caactgctac ttccccctgc agagctacgg 660

cttccagccc accaacggcg tgggctacca gccctaccgc gtggtggtgc tgagcttcga 720

gctgctgcac gcccccgcca ccgtgtgcgg ccccaagaag tccaccaacc tggtgaagaa 780

caagtgcgtg aacttccacc accaccacca ccactaaggc tcagcaacaa cagcagcaga 840

agtctcaaca tcagacatca gttaattata tgcaatcaaa ctgacaaagc ttgtgaaaga 900

atgttctgaa ataaacattt taaccattaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gaagagc 1057

<210> 4

<211> 1057

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccatgg tgttcgtgtt 60

cctggtgctg ctgcccctgg tgagcagctt caccgtggag aagggcatct accagaccag 120

caacttccgc gtgcagccca ccgagagcat cgtgcgcttc cccaacatca ccaacctgtg 180

ccccttcggc gaggtgttca acgccacccg cttcgccagc gtgtacgcct ggaaccgcaa 240

gcgcatcagc aactgcgtgg ccgactacag cgtgctgtac aacagcgcca gcttcagcac 300

cttcaagtgc tacggcgtga gccccaccaa gctgaacgac ctgtgcttca ccaacgtgta 360

cgccgacagc ttcgtgatcc gcggcgacga ggtgcgccag atcgcccccg gccagaccgg 420

caagatcgcc gactacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg 480

gaacagcaac aacctggaca gcaaggtggg cggcaactac aactacctgt accgcctgtt 540

ccgcaagagc aacctgaagc ccttcgagcg cgacatcagc accgagatct accaggccgg 600

cagcaccccc tgcaacggcg tggagggctt caactgctac ttccccctgc agagctacgg 660

cttccagccc accaacggcg tgggctacca gccctaccgc gtggtggtgc tgagcttcga 720

gctgctgcac gcccccgcca ccgtgtgcgg ccccaagaag tccaccaacc tggtgaagaa 780

caagtgcgtg aacttccacc accaccacca ccactaagct ggagcctcgg tggccatgct 840

tcttgcccct tgggcctccc cccagcccct cctccccttc ctgcacccgt acccccgtgg 900

tctttgaata aagtctgagt gggcggcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gaagagc 1057

<210> 5

<211> 1060

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cacaggctct ttttttgcag aagcttaaaa taaacgctca gctttgacca tggtgttcgt 60

gttcctggtg ctgctgcccc tggtgagcag cttcaccgtg gagaagggca tctaccagac 120

cagcaacttc cgcgtgcagc ccaccgagag catcgtgcgc ttccccaaca tcaccaacct 180

gtgccccttc ggcgaggtgt tcaacgccac ccgcttcgcc agcgtgtacg cctggaaccg 240

caagcgcatc agcaactgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag 300

caccttcaag tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt 360

gtacgccgac agcttcgtga tccgcggcga cgaggtgcgc cagatcgccc ccggccagac 420

cggcaagatc gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc 480

ctggaacagc aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtaccgcct 540

gttccgcaag agcaacctga agcccttcga gcgcgacatc agcaccgaga tctaccaggc 600

cggcagcacc ccctgcaacg gcgtggaggg cttcaactgc tacttccccc tgcagagcta 660

cggcttccag cccaccaacg gcgtgggcta ccagccctac cgcgtggtgg tgctgagctt 720

cgagctgctg cacgcccccg ccaccgtgtg cggccccaag aagtccacca acctggtgaa 780

gaacaagtgc gtgaacttcc accaccacca ccaccactaa ggctcagcag taacagtagc 840

agaagtttgg acatcagaca tcagttaatg acaaacaatc aaactgacac agcttgtgaa 900

agaatgttct gaaataaaca tttttaaaat taaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagaagagc 1060

<210> 6

<211> 1061

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgttcg 60

tgttcctggt gctgctgccc ctggtgagca gcttcaccgt ggagaagggc atctaccaga 120

ccagcaactt ccgcgtgcag cccaccgaga gcatcgtgcg cttccccaac atcaccaacc 180

tgtgcccctt cggcgaggtg ttcaacgcca cccgcttcgc cagcgtgtac gcctggaacc 240

gcaagcgcat cagcaactgc gtggccgact acagcgtgct gtacaacagc gccagcttca 300

gcaccttcaa gtgctacggc gtgagcccca ccaagctgaa cgacctgtgc ttcaccaacg 360

tgtacgccga cagcttcgtg atccgcggcg acgaggtgcg ccagatcgcc cccggccaga 420

ccggcaagat cgccgactac aactacaagc tgcccgacga cttcaccggc tgcgtgatcg 480

cctggaacag caacaacctg gacagcaagg tgggcggcaa ctacaactac ctgtaccgcc 540

tgttccgcaa gagcaacctg aagcccttcg agcgcgacat cagcaccgag atctaccagg 600

ccggcagcac cccctgcaac ggcgtggagg gcttcaactg ctacttcccc ctgcagagct 660

acggcttcca gcccaccaac ggcgtgggct accagcccta ccgcgtggtg gtgctgagct 720

tcgagctgct gcacgccccc gccaccgtgt gcggccccaa gaagtccacc aacctggtga 780

agaacaagtg cgtgaacttc caccaccacc accaccacta agctggagcc tcggtggcca 840

tgcttcttgc cccttgggcc tccccccagc ccctcctccc cttcctgcac ccgtaccccc 900

gtggtctttg aataaagtct gagtgggcgg caaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagaagag c 1061

<210> 7

<211> 1071

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccatgg tgttcgtgtt 60

cctggtgctg ctgcccctgg tgagcagctt caccgtggag aagggcatct accagaccag 120

caacttccgc gtgcagccca ccgagagcat cgtgcgcttc cccaacatca ccaacctgtg 180

ccccttcggc gaggtgttca acgccacccg cttcgccagc gtgtacgcct ggaaccgcaa 240

gcgcatcagc aactgcgtgg ccgactacag cgtgctgtac aacagcgcca gcttcagcac 300

cttcaagtgc tacggcgtga gccccaccaa gctgaacgac ctgtgcttca ccaacgtgta 360

cgccgacagc ttcgtgatcc gcggcgacga ggtgcgccag atcgcccccg gccagaccgg 420

caagatcgcc gactacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg 480

gaacagcaac aacctggaca gcaaggtggg cggcaactac aactacctgt accgcctgtt 540

ccgcaagagc aacctgaagc ccttcgagcg cgacatcagc accgagatct accaggccgg 600

cagcaccccc tgcaacggcg tggagggctt caactgctac ttccccctgc agagctacgg 660

cttccagccc accaacggcg tgggctacca gccctaccgc gtggtggtgc tgagcttcga 720

gctgctgcac gcccccgcca ccgtgtgcgg ccccaagaag tccaccaacc tggtgaagaa 780

caagtgcgtg aacttccacc accaccacca ccactaaacc agcctcaaga acacccgaat 840

ggagtctcta agctacataa taccaactta cactttacaa aatgttgtcc cccaaaatgt 900

agccattcgt atctgctcct aataaaaaga aagtttcttc acaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagaagag c 1071

<210> 8

<211> 1084

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgttcg 60

tgttcctggt gctgctgccc ctggtgagca gcttcaccgt ggagaagggc atctaccaga 120

ccagcaactt ccgcgtgcag cccaccgaga gcatcgtgcg cttccccaac atcaccaacc 180

tgtgcccctt cggcgaggtg ttcaacgcca cccgcttcgc cagcgtgtac gcctggaacc 240

gcaagcgcat cagcaactgc gtggccgact acagcgtgct gtacaacagc gccagcttca 300

gcaccttcaa gtgctacggc gtgagcccca ccaagctgaa cgacctgtgc ttcaccaacg 360

tgtacgccga cagcttcgtg atccgcggcg acgaggtgcg ccagatcgcc cccggccaga 420

ccggcaagat cgccgactac aactacaagc tgcccgacga cttcaccggc tgcgtgatcg 480

cctggaacag caacaacctg gacagcaagg tgggcggcaa ctacaactac ctgtaccgcc 540

tgttccgcaa gagcaacctg aagcccttcg agcgcgacat cagcaccgag atctaccagg 600

ccggcagcac cccctgcaac ggcgtggagg gcttcaactg ctacttcccc ctgcagagct 660

acggcttcca gcccaccaac ggcgtgggct accagcccta ccgcgtggtg gtgctgagct 720

tcgagctgct gcacgccccc gccaccgtgt gcggccccaa gaagtccacc aacctggtga 780

agaacaagtg cgtgaacttc caccaccacc accaccacta agctcgcttt cttgctgtcc 840

aatttctatt aaaggttcct ttgttcccta agtccaacta ctaaactggg ggatattatg 900

aagggccttg agcatctgga ttctgcctaa taaaaaacat ttattttcat tgcaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagaa 1080

gagc 1084

<210> 9

<211> 1698

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccatgg tgttcgtgtt 60

cctggtgctg ctgcccctgg tgagcagctt caccgtggag aagggcatct accagaccag 120

caacttccgc gtgcagccca ccgagagcat cgtgcgcttc cccaacatca ccaacctgtg 180

ccccttcggc gaggtgttca acgccacccg cttcgccagc gtgtacgcct ggaaccgcaa 240

gcgcatcagc aactgcgtgg ccgactacag cgtgctgtac aacagcgcca gcttcagcac 300

cttcaagtgc tacggcgtga gccccaccaa gctgaacgac ctgtgcttca ccaacgtgta 360

cgccgacagc ttcgtgatcc gcggcgacga ggtgcgccag atcgcccccg gccagaccgg 420

caagatcgcc gactacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg 480

gaacagcaac aacctggaca gcaaggtggg cggcaactac aactacctgt accgcctgtt 540

ccgcaagagc aacctgaagc ccttcgagcg cgacatcagc accgagatct accaggccgg 600

cagcaccccc tgcaacggcg tggagggctt caactgctac ttccccctgc agagctacgg 660

cttccagccc accaacggcg tgggctacca gccctaccgc gtggtggtgc tgagcttcga 720

gctgctgcac gcccccgcca ccgtgtgcgg ccccaagaag tccaccaacc tggtgaagaa 780

caagcgcgtg cagcccaccg agagcatcgt gcgcttcccc aacatcacca acctgtgccc 840

cttcggcgag gtgttcaacg ccacccgctt cgccagcgtg tacgcctgga accgcaagcg 900

catcagcaac tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt 960

caagtgctac ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc 1020

cgacagcttc gtgatccgcg gcgacgaggt gcgccagatc gcccccggcc agaccggcaa 1080

gatcgccgac tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa 1140

cagcaacaac ctggacagca aggtgggcgg caactacaac tacctgtacc gcctgttccg 1200

caagagcaac ctgaagccct tcgagcgcga catcagcacc gagatctacc aggccggcag 1260

caccccctgc aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt 1320

ccagcccacc aacggcgtgg gctaccagcc ctaccgcgtg gtggtgctga gcttcgagct 1380

gctgcacgcc cccgccaccg tgtgcggccc caagaagtcc accaacctgg tgaagaacaa 1440

gtaaaccagc ctcaagaaca cccgaatgga gtctctaagc tacataatac caacttacac 1500

tttacaaaat gttgtccccc aaaatgtagc cattcgtatc tgctcctaat aaaaagaaag 1560

tttcttcaca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa agaagagc 1698

<210> 10

<211> 1929

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccatgg tgttcgtgtt 60

cctggtgctg ctgcccctgg tgagcagcca gtgcgtgaac ctgaccaccc gcacccagct 120

gccccccgcc tacaccaaca gcttcacccg cggcgtgtac taccccgaca aggtgttccg 180

cagcagcgtg ctgcacagca cccaggacct gttcctgccc ttcttcagca acgtgacctg 240

gttccacgcc atccacgtga gcggcaccaa cggcaccaag cgcttcgaca accccgtgct 300

gcccttcaac gacggcgtgt acttcgccag caccgagaaa agcaacatca tccgcggctg 360

gatcttcggc accaccctgg acagcaagac ccagagcctg ctgatcgtga acaacgccac 420

caacgtggtg atcaaggtgt gcgagttcca gttctgcaac gaccccttcc tgggcgtgta 480

ctaccacaag aacaacaaga gctggatgga gagcgagttc cgcgtgtaca gcagcgccaa 540

caactgcacc ttcgagtacg tgagccagcc cttcctgatg gacctggagg gcaagcaggg 600

caacttcaag aacctgcgcg agttcgtgtt caagaacatc gacggctact tcaagatcta 660

cagcaagcac acccccatca acctggtgcg cgacctgccc cagggcttca gcgccctgga 720

gcccctggtg gacctgccca tcggcatcaa catcacccgc ttccagaccc tgctggccct 780

gcaccgcagc tacctgaccc ccggcgacag cagcagcggc tggaccgccg gcgccgccgc 840

ctactacgtg ggctacctgc agccccgcac cttcctgctg aagtacaacg agaacggcac 900

catcaccgac gccgtggact gcgccctgga ccccctgagc gagaccaagt gcaccctgaa 960

gtccttcacc gtggagaagg gcatctacca gaccagcaac ttccgcgtgc agcccaccga 1020

gagcatcgtg cgcttcccca acatcaccaa cctgtgcccc ttcggcgagg tgttcaacgc 1080

cacccgcttc gccagcgtgt acgcctggaa ccgcaagcgc atcagcaact gcgtggccga 1140

ctacagcgtg ctgtacaaca gcgccagctt cagcaccttc aagtgctacg gcgtgagccc 1200

caccaagctg aacgacctgt gcttcaccaa cgtgtacgcc gacagcttcg tgatccgcgg 1260

cgacgaggtg cgccagatcg cccccggcca gaccggcaag atcgccgact acaactacaa 1320

gctgcccgac gacttcaccg gctgcgtgat cgcctggaac agcaacaacc tggacagcaa 1380

ggtgggcggc aactacaact acctgtaccg cctgttccgc aagagcaacc tgaagccctt 1440

cgagcgcgac atcagcaccg agatctacca ggccggcagc accccctgca acggcgtgga 1500

gggcttcaac tgctacttcc ccctgcagag ctacggcttc cagcccacca acggcgtggg 1560

ctaccagccc taccgcgtgg tggtgctgag cttcgagctg ctgcacgccc ccgccaccgt 1620

gtgcggcccc aagaagtcca ccaacctggt gaagaacaag tgcgtgaact tctaaaccag 1680

cctcaagaac acccgaatgg agtctctaag ctacataata ccaacttaca ctttacaaaa 1740

tgttgtcccc caaaatgtag ccattcgtat ctgctcctaa taaaaagaaa gtttcttcac 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920

aagaagagc 1929

<210> 11

<211> 2061

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccatgg tgttcgtgtt 60

cctggtgctg ctgcccctgg tgagcagcca gtgcgtgaac ctgaccaccc gcacccagct 120

gccccccgcc tacaccaaca gcttcacccg cggcgtgtac taccccgaca aggtgttccg 180

cagcagcgtg ctgcacagca cccaggacct gttcctgccc ttcttcagca acgtgacctg 240

gttccacgcc atccacgtga gcggcaccaa cggcaccaag cgcttcgaca accccgtgct 300

gcccttcaac gacggcgtgt acttcgccag caccgagaaa agcaacatca tccgcggctg 360

gatcttcggc accaccctgg acagcaagac ccagagcctg ctgatcgtga acaacgccac 420

caacgtggtg atcaaggtgt gcgagttcca gttctgcaac gaccccttcc tgggcgtgta 480

ctaccacaag aacaacaaga gctggatgga gagcgagttc cgcgtgtaca gcagcgccaa 540

caactgcacc ttcgagtacg tgagccagcc cttcctgatg gacctggagg gcaagcaggg 600

caacttcaag aacctgcgcg agttcgtgtt caagaacatc gacggctact tcaagatcta 660

cagcaagcac acccccatca acctggtgcg cgacctgccc cagggcttca gcgccctgga 720

gcccctggtg gacctgccca tcggcatcaa catcacccgc ttccagaccc tgctggccct 780

gcaccgcagc tacctgaccc ccggcgacag cagcagcggc tggaccgccg gcgccgccgc 840

ctactacgtg ggctacctgc agccccgcac cttcctgctg aagtacaacg agaacggcac 900

catcaccgac gccgtggact gcgccctgga ccccctgagc gagaccaagt gcaccctgaa 960

gtccttcacc gtggagaagg gcatctacca gaccagcaac ttccgcgtgc agcccaccga 1020

gagcatcgtg cgcttcccca acatcaccaa cctgtgcccc ttcggcgagg tgttcaacgc 1080

cacccgcttc gccagcgtgt acgcctggaa ccgcaagcgc atcagcaact gcgtggccga 1140

ctacagcgtg ctgtacaaca gcgccagctt cagcaccttc aagtgctacg gcgtgagccc 1200

caccaagctg aacgacctgt gcttcaccaa cgtgtacgcc gacagcttcg tgatccgcgg 1260

cgacgaggtg cgccagatcg cccccggcca gaccggcaag atcgccgact acaactacaa 1320

gctgcccgac gacttcaccg gctgcgtgat cgcctggaac agcaacaacc tggacagcaa 1380

ggtgggcggc aactacaact acctgtaccg cctgttccgc aagagcaacc tgaagccctt 1440

cgagcgcgac atcagcaccg agatctacca ggccggcagc accccctgca acggcgtgga 1500

gggcttcaac tgctacttcc ccctgcagag ctacggcttc cagcccacca acggcgtggg 1560

ctaccagccc taccgcgtgg tggtgctgag cttcgagctg ctgcacgccc ccgccaccgt 1620

gtgcggcccc aagaagtcca ccaacctggt gaagaacaag tgcgtgaact tcggcagcgc 1680

cagcgacgtg gacctgggcg acatcagcgg catcaacgcc agcgtggtga acatccagaa 1740

ggagatcgac cgcctgaacg aggtggccaa gaacctgaac gagagcctga tcgacctgca 1800

ggagtaaacc agcctcaaga acacccgaat ggagtctcta agctacataa taccaactta 1860

cactttacaa aatgttgtcc cccaaaatgt agccattcgt atctgctcct aataaaaaga 1920

aagtttcttc acaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040

aaaaaaaaaa aaaagaagag c 2061

<210> 12

<211> 2574

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccatgg tgttcgtgtt 60

cctggtgctg ctgcccctgg tgagcagcca gtgcgtgaac ctgaccaccc gcacccagct 120

gccccccgcc tacaccaaca gcttcacccg cggcgtgtac taccccgaca aggtgttccg 180

cagcagcgtg ctgcacagca cccaggacct gttcctgccc ttcttcagca acgtgacctg 240

gttccacgcc atccacgtga gcggcaccaa cggcaccaag cgcttcgaca accccgtgct 300

gcccttcaac gacggcgtgt acttcgccag caccgagaaa agcaacatca tccgcggctg 360

gatcttcggc accaccctgg acagcaagac ccagagcctg ctgatcgtga acaacgccac 420

caacgtggtg atcaaggtgt gcgagttcca gttctgcaac gaccccttcc tgggcgtgta 480

ctaccacaag aacaacaaga gctggatgga gagcgagttc cgcgtgtaca gcagcgccaa 540

caactgcacc ttcgagtacg tgagccagcc cttcctgatg gacctggagg gcaagcaggg 600

caacttcaag aacctgcgcg agttcgtgtt caagaacatc gacggctact tcaagatcta 660

cagcaagcac acccccatca acctggtgcg cgacctgccc cagggcttca gcgccctgga 720

gcccctggtg gacctgccca tcggcatcaa catcacccgc ttccagaccc tgctggccct 780

gcaccgcagc tacctgaccc ccggcgacag cagcagcggc tggaccgccg gcgccgccgc 840

ctactacgtg ggctacctgc agccccgcac cttcctgctg aagtacaacg agaacggcac 900

catcaccgac gccgtggact gcgccctgga ccccctgagc gagaccaagt gcaccctgaa 960

gtccttcacc gtggagaagg gcatctacca gaccagcaac ttccgcgtgc agcccaccga 1020

gagcatcgtg cgcttcccca acatcaccaa cctgtgcccc ttcggcgagg tgttcaacgc 1080

cacccgcttc gccagcgtgt acgcctggaa ccgcaagcgc atcagcaact gcgtggccga 1140

ctacagcgtg ctgtacaaca gcgccagctt cagcaccttc aagtgctacg gcgtgagccc 1200

caccaagctg aacgacctgt gcttcaccaa cgtgtacgcc gacagcttcg tgatccgcgg 1260

cgacgaggtg cgccagatcg cccccggcca gaccggcaag atcgccgact acaactacaa 1320

gctgcccgac gacttcaccg gctgcgtgat cgcctggaac agcaacaacc tggacagcaa 1380

ggtgggcggc aactacaact acctgtaccg cctgttccgc aagagcaacc tgaagccctt 1440

cgagcgcgac atcagcaccg agatctacca ggccggcagc accccctgca acggcgtgga 1500

gggcttcaac tgctacttcc ccctgcagag ctacggcttc cagcccacca acggcgtggg 1560

ctaccagccc taccgcgtgg tggtgctgag cttcgagctg ctgcacgccc ccgccaccgt 1620

gtgcggcccc aagaagtcca ccaacctggt gaagaacaag cgcgtgcagc ccaccgagag 1680

catcgtgcgc ttccccaaca tcaccaacct gtgccccttc ggcgaggtgt tcaacgccac 1740

ccgcttcgcc agcgtgtacg cctggaaccg caagcgcatc agcaactgcg tggccgacta 1800

cagcgtgctg tacaacagcg ccagcttcag caccttcaag tgctacggcg tgagccccac 1860

caagctgaac gacctgtgct tcaccaacgt gtacgccgac agcttcgtga tccgcggcga 1920

cgaggtgcgc cagatcgccc ccggccagac cggcaagatc gccgactaca actacaagct 1980

gcccgacgac ttcaccggct gcgtgatcgc ctggaacagc aacaacctgg acagcaaggt 2040

gggcggcaac tacaactacc tgtaccgcct gttccgcaag agcaacctga agcccttcga 2100

gcgcgacatc agcaccgaga tctaccaggc cggcagcacc ccctgcaacg gcgtggaggg 2160

cttcaactgc tacttccccc tgcagagcta cggcttccag cccaccaacg gcgtgggcta 2220

ccagccctac cgcgtggtgg tgctgagctt cgagctgctg cacgcccccg ccaccgtgtg 2280

cggccccaag aagtccacca acctggtgaa gaacaagtaa accagcctca agaacacccg 2340

aatggagtct ctaagctaca taataccaac ttacacttta caaaatgttg tcccccaaaa 2400

tgtagccatt cgtatctgct cctaataaaa agaaagtttc ttcacaaaaa aaaaaaaaaa 2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagaa gagc 2574

<210> 13

<211> 4173

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccggat ccgccatgtt 60

cgtgttcctg gtgctgctgc ccctggtgag cagccagtgc gtgaacctga ccacccgcac 120

ccagctgccc cccgcctaca ccaacagctt cacccgcggc gtgtactacc ccgacaaggt 180

gttccgcagc agcgtgctgc acagcaccca ggacctgttc ctgcccttct tcagcaacgt 240

gacctggttc cacgccatcc acgtgagcgg caccaacggc accaagcgct tcgacaaccc 300

cgtgctgccc ttcaacgacg gcgtgtactt cgccagcacc gagaagtcca acatcatccg 360

cggctggatc ttcggcacca ccctggacag caagacccag agcctgctga tcgtgaacaa 420

cgccaccaac gtggtgatca aggtgtgcga gttccagttc tgcaacgacc ccttcctggg 480

cgtgtactac cacaagaaca acaagagctg gatggagagc gagttccgcg tgtacagcag 540

cgccaacaac tgcaccttcg agtacgtgag ccagcccttc ctgatggacc tggagggcaa 600

gcagggcaac ttcaagaacc tgcgcgagtt cgtgttcaag aacatcgacg gctacttcaa 660

gatctacagc aagcacaccc ccatcaacct ggtgcgcgac ctgccccagg gcttcagcgc 720

cctggagccc ctggtggacc tgcccatcgg catcaacatc acccgcttcc agaccctgct 780

ggccctgcac cgcagctacc tgacccccgg cgacagcagc agcggctgga ccgccggcgc 840

cgccgcctac tacgtgggct acctgcagcc ccgcaccttc ctgctgaagt acaacgagaa 900

cggcaccatc accgacgccg tggactgcgc cctggacccc ctgagcgaga ccaagtgcac 960

cctgaagtcc ttcaccgtgg agaagggcat ctaccagacc agcaacttcc gcgtgcagcc 1020

caccgagagc atcgtgcgct tccccaacat caccaacctg tgccccttcg gcgaggtgtt 1080

caacgccacc cgcttcgcca gcgtgtacgc ctggaaccgc aagcgcatca gcaactgcgt 1140

ggccgactac agcgtgctgt acaacagcgc cagcttcagc accttcaagt gctacggcgt 1200

gagccccacc aagctgaacg acctgtgctt caccaacgtg tacgccgaca gcttcgtgat 1260

ccgcggcgac gaggtgcgcc agatcgcccc cggccagacc ggcaagatcg ccgactacaa 1320

ctacaagctg cccgacgact tcaccggctg cgtgatcgcc tggaacagca acaacctgga 1380

cagcaaggtg ggcggcaact acaactacct gtaccgcctg ttccgcaaga gcaacctgaa 1440

gcccttcgag cgcgacatca gcaccgagat ctaccaggcc ggcagcaccc cctgcaacgg 1500

cgtggagggc ttcaactgct acttccccct gcagagctac ggcttccagc ccaccaacgg 1560

cgtgggctac cagccctacc gcgtggtggt gctgagcttc gagctgctgc acgcccccgc 1620

caccgtgtgc ggccccaaga agtccaccaa cctggtgaag aacaagtgcg tgaacttcaa 1680

cttcaacggc ctgaccggca ccggcgtgct gaccgagagc aacaagaagt tcctgccctt 1740

ccagcagttc ggccgcgaca tcgccgacac caccgacgcc gtgcgcgacc cccagaccct 1800

ggagatcctg gacatcaccc cctgcagctt cggcggcgtg agcgtgatca cccccggcac 1860

caacaccagc aaccaggtgg ccgtgctgta ccaggacgtg aactgcaccg aggtgcccgt 1920

ggccatccac gccgaccagc tgacccccac ctggcgcgtg tacagcaccg gcagcaacgt 1980

gttccagacc cgcgccggct gcctgatcgg cgccgagcac gtgaacaaca gctacgagtg 2040

cgacatcccc atcggcgccg gcatctgcgc cagctaccag acccagacca acagcccccg 2100

ccgcgcccgc agcgtggcca gccagagcat catcgcctac accatgagcc tgggcgccga 2160

gaacagcgtg gcctacagca acaacagcat cgccatcccc accaacttca ccatcagcgt 2220

gaccaccgag atcctgcccg tgagcatgac caagaccagc gtggactgca ccatgtacat 2280

ctgcggcgac agcaccgagt gcagcaacct gctgctgcag tacggcagct tctgcaccca 2340

gctgaaccgc gccctgaccg gcatcgccgt ggagcaggac aagaacaccc aggaggtgtt 2400

cgcccaggtg aagcagatct acaagacccc ccccatcaag gacttcggcg gcttcaactt 2460

cagccagatc ctgcccgacc ccagcaagcc cagcaagcgc agcttcatcg aggacctgct 2520

gttcaacaag gtgaccctgg ccgacgccgg cttcatcaag cagtacggcg actgcctggg 2580

cgacatcgcc gcccgcgacc tgatctgcgc ccagaagttc aacggcctga ccgtgctgcc 2640

ccccctgctg accgacgaga tgatcgccca gtacaccagc gccctgctgg ccggcaccat 2700

caccagcggc tggaccttcg gcgccggcgc cgccctgcag atccccttcg ccatgcagat 2760

ggcctaccgc ttcaacggca tcggcgtgac ccagaacgtg ctgtacgaga accagaagct 2820

gatcgccaac cagttcaaca gcgccatcgg caagatccag gacagcctga gcagcaccgc 2880

cagcgccctg ggcaagctgc aggacgtggt gaaccagaac gcccaggccc tgaacaccct 2940

ggtgaagcag ctgagcagca acttcggcgc catcagcagc gtgctgaacg acatcctgag 3000

ccgcctggac aaggtggagg ccgaggtgca gatcgaccgc ctgatcaccg gccgcctgca 3060

gagcctgcag acctacgtga cccagcagct gatccgcgcc gccgagatcc gcgccagcgc 3120

caacctggcc gccaccaaga tgagcgagtg cgtgctgggc cagagcaagc gcgtggactt 3180

ctgcggcaag ggctaccacc tgatgagctt cccccagagc gccccccacg gcgtggtgtt 3240

cctgcacgtg acctacgtgc ccgcccagga gaagaacttc accaccgccc ccgccatctg 3300

ccacgacggc aaggcccact tcccccgcga gggcgtgttc gtgagcaacg gcacccactg 3360

gttcgtgacc cagcgcaact tctacgagcc ccagatcatc accaccgaca acaccttcgt 3420

gagcggcaac tgcgacgtgg tgatcggcat cgtgaacaac accgtgtacg accccctgca 3480

gcccgagctg gacagcttca aggaggagct ggacaagtac ttcaagaacc acaccagccc 3540

cgacgtggac ctgggcgaca tcagcggcat caacgccagc gtggtgaaca tccagaagga 3600

gatcgaccgc ctgaacgagg tggccaagaa cctgaacgag agcctgatcg acctgcagga 3660

gctgggcaag tacgagcagt acatcaagtg gccctggtac atctggctgg gcttcatcgc 3720

cggcctgatc gccatcgtga tggtgaccat catgctgtgc tgcatgacca gctgctgcag 3780

ctgcctgaag ggctgctgca gctgcggcag ctgctgcaag ttcgacgagg acgacagcga 3840

gcccgtgctg aagggcgtga agctgcacta caccggcagg aagaggagtc acgcaggcta 3900

ccagactatc tagggtacca ccagcctcaa gaacacccga atggagtctc taagctacat 3960

aataccaact tacactttac aaaatgttgt cccccaaaat gtagccattc gtatctgctc 4020

ctaataaaaa gaaagtttct tcacaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140

aaaaaaaaaa aaaaaaaaaa aaaaaagaag agc 4173

Claims

1. The mRNA is mRNA-1096, the mRNA-1096 comprises a5' -UTR8, an mRNA of a target gene, a 3' -UTR8 and a Poly A which are connected in sequence, and the sequence of the 5' -UTR8 is obtained by replacing T in the 1 st to 50 th positions of the sequence 8 in a sequence table with U; the sequence of the 3' -UTR7 is obtained by replacing T in positions 822 to 956 of the sequence 8 in a sequence table with U; the sequence of the Poly A is obtained by replacing T in 957-1076 th positions of a sequence 8 in a sequence table with U.

2. The use of the mRNA-1096 backbone sequence of claim 1, in any one of:

1) Preparing an mRNA vaccine;

2) Preparing mRNA antibody;

3) Preparing mRNA cell factors;

4) Preparing related medicines used in mRNA therapy.

3. A vaccine, characterized in that the active component of the vaccine is mRNA-1096 of claim 1, and the mRNA of the target gene is a sequence obtained by replacing T at positions 51 to 821 of a sequence 8 in a sequence table with U.

4. The vaccine of claim 3, wherein the mRNA has a Cap structure attached to its 5' end.

5. Use of a vaccine according to any one of claims 3 to 4 in the manufacture of a novel coronary pneumonia vaccine product.

6. A biomaterial associated with the mRNA of claim 1 or the vaccine of any one of claims 3 to 4, wherein the biomaterial is any one of B1) to B6):

b1 A nucleic acid molecule encoding the mRNA-1096 of claim 1;

7. The biomaterial according to claim 6, wherein the recombinant vector is a recombinant vector in which the sequence shown in sequence 8 in the sequence table is inserted between NheI-XhoI sites of the vector pcdna3.1 (+) while leaving other sites of the vector unchanged.

8. The biomaterial according to claim 7, wherein the recombinant bacterium is transformed into E.coli competent cells of the recombinant plasmid of claim 6.

9. Use of the biomaterial of any one of claims 6 to 8 in the manufacture of a vaccine product, said vaccine being a novel coronavirus vaccine.