CN115232826B - New coronavirus vaccine based on 1096 skeleton sequence - Google Patents
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Abstract
The application relates to a 1096 framework sequence-based vaccine, which provides a novel vaccine for preventing and controlling infection of novel coronaviruses and provides technical references for research and development of other mRNA-based vaccines, antibodies, cytokines and the like. The active ingredient 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 822 th to 956 th positions of a sequence 8 in a sequence table with U; the sequence of Poly A is obtained by replacing T in positions 957 to 1076 of the sequence 8 in the sequence table with U.
Description
Technical Field
The application relates to the field of nucleic acid vaccines, in particular to a novel coronavirus vaccine based on 1096 framework sequences.
Background
The recently emerging mRNA vaccine is one of the novel nucleic acid vaccines. The gene coding script of the target antigen is directly immunized, and then the target antigen protein is synthesized by the organism, so that humoral immunity and cellular immune response can be simultaneously and rapidly induced. The mRNA vaccine is conceptually more advantageous than the traditional inactivated vaccine, attenuated vaccine, and the novel subunit vaccine, viral vector vaccine and DNA vaccine: 1) The mRNA vaccine technology has high research and development speed, can complete GMP production and quality control within three months, and is beneficial to emergency prevention and control of epidemic of sudden infectious diseases; 2) mRNA vaccines are relatively safe, are themselves non-infectious, and do not require entry into the nucleus, with no risk of gene alteration. The mRNA vaccine is subjected to enzymolysis and clearance after being translated in vivo at a short pause and a moment; 3) The mRNA vaccine has stronger immunogenicity, can induce stronger humoral immune response and cellular immune response, thus not only being capable of preventing toxicity outside cells, but also being capable of being disinfected inside cells; 4) The mRNA vaccine research and development preparation platform has the advantages of universality, low biosafety risk and strong industrialization capability, and can realize standardized and safe production of vaccines. Only the nucleotide sequence of mRNA needs to be changed to realize large-scale production, and other production equipment and condition items do not need to be changed.
The research and development of novel coronavirus mRNA vaccine with independent intellectual property is not only corresponding to the significance of the current epidemic situation, but also enriches the vaccine reserve of the normalized prevention and control. Successful development of new coronamrna vaccines depends largely on optimization of their own sequences. The sequence of an mRNA vaccine except for the antigen gene coding region can be considered as the backbone of the mRNA vaccine. Optimizing the skeleton sequence of mRNA vaccine can raise antigen expression efficiency, and can realize the fast research and development of different mRNA vaccines to treat the sudden and viral variation of different infectious diseases.
Disclosure of Invention
The application aims to provide mRNA based on 1096 framework sequences and application thereof.
The application 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 822 th to 956 th positions of a sequence 8 in a sequence table with U; the sequence of Poly A is obtained by replacing T in positions 957 to 1076 of the sequence 8 in the sequence table with U.
The application also claims an mRNA-1096 framework sequence, wherein the 1096 framework sequence comprises 5' -UTR8 connected to the 5' -end of mRNA of a target gene and 3' -UTR8 and Poly A connected to the 3' -end of a gene coding region of the target mRNA, 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 822 th to 956 th positions of a sequence 8 in a sequence table with U; the sequence of Poly A is obtained by replacing T in positions 957 to 1076 of the sequence 8 in the sequence table with U. And the application of the mRNA-1096 framework sequence in any of the following is also within the scope of the application:
1) Preparing an mRNA vaccine;
2) Preparing mRNA antibody;
3) Preparing mRNA cytokines;
4) Related drugs used in mRNA therapies are prepared.
The mRNA-1096 skeleton sequence can be applied to different fields according to different properties of the mRNA of the target gene, wherein the mRNA of the target gene in the corresponding mRNA-1096 skeleton is different in the preparation of an mRNA vaccine, the preparation of an mRNA antibody, the preparation of an mRNA cytokine or other applications.
The application 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 in 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 use of the mRNA or vaccine described above in the preparation of novel coronatine vaccine products is also within the scope of the present application.
The application 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 said mRNA;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
b4 A recombinant plasmid containing the nucleic acid molecule of B1) or a recombinant plasmid containing the expression cassette of B2);
b5 A recombinant bacterium comprising the nucleic acid molecule of B1), a recombinant bacterium comprising the expression cassette of B2), or a recombinant bacterium comprising the recombinant vector of B3);
b5 A transgenic cell line comprising the nucleic acid molecule of B1), or a transgenic cell line comprising the expression cassette of B2), or a transgenic cell line comprising the recombinant vector of B3).
The recombinant vector is obtained by inserting a sequence shown as a sequence 8 in a sequence table between NheI-XhoI sites of a vector pcDNA3.1 (+) and keeping other sites of the vector unchanged.
Wherein, the recombinant bacterium converts the competent cell DH5 alpha of the escherichia coli of the recombinant plasmid.
The application of the biological material in preparing vaccine products is also in the protection scope of the application, and further, the vaccine is a novel coronavirus vaccine.
The application has the beneficial effects that: provides a novel vaccine for preventing and controlling the infection of novel coronaviruses, and provides technical references for research and development of other vaccines, antibodies, cytokines and the like based on mRNA.
Drawings
Fig. 1 is a schematic structural diagram of the vaccine of the present application.
FIG. 2 is a schematic diagram of the structure of a recombinant vector according to an embodiment of the present application.
FIG. 3 is a schematic diagram of the structure of 13 vaccines in example 1 of the present application.
FIG. 4 is a graph showing the results of mass analysis in example 1 of the present application.
FIG. 5 is a graph showing the results of detection of the expression of a transfected protein by cells in example 1 of the present application.
FIG. 6 is an evaluation of in vivo efficacy of the novel coronamrna vaccine of example 2 of the present application in mice.
FIG. 7 shows the immunization of mouse serum novel coronavirus neutralizing antibodies (NTs) with the novel coronavirus mRNA-1096, mRNA-1710, mRNA-2568 and mRNA-4185 vaccine sequences of example 2 of the present application 50 ) And (3) the situation.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the specifications of the products. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 sequence design, preparation of novel coronavirus mRNA and in vitro detection of antigen expression in various cells
1. Sequence design of novel crown mRNA vaccine
The sequence design of the novel crown mRNA vaccine includes the backbone code sequence of mRNA: 5'-Cap (5' -Cap); a5 '-untranslated region (5' -UTR); the 3 '-untranslated region (3' -UTR) and the Poly A tail (Poly A) and the gene coding region (CDS) structural sequences of mRNA are shown in FIG. 1. The CDS of the novel crown mRNA is composed of triplet codons from the initiation codon AUG to the termination codon, and the amino acid sequence of different novel crown antigen proteins is determined. To achieve in vitro transcription of mRNA, a novel crown mRNA sequence template is constructed between NheI-XhoI sites in the multiple cloning site region of the vector pcDNA3.1 (+) to obtain a recombinant plasmid, and in vitro transcription is initiated by T7 promoter sequence on the vector sequence under the action of T7 transcriptase, as shown in FIG. 2. Thus, the design optimization resulted in 13 different new crown mRNA vaccine sequences, as shown in FIG. 3; the sequences of the 13 mRNAs are shown in the sequence table:
1. sequence 1 (mRNA-1029):
from the 5' end, 1 to 49 are 5' -UTR1, 50 to 820 are CDS (306-318 AA and RBD regions encoding novel crown S protein NTD), 821 to 889 are 3' -UTR1, 890 to 1009 are PolyA tails.
2. Sequence 2 (mRNA-1060):
from the 5' end, 1 to 37 are 5' -UTR2, 38 to 808 are CDS (306-318A A and RBD regions of NTD encoding novel coronal S proteins), 809 to 920 are 3' -UTR2, 921 to 1040 are Poly A tails.
3. Sequence 3 (mRNA-1069):
from the 5' end, 1 to 46 are 5' -UTR3, 47 to 817 are CDS (306-318A A and RBD region of NTD encoding novel coronal S protein), 818 to 929 are 3' -UTR3, 930 to 1049 are Poly A tails.
4. Sequence 4 (mRNA-1069H):
from the 5' end, 1 to 46 are 5' -UTR4, 47 to 817 are CDS (306-318A A and RBD region of NTD encoding novel coronal S protein), 818 to 929 are 3' -UTR4, 930 to 1049 are Poly A tails.
5. Sequence 5 (mRNA-1072):
from the 5' end, 1 to 49 are 5' -UTR5, 50 to 820 are CDS (306-318A A and RBD region of NTD encoding novel crown S protein), 821 to 932 are 3' -UTR5, 933 to 1052 are Poly A tails.
6. Sequence 6 (mRNA-1073):
from the 5' end, 1 to 50 are 5' -UTR6, 51 to 821 are CDS (306-318A A and RBD regions of NTD encoding novel crown S proteins), 822 to 933 are 3' -UTR6, 934 to 1053 are Poly A tails.
7. Sequence 7 (mRNA-1083):
from the 5' end, 1 to 46 are 5' -UTR7, 47 to 817 are CDS (306-318A A and RBD regions of NTD encoding novel crown S proteins), 818 to 943 are 3' -UTR7, 944 to 1063 are Poly A tails.
8. Sequence 8 (mRNA-1096):
from the 5' end, 1 to 50 are 5' -UTR8, 51 to 821 are CDS (306-318A A and RBD regions of NTD encoding novel coronal S proteins), 822 to 956 are 3' -UTR8, 957 to 1076 are Poly A tails.
9. Sequence 9 (mRNA-1710):
from the 5' end, 1 to 46 are 5' -UTR7, 47 to 1444 are CDS (306-318 AA of NTD encoding novel crown S protein and two-segment receptor binding region RBD), 1445 to 1570 are 3' -UTR7, and 1571 to 1690 are Poly A tails.
10. Sequence 10 (mRNA-1941):
from the 5' end, 1 to 46 are 5' -UTR7, 47 to 1675 are CDS (15-318A A of NTD encoding novel coronal S protein and receptor binding domain RBD), 1676 to 1801 are 3' -UTR7, 1802 to 1921 are Poly A tails.
11. Sequence 11 (mRNA-2073):
from the 5' end, 1 to 46 are 5' -UTR7, 47 to 1807 are CDS (15-318A A, receptor binding regions RBD and HR2, encoding NTD of the novel crown S protein), 1808 to 1933 are 3' -UTR7, and 1934 to 2053 are Poly A tails.
12. Sequence 12 (mRNA-2586):
from the 5' end, 1 st to 46 th are 5' -UTR7, 47 th to 2320 th are CDS (15-318 th A A th and two-segment receptor binding region RBD encoding NTD of novel crown S protein), 2321 st to 2446 th are 3' -UTR7, 2447 th to 2566 th are Poly A tails.
13. Sequence 13 (mRNA-4185):
from the 5' end, 1 to 46 are 5' -UTR7, 47 to 3913 are CDS (encoding a novel coronal S protein), 3920 to 4045 are 3' -UTR7, and 4046 to 4165 are Poly a tails.
2. In vitro synthesis of novel coronal mRNA vaccines
1. The designed 13 new crown mRNA vaccine sequences were DNA template synthesized and verified by sequencing.
2. Amplifying the DNA template synthesized in the step 1:
the DNA templates of 13 mRNAs were ligated between NheI-XhoI sites of the vector pcDNA3.1 (+)The obtained 13 recombinant plasmids are 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 respectively. The 13 recombinant plasmids are respectively transformed into competent cells DH5 alpha, and a large amount of amplified thalli are obtained by culturing host escherichia coli. The recombinant plasmids amplified in the amplified cells were then extracted by means of endotoxin-free plasmid large extraction kit (Tiangen Biotechnology (Beijing), inc., DP 117), respectively. Linearizing the amplified recombinant plasmids respectively: the extracted recombinant plasmid is subjected to enzyme tangential digestion by using XbaI and SapI, and is purified to obtain a template for in vitro synthesis of mRNA, and Qubit is adopted TM dsDNA BR Assay Kit (Invitrogen, Q32850) kit.
3. Preparing a reaction system shown in Table 1 by taking the DNA amplified in the step 2 as an in vitro synthesis template of mRNA, incubating for 1h at 37 ℃ for in vitro transcription to obtain a large amount of in vitro transcribed RNA, and adopting Qubit TM dsDNA BR Assay Kit (Invitrogen, Q32850) kit.
TABLE 1 in vitro transcription Using T7-FlashScribe TM Transmission kit (Cellscript, C-ASF 3507)
4. Purifying the in vitro transcribed RNA product of the step 3: 1 μl RNase-Free DNase I was added to the transcription reaction system and incubated at 37deg.C for 15min to remove the DNA template from the in vitro transcription product system to obtain a transcription product. And purifying the obtained transcription product, wherein the purification method is as follows:
(1) Addition of RNase-Free H to transcript 2 O makes up 200 μl;
(2) 200 μl of the mixture A (water saturated phenol: chloroform: isoamyl alcohol, v: v, 25:24:1) was added, vortexed for 10s, centrifuged at 13800 Xg for 5min at 4deg.C, and then the upper water phase in the tube was transferred to a new tube;
(3) Adding an equal volume of mixed solution B (chloroform: isoamyl alcohol, v: v, 24:1) into a new tube, swirling for 10s, centrifuging at 4 ℃ and 13800 Xg for 5min, and then transferring the upper water phase in the tube into the new tube;
(4) An equal volume of 5M ammonium acetate solution was added to the fresh tube, and after vortexing, the mixture was left on ice for 15min, centrifuged at 4℃at 13800 Xg for 15min, and the supernatant was discarded.
(5) Adding 70% ice ethanol to clean RNA, and discarding 70% ethanol; adding appropriate amount of RNase-Free water (Solarbio, R1600), and re-suspending with Qubit TM The RNA BR Assay Kit (Invitrogen, Q10211) Kit was quantified.
5. And (3) performing mRNA capping reaction on the RNA transcription purification product obtained in the step (4), wherein the specific steps are as follows:
(1) RNA denaturation: 60. Mu.g of the transcription purified product was taken, incubated at 65℃for 15min for denaturation treatment, and then transferred to ice.
(2) mRNA capping reaction after adding RNA denatured product, the reaction system is prepared as shown in Table 2, and incubated at 37 ℃ for 0.5h, thus obtaining the capping product of mRNA with Cap 0.
TABLE 2 preparation of Cap 0 capping reaction System for mRNA Script Cap TM Cap 1 supporting System kit (Cellscript, C-SCCS 1710).
mRNA capping product purification: and (4) the same as the step (4).
Mass analysis of mRNA
The synthetic mRNA was mass analyzed using an Agilent 2100 biological analyzer and an RNA Nano 6000Assay Kit (Aglient, 5067-1511) as follows:
(1) mRNA denaturation: mRNA and mRNA Ladder were denatured at 70℃for 2min, then immediately ice-bathed.
(2) Preparing gel: RNA gel matrix was added to the filter tube as described, centrifuged at 1500 Xg for 10min at room temperature and stored at 4℃for further use.
(3) Preparing a gel-dye mixture: the RNA dye was equilibrated for 30min in the absence of light, then vortexed for several seconds, the gel dye mixture was prepared in a 65:1 ratio after transient centrifugation, the mixture was vortexed and mixed well, and centrifuged at 13000 Xg for 10min at room temperature.
(4) Loading gel-dye mixture: the chip preparation clamp was adjusted to the top-most position prior to using the RNA nano chip. The RNA chip was placed in the chip well and 9. Mu.l of gel-dye mixture (without bubbles) was added to the wells marked with black G; closing the glue injector when the piston of the injector is at the position of 1ml, pressing the injector, fixing the injector for 30s by using a clamp, then loosening the injector, and pulling the piston back to the position of 1ml after 5 s; the injector was opened and 9 μl of the gel-dye mixture was added to the other wells in which white G was noted.
(5) Loading a Marker: mu.l RNA Marker was added to each of all the sample wells and the ladder wells.
(6) Loading Ladder with mRNA: mu.l of ladder was added to the wells marked with the ladder pattern, mRNA was added to the remaining 12 wells (unused wells were replaced with RNase-free water), the chip was placed on a chip vortex for 1min with 2400r/min shaking, and then the chip was placed in an Agilent 2100 instrument for 5min for detection.
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 were consistent with the target band. Sequencing showed that each product obtained was identical to the mRNA sequence designed in step one.
3. mRNA cell transfection protein expression detection
1. Inoculating cells: 293T cells (ATCC) were seeded in 6-well plates, 1X 10 per well 6 Individual cells, 37 ℃,5% co 2 The transfection can be performed in the incubator until the cells reach 80-90% confluence.
2. Preparing a transfection complex: the transfection complexes of 13 mRNAs were prepared according to the following formulation.
By using-mRNA Transfection kit(Mirus，MIR 2250)：250μl Opti-MEM+2.5 μg mRNA+5μl mRNA boost reagent+5μl TransAnd (3) mixing the IT-mRNA reagent uniformly, and standing at room temperature for 2-5min to form a transfection complex.
3. Transfected cells: 13 transfection complexes were added dropwise to cells and shaken up and down and left and right to uniformly distribute the transfection complexes at 37℃with 5% CO 2 Cells were harvested after 18h incubation, and cell culture medium was not required to be changed before and after transfection.
4. Extracting total cell protein: after cells were washed twice with PBS, cells were lysed thoroughly by vortexing with cell lysate RIPA (Jin Pulai, P06M 11) +protease inhibitor 100× (Jin Pulai, P01C 01). After 30min of ice bath, the supernatant was centrifuged at 13800 Xg for 15min at 4 ℃.
5. Quantification of total cellular protein: total protein in cell lysis supernatants was quantified using BCA protein quantification kit (Jin Pulai, P06M 16). Mixing cell lysis supernatant with BCA working solution, incubating at 37deg.C for 45min, A 562 nm Absorbance was measured and total cell protein concentration was calculated.
6.Western blotting (WB) detection of target protein expression: precast gel Bolt by utilizing protein electrophoresis TM Total protein (30 μg) was isolated by electrophoresis (200V, 22 min) of 4 to 12%, bis-Tris,1.0mm,Mini Protein Gel (Invitrogen, NW04120 BOX). The isolated proteins on the gels were transferred to an iBlot 2Transfer Stacks,PVDF (Invitrogen, IB 24001) membrane under gradient voltage (20 v,1min; 23v,4min;25v,2 min), and then incubated at room temperature in 1×tbst with 5% nonfat milk powder, 20r/min, and blocked for 1h. A new coronavirus spike protein receptor binding domain RBD Rabbit polyclonal antibody diluent (1:2000), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody, rabbit PAb, antigen Affinity Purified (Yinqiao Shenzhou, 40592-T62), 20r/min, and shaking at room temperature for 2h. Membranes were washed with 1 XTBST, incubated at 60r/min for 10min at room temperature, and repeated 3 times to thoroughly remove primary antibody residues. The secondary antibody adopts Goat Anti-rabbit IgG secondary antibody diluent marked by horseradish peroxidase (HRP) (1:10000), goat Anti-Rabbit IgG Secondary Antibody (HRP) (SSA 004, yiqiaoshenzhou) and 20r/min, and shaking culture is carried out for 1h at room temperature. The membrane was washed with 1 XTBST, shaken at room temperature for 10min at 60r/min, and repeated 3 times to completely remove the secondary antibody residue. Super-sensitivity chromogenic kit (assist in holy) using ECL chemistryBiology, 36208ES 60) was incubated with the membrane at room temperature for 3min in the dark and HRP-labeled antibody-bound antigen was detected in a chemiluminescent instrument. Revealing bands and protein markers by exposure, pageRuler TM Prestained Protein Ladder (Invitrogen, 26617) band alignment verifies expression of the antigen protein of interest. Membrane regeneration solution (soribao, SW 3020) was used to remove antibodies on the membrane, and after the membrane was again blocked, reference antibodies were incubated to detect the uniformity of the protein loading amount. One antibody used β -actin Rabbit mAb (1:5000), ACTB Rabbit mAb (abclon, AC 038), and the second antibody used horseradish peroxidase (HRP) -labeled Goat Anti-Rabbit IgG secondary antibody diluent (1:10000), gold Anti-Rabbit IgG Secondary Antibody (HRP) (SSA 004, san-a-se).
The results are shown in fig. 5, which shows that: mRNA-1029, mRNA-1060, mRNA-1069H, mRNA-1072, mRNA-1073, mRNA-1083, mRNA-1096 transfected HEK-293t cells (FIG. 5-1), WB detected expression of the antigen of interest; 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 were transfected into HEK-293t cells, and WB detected expression of the antigen of interest. mRNA-1083, mRNA-1096, mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568 and mRNA-4185 were selected as candidate sequences for in vivo efficacy evaluation.
Example 2 case of antibody production after immunization of mice with New coronal mRNA vaccine
1. Preparation of efficient delivery formulation of novel crown mRNA vaccine
Preparation of formulations New crown mRNA-LPPs lipopolymer vaccines were prepared in concert with Shanghai microorganisms.
2. Evaluation of in vivo efficacy of novel coronal mRNA vaccine
The experimental animals BALB/c mice (female, 6-8 weeks, 16-18g, beijing Bei Fu) were randomly divided as shown in Table 3 (5 per group) and the mice were immunized by intramuscular injection. Sufficient amounts of mouse serum were obtained on day 10 after each immunization using orbital bleeding. Detecting the generation and titer of RBD specific antibodies in serum of the immunized mice by ELISA; and detecting the generation and titer of the pseudovirus neutralizing antibodies in the serum of the immunized mice by using the novel coronavirus S protein pseudovirus, and evaluating the in vivo immune efficacy of the novel coronavirus mRNA vaccine.
ELISA method for detecting RBD specific antibody in serum of immunized mice comprises the following steps:
(1) Coating: RBD protein (40592-VNAH, yinqiao) was diluted to 1 ng/. Mu.l with carbonate buffer (50 mM, pH 9.6, 0.22 μm filter). 100 mug RBD protein diluent is added into each hole of a 96-well plate, and the mixture is sealed and subjected to 4 ℃ overnight;
(2) Washing the plate: after coating overnight, the protein coating solution was removed by pouring the 96-well plate, 200. Mu.L of washing solution (1 XTBS containing 0.2% Tween-20) was added to each well, gently shaken by hand for 30s and then dried on paper, and repeated 6 times;
(3) Closing: mu.l of blocking solution (1 XTBS with 2% BSA) was added to each well and incubated for 2h at 37 ℃;
(4) Washing the plate: the blocking solution was removed by pouring the 96-well plate, 200. Mu.l of washing solution (1 XTBS with 0.2% Tween-20) was added to each well, gently shaken by hand for 30s and then dried on paper, and repeated 6 times;
(5) Incubating primary antibodies: the serum of immunized mice was subjected to 10-fold gradient dilution with antibody dilution (washing solution containing 0.5% BSA) to obtain 10 -1 To 10 -6 Serum of different dilutions was added to each well with 100 μl of serum dilution and incubated for 2h at 37deg.C in incubator;
(6) Washing the plate: the serum dilutions were removed by pouring the 96-well plate, 200. Mu.L of wash solution (1 XTBS with 0.2% Tween-20) was added to each well, gently shaken by hand for 30s and then dried on paper, and repeated 6 times;
(7) Incubating a secondary antibody: horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (Biyun Tian, A0216) was 250-fold diluted with antibody diluent (washing solution containing 0.5% BSA) to obtain secondary antibody diluent, 100 μl of secondary antibody diluent was added to each well, and incubated for 1H at 37deg.C in an incubator;
(8) Washing the plate: pouring the 96-well plate to remove the secondary antibody diluent, adding 200 μl of washing solution (1 XTBS containing 0.2% Tween-20) to each well, gently shaking for 30s, and drying on paper for 6 times;
(9) Color development: adding 100 μl TMB substrate (radix Sangusorbae) into each well, and incubating at room temperature in dark place for 20min;
(10) Terminating the color development: 50 mu L of 2M H are added to each well 2 SO 4 Detection of A on an ELISA apparatus 450 OD value of (d);
(11) Serum antibody titer determination: a certain dilution serum OD/negative control OD is more than or equal to 2.1, the next dilution ratio OD/negative control OD is less than 2.1, and the dilution ratio is the corresponding antibody titer of the serum sample (calculated as 0.05 if the negative control OD is less than 0.05).
Table 3: intramuscular injection vaccine sequence and immune condition of mice
The results are shown in fig. 6: RBD-specific IgG antibodies were detected in the serum of immunized mice of mRNA-1096, mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568 and mRNA-4185. Wherein mRNA-1096, mRNA-1710 immunized mice have a priming serum titer of about 1:10000.
the novel coronavirus S protein pseudovirus detects the condition of a pseudovirus neutralizing antibody in serum of an immunized mouse, and the specific detection method is as follows:
(1) Cell plating: the day before the experiment, HEK293T-ACE2 cells to be infected (Yesen Biotech, HB 200710) were inoculated in 96-well cell culture plates at an inoculum size of about 1X 10 4 The next day, pseudovirus infection was performed when the cell density was around 30%. HEK293T-ACE2 cell culture broth is 90% DMEM+10% FBS+1% diabody+0.75 μg/mL puromycin;
(2) Immune serum inhibits pseudoviral infection: the serum of immunized mice is subjected to 10-time gradient dilution by using cell culture solution to obtain 10 -1 To 10 -6 Serum at different dilutions. Frozen pseudoviruses (titres 1X 10) 6 TU/mL, 0.5. Mu.l) (Yeasen Biotech, HB 200707) was thawed on ice and mixed with 100. Mu.l of diluted serum, incubated for 1h at 37℃and then cultured cells were added. Changing fresh culture medium to continue culturing after virus infection for 6 h;
(3) Infection detection: 48h after the replacement of the pseudovirus in the cell infection, the green fluorescent protein expression was observed by a fluorescence microscope and the luciferase activity was detected using a firefly luciferase reporter gene detection kit (Yeasen Biotech, HB 181218).
(4) Serum pseudovirus neutralizing antibody titre (NT) 50 ) And (3) detection: NT 50 Defined as a 50% reduction in fluorescence relative to light units (RLUs) in the pseudovirus control group. 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 immune mouse serum antibodies of mRNA-1096, mRNA-1710, mRNA-2568 and mRNA-4185 all have the novel coronas protein pseudovirus neutralizing activity. The neutralizing antibodies (NT) of the novel coronas protein pseudovirus of the primary and secondary serum of the mRNA vaccine 50 ) Titer statistics are given in Table 4, the neutralizing antibodies (NT) against the novel coronas protein pseudovirus of the secondary mouse serum of mRNA-1096, mRNA-1710, mRNA-2568 50 ) The titer is superior to that of the di-immune mouse serum pseudovirus NT of RBD-mRNA-LNPs vaccine of New York blood center at the same dosage 50 Titer (1:10000). Wherein mRNA-1096 is used for priming mouse serum pseudovirus NT 50 Titers all exceeded 1:20000, indicating that vaccine mRNA-1096 can promote induction of adaptive immune responses in vivo.
TABLE 4 New coronamRNA-1096, mRNA-1710, mRNA-2568 and mRNA-4185 vaccine sequences immunization of mouse serum New coronavirus neutralizing antibodies (NT) 50 ) Titer.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> military medical institute of the military academy of China's civil liberation army
<120> a novel coronavirus vaccine based on 1096 backbone sequence
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1017
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<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 (9)
1. An mRNA, which is characterized in that the mRNA is mRNA-1096, the mRNA-1096 comprises 5' -UTR8, mRNA of a target gene, 3' -UTR8 and Poly A which are connected in sequence, 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 822 th to 956 th positions of a sequence 8 in a sequence table with U; the sequence of Poly A is obtained by replacing T in positions 957 to 1076 of the sequence 8 in the sequence table with U.
2. Use of the mRNA-1096 of claim 1 in any of the following:
1) Preparing an mRNA vaccine;
2) mRNA antibodies were prepared.
3. A vaccine, characterized in that the active ingredient of the vaccine is mRNA-1096 described in claim 1, and the mRNA of the target gene is a sequence obtained by replacing T in 51 th to 821 th positions 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 the 5' end.
5. Use of the vaccine of any one of claims 3-4 in the preparation of a novel coronatine vaccine product.
6. A biomaterial associated with the mRNA of claim 1 or the vaccine of any one of claims 3-4, characterized in that the biomaterial is any one of B1) -B6):
b1 A nucleic acid molecule encoding the mRNA-1096 of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
b4 A recombinant plasmid containing the nucleic acid molecule of B1) or a recombinant plasmid containing the expression cassette of B2);
b5 A recombinant bacterium comprising the nucleic acid molecule of B1), a recombinant bacterium comprising the expression cassette of B2), or a recombinant bacterium comprising the recombinant vector of B3);
b5 A transgenic cell line comprising the nucleic acid molecule of B1), or a transgenic cell line comprising the expression cassette of B2), or a transgenic cell line comprising the recombinant vector of B3).
7. The biomaterial according to claim 6, wherein the recombinant vector is a recombinant vector in which the sequence shown as sequence 8 in the sequence table is inserted between NheI- -XhoI sites of the vector pcDNA3.1 (+) and other sites of the vector are maintained unchanged.
8. The biomaterial according to claim 7, wherein the recombinant bacterium is transformed with competent cells of escherichia coli of the recombinant plasmid according to claim 6.
9. Use of the biomaterial according to any one of claims 6-8 for the preparation of a vaccine product, said vaccine being a novel coronavirus vaccine.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110714015A (en) * | 2019-10-29 | 2020-01-21 | 珠海丽凡达生物技术有限公司 | mRNA rabies vaccine |
| CN111218458A (en) * | 2020-02-27 | 2020-06-02 | 珠海丽凡达生物技术有限公司 | mRNAs encoding SARS-CoV-2 virus antigen and vaccine and preparation method of vaccine |
| CN111330003A (en) * | 2020-03-23 | 2020-06-26 | 翁炳焕 | Preparation method of novel coronary pneumonia antisense RNA multivalent vaccine |
| CN111607613A (en) * | 2020-05-19 | 2020-09-01 | 深圳市新合生物医疗科技有限公司 | Plasmid vector for expressing mRNA of cellular immune vaccine and construction method and application thereof |
| CN111821433A (en) * | 2020-02-06 | 2020-10-27 | 深圳市瑞吉生物科技有限公司 | mRNA vaccine and synthetic method and kit thereof |
| CN112379090A (en) * | 2021-01-07 | 2021-02-19 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus antibody detection kit and preparation method and application thereof |
| CN115232825A (en) * | 2021-04-22 | 2022-10-25 | 中国人民解放军军事科学院军事医学研究院 | Novel coronavirus vaccine based on 1083 framework |
-
2021
- 2021-04-22 CN CN202110434369.3A patent/CN115232826B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110714015A (en) * | 2019-10-29 | 2020-01-21 | 珠海丽凡达生物技术有限公司 | mRNA rabies vaccine |
| CN111821433A (en) * | 2020-02-06 | 2020-10-27 | 深圳市瑞吉生物科技有限公司 | mRNA vaccine and synthetic method and kit thereof |
| CN111218458A (en) * | 2020-02-27 | 2020-06-02 | 珠海丽凡达生物技术有限公司 | mRNAs encoding SARS-CoV-2 virus antigen and vaccine and preparation method of vaccine |
| CN111330003A (en) * | 2020-03-23 | 2020-06-26 | 翁炳焕 | Preparation method of novel coronary pneumonia antisense RNA multivalent vaccine |
| CN111607613A (en) * | 2020-05-19 | 2020-09-01 | 深圳市新合生物医疗科技有限公司 | Plasmid vector for expressing mRNA of cellular immune vaccine and construction method and application thereof |
| CN112379090A (en) * | 2021-01-07 | 2021-02-19 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus antibody detection kit and preparation method and application thereof |
| CN115232825A (en) * | 2021-04-22 | 2022-10-25 | 中国人民解放军军事科学院军事医学研究院 | Novel coronavirus vaccine based on 1083 framework |
Non-Patent Citations (3)
| Title |
|---|
| COVID-19 mRNA疫苗关键技术与产品进展分析;葛华等;《军事医学》;第44卷(第4期);全文 * |
| Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines;Kellie D. Nance等;《ACS Cent. Sci.》;第7卷(第5期);全文 * |
| PREDICTED: Pan paiscus hemoglobin subunit beta (LOC100976465), mRNA;NCBI;《GenBank》;全文 * |
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