CN114940999A

CN114940999A - Vaccine preparation method based on 1083 framework

Info

Publication number: CN114940999A
Application number: CN202110434216.9A
Authority: CN
Inventors: 杨静; 王升启; 李蕾; 王鑫; 龙晋蓉; 桑野; 曹艺明; 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: 2022-08-26
Anticipated expiration: 2041-04-22
Also published as: CN114940999B

Abstract

The invention discloses a vaccine preparation method based on 1083 skeleton, which comprises the steps of inserting mRNA of a target gene into 1083 skeleton to obtain a vaccine active substance sequence, wherein the 1083 skeleton comprises 5 ' -UTR7 connected to the 5 ' end of the gene coding region of the target mRNA, and 3 ' -UTR7 and Poly A connected to the 3 ' end of the gene coding region of the target mRNA, and the sequence of the 5 ' -UTR7 is a sequence obtained by replacing T in the 1 st to 46 th sequences of 7 in a sequence table with U; the sequence of the 3' -UTR7 is a sequence obtained by replacing T in 818-943 of a sequence 7 in a sequence table with U; the sequence of the Poly A is a sequence obtained by replacing T in 944 to 1063 of the sequence 7 in the sequence table with U.

Description

Vaccine preparation method based on 1083 framework

Technical Field

The invention relates to the field of nucleic acid vaccines, in particular to a vaccine preparation method based on 1083 frameworks.

Background

The recently emerging mRNA vaccines are one of the novel nucleic acid vaccines. It can directly immunize the gene coding script of target antigen, and then the target antigen protein is synthesized by body self, so that it 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 mRNA vaccine technology has high research and development speed, can complete GMP production and quality control within three months, and is favorable for emergency prevention and control of epidemic of the paroxysmal infectious diseases; 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 transiently stayed in the body to realize that the antigen protein is removed by enzymolysis after being translated; 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 preparation platform has the advantages of universality, low biological safety risk and strong industrialization capability, and can realize the standardized and safe production of the vaccine. 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 novel coronavirus mRNA vaccines with independent intellectual property rights in China not only has important significance for the current epidemic situation, but also can enrich the stock of normalized prevention and control vaccines. Successful development of a new corona mRNA vaccine depends largely on optimization of its own sequence. The sequence of the mRNA vaccine except the coding region of the antigen gene can be regarded as the framework of the 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 a method for preparing mRNA based on 1083 skeleton.

The invention provides an mRNA preparation method, which comprises the step of inserting mRNA of a target gene into a 1083 skeleton to obtain an active component of an mRNA vaccine, wherein the 1083 skeleton on the skeleton comprises a 5 ' -UTR7 connected to the 5 ' end of a gene coding region of the target mRNA and a3 ' -UTR7 and Poly A connected to the 3 ' end of the gene coding region of the target mRNA, and the sequence of the 5 ' -UTR7 is a sequence obtained by replacing T in the 1 st to 46 th sequences of 7 in a sequence table with U; the sequence of the 3' -UTR7 is a sequence obtained by replacing T in 818 to 943 of the sequence 7 in a sequence table with U; the sequence of the Poly A is a sequence obtained by replacing T in 944 to 1063 of the sequence 7 in the sequence table with U.

Wherein the vaccine is a novel coronary pneumonia vaccine or a rabies virus vaccine.

Wherein the mRNA of the target gene is any one of the following (1) to (6):

(1) a sequence obtained by replacing T in the 47 th to 817 th positions of the sequence 7 in the sequence table with U;

(2) a sequence obtained by replacing T in 47 th to 1444 th positions of the sequence 9 in the sequence table with U;

(3) a sequence obtained by replacing T in the 47 th to 1675 th positions of the sequence 10 in the sequence table with U;

(4) a sequence obtained by replacing T in the 47 th to 1807 th positions of the sequence 11 in the sequence table with U;

(5) a sequence obtained by replacing T in the 47 th to 2320 th positions of the sequence 12 in the sequence table with U;

(6) a sequence obtained by replacing T in the 56 th to 3913 th positions of the sequence 13 in the sequence table with U;

(7) a sequence obtained by replacing T in the 47 th to 1636 th positions of the sequence 14 in the sequence table with U.

The gene for coding the vaccine is introduced into a vector to obtain a recombinant vector, and the recombinant vector is transformed into a recombinant bacterium and then is subjected to in vitro transcription to obtain the vaccine.

Wherein the recombinant vector is any one of the following (1) to (6):

(1) inserting the sequence shown in the sequence 7 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the sequences of other sites of the vector unchanged;

(2) inserting a sequence shown in a sequence 9 in a sequence table into NheI-XhoI sites of a vector pcDNA3.1(+), and keeping other sites of the vector unchanged;

(3) inserting the sequence shown in the sequence 10 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the sequences of other sites of the vector unchanged;

(4) inserting the sequence shown in the sequence 11 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the sequences of other sites of the vector unchanged;

(5) inserting the sequence shown in the sequence 12 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the other sites of the vector unchanged;

(6) the sequence shown in the sequence 13 in the sequence table is inserted between NheI-XhoI sites of the vector pcDNA3.1(+), and the sequences of other sites of the vector are kept unchanged.

(7) The sequence shown in the sequence 14 in the sequence table is inserted between NheI-XhoI sites of the vector pcDNA3.1(+), and the sequences of other sites of the vector are kept unchanged.

Wherein the recombinant bacterium is an Escherichia coli competent cell DH5 alpha transformed with the recombinant plasmid.

The use of the above method for the preparation of a vaccine is also intended to be within the scope of the present invention.

Wherein the vaccine is a novel coronavirus vaccine or a rabies virus vaccine.

The beneficial effects of the invention are that the invention provides a method for screening the vaccine by using the 1083 framework of the optimized mRNA vaccine framework sequence, which can improve the mRNA expression efficiency, and can realize the rapid research and development of different mRNA vaccines, mRNA antibodies, mRNA-based cytokines or proteins and the like so as to deal with the sudden change of different infectious diseases and virus variation, or treat tumor-related diseases, rare diseases, cardiovascular diseases, cytokine-deficient diseases and the like.

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 blood-freshening Coronavirus of mice immunized with the neocorona mRNA-1083, mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568 and mRNA-4185 vaccine sequences of example 2 of the present invention (NT) ₅₀ ) The situation is.

FIG. 8 shows the neutralizing antibody against rabies virus in the serum of mice immunized with the mRNA-1902P sequence of rabies virus mRNA vaccine in accordance with an embodiment of the present invention.

FIG. 9 shows the body weight of a rabies virus mRNA vaccine mRNA-1902P sequence immunized mouse after challenge in accordance with an embodiment of the present invention;

FIG. 10 shows the survival of rabies virus mRNA vaccine mRNA-1902P sequence immunized mice after challenge in accordance with an embodiment of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present 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 and rabies virus mRNA and antigen expression assay of various cells in vitro

Sequence design of mRNA vaccine for Xinguan and rabies

Sequence design for the new coronary mRNA vaccine included the backbone code sequence for 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. To achieve in vitro transcription of mRNA, a new crown mRNA sequence template was constructed between NheI- -XhoI sites in the multiple cloning site region of pcDNA3.1(+) to obtain a recombinant plasmid, and in vitro transcription was initiated by T7 transcriptase using the T7 promoter sequence, as shown in FIG. 2. Thus, design optimization resulted in 14 different novel crown mRNA vaccine sequences, as shown in fig. 3. (ii) a The sequences of the 14 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 tails from 890 to 1009 from 50.

2. Sequence 2 (mRNA-1060):

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

3. Sequence 3 (mRNA-1069):

5 ' -UTR3 from 1 to 46 at the 5 ' end, CDS (306-318 AA and RBD region of NTD encoding novel crown S protein) from 47 to 817, 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 at the 5 ' end, CDS (306-318 AA and RBD region of NTD encoding new crown S protein) from 47 to 817, 3 ' -UTR4 from 818 to 929 and Poly A tail from 930 to 1049.

5. Sequence 5 (mRNA-1072):

5 ' -UTR5 from 1 to 49 at the 5 ' end, CDS (306-318 AA and RBD region of NTD encoding new crown S protein) from 50 to 820, 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 (306-318 AA and RBD region encoding NTD of the novel crown S protein) from 51 to 821, 3 ' -UTR6 from 822 to 933, and Poly A tails from 934 to 1053.

7. Sequence 7 (mRNA-1083):

5 ' -UTR7 from 1 to 46 at the 5 ' end, CDS (306-318 AA and RBD region encoding NTD of 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 1 to 50 at the 5 ' end, CDS (306-318 AA and RBD region encoding NTD of the novel crown S protein) from 51 to 821, 3 ' -UTR8 from 822 to 956, and Poly A tail from 957 to 1076.

9. Sequence 9 (mRNA-1710):

5 ' -UTR7 from 1 to 46 at the 5 ' end, CDS (NTD encoding the novel crown S protein and two receptor binding regions RBD) from 47 to 1444, 3 ' -UTR7 from 1445 to 1570, and Poly A tails from 1571 to 1690.

10. Sequence 10 (mRNA-1941):

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

11. Sequence 11 (mRNA-2073):

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

12. Sequence 12 (mRNA-2586):

5 ' -UTR7 from 1 to 46 at the 5 ' end, CDS (15-318 AA encoding NTD of the novel crown S protein and two segments of receptor binding region RBD) from 47 to 2320, 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 at 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.

14. Sequence 14(mRNA-1902P)

5 ' -UTR7 from 1 to 46 of the 5 ' end, CDS from 47 to 1636 (encoding rabies virus CTN-1 strain G protein Genbank accession number ACR39382.1), 3 ' -UTR from 1637 to 1762, and Poly A tails from 1763 to 1882.

II, in vitro synthesis of novel crown mRNA vaccine and rabies virus mRNA

1. The designed 13 new crown mRNA vaccines and 1 rabies virus mRNA sequence are subjected to DNA template synthesis and verified by sequencing.

2. Amplifying the DNA template synthesized in the step 1: the DNA template was ligated between NheI- -XhoI sites of vector pcDNA3.1(+) to obtain a recombinant plasmid. The recombinant plasmid was transformed into competent cell DH 5. alpha. and cultured in host E.coli to obtain a large amount of amplified cells. The recombinant plasmid amplified in the amplified cells was extracted with the aid of an endotoxin-free plasmid macroextraction kit (Tiangen Biotech, Beijing, Ltd., DP 117). Linearizing the amplified recombinant plasmid: 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-ASF3507)

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) adding 200 μ l of mixture A (water saturated phenol: chloroform: isoamyl alcohol, v: v: v, 25:24:1), vortexing for 10s, centrifuging at 13800 Xg for 5min at 4 deg.C, and transferring the upper aqueous phase to a new tube;

(3) adding equal volume of mixed solution B (chloroform: isoamyl alcohol, v: v, 24:1) into the new tube, vortexing for 10s, centrifuging at 13800 Xg for 5min at 4 ℃, 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 a proper amount of RNase-Free water (Solarbio, R1600) for heavy suspension, 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 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 for mRNA reaction System formulation with 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 analysis of the synthesized mRNA is carried out by adopting Agilent 2100 bioanalyzer and RNA Nano 6000Assay Kit (Aglient, 5067-:

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

(2) Preparing a gel: the 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 is balanced for 30min in the dark, then vortexed for several seconds, and after instantaneous centrifugation, a gel dye mixture is prepared in a ratio of 65:1, the mixture is vortexed and mixed evenly, and centrifuged at 13000 Xg for 10min at room temperature.

(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 μ l gel-dye mixture (without air bubble) into the hole marked black G; closing the glue injector when the injector piston is at the position of 1ml, pressing the injector, fixing the injector by using a clamp for 30s, then loosening the injector, 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, and 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.

Third, mRNA cell transfection protein expression detection

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

2. Preparing a transfection complex: 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. Transfection of cells: the transfection complex was added dropwise to the cells and shaken up and down to distribute the transfection complex uniformly at 37 ℃ with 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 (copaiy, P06M11) + protease inhibitor 100 × (copaiy, P01C01) 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 quantitated using BCA protein quantitation kit (Kirpley, P06M 16). Mixing cell lysis supernatant with BCA working solution, incubating at 37 deg.C for 45min, A _562nm 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 isolated by electrophoresis (200V, 22min) using 4to 12%, Bis-Tris,1.0mm, Mini Protein Gel (Invitrogen, NW04120 BOX). The protein isolates on the gel were transferred to iBlot 2Transfer Stacks, PVDF (Invitrogen, IB24001) membranes under a gradient voltage (20V, 1 min; 23V, 4 min; 25V, 2min) and then dried in 5% skim milkShake-culturing in 1 × TBST at room temperature, 20r/min, and blocking for 1 h. The primary Antibody adopts RBD Rabbit polyclonal Antibody diluent (1:2000) of a new coronavirus Spike protein receptor binding region, SARS-CoV-2(2019-nCoV) Spike RBD Antibody, Rabbit PAb, Antigen Affinity purification (40592-T62, Chinesian, Chinesen, 20r/min, and is cultured for 2h at room temperature. The membrane was washed with 1 XTSST 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 (1:10000) labeled with horseradish peroxidase (HRP), Goat Anti-Rabbit IgG Secondary Antibody (HRP) (SSA 004, Chiyoho) at 20r/min, and incubated at room temperature for 1 h. The membrane was washed with 1 XTSST 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-chromogenic kit (assist in san-Jose biose, 36208ES60) incubated with membrane for 3min at room temperature in the dark. Visualization of bands and protein marker by exposure, PageRuler ^TM The expression of the target antigen Protein was confirmed by alignment of the Prestained Protein Ladder (Invitrogen, 26617) bands. The antibody on the membrane is removed by using membrane regeneration liquid (Solebao, SW3020), the membrane is sealed again, and then the internal reference antibody is incubated to detect the consistency of the amount of the protein loaded. The primary Antibody was a dilution of beta-actin Rabbit monoclonal Antibody (1:5000), ACTB Rabbit mAb (Abclonal, AC038), Secondary Antibody was a dilution of Goat Anti-Rabbit IgG Secondary Antibody labeled with horseradish peroxidase (HRP) (1:10000), Goat Anti-Rabbit IgG Secondary Antibody (HRP) (SSA 004, Chin. Yi.).

The results are shown in FIG. 5, showing: HEK-293t cells (shown in figure 5-1) were transfected by mRNA-1029, mRNA-1060, mRNA-1069H, 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 were used as candidate sequences of the novel corona mRNA vaccine for evaluation of in vivo efficacy. After transfection of HEK-293t cells with the rabies G protein mRNA-1902P vaccine designed based on the backbone code sequence of mRNA-1083, expression of the antigen of interest, rabies G protein, was detected with WB primary Antibody against Monoclonal Antibody-viruses (Hytest,3R7-4F1) and with Goat Anti-Mouse IgG Secondary Antibody (HRP) (SSA 007, Hooka), as well as further verified the versatility of the backbone code sequence based on mRNA-1083 (FIGS. 5-4).

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

Preparation method of efficient delivery preparation of new coronary mRNA vaccine

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

Second, evaluation of in vivo efficacy of New coronary mRNA vaccine

Experimental animals BALB/c mice (female, 6-8 weeks, 16-18g, Beijing sbefu) were randomly assigned as shown in Table 3 (5 mice per group), and the mice were immunized by intramuscular injection. Adequate mouse sera were obtained by orbital bleeding 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 titer of a pseudovirus neutralizing antibody in the serum of an immunized mouse by using the new corona S protein pseudovirus, and evaluating the in vivo immune 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, Chin) was diluted to1 ng/. mu.l with 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 coating overnight, pouring a 96-well plate to remove protein coating solution, 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;

(3) and (3) sealing: add 200. mu.l blocking solution (1 × TBS with 2% BSA) to each well, incubate 2h at 37 deg.C in incubator;

(4) washing the plate: pouring 96-well plate to remove blocking solution, adding 200 μ l of washing solution (1 × TBS containing 0.2% Tween-20) per well, shaking gently by hand for 30s, patting dry on paper, and repeating for 6 times;

(5) incubating the primary antibody: the serum of the immunized mouse was diluted 10-fold with an antibody diluent (washing solution containing 0.5% BSA) to obtain 10 ^-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 by 250 times with an antibody diluent (a washing solution containing 0.5% BSA) to obtain a secondary antibody diluent, adding 100 μ L of the secondary antibody diluent into each hole, and incubating for 1H at 37 ℃ in an incubator;

(8) washing the plate: pouring a 96-well plate to remove the secondary antibody diluent, adding 200 ul of washing solution (1 xTBS containing 0.2% Tween-20) into each well, shaking lightly 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 20 min;

(10) and (3) stopping color development: add 50. mu.L of 2M H per well ₂ SO ₄ Detecting 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 with mRNA-1083, mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568 and mRNA-4185. Wherein the primary immune serum titer of mRNA-1083 and mRNA-1710 immunized mice reaches about 1: 10000; the serum titer of the hyperimmune serum reaches about 1:1000000, which is better than the serum RBD-specific IgG antibody level of the RBD-mRNA-LNPs vaccine in New York blood center (1:230000) under the same dose.

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: the day before the experiment, HEK293T-ACE2 cells (Yeasen Biotech, HB200710) to be infected were seeded in 96-well cell culture plates at an inoculum size of approximately 1X 10 ⁴ One cell/well, next day, pseudoviral infection was performed with cell density around 30%. The HEK293T-ACE2 cell culture solution is 90% DMEM + 10% FBS + 1% double antibody +0.75 mu g/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, HB200707) was thawed on ice, mixed with 100. mu.l of the diluted serum, incubated at 37 ℃ for 1h in an 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: at 48h after the cells were infected with pseudovirus and the fluid was changed, the expression of green fluorescent protein was observed by fluorescence microscopy and luciferase activity was detected using a firefly luciferase reporter assay kit (Yeasen Biotech, HB 181218).

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

The results are shown in FIG. 7: the serum antibodies of the immunized mice of mRNA-1083, mRNA-1710, mRNA-2568 and mRNA-4185 all have the neutralizing activity of the novel crown S protein pseudovirus. Primary and secondary serum neutralizing antibodies (NT) against the mRNA vaccine against the new crown S protein pseudovirus ₅₀ ) Titers were counted in Table 4 for neutralizing antibodies against the Bisaran mouse blood-freshening coronavirus protein pseudovirus (NT) of mRNA-1083, mRNA-1710, mRNA-2568 ₅₀ ) The titer is better than that of the same dosageDiimmune mouse serum pseudovirus NT for RBD-mRNA-LNPs vaccine in New York blood center ₅₀ Titre (1: 10000). Wherein mRNA-1083 primes mouse serum pseudovirus NT ₅₀ The titer exceeds 1: 20000; erinized mouse serum pseudovirus NT ₅₀ The titer exceeded 1: 100000. Further, mRNA-1083 based framework code sequences can facilitate the induction of adaptive immune responses in vivo.

TABLE 4 neutralizing antibody against blood-refreshing coronavirus of mice immunized with neocorona mRNA-1083, mRNA-1710, mRNA-1941, mRNA-2073, mRNA-2568, and mRNA-4185 vaccine sequences (NT) ₅₀ ) The titer.

Example 3 investigation of neutralizing antibody production and immunoprotection following immunization of mice with rabies mRNA vaccine

Preparation of efficient delivery preparation of rabies mRNA vaccine

Preparation of the preparation rabies mRNA-1902P-LPPs lipid polymer vaccine was prepared by cooperation with Shanghai microbial corporation.

Second, in vivo efficacy evaluation of rabies mRNA vaccine

The experimental animals BALB/c mice (female, 6-8 weeks, 16-18g, Beijing sbefu) were randomly divided into mRNA-1902P immunization group (immunization dose of mRNA-1902P ═ 30. mu.g/mouse) and negative control group (6 mice per group), and the mice were immunized by intramuscular injection. Immunizations were performed twice, each with a 14 day interval. Adequate mouse serum was obtained by orbital bleeds on day 10 after each immunization. The neutralizing antibody titer of the rabies virus of the serum of the immunized mice is detected by a FAVN method recognized by WHO, and the in vivo immunization efficacy of the rabies mRNA vaccine is evaluated.

And (3) detecting the rabies virus neutralizing antibody of the mouse serum according to the standard GB/T34739-2017. The FAVN experiment was performed after inactivation of the mouse serum samples at 56 ℃ for 30 min:

add 100 mul culture medium into each hole of the culture plate, use 50 mul multi-channel liquid-moving machine to treat serum sample and standard serum contrast to carry on 3 times of dilution continuously, each sample dilution to be examined sets 2 holes, each dilution of standard serum contrast sets 4 holes. Discard 50. mu.l of the aspirated liquid from the last row of wells.

The plates were plated with 150. mu.l of medium per well and virus was serially diluted 4-fold using a 50. mu.l multi-channel pipette, 2 wells per dilution. Discard 50. mu.l of the aspirated liquid from the last row of wells.

Meanwhile, a cell control and a culture medium control are arranged in a hollow white area of the culture plate, 200 mu l of culture medium is added into each hole of the culture medium control area, and 150 mu l of culture medium is added into each hole of the cell control area.

Adding 50 μ l virus into all the wells of the serum sample well and the serum control well, attacking virus strain CVS-11 with rabies standard, and culturing the virus by BHK-21 milk hamster kidney continuous cell line proliferation with titer not less than 10 ⁵ TCID 50/ml. Placing in 37 deg.C incubator, neutralizing and culturing for 60 min.

The density of the BHK-21 cell suspension was adjusted to 4.0X 10 ⁵ One per ml. In addition to the media control wells, 50. mu.l of cell suspension was added per well. The culture plate was incubated at 37 ℃ in a 5% carbon dioxide incubator for 48 h.

The liquid in the plate was discarded in 0.1% sodium hydroxide solution. The plate was immersed directly into the first fixative vessel, taken out after a while, and the liquid in the wells was discarded back into the fixative. Immersing the culture plate in a second container of fixative, discarding the liquid in the well back to the fixative after 20min, and drying by self-heating or blotting with absorbent paper.

FITC-labeled rabies virus nucleoprotein fluorescent antibody (Milipore5500) was diluted with 0.01M PBS (pH7.4) to working concentration and added to the detection cell plate at 50. mu.l/well, and incubated at 37 ℃ for 60 min. The labeled antibody was discarded in 0.1% hydroxide solution, washed 2 times with 0.01M PBS, and dried by pouring on absorbent paper.

Staining was observed by fluorescence microscopy, observing all fields per well, with "+" indicating more than 1 fluorescent cell, indicating unneutralized virus. The cell well without fluorescent particle labeling is "-". Substituting the observed detection result parameters into the following formula to calculate the neutralizing antibody titer (E) of the serum to be detected, wherein the unit is IU/ml, E is 0.5(At/Ac)×3 ^(Bt-Bc)/4 . In the formula, At is the maximum dilution multiple of the serum without fluorescent staining in the whole array of holes of the serum to be detected; ac is the maximum dilution multiple of serum without fluorescent staining of positive standard substance whole column; bt is the number of holes of the serum to be detected which appear without fluorescent staining after At dilution; bc is the number of wells of the standard that appeared after Ac dilution without fluorescent staining.

The results are shown in FIG. 8: the rabies virus neutralizing antibody titer of the mouse serum after two times of rabies mRNA-1902P vaccine immunization is 30.77-121.5IU/ml, which is 61.54-243 times of the WHO recognized effective protective antibody threshold (0.5 IU/ml).

Third, investigation of immune protection effect of rabies mRNA vaccine

Rabies virus challenge experiments were performed on mice 43 days after priming. Mice were injected intracerebrally with 25 μ l, virus titer 50LD ₅₀ Rabies strain BD 06. The body weight of the mice was recorded before and after challenge, and the life status was observed.

The results are shown in FIG. 9: after challenge, the rabies mRNA-1902P vaccine immunized mice basically maintain the weight before challenge, and the weight of the negative control group mice shows a significant downward trend starting at the fourth day after challenge. The rabies mRNA vaccine mRNA-1902P has good immune protection effect.

The results are shown in FIG. 10: after challenge, all mice immunized with rabies mRNA-1902P vaccine survived, while the negative control mice died 3, 2, and 1 mice on

days

6, 7, and 8 after challenge, respectively. Further proves that the rabies mRNA vaccine mRNA-1902P has good immune protection effect.

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 examples, it will be appreciated that the invention may 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

<110> military medical research institute of military science institute of people's liberation force of China

<120> vaccine preparation method based on 1083 framework

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1009

<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 aaaaaaaaa 1009

<210> 2

<211> 1040

<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 1040

<210> 3

<211> 1049

<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 aaaaaaaaa 1049

<210> 4

<211> 1049

<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 aaaaaaaaa 1049

<210> 5

<211> 1052

<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 aa 1052

<210> 6

<211> 1053

<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 aaa 1053

<210> 7

<211> 1063

<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 aaa 1063

<210> 8

<211> 1076

<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 aaaaaa 1076

<210> 9

<211> 1690

<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 1690

<210> 10

<211> 1921

<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

a 1921

<210> 11

<211> 2053

<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 aaa 2053

<210> 12

<211> 2566

<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 aaaaaa 2566

<210> 13

<211> 4165

<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 aaaaa 4165

<210> 14

<211> 1882

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

acttgttctt tttgcagaag ctcagaataa acgctcaact ttggccggat ccgccatgat 60

tcctcaagct ctgttgtttg tacctcttct ggtttttcca ttgtgtttcg ggaaattccc 120

catttacacg ataccagaca aactcggccc ctggagtccc atcgatatac atcacctcag 180

ctgtccgaac aatctggttg tggaggacga aggatgtacc aatctgtcag gattctcata 240

catggagctt aaagtaggat atatttcggc cataaaggtg aacgggttca cttgtacggg 300

tgtggtaacg gaagcagaaa cctacactaa ctttgtcggt tatgtcacca ccacgtttaa 360

gagaaagcac ttccgaccaa caccggatgc atgcagatca gcatacaatt ggaagatggc 420

aggtgacccc agatatgaag agtctctgca caatccctat cctgattatc attggctccg 480

gactgtaaaa accaccaaag agtctgttgt tatcatatct ccaagtgtgg cagacttaga 540

cccgtacgat aaatcacttc attcgagagt ttttcctaga ggaaaatgct caggaataac 600

ggtgtcttct gcctactgct ctaccaacca tgattatacc atctggatgc ctgaaaatcc 660

tagactgggg acctcttgtg atattttcac caacagcaga gggaagagag catccaaagg 720

gagcaagacc tgtggatttg tggatgagag aggcttgtac aaatctctaa aaggagcatg 780

caaactgaag ctgtgtggag ttcttggact taggcttatg gacggaacct gggtcgcgat 840

tcagacatca aacgagacca agtggtgccc tcctgatcaa ctagtgaatc tacatgactt 900

tcattcagat gagattgaac atcttgttgt ggaggagttg gttaagaaga gggaggagtg 960

cctggatgca ctggagtcca tcatgaccac caagagcgtg agcttccgcc gcctgagcca 1020

cctgcgcaag ctggtgcccg gcttcggcaa ggcctacacc atcttcaaca agaccctgat 1080

ggaggccgac gcccactaca agagcgtgcg cacctggaac gagatcatcc ccagcaaggg 1140

ctgcctgcgc gtgggcggcc gctgccaccc ccacgtgaac ggcgtgttct tcaacggcat 1200

catcctgggc cccgacggcc acgtgctgat ccccgagatg cagagcagcc tgctgcagca 1260

gcacatggag ctgctggaga gcagcgtgat ccccctgatg caccccctgg ccgaccccag 1320

caccgtgttc aaggacggcg acgaggtgga ggacttcgtg gaggtgcacc tgcccgacgt 1380

gcacaagcag gtgagcggcg tggacctggg cctgcccaac tggggcaagg acgtgctgat 1440

gggcgccggc gtgctgaccg ccctgatgct gatgatcttc ctgatgacct gctgccgccg 1500

caccaaccgc gccgagagca tccagcacag cctgggcgag accggccgca aggtgagcgt 1560

gaccagccag agcggccgcg tgatcagcag ctgggagagc tacaagagcg gcggcgagac 1620

caagctgtaa ggtaccacca gcctcaagaa cacccgaatg gagtctctaa gctacataat 1680

accaacttac actttacaaa atgttgtccc ccaaaatgta gccattcgta tctgctccta 1740

ataaaaagaa agtttcttca caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aa 1882

Claims

1. A method for preparing an mRNA vaccine, which is characterized by comprising the step of inserting mRNA of a target gene into a 1083 skeleton to obtain a vaccine active substance sequence, wherein the 1083 skeleton comprises 5 ' -UTR7 connected to the 5 ' end of a gene coding region of the target mRNA and 3 ' -UTR7 and Poly A connected to the 3 ' end of the gene coding region of the target mRNA, and the sequence of the 5 ' -UTR7 is a sequence obtained by replacing T in the 1 st to 46 th sequences of the 7 in a sequence table with U; the sequence of the 3' -UTR7 is a sequence obtained by replacing T in 818 to 943 of the sequence 7 in a sequence table with U; the sequence of the Poly A is a sequence obtained by replacing T in 944 to 1063 of the sequence 7 in the sequence table with U.

2. The method according to claim 1, wherein the mRNA of the target gene is any one of the following (1) to (6):

(3) a sequence obtained by replacing T at positions 47 to 1675 of the sequence 10 in the sequence table with U;

(6) a sequence obtained by replacing T in the 56 th to 3913 th sites of the sequence 13 in the sequence table with U;

(7) a sequence obtained by replacing T in the 47 th to 1636 th positions of the sequence 14 in the sequence listing with U.

3. The method according to claim 2, wherein the gene encoding the vaccine is introduced into a vector to obtain a recombinant vector, and the recombinant vector is transformed into a recombinant bacterium and then transcribed in vitro to obtain the vaccine.

4. The method according to claim 3, wherein the recombinant vector is any one of the following (1) to (6):

(2) inserting the sequence shown in the sequence 9 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the sequences of other sites of the vector unchanged;

(4) inserting the sequence shown in the sequence 11 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the other sites of the vector unchanged;

(5) inserting the sequence shown in the sequence 12 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the sequences of other sites of the vector unchanged;

(6) inserting the sequence shown in the sequence 13 in the sequence table into the NheI-XhoI sites of the vector pcDNA3.1(+), and keeping the other sites of the vector unchanged;

5. The method according to claim 4, wherein the recombinant bacterium is an E.coli competent cell transformed with the recombinant plasmid of claim 5.

6. Use of the method of any one of claims 1 to 5 for the preparation of a vaccine.

7. The use according to claim 1, wherein the vaccine is a novel coronavirus vaccine or a rabies virus vaccine.