CN113754781B - Vaccine based on mRNA and aiming at coronavirus and preparation method thereof - Google Patents

Abstract

The invention relates to the field of medicines, in particular to a vaccine aiming at coronavirus based on mRNA and a preparation method thereof, wherein the vaccine comprises the following components in parts by weight: liposome-encapsulated mRNA-RBD + NTD + RBD vaccines; the mRNA is designed according to the genome of Venezuelan equine encephalomyelitis virus TC83 in alphavirus family; mRNA-RBD + NTD + RBD contains a secretion signal peptide, a new coronary pneumonia Spike protein gene receptor binding Region (RBD) + an N end region (NTD) + a receptor binding Region (RBD) + a Foldon sequence; the invention can express coronavirus antigen by using one mRNA, has strong immunogenicity, can relieve the pain of patients and can achieve the immune effect.

Description

Vaccine based on mRNA and aiming at coronavirus and preparation method thereof

Technical Field

The invention relates to the field of medicine, in particular to a vaccine aiming at coronavirus based on mRNA and a preparation method thereof.

Background

Coronaviruses are a large family of viruses that cause severe diseases such as the common cold and even Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), COVID19, etc. Common signs of infection are respiratory symptoms, fever, cough, shortness of breath, dyspnea, and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. Coronavirus is a zoonosis virus that is transmitted between animals and humans. Detailed investigations have shown that severe acute respiratory syndrome coronavirus is transmitted from a paguma larvata to a human, and that middle east respiratory syndrome coronavirus is transmitted from a dromedary camel to a human. In addition, there are some known coronaviruses that are transmitted in animals, but do not yet infect humans. Novel coronavirus SARS-CoV-2 is a novel coronavirus strain that has not been previously discovered in humans. The severe infectious disease COVID19 caused by SARS-CoV-2. SARS-CoV-2 has the characteristics of good infectivity and high lethality rate, and by 5 months in 2021, SARS-CoV-2 has caused infection of 1 hundred million, 5 million and more than ten thousand people and death of 3 million and more people all over the world. And new coronaviruses are in constant variation and have become more prevalent, more severe in patient condition, and longer in duration. The british variant b.1.1.7, the south african variant b.1.351, the brazil variant p.1 and the indian variant b.1.617, which appeared in 2021, all caused epidemic rebound in local areas. According to the existing data, the coronavirus represented by the new coronavirus is likely to become a long-existing and annual epidemic virus. An effective vaccine is the best means to combat coronavirus infection.

The SARS-CoV-2 virus infection process is mainly the recognition of angiotensin converting enzyme 2(ACE2) receptor mediated by Spike protein, cell attachment and fusion. The Spike protein of SARS-CoV-2 virus is composed of N-terminal signal peptide, S1 subunit and S2 subunit, the S1 subunit is responsible for receptor binding, and the S2 subunit is responsible for membrane fusion. The S1 subunit contains an N-terminal NTD and a coronavirus receptor binding domain RBD, wherein RBD is primarily responsible for binding of the virus to the receptor, and the S2 subunit contains HR domains, including HR1 and HR2, that are closely associated with viral fusion. Therefore, the method for generating antibodies through vaccination to neutralize S1 subunit, particularly the receptor binding region RBD of coronavirus, so as to block the binding of SARS-CoV-2 virus and ACE2 receptor, and further induce cell fusion is an effective way for blocking virus infection and spread.

Administration of lipid nanoparticles LNP by IV injection resulted in an increase in neutrophils and monocytes in male rats, and microscopic changes in liver and mild increases in liver enzymes (ALT and AST) were observed. In female monkeys, LNP induces little and transient complement activation. LNP induces mild to moderate and reversible pro-inflammatory responses. These phenomena can be used to reduce the toxicity of LNP-induced immune activation by reducing the dose or by reducing the frequency of administration.

The existing new crown mRNA vaccine adopts a spike protein S gene full-length sequence, the molecular weight of the expressed protein is larger, the proportion of antigenic determinants is lower, the effective antigenic molecules expressed and presented are fewer, the continuous virus gene mutation can not be effectively considered, and the vaccine is not beneficial to quick upgrade and update.

Therefore, it is of great practical significance to provide a novel mRNA-based vaccine against coronavirus and a preparation method thereof.

Disclosure of Invention

In view of the above, the invention provides a vaccine against coronavirus based on mRNA and a preparation method thereof, wherein a sequence of a coronavirus Receptor Binding Domain (RBD) + N-terminal domain (NTD) + Receptor Binding Domain (RBD) + Foldon gene is used to express a secretory signal peptide, so that the vaccine has strong immunogenicity, can relieve pain of patients and achieve the immune effect of the vaccine, and simultaneously can express RBD domain structural proteins of different new crown variant strains and can also prepare multivalent vaccines to have the protection effect on different variant strains.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an antigen fragment, which comprises a secretion signal peptide, a Spike protein receptor binding Region (RBD) of the neocoronary pneumonia, an N-terminal region (NTD), a receptor binding Region (RBD) and a Foldon sequence gene.

In some embodiments of the invention, the antigenic fragment has:

(I) an amino acid sequence shown as SEQ ID No. 1;

or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I);

or

(III) an amino acid sequence which has at least 90% sequence identity with the sequence of (I) or (II) and which is functionally identical or similar to the amino acid sequence of (I).

In some embodiments of the invention, more than one of said one or more substitutions, deletions or additions is 2, 3, 4, 5, 6, 7 or 8.

The invention also provides nucleic acid molecules encoding the antigenic fragments of claim,

in some embodiments of the invention, the nucleic acid molecule has:

(I) a nucleotide sequence shown as SEQ ID No. 2;

or

(II) as shown in the complementary nucleotide sequence of the nucleotide sequence shown in any SEQ ID No. 2; or

(III) as represented by a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;

or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III);

or

(V) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of (I), (II), (III) or (IV).

In addition, the invention also provides a recombinant expression vector, which comprises the nucleic acid molecule and a self-replicating vector.

In some embodiments of the invention, the self-replicating vector comprises a TC-83 self-replicating vector.

The invention also provides a preparation method of the recombinant expression vector, which comprises the following steps:

step a: synthesizing RBD + NTD + RBD gene segments;

step b: constructing a TC-83 self-replicating vector;

obtaining a self-replicating mRNA sequence from a genome of an alphavirus family; the self-replicating mRNA sequence comprises a gene encoding an alphavirus self-replicating component, lacking a gene that produces a structural protein having an infectious alphavirus particle; amplifying a plasmid constructed by a self-replicating mRNA sequence as a template, and synthesizing to obtain the TC-83 self-replicating vector; in some embodiments of the invention, the TC-83 self-replicating vector has the sequence shown in SEQ ID No. 3;

step c: preparing a recombinant plasmid JCXH-106;

inserting the RBD + NTD + RBD gene carrying ApaI at the upstream and NotI at the downstream into the ApaI and NotI enzyme cutting sites of a TC-83 replication vector to obtain a recombinant plasmid JCXH-106; in some embodiments of the invention, the TC-83 self-replicating vector has the sequence shown in SEQ ID No. 4.

More importantly, the invention also provides application of the antigen fragment, the nucleic acid molecule, the expression vector and the expression vector prepared by the preparation method in preparing vaccines for preventing coronavirus or medicines for treating coronavirus.

Most importantly, the invention also provides a vaccine which comprises the antigen fragment, the nucleic acid molecule, the expression vector prepared by the preparation method and pharmaceutically acceptable auxiliary materials; or directly or indirectly preparing the vaccine from the antigen fragment, the nucleic acid molecule, the expression vector and the expression vector prepared by the preparation method.

The traditional inactivated vaccine and recombinant protein vaccine have long production period and complex process and cannot meet the large-scale inoculation requirement of the sudden large-scale epidemic. mRNA is a rapid response vaccine development platform for outbreak epidemic situations. The mRNA (messenger ribonucleic acid) vaccine developed by modern and BioNTech companies in recent years has initiated clinical trials, providing preliminary data on the safety and efficacy of mRNA vaccines. The mRNA vaccine is synthesized by taking linearized plasmid DNA as a template and performing enzyme transcription reaction in vitro, and the synthesis strategy avoids the problems of living cell culture production mode, safety, complex production process and the like which need to be considered. The mRNA vaccine platform has the characteristics of safety, effectiveness, short production period and simple process, thereby being particularly suitable for dealing with the explosive epidemic situation.

The invention also provides a preparation method of the vaccine, which comprises the following steps:

step a: synthesizing an RBD + NTD + RBD gene segment;

step b: constructing a TC-83 self-replicating vector;

obtaining a self-replicating mRNA sequence from a genome of an alphavirus family; the self-replicating mRNA sequence comprises a gene encoding an alphavirus self-replicating component, lacking a gene that produces a structural protein having an infectious alphavirus particle; amplifying a plasmid constructed by a self-replicating mRNA sequence as a template, and synthesizing to obtain the TC-83 self-replicating vector;

step c: preparing a recombinant plasmid JCXH-106;

inserting the RBD + NTD + RBD gene carrying ApaI at the upstream and NotI at the downstream into the ApaI and NotI enzyme cutting sites of a TC-83 replication vector to obtain a recombinant plasmid JCXH-106;

step d: linearizing JCXH-106 plasmid by restriction endonuclease;

step e: carrying out in-vitro transcription reaction on the linearized JCXH-106 plasmid, degrading the template DNA, and finally adding a 7-methylated guanylic acid cap structure to the 5' end of the transcribed mRNA;

step f: and e, mixing mRNA with lipid, wrapping the mRNA into LNP to obtain an mRNA-LNP compound, concentrating and changing the solution to the preparation solution prepared in the step e to obtain the vaccine.

The invention expresses RBD + NTD + RBD tandem protein at the same time, compared with singly expressing RBD, because the antigen selection is a multi-target, the invention is a better antigen selection strategy, the structure of the invention can obviously enhance the immunogenicity of the RBD, and increases the SARS-CoV-2 neutralizing antibody generated in the body of a mouse. Thus, a protective effect can be achieved with a smaller vaccine dose. The dosage of the liposome is correspondingly reduced because of the reduction of the inoculation dosage, so that the cytotoxicity generated by the liposome is reduced, and the vaccine toxicity is reduced while the immune effect is ensured.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a map of the structure of mRNA-RBD + NTD + RBD plasmid of the present invention, containing kanamycin resistance gene (Kan), T7 promoter gene, nsp gene, RBD + NTD + RBD gene, polyA gene;

FIG. 2 is a diagram showing the results of electrophoresis after in vitro transcription of mRNA-RBD + NTD + RBD according to the present invention; wherein, Lane A is mRNA-RBD + NTD + RBD in vitro transcription size, Lane M is RNAlader;

FIG. 3 shows a graph of expression of RBD + NTD + RBD protein after transfection of BHK cells in vitro with mRNA-RBD + NTD + RBD according to the present invention by Western detection;

FIG. 4 is a graph showing the results of the antibody titer against RBD induced after immunization of mice with mRNA-RBD + NTD + RBD according to the present invention by ELISA assay; wherein, fig. 4A shows the change in antibody (over time) during immunization of animal a 4; FIG. 4B shows the antibody changes (over time) during immunization of animal A11;

FIG. 5 shows the results of plasmid sequencing.

Detailed Description

The invention discloses a vaccine aiming at coronavirus based on mRNA and a preparation method thereof, and a person skilled in the art can realize the vaccine by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In order to achieve the above object, the present invention adopts the following technical solutions:

an mRNA-based vaccine against coronavirus, comprising: liposome-encapsulated mRNA-RBD + NTD + RBD; the mRNA is designed from the genome of the alphavirus. mRNA-RBD + NTD + RBD contains a secretory signal peptide, a Spike protein receptor binding Region (RBD) of the new coronary pneumonia, an N end region (NTD) + a receptor binding Region (RBD) + a Foldon sequence gene.

Viruses of the alphavirus family include: venezuelan Equine encephalomyelitis virus (TC83 Venezuelan equines encephalitisis virus, VEEV), sindbis virus (Sin-dbis virus), Chikungunya virus (Chikungunya virus), Eastern Equine encephalomyelitis virus (Eastern Equine encphalis-tis virus), Western Equine encephalomyelitis virus (Western equiencephalitis virus), mayaru virus (Mayarovirus), shenglisen forest virus (semlikriest virus), Venezuelan Equine encephalomyelitis virus (Venezuelan equines encephalitisis virus), and the like; as a preferred example, Venezuelan Equine encephalomyelitis Virus (TC83 Venezuelan Equisene Encephalitis Virus, VEEV) is used. It should be noted that: the alphavirus family is not exhaustive, and any virus designed to obtain the Spike protein Receptor Binding Domain (RBD) + N Terminal Domain (NTD) + Receptor Binding Domain (RBD) + Foldon sequence gene of mRNA for the new coronary pneumonia is within the scope of the present invention.

The foregoing mRNA-based vaccine against coronavirus, the mRNA comprising: conventional mRNA or self-replicating mRNA. There are two major routes to existing mRNA vaccine platform technology, one is based on conventional mRNA and the other is based on self-replicating mRNA; both routes can be applied to the present invention. After the self-replicating mRNA vaccine is inoculated into an animal body, a self sequence can be replicated by taking the self as a template, so that compared with the conventional mRNA vaccine, the self-replicating mRNA vaccine requires less inoculation dosage, and an adjuvant effect formed by an immune response induced during self replication can induce stronger immune response and further enhance humoral and cellular immune responses; thus, as a preferred option, self-replicating mRNA is synthesized in vitro by enzymatic transcription using linearized plasmid DNA as a template.

The mRNA-based vaccines described above against coronavirus are synthesized by self-replicating mRNA using linearized plasmid DNA as a template and performing an enzymatic transcription reaction in vitro.

A method for preparing an mRNA-based vaccine against coronavirus, comprising the steps of:

the steps for preparing the mRNA-RBD + NTD + RBD preparation include:

step a, synthesizing RBD + NTD + RBD gene segments:

inquiring a Spike amino acid sequence and designing a secretion signal peptide, wherein the sequence of a receptor binding Region (RBD) + an N end region (NTD) + a receptor binding Region (RBD) + a Foldon gene is optimized according to a human preference codon table to obtain an optimized sequence, a promoter and an ApaI enzyme digestion site GGGCCC are added to the upstream of the optimized sequence, a NotI enzyme digestion site GCGGCCGC is added to the downstream of the optimized sequence, and the designed sequence is synthesized to obtain the secretion signal peptide; the gene sequence is shown as SEQ ID No.2 in the sequence table;

step b, constructing a TC-83 self-replicating vector:

designing a self-replicating mRNA sequence according to a genome of an alphavirus family, wherein the self-replicating mRNA sequence comprises a gene capable of coding an alphavirus self-replicating component and lacks a gene capable of manufacturing a structural protein of infectious alphavirus particles, a plasmid constructed by the self-replicating mRNA sequence is used as a template for amplification, and the designed sequence is obtained through synthesis;

in some embodiments of the invention, the self-replicating mRNA is designed from the genome of Venezuelan Equine encephalomyelitis Virus (TC83 Venezuelan Equine enchaitis Virus, VEEV) in the alphavirus family, which contains genes encoding the alphavirus self-replicating components but lacks structural proteins encoding the infectious alphavirus particles, and the constructed plasmid is amplified as a template, and the designed sequence is directly obtained by synthesis, and the gene sequence is represented by SEQ ID No.3 in the sequence table;

step c, preparing a recombinant plasmid JCXH-106:

inserting an upstream RBD + NTD + RBD gene carrying ApaI and a downstream RBD + NTD gene carrying NotI between ApaI and NotI enzyme cutting sites of a TC-83 replication vector to obtain a recombinant plasmid JCXH-106 (shown in figure 1), wherein the gene sequence is shown as SEQ ID No.4 in a sequence table;

step d, carrying out linearization on the JCXH-106 plasmid by using restriction enzyme digestion;

step e, linearization of JCXH-106 plasmid first step in vitro transcription reaction (IVT) using T7 RNA polymerase, second step in degradation of template DNA using Turbo DNase, third step in addition of 7-methylated guanylate Cap structure (called Cap0) to 5' end of transcribed mRNA using capping enzyme (see FIG. 2);

step f, mRNA is rapidly mixed with lipids (consisting of 1, 2-dimethylsilyl-rac-3-methoxypolyethylene glycol-2000(DMG-PEG2000), cholesterol, Distearoylphosphatidylcholine (DSPC) and cationic lipid DLinDMA by a Nanolasembler mixer, causing precipitation of lipids and entrapment of the charged mRNA into LNP.

The application method of the new crown RBD + NTD + RBD high-expression mRNA-RBD + NTD + RBD based on Venezuelan Equise Encephalitis Virus (VEEV) in alphavirus family adopts a prime-boost administration application mode of vaccine; prime-boost administration of the vaccine used mRNA-RBD + NTD + RBD.

In the preparation method of the mRNA-based combined vaccine for the coronavirus and the influenza virus, the restriction enzyme is BspQI enzyme.

In the preparation method for the mRNA-based coronavirus, the linearized JCXH-106 plasmid is subjected to in vitro transcription reaction by using T7 RNA polymerase, the template DNA is degraded by using Turbo DNase, and a 7-methylated guanylic acid cap structure is added to the 5' end of the transcribed mRNA by using a capping enzyme.

In the preparation of the aforementioned mRNA-based vaccine against coronavirus, lipids include: PEG2000, cholesterol, distearoylphosphatidylcholine, cationic lipid DLinDMA.

In the preparation method of the mRNA-based combined vaccine for coronavirus and influenza virus, the mRNA and the lipid are mixed by using a Nanolasemblr mixer.

The administration mode of the vaccine is first injection and boosting injection, and the number of boosting injections is determined according to the needs of patients.

It should be noted that: mRNA-RBD + NTD + RBD in vitro transcription mRNA product, and the specification has described in detail the whole process of in vitro transcription and liposome encapsulation, any company or individual can prepare the in vitro transcription liposome encapsulated mRNA vaccine according to the steps in the specification.

The invention has the advantages that:

the invention can express coronavirus antigen by using one mRNA, has strong immunogenicity, can relieve the pain of patients and can achieve the immune effect;

based on the fact that the self-replicating mRNA vaccine has strong immunogenicity in animal experiments and can replicate self sequences by taking the self as a template, compared with the conventional mRNA vaccine, the self-replicating mRNA vaccine requires less inoculation dose, and adjuvant effect formed by immune response induced during self replication can induce stronger immune response and further enhance humoral and cellular immune response;

the self-replicating mRNA is synthesized by taking linearized plasmid DNA as a template and performing enzyme transcription reaction in vitro, and the synthesis strategy avoids the problems of safety, complex production process and the like of a living cell culture production mode which needs to be considered;

the method has simple process, and mRNA vaccine can be prepared only by in vitro transcription and liposome encapsulation;

mRNA is a universal vaccine platform, can select new antigen sequences according to annual or seasonal variation of strains, and can rapidly develop new vaccines under the condition of not changing a process flow.

In the mRNA-based vaccine against coronavirus and the preparation method thereof, the used raw materials and reagents can be purchased from the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 RBD + NTD + RBD Gene fragment Synthesis

The protein genes of Spike (SARS-CoV-2) and British variant B.1.1.7, south African variant B.1.351, Brazilian variant P.1 and Indian variant B.1.617 are inquired from NCBI, and then secretion signal peptides, receptor binding Region (RBD) + N terminal region (NTD) + receptor binding Region (RBD) + Foldon sequence gene are designed, then corresponding optimization is carried out according to human codons, ApaI restriction enzyme cutting sites and promoters are added at the upstream, NotI restriction enzyme cutting sites are added at the downstream, and finally DNA sequences are directly obtained by synthesis (obtained in a mode that the company provides cloning plasmids pUC57-RBD + NTD + RBD), (the sequence is shown as SEQ ID No. 2). The obtained amino acid sequence is shown as SEQ ID No. 1.

EXAMPLE 2 construction of TC-83 self-replicating vectors

Self-replicating mrnas are designed from the genome of Venezuelan Equine encephalomyelitis Virus (TC83 Venezuelan escherichia Virus, VEEV) in alphavirus family, contain genes encoding self-replicating components of alphavirus, but lack structural proteins encoding for the production of infectious alphavirus particles, and are amplified as templates in the constructed plasmids, and the designed sequences are directly obtained by synthesis (sequences shown in seq id No. 3).

EXAMPLE 3 preparation of recombinant plasmid JCXH-106

pUC57-RBD + NTD + RBD and TC-83 self-replicating vectors were double digested with Apa I and Not I, 20. mu.L: pUC57-RBD + NTD + RBD or TC-83 self-replicating vector < 1. mu.g, ApaI 1. mu.L, NotI 1. mu.L, 10 × CutSmart Buffer 2. mu.L, ddH ₂ O make up the system to 20. mu.L. Carrying out water bath at 25 ℃ for 1h to cut the plasmid, then carrying out water bath at 37 ℃ for 1h to cut the plasmid, adding 0.5 mu L of CIP into the vector fragment, and carrying out water bath at 37 ℃ for 30min to dephosphorize. Mixing the enzyme digestion mixture with 6 × Loading Buffer, and electrifyingThe fragment (pUC57-RBD + NTD + RBD recovery fragment RBD + NTD + RBD gene fragment 2574bp in length; TC-83 self-replicating vector recovery fragment about 9.5Kb in length) was recovered by electrophoresis (1% agarose, 94V) and gel electrophoresis, and eluted with 30. mu.L of elusion Buffer.

Respectively taking an RBD + NTD + RBD gene fragment and a TC-83 self-replicating vector fragment according to a connection system: vector 50ng, insert moles: vector fragment molar ═ 5:1, T4Ligase 1. mu.L, 10 XLigase Buffer 5. mu.L, ddH ₂ O make up the system to 10. mu.l and attach for 1h at 22 ℃. The ligation product was gently mixed with competent cells of E.coli DH 5. alpha. in a volume of 1:10, ice-cooled for 30min, heat shocked at 42 ℃ for 45s, ice-cooled for 3min, and added with 500. mu.L of preheated SOB broth, mixed well, cultured at 37 ℃ and 180rpm for 1h, spread on LK plates (LB-Kan plates: LB plates containing 50. mu.g/mL Kan), and cultured at 37 ℃ for 16-20 h.

And (3) PCR screening: amplifying a target band Spike by using bacterial plasmids extracted by a boiling method as a template, wherein an upstream primer F: 5'-TATGGCCATGACTACTCTAGCTA-3', downstream primer R: 5'-GGGAAACGCCTGGTATCTTT-3', the reaction circulation conditions are as follows: 94 ℃ 3min → (94 ℃ 1min, 47 ℃ 30s, 72 ℃ 3min) × 30 cycles → 72 ℃ 10min → 4 ℃; electrophoretic Observation of PCR results (electrophoresis conditions: 1% agarose gel; 90V, loading: 5. mu.l PCR product), expected results: the RBD + NTD + RBD fragment is approximately 2977 bp.

Enzyme digestion verification: plasmids were extracted from positive bacteria screened and verified by PCR, digested with Apa I and Not I according to the above digestion system, and the digested mixture was mixed with 6 × Loading Buffer for electrophoresis (1% agarose, 94V).

Sequencing and verifying: the plasmid which is expected by the extraction PCR and the enzyme digestion verification is sent to a sequencing company for sequencing (the sequencing result is shown in figure 5). Coli carrying the positive plasmid, which was completely correct after sequencing verification, was stored at-80 ℃.

And (3) sequence alignment results: query is the design sequence and subject is the sequencing result sequence, NCBI two-sequences blast. The results indicated that the antigen-coding region from the start codon to the stop codon was completely correct. The sequence is shown in SEQ ID No. 4.

Example 4 linearization and recovery of JCXH-106 plasmid by BspQI digestion

JCXH-106 plasmid 10. mu.g, BspQI 1. mu.L, 10 XNEBuffer 3.15. mu.L, ddH ₂ O, the system is complemented to 50 mu L, and the plasmid is cut by enzyme in water bath at 50 ℃ for 1 h. The cleavage mixture was mixed with 6 Xloading Buffer, electrophoresed (1% agarose, 94V) and the corresponding length of the fragment recovered in gel (JCXH-106 plasmid used, length about 12Kb), 30. mu.L of Elution Buffer Elution.

Example 5

After linearization JCXH-106 plasmid was prepared by using an in vitro transcription reaction (IVT) initiated with T7 RNA polymerase, Turbo DNase enzyme to degrade template DNA, and capping enzyme (Vaccidia capping enzyme) to add 7-methylguanylate Cap structure (called Cap0) to the 5' end of transcribed mRNA as follows.

10 Xreaction Buffer 2. mu.L, NTP 0.5mM each, linearized JCXH-1021. mu.g, T7 RNA Polymerase 2. mu.L, water supplemented to 20. mu.L, reacted at 30 ℃ for 1 hour and added, 1. mu.L of TURBO ^TM DNase, 4. mu.L of 10X clamping Buffer, 2. mu.L of GTP (10mM), 2. mu.L of SAM (2mM), 2. mu.L of Vaccinia clamping Enzyme, supplemented with water in a total volume of 40. mu.L, reacted at 30 ℃ for 1 hour. Then, water was added to 200. mu.L, and 120. mu.L of 7.5M lithium chloride was added thereto, and the mixture was left to stand at-20 ℃ for 30 minutes, then centrifuged at 14000g and 4 ℃ for 30 minutes, the supernatant was discarded, the precipitate was washed with 70% ethanol, centrifuged at 14000g and 4 ℃ for 5 minutes, the supernatant was discarded, and then air-dried for 5 minutes, and dissolved in 40. mu.L of water. After quantitation by spectrophotometer, 400ng was mixed with 10. mu.L of northern Max-Gly Sample Loading Dye, incubated at 50 ℃ for 30 minutes, and electrophoresed with northern Max-Gly Gel Prep/Running buffer (1% agarose, 70V). The electrophoresis picture is shown in FIG. 2, and the molecular weight size is expected.

Example 6 mRNA-RBD + NTD + RBD with lipid encapsulation

The mRNA is rapidly mixed with lipids (including 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000(DMG-PEG2000), cholesterol, Distearoylphosphatidylcholine (DSPC) and cationic lipids, dissolved in alcohol) by a Nanolasemblr mixer, causing precipitation of the lipids and entrapment of the charged mRNA into the LNP. The mRNA-LNP complex is then reconstituted by concentration and exchange into formulation solution.

Example 7 Westernblot assay for mRNA-RBD + NTD + RBD expression in cells

BHK-21 cells purchased from Shanghai cell center were subcultured until the number of cells was sufficient, trypsinized to 6-well plates per well of the cell culture, and plated overnight in a CO2 incubator at 37 ℃. The next day, liposome-encapsulated mRNA-RBD + NTD + RBD was transfected into the plated BHK-21 cells, and after 72h of culture, the cells were lysed to collect a protein sample. The specific method comprises the following steps:

1) cell culture and plating: inoculating the recovered BHK-21 cells into a culture bottle of 75cm2, wherein the culture medium is DMEM high-sugar medium + 5% double antibody + 10% fetal bovine serum, digesting the cells by pancreatin when the confluence of the cells at the bottom of the bottle reaches more than 80%, and counting. Appropriate number of cells were plated in 6-well cell culture plates at 37 ℃ with CO ₂ The incubator was overnight.

2) Transfection of Liposomal mRNA into BHK-21 cells: the medium in the well-spread 6-well plates was blotted dry, washed once with PBS buffer, and liposome-encapsulated mRNA was mixed with 1mL Opti-MEM medium, added to the washed 6-well plates, and incubated at 37 ℃ in CO ₂ The incubator was supplemented 6h later with 1mL of DMEM high-glucose medium containing 20% fetal bovine serum. CO at 37 deg.C ₂ And (5) culturing for 72h in an incubator.

3) Processing of protein samples: after 72 hours, 200. mu.L of cell lysate and 1% PMSF were added to BHK-21 cells, and the mixture was left on ice for 5 minutes, centrifuged at 14000g for 5 minutes, and the supernatant was transferred to a fresh centrifuge tube and then added to a metal bath of 5 XSDS 95 ℃ for 12 minutes. Placing at-20 deg.C for use.

4) Western blot detection:

electrophoresis: the concentration of polyacrylamide gel is 6%, the loading amount of protein is 20 μ L, the electrophoresis condition of lamination gel is 150V and 10min, and the electrophoresis condition of gel is 200V and 30 min.

Electric conversion: using a nitrocellulose membrane, membranes were spun at 100V for 1 hour.

And (3) sealing: 5% BSA was prepared as blocking solution using 1 XPBST and blocked overnight at 4 ℃.

Primary antibody incubation: SARA-Cov-2(2019-nCov) spike antibody was reacted with 1: 500 were diluted and incubated at room temperature for 2 hours. Wash 3 times with 1X PBST for 5 minutes each.

And (3) secondary antibody incubation: and (3) diluting the secondary antibody by using a blocking solution at a dilution ratio of 1: 10000, incubation at room temperature for 1 hour. Wash 3 times with 1X PBST for 5 minutes each.

And (3) developing: developer solution A: solution B is 1:1, developing in an imaging system.

The experimental result is shown in figure 3, figure 3 is an RBD + NTD + RBD protein expression graph after mRNA-RBD + NTD + RBD transfects BHK cells in vitro, and the WB result shows that the corresponding target protein can be efficiently expressed after the cells are transfectd in vitro;

as shown in FIG. 3, the expression of RBD + NTD + RBD protein was better after in vitro transfection of BHK cells with mRNA-RBD + NTD + RBD.

Example 8 mRNA-RBD + NTD + RBD vaccine immunization of mice induced anti-Spike antibody titer assays

After 6-8 weeks old BALB/c female mice are adaptively fed for 7 days, 5 mRNA-RBD + NTD + RBD groups and 5 mRNA-RBD groups are respectively immunized with mRNA-RBD + NTD + RBD or mRNA-RBD vaccines, 3 mu g of the vaccine is injected, and after 28 days, the dose of 3 mu g of the vaccine is further strengthened and injected. Blood was taken on day 0, day 7 post-injection, day 14, day 21, day 28, day 35, and day 42, respectively, to test for RBD antibody titer.

The antibody titer detection method is specifically characterized in that each hole of a 96-hole enzyme label plate is coated with 50ng of Spike RBD structural domain protein overnight at room temperature, the next day is washed with PBST for 3 times, the hole is sealed with 5% milk, after 37 ℃ for 1 hour, the hole is washed with PBST for 3 times, mouse serum with corresponding dilution ratio is added, after 37 ℃ for 1 hour, the hole is washed with PBST for 3 times, anti-mouse igg heavy chain light chain HRP is added, after 37 ℃ for 1 hour, the hole is washed with PBST for 3 times, and finally, color developing liquid is added for developing.

The experimental results are shown in fig. 4; the anti-RBD antibody titers in the mouse sera.

In FIG. A, A4-0, A4-7, A4-14, A4-21, A4-28, A4-28+7, and A4-28+15 were taken on days 0, 7, 14, 21, 28, 35, and 42, respectively, and 5 mice were administered with a dose of 3. mu.g in the mRNA-RBD + NTD + RBD group; in FIG. B, A11-0, A11-7, A11-14, A11-21, A11-28, A11-28+7, and A11-28+14 were taken on days 0, 7, 14, 21, 28, 35, and 42, respectively, and 5 mice were administered with a dose of 3. mu.g in the mRNA-RBD group.

TABLE 1 data of FIG. 4

A11	A11-0	A11-7	A11-14	A11-21	A11-28	A11-28+7	A11-28+14
								1∶20	0.7841	1.5896	2.3049	4.2413	4.6864	5.3017	4.9129
1∶40	0.3833	0.7674	1.0432	3.5511	3.9547	4.8432	4.8687
								1∶80	0.2369	0.4684	0.5795	2.5354	3.0429	3.9686	4.9203
1∶160	0.1495	0.2904	0.3305	1.3958	2.0929	3.1179	4.8089
								1∶320	0.1081	0.1898	0.1915	0.7526	1.2325	2.0836	4.0823
1∶640	0.0874	0.1185	0.123	0.4195	0.7089	1.2696	3.3942
								1∶1280	0.0791	0.0912	0.0877	0.2589	0.4041	0.7093	2.4264
1∶2560	0.0704	0.0819	0.0777	0.1512	0.2428	0.3931	1.4518
								1∶5120	0.068	0.0709	0.07	0.1065	0.1409	0.233	0.8038
1∶10240	0.0681	0.0714	0.0727	0.084	0.1032	0.1409	0.4333
								1∶20480	0.0686	0.0669	0.0673	0.0743	0.0923	0.1081	0.214
1∶40960	0.0671	0.0691	0.071	0.0733	0.0878	0.082	0.1339
A4	A4-0	A4-7	A4-14	A4-21	A4-28	A4-28+7	A4-28+15
								1∶20	0.5369	0.9762	2.3295	4.5049	3.8598	5.713	4.8823
1∶40	0.3251	0.5791	1.3905	3.4451	2.8469	6	4.3877
								1∶80	0.2053	0.3462	0.7881	2.3778	1.7749	5.6365	4.6686
1∶160	0.1287	0.2085	0.3998	1.298	1.0783	5.6487	4.5662
								1∶320	0.0919	0.1372	0.2307	0.6464	0.6095	5.0958	4.5491
1∶640	0.0796	0.0909	0.1509	0.379	0.2974	5.0762	4.2721
								1∶1280	0.0591	0.0733	0.0959	0.1993	0.183	4.8246	3.5518
1∶2560	0.0534	0.0636	0.0749	0.1294	0.1158	4.4342	3.1621
								1∶5120	0.0535	0.059	0.0619	0.0936	0.0901	3.5711	2.2865
1∶10240	0.0571	0.0547	0.0545	0.0763	0.0748	2.4539	1.6216
								1∶20480	0.0551	0.0515	0.0526	0.0718	0.0645	1.5422	1.0141
1∶40960	0.0613	0.0551	0.0539	0.069	0.0667	0.9435	0.6323

Therefore, mRNA-RBD + NTD + RBD can induce and generate better antibody reaction in animals compared with mRNA-RBD vaccine, the specific enzyme-labeled antibody of the new structure after the new structure antibody level is strengthened is more than 1: 40960, while the specific enzyme-labeled antibody of the new structure after the conventional RBD is strengthened is 1: 10240-4 times, and the new structure antibody level is more than 4 times.

Experiments show that the vaccine obtained by the method can be well expressed in cells and can induce and generate better antibody response in animals.

The invention can express the antigen of coronavirus by using one mRNA, has strong immunogenicity, can relieve the pain of patients, can achieve the immune effect, and has good application prospect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Chengdu European forest Biotechnology GmbH; jiachen West sea (Hangzhou) Biotechnology Co Ltd

<120> mRNA-based vaccine against coronavirus and method for preparing the same

<130> MP21020191

<160> 4

<170> SIPOSequenceListing 1.0

<210> 4

<211> 832

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu

1 5 10 15

Gly Met Leu Val Ala Ser Val Leu Ala Gly Gly Ser Gly Gly Gly Gly

20 25 30

Ser Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

35 40 45

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

50 55 60

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

65 70 75 80

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

85 90 95

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

100 105 110

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

115 120 125

Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

130 135 140

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

145 150 155 160

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

165 170 175

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

180 185 190

Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro

195 200 205

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro

210 215 220

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

225 230 235 240

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

245 250 255

Asn Phe Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Asn Leu Thr

260 265 270

Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly

275 280 285

Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr

290 295 300

Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala

305 310 315 320

Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val

325 330 335

Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn

340 345 350

Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln

355 360 365

Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys

370 375 380

Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys

385 390 395 400

Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala

405 410 415

Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu

420 425 430

Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys

435 440 445

Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn

450 455 460

Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val

465 470 475 480

Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala

485 490 495

Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr

500 505 510

Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe

515 520 525

Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys

530 535 540

Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Gly Gly

545 550 555 560

Ser Gly Gly Gly Gly Ser Gly Gly Arg Val Gln Pro Thr Glu Ser Ile

565 570 575

Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

580 585 590

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

595 600 605

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

610 615 620

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

625 630 635 640

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

645 650 655

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn

660 665 670

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

675 680 685

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

690 695 700

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

705 710 715 720

Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe

725 730 735

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly

740 745 750

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

755 760 765

His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val

770 775 780

Lys Asn Lys Cys Val Asn Phe Gly Gly Ser Leu Gly Gly Gly Gly Ser

785 790 795 800

Gly Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

805 810 815

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

820 825 830

<210> 2

<211> 2502

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggccccta tgggcagcct gcagcccctg gccaccctgt acctgctggg catgctggtg 60

gccagcgtgc tggccggcgg cagcggcggc ggcggcagcg gatccagggt gcagcccacc 120

gagagcatcg tgcggttccc caacatcacc aacctgtgcc ccttcggcga ggtgtttaat 180

gccaccaggt ttgccagcgt gtacgcctgg aatagaaaaa gaatttctaa ttgtgtggcc 240

gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta cggcgtgtcc 300

ccaacaaaac tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatccgc 360

ggcgacgagg tgcggcagat cgcccccggc cagaccggca acatcgccga ctacaactac 420

aagctgcccg acgacttcac cggctgcgtg atcgcctgga attctaataa tctggacagc 480

aaggtgggcg gcaactacaa ctacctgtac agactgttta gaaaatctaa tctgaagccc 540

ttcgagaggg acatcagcac cgagatctac caggccggca gcaccccatg taatggagtg 600

aagggcttca actgctactt ccccctgcag agctacggct tccagcccac ctacggcgtg 660

ggctaccagc cctacagagt ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc 720

gtgtgcggcc ccaagaagtc taccaacctg gtgaagaaca agtgcgtgaa cttcggcggc 780

agcggcggcg gcggcagcgg cggcgtgaac ctgaccacca ggacacagct gcccccagcc 840

tacaccaaca gcttcaccag gggcgtgtac tacccagata aagtgtttag aagttctgtg 900

ctgcacagca cccaggacct gtttctgccc ttcttcagca acgtgacctg gtttcatgcc 960

atccacgtgt ccggcaccaa cggcaccaag aggtttgata atccagtgct gcccttcaac 1020

gacggcgtgt acttcgccag caccgagaag tcaaacatca tccgcggctg gatcttcggc 1080

accaccctgg acagcaagac ccagagcctg ctgatcgtga acaacgccac caacgtggtg 1140

atcaaggtgt gcgagttcca gttttgtaat gatccttttc tgggcgtgta ctaccacaag 1200

aacaacaaga gctggatgga gagcgagttc cgggtgtaca gcagcgccaa caactgcacc 1260

ttcgagtacg tgtcccagcc cttcctgatg gatctggagg gcaagcaggg caacttcaag 1320

aacctgcggg agttcgtgtt taaaaatatt gatggatatt ttaaaattta ttctaaacat 1380

actccaatta atctggtgcg ggacctgccc cagggcttca gcgccctgga gcccctggtg 1440

gacctgccca tcggcatcaa catcaccagg tttcagaccc tgctggccct gcacaggagc 1500

tacctgaccc caggagacag cagcagcggc tggacagccg gcgccgccgc ctactacgtg 1560

ggctacctgc agcccaggac atttctgctg aagtacaacg agaacggcac catcaccgac 1620

gccgtggact gcgccctgga ccccctgagc gagaccaagt gcaccctgaa atctggcggc 1680

agcggcggcg gcggcagcgg cggacgggtg cagcccaccg agagcatcgt gcggttcccc 1740

aacatcacca acctgtgccc cttcggcgag gtgttcaacg ccacccggtt cgccagcgtg 1800

tacgcctgga accggaagcg gatcagcaac tgcgtggccg actacagcgt gctgtacaac 1860

agcgccagct tcagcacctt caagtgctac ggcgtgagcc ccaccaagct gaacgacctg 1920

tgcttcacca acgtgtacgc cgacagcttc gtgatccggg gcgacgaggt gcggcagatc 1980

gcccccggcc agaccggcac catcgccgac tacaactaca agctgcccga cgacttcacc 2040

ggctgcgtga tcgcctggaa cagcaacaac ctggacagca aggtgggcgg caactacaac 2100

tacctgtacc ggctgttccg gaaatcaaac ctgaagccct tcgagcggga catcagcacc 2160

gagatctacc aggccggcag caccccctgc aacggcgtga agggcttcaa ctgctacttc 2220

cccctgcaga gctacggctt ccagcccacc tacggcgtgg gctaccagcc ctaccgggtg 2280

gtggtgctga gcttcgagct gctgcacgcc cccgccaccg tgtgcggccc caagaaaagt 2340

accaacctgg tgaagaacaa gtgcgtgaac ttcggcggca gcctgggcgg cggcggcagc 2400

ggcagcgcca tcggcggcta catccccgag gccccccggg acggccaggc ctacgtgcgg 2460

aaggacggcg agtgggtgct gctgagcacc ttcctgtgat aa 2502

<210> 3

<211> 9636

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 60

ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 120

aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 180

tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 240

gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 300

gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 360

aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 420

ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480

aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 540

ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 600

agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 660

cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 720

ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 780

ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 840

tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 900

tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 960

cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1020

tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080

tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1140

tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1200

tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1260

ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1320

acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380

atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1440

caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1500

acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1560

tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1620

tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680

aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1740

ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1800

taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1860

tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1920

ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980

gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2040

aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2100

ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2160

cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2220

gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280

aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2340

ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2400

ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2460

ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2520

tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580

gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2640

cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2700

aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2760

aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2820

ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880

tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2940

taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3000

cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3060

agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3120

tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3180

cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3240

gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3300

cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3360

cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3420

gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3480

tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3540

gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3600

tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3660

tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3720

agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3780

tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3840

gcatcattgg tgctatagcg cggctgttca agttttcccg ggtatgcaaa ccgaaatcct 3900

cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3960

acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4020

aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080

gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4140

tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4200

tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4260

cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4320

acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4380

acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4440

cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4500

ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4560

atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4620

caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680

atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4740

tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4800

aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4860

gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4920

tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980

caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5040

acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5100

cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5160

aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5220

aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280

ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5340

gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5400

gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5460

gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5520

caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5580

ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5640

ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5700

catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5760

cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5820

tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5880

acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5940

ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6000

tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6060

cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6120

ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180

ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6240

ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6300

ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6360

cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6420

ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6480

aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6540

taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6600

aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6660

cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6720

acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6780

tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6840

acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6900

tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6960

aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7020

tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080

cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7140

acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7200

aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7260

gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7320

aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380

gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7440

tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7500

gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7560

gtaaggcgcg cccacccagc ggccgcatac agcagcaatt ggcaagctgc ttacatagaa 7620

ctcgcggcga ttggcatgcc gccttaaaat ttttatttta tttttctttt cttttccgaa 7680

tcggattttg tttttaatat ttcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 7740

aaaagaagag ctagggataa cagggtaatt gagcaaaagg ccagcaaaag gccaggaacc 7800

gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 7860

aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 7920

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 7980

tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 8040

tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 8100

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 8160

tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 8220

ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 8280

tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 8340

aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 8400

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 8460

aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 8520

ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 8580

acagttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat 8640

caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt 8700

tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac 8760

aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga 8820

cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag 8880

gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg 8940

attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa 9000

tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag 9060

gatattcttc taatacctgg aatgctgttt tcccagggat cgcagtggtg agtaaccatg 9120

catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc 9180

agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca 9240

gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc 9300

cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc 9360

gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt 9420

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 9480

aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 9540

aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 9600

tagggataac agggtaatta atacgactca ctatag 9636

<210> 4

<211> 12120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 60

ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 120

aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 180

tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 240

gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 300

gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 360

aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 420

ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480

aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 540

ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 600

agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 660

cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 720

ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 780

ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 840

tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 900

tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 960

cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1020

tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080

tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1140

tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1200

tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1260

ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1320

acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380

atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1440

caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1500

acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1560

tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1620

tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680

aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1740

ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1800

taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1860

tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1920

ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980

gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2040

aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2100

ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2160

cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2220

gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280

aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2340

ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2400

ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2460

ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2520

tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580

gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2640

cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2700

aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2760

aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2820

ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880

tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2940

taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3000

cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3060

agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3120

tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3180

cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3240

gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3300

cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3360

cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3420

gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3480

tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3540

gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3600

tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3660

tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3720

agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3780

tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3840

gcatcattgg tgctatagcg cggctgttca agttttcccg ggtatgcaaa ccgaaatcct 3900

cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3960

acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4020

aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080

gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4140

tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4200

tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4260

cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4320

acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4380

acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4440

cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4500

ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4560

atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4620

caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680

atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4740

tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4800

aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4860

gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4920

tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980

caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5040

acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5100

cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5160

aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5220

aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280

ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5340

gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5400

gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5460

gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5520

caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5580

ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5640

ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5700

catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5760

cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5820

tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5880

acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5940

ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6000

tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6060

cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6120

ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180

ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6240

ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6300

ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6360

cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6420

ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6480

aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6540

taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6600

aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6660

cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6720

acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6780

tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6840

acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6900

tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6960

aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7020

tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080

cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7140

acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7200

aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7260

gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7320

aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380

gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7440

tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7500

gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7560

gatggcccct atgggcagcc tgcagcccct ggccaccctg tacctgctgg gcatgctggt 7620

ggccagcgtg ctggccggcg gcagcggcgg cggcggcagc ggatccaggg tgcagcccac 7680

cgagagcatc gtgcggttcc ccaacatcac caacctgtgc cccttcggcg aggtgtttaa 7740

tgccaccagg tttgccagcg tgtacgcctg gaatagaaaa agaatttcta attgtgtggc 7800

cgactacagc gtgctgtaca acagcgccag cttcagcacc ttcaagtgct acggcgtgtc 7860

cccaacaaaa ctgaacgacc tgtgcttcac caacgtgtac gccgacagct tcgtgatccg 7920

cggcgacgag gtgcggcaga tcgcccccgg ccagaccggc aacatcgccg actacaacta 7980

caagctgccc gacgacttca ccggctgcgt gatcgcctgg aattctaata atctggacag 8040

caaggtgggc ggcaactaca actacctgta cagactgttt agaaaatcta atctgaagcc 8100

cttcgagagg gacatcagca ccgagatcta ccaggccggc agcaccccat gtaatggagt 8160

gaagggcttc aactgctact tccccctgca gagctacggc ttccagccca cctacggcgt 8220

gggctaccag ccctacagag tggtggtgct gagcttcgag ctgctgcacg cccccgccac 8280

cgtgtgcggc cccaagaagt ctaccaacct ggtgaagaac aagtgcgtga acttcggcgg 8340

cagcggcggc ggcggcagcg gcggcgtgaa cctgaccacc aggacacagc tgcccccagc 8400

ctacaccaac agcttcacca ggggcgtgta ctacccagat aaagtgttta gaagttctgt 8460

gctgcacagc acccaggacc tgtttctgcc cttcttcagc aacgtgacct ggtttcatgc 8520

catccacgtg tccggcacca acggcaccaa gaggtttgat aatccagtgc tgcccttcaa 8580

cgacggcgtg tacttcgcca gcaccgagaa gtcaaacatc atccgcggct ggatcttcgg 8640

caccaccctg gacagcaaga cccagagcct gctgatcgtg aacaacgcca ccaacgtggt 8700

gatcaaggtg tgcgagttcc agttttgtaa tgatcctttt ctgggcgtgt actaccacaa 8760

gaacaacaag agctggatgg agagcgagtt ccgggtgtac agcagcgcca acaactgcac 8820

cttcgagtac gtgtcccagc ccttcctgat ggatctggag ggcaagcagg gcaacttcaa 8880

gaacctgcgg gagttcgtgt ttaaaaatat tgatggatat tttaaaattt attctaaaca 8940

tactccaatt aatctggtgc gggacctgcc ccagggcttc agcgccctgg agcccctggt 9000

ggacctgccc atcggcatca acatcaccag gtttcagacc ctgctggccc tgcacaggag 9060

ctacctgacc ccaggagaca gcagcagcgg ctggacagcc ggcgccgccg cctactacgt 9120

gggctacctg cagcccagga catttctgct gaagtacaac gagaacggca ccatcaccga 9180

cgccgtggac tgcgccctgg accccctgag cgagaccaag tgcaccctga aatctggcgg 9240

cagcggcggc ggcggcagcg gcggacgggt gcagcccacc gagagcatcg tgcggttccc 9300

caacatcacc aacctgtgcc ccttcggcga ggtgttcaac gccacccggt tcgccagcgt 9360

gtacgcctgg aaccggaagc ggatcagcaa ctgcgtggcc gactacagcg tgctgtacaa 9420

cagcgccagc ttcagcacct tcaagtgcta cggcgtgagc cccaccaagc tgaacgacct 9480

gtgcttcacc aacgtgtacg ccgacagctt cgtgatccgg ggcgacgagg tgcggcagat 9540

cgcccccggc cagaccggca ccatcgccga ctacaactac aagctgcccg acgacttcac 9600

cggctgcgtg atcgcctgga acagcaacaa cctggacagc aaggtgggcg gcaactacaa 9660

ctacctgtac cggctgttcc ggaaatcaaa cctgaagccc ttcgagcggg acatcagcac 9720

cgagatctac caggccggca gcaccccctg caacggcgtg aagggcttca actgctactt 9780

ccccctgcag agctacggct tccagcccac ctacggcgtg ggctaccagc cctaccgggt 9840

ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc gtgtgcggcc ccaagaaaag 9900

taccaacctg gtgaagaaca agtgcgtgaa cttcggcggc agcctgggcg gcggcggcag 9960

cggcagcgcc atcggcggct acatccccga ggccccccgg gacggccagg cctacgtgcg 10020

gaaggacggc gagtgggtgc tgctgagcac cttcctgtga taacggccgc atacagcagc 10080

aattggcaag ctgcttacat agaactcgcg gcgattggca tgccgcctta aaatttttat 10140

tttatttttc ttttcttttc cgaatcggat tttgttttta atatttcaaa aaaaaaaaaa 10200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaga agagctaggg ataacagggt aattgagcaa 10260

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 10320

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 10380

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 10440

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 10500

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 10560

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 10620

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 10680

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 10740

acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 10800

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 10860

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 10920

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 10980

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 11040

gtatatatga gtaaacttgg tctgacagtt agaaaaactc atcgagcatc aaatgaaact 11100

gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg 11160

aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga 11220

ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat 11280

caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca 11340

tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat 11400

caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt 11460

taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat 11520

caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccag 11580

ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg 11640

gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg 11700

caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc 11760

gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat 11820

cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc 11880

tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 11940

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 12000

gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 12060

ggcgtatcac gaggcccttt cgtctaggga taacagggta attaatacga ctcactatag 12120

Claims (8)

1. An antigenic fragment, characterized in that it has the amino acid sequence shown as SEQ ID No. 1.

2. A nucleic acid molecule encoding the antigenic fragment of claim 1.

3. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule has the nucleotide sequence set forth in SEQ ID No. 2.

4. A recombinant expression vector comprising the nucleic acid molecule of claim 2 or 3 and a self-replicating vector.

5. The method for producing a recombinant expression vector according to claim 4, comprising the steps of:

step a: synthesizing RBD + NTD + RBD gene segments;

step b: constructing a TC-83 self-replicating vector;

obtaining a self-replicating mRNA sequence from a genome of an alphavirus family; the self-replicating mRNA sequence comprises a gene encoding an alphavirus self-replicating component, lacking a gene that produces a structural protein having an infectious alphavirus particle; amplifying a plasmid constructed by a self-replicating mRNA sequence as a template, and synthesizing to obtain the TC-83 self-replicating vector;

step c: preparing a recombinant plasmid JCXH-106;

inserting the RBD + NTD + RBD gene carrying ApaI at the upstream and NotI at the downstream into the enzyme cutting sites of ApaI and NotI of the TC-83 replication vector to obtain the recombinant plasmid JCXH-106.

6. Use of the antigenic fragment according to claim 1, the nucleic acid molecule according to claim 2 or 3, the expression vector according to claim 4 or the expression vector obtained by the preparation method according to claim 5 for the preparation of a vaccine for the prevention of coronaviruses or for the preparation of a medicament for the treatment of coronaviruses.

7. Vaccine comprising the antigenic fragment of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4, the expression vector produced by the method of claim 5, and a pharmaceutically acceptable excipient; or

The vaccine is prepared directly or indirectly from the antigen fragment of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4, or the expression vector prepared by the preparation method of claim 5.

8. The method of preparing the vaccine of claim 7, comprising the steps of:

step a: synthesizing RBD + NTD + RBD gene segments;

step b: constructing a TC-83 self-replicating vector;

obtaining a self-replicating mRNA sequence from a genome of an alphavirus family; the self-replicating mRNA sequence comprises a gene encoding an alphavirus self-replicating component, lacking a gene that produces a structural protein having an infectious alphavirus particle; amplifying a plasmid constructed by a self-replicating mRNA sequence as a template, and synthesizing to obtain the TC-83 self-replicating vector;

step c: preparing a recombinant plasmid JCXH-106;

inserting the RBD + NTD + RBD gene carrying ApaI at the upstream and NotI at the downstream into the ApaI and NotI enzyme cutting sites of a TC-83 replication vector to obtain a recombinant plasmid JCXH-106;

step d: linearizing JCXH-106 plasmid by restriction endonuclease;

step e: carrying out in-vitro transcription reaction on the linearized JCXH-106 plasmid, degrading the template DNA, and finally adding a 7-methylated guanylic acid cap structure to the 5' end of the transcribed mRNA;

step f: and e, mixing mRNA and lipid, wrapping the mRNA into LNP to obtain an mRNA-LNP compound, and concentrating and changing the solution to the preparation solution prepared in the step e to obtain the vaccine.

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