CN117625652A - Novel coronavirus variant mRNA vaccine and application thereof - Google Patents
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- CN117625652A CN117625652A CN202211064993.XA CN202211064993A CN117625652A CN 117625652 A CN117625652 A CN 117625652A CN 202211064993 A CN202211064993 A CN 202211064993A CN 117625652 A CN117625652 A CN 117625652A
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Abstract
The invention relates to the field of nucleic acid vaccines, and the inventor obtains an optimized omicron (B.1.1.529) S glycoprotein mRNA sequence after transcription by optimizing a novel coronary variant omicron (B.1.1.529) S glycoprotein coding region. The mRNA after transcription optimization has more stable structure, higher translation efficiency of S glycoprotein delivered to mammals and human bodies and can induce immune response in vivo. The mRNA nucleic acid molecule of the invention can be prepared into liposome pharmaceutical compositions for controlling, preventing and treating coronavirus infection diseases.
Description
Technical Field
The invention relates to the technical field of vaccine development, in particular to mRNA nucleic acid molecules for controlling, preventing and treating coronavirus infection, a pharmaceutical composition containing the mRNA nucleic acid molecules and application thereof.
Background
Coronavirus (CoV) is a zoonotic virus that is widely found in nature, and was originally discovered and isolated in mice. CoV belongs to the order of the mantle viridae (Nidovirales), the subgenoles Coronaviridae (cornidovirrineae), the family Coronaviridae (cornoavidae), the subfamily orthocoronaviridae (orthoviroavirinae). The orthocoronaviridae are divided into 47 genera of coronaviridae a (alphacorenavirus, α -CoV), coronaviridae b (betacorenavirus, β -CoV), coronaviridae c (Gammacoronavirus, γ -CoV) and delta coronaviridae (deltacorenavirus, δ -CoV) 4, of which α -CoV is divided into 19 subgenera, according to the unique conserved 5 domains in all viruses of the order of the set of viruses, in combination with phylogenetic and genomic structures; beta-CoV is divided into 16 subgenera 5; delta-CoV split 3 subgenera 7; gamma-CoV is divided into 3 subgenera 5.
CoV is an enveloped single-stranded sense (+) non-segmented RNA virus, approximately 100nm in diameter, with a genome of about 26-32 kb. The genome has a cap structure at the 5 'end and a poly (A) structure at the 3' end; both the 5 'and 3' ends have untranslated regions (untranslated regions, UTR) which contain cis-regulatory elements that play an important role in replication and transcription of the viral genome. The 2/3 region of the 5' end of the genome encodes 2 large open reading frames (open reading frame, ORF): ORF1a and ORF1b, translated into 2 large polyproteins 1ab (PP 1 ab), PP1ab is cleaved by the viral chymotrypsin-like protease 3 chymmotrypsin-like protease 3,3CL-pro and papain-like protease (PLpro) into 16 nonstructural proteins (non-structural protein, nsp) that are primarily involved in replication and transcription of the virus; the genome of the other 1/3 region encodes 4 major structural proteins: spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N); among the S and N genes are genes encoding accessory proteins designated ORF 3a, 6, 7a, 8 and 10, respectively, which are essential components of viral particles. Wherein, S glycoprotein is located on the surface of virus to form a rod-shaped structure, and is one of main antigen proteins of virus, and is a main gene for typing.
Like all coronaviruses, SARS-CoV-2 utilizes S glycoprotein to facilitate entry into host cells. The protein contains two important functional domains: one is the S1 Receptor Binding Domain (RBD) and the other is the S2 domain that mediates fusion of the viral and host cell membranes.
The S glycoprotein first binds to human angiotensin 2 (hACE 2) expressing surface receptors of host cells using the S1 receptor binding domain. Next, the S1 receptor binding domain is shed from the viral surface, facilitating fusion of the S2 domain with the host cell membrane. This process requires activation of the S glycoprotein, which is accomplished by cleavage at two sites (S1/S2 and S2') by proteases Furin and TMPRSS 2. Cleavage of Furin at the S1/S2 site can result in a conformational change in the viral S glycoprotein, thereby exposing the RBD domain and/or S2 domain. Researchers believe that TMPRSS2 cleavage of the SARS-CoV-2S glycoprotein can promote fusion of the viral capsid with the host cell, thereby allowing the virus to enter the host cell.
Exposure of the RBD domain in the S1 protein subunit can result in conformational instability of the subunit. Thus, during binding, conformational rearrangement of the subunit occurs in two states, referred to as the up-conformation and the down-conformation, respectively. Wherein the down state will transiently hide the RBD domain and the up state will expose the RBD domain, which will eventually temporarily place the protein subunit in an unstable state. In trimeric S glycoproteins, only one of the three RBD domains binds to a host cell surface receptor in an accessible conformation.
SARS-CoV-2S glycoprotein, ACE2 and Furin and TMPRSS2 enzymes play an important role in binding and mediating viral entry into host cells and thus can be a key target for covd drug development and viral inhibition, however, vaccines play a very critical role in preventing or blocking the transmission of novel coronaviruses.
The new coronavirus vaccines currently existing on the market have a number of drawbacks in their general design and optimization of the S glycoprotein mRNA sequence, in particular: 1) mRNA sequences are poorly stable and are easily degraded by intracellular enzymes in cells, resulting in lower levels of S glycoprotein effective antigen expression. 2) The mRNA sequence is translated with low efficiency, resulting in a low amount of effective antigen translated into S glycoprotein in the cell, which affects the effective activation of the immune system. Thus, it is currently desirable to provide an optimized design of S glycoprotein mRNA sequences for novel coronavirus vaccines.
In view of this, the present invention has been made.
Summary of The Invention
The present invention provides nucleic acid molecules useful for the prevention, control and treatment of novel coronavirus variants omicron (b.1.1.529), the optimized nucleic acid molecules comprising a coding region, wherein said coding region comprises one or more open reading frames (open reading frame, ORFs), and wherein at least one ORF encodes an S glycoprotein of coronavirus omicron (b.1.1.529), said S glycoprotein having the protein sequence shown in SEQ ID NO: 1. The optimized nucleotide sequence ensures that the transcribed mRNA structure is more stable, and the translation efficiency of target proteins in mammals and human bodies is higher.
In some embodiments, the invention provides an optimized nucleic acid molecule comprising an S glycoprotein encoding a coronavirus omicron (b.1.1.529), wherein the coding region comprises one or more open reading frames (open reading frame, ORFs), and wherein at least one ORF encodes an S glycoprotein of coronavirus omicron (b.1.1.529) having a protein sequence as shown in SEQ ID No. 1, but having a nucleotide sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence shown in SEQ ID No. 2.
In another embodiment, the invention provides an optimized nucleic acid molecule comprising an S glycoprotein encoding coronavirus omicron (B.1.1.529), the sequence optimization comprising adjusting for codon preference when expressed in humans, increasing GC content of the sequence, etc., the sequence of which is shown in SEQ ID NO:3-SEQ ID NO: 5.
In another embodiment, the optimized DNA coding gene is used as a template for transcription to obtain an mRNA molecule for coding the S glycoprotein of the novel coronal variant omicron (B.1.1.529), and the sequence of the mRNA molecule is shown as SEQ ID NO. 6-SEQ ID NO. 8. The optimized nucleotide sequence enables the transcribed mRNA structure to be more stable, the translation efficiency of target proteins in mammals and human bodies to be higher, the technical problems that mRNA translation efficiency is poor, stability is to be improved and the like in the prior art can be solved, and an organism can be efficiently induced to generate immune response, so that the problems of low antibody titer, small effective antigen quantity and high production cost of a novel coronary variant strain omicron after immunization in the prior art are improved.
In another embodiment, the present disclosure provides an mRNA nucleic acid molecule comprising:
(i) A 5 'untranslated region (5' -UTR);
(ii) A CDS, wherein the CDS comprises an Open Reading Frame (ORF) encoding a coronavirus omicron (b.1.1.529) antigen S glycoprotein capable of inducing an immune response comprising a nucleotide sequence as set forth in SEQ ID No. 6-SEQ ID No. 8;
(iii) 3 '-untranslated region (3' -UTR).
In another embodiment, the mRNA nucleic acid molecule further comprises a 5 'untranslated region (5' utr). Optionally, wherein the 5' UTR comprises the sequence shown as SEQ ID NO 9-SEQ ID NO 11.
In another embodiment, the mRNA nucleic acid molecule further comprises a 3 'untranslated region (3' utr). Optionally, wherein the 3' UTR comprises the sequence of SEQ ID NO. 12-SEQ ID NO. 14.
In another embodiment, the mRNA nucleic acid molecules of the present invention further comprise a native 5' -cap structure or analog thereof produced by an endogenous process. Modification of the 5' -cap can increase the stability of the nucleic acid molecule, increase its half-life and can increase translation efficiency. Modifications to the native 5' -cap structure include 2' -O-methylation at the 5' -end of the polynucleotide and/or at the ribose 2' -hydroxy group of the 5' -end nucleic acid. The 5' -cap analogue may optionally be selected from m7G (5 ') ppp (5 ') (2 ' OMeA) pG, 3' -O-Me-m7G (5 ') ppp (5 ') G; g (5 ') ppp (5') A; g (5 ') ppp (5') G; m7G (5 ') ppp (5') A; m7G (5 ') ppp (5') G, etc.
In another embodiment, the mRNA nucleic acid molecule further comprises a poly-a tail or polyadenylation signal, optionally having a length of 80 to 180 nucleotides. In a preferred embodiment, the 3' -polyadenylation sequence (polyA) is preferably 80-180A with a linker sequence in between, as shown in SEQ ID NO. 15; more preferably 80-160A's, still more preferably 130A's, as shown in SEQ ID NO. 16.
In another preferred embodiment, the CDS comprises a signal peptide sequence as shown in SEQ ID NO. 17-SEQ ID NO. 19 and a mature peptide sequence as shown in SEQ ID NO. 1.
In another embodiment, the mRNA nucleic acid molecule further comprises one or more functional nucleotide analog modifications selected from the group consisting of pseudouridine (ψ), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e 1 ψ), 5-methoxy-uridine (mo 5U) and 5-methylcytosine (m 5C). In some embodiments, the presently disclosed mRNA includes pseudo-uridine substitutions at one or more or all uridine positions of the nucleic acid.
In another embodiment, the invention provides a ribonucleic acid (RNA) comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA), wherein CDS comprises an Open Reading Frame (ORF) encoding coronavirus omicron (b.1.1.529) antigen S glycoprotein capable of inducing an immune response. Wherein the 5' -UTR further comprises a Kozak sequence. The addition of 5 '-cap structure, 5' -UTR, kozak, 3 '-poly A sequence (polyA) and 3' -UTR sequence elements can further improve the stability of the sequence and avoid degradation. In another preferred embodiment, the CDS may further comprise a signal peptide sequence. The purpose of adding the signal peptide is to enhance the translation of the protein, improve the translation stability and promote better folding of the protein. The preferred signal peptide sequence is shown in SEQ ID NO. 17-SEQ ID NO. 19.
In another preferred embodiment, the invention provides an mRNA nucleic acid molecule comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA) as shown in SEQ ID NO. 20-SEQ ID NO. 22.
In a particularly preferred embodiment, the inventors have achieved a reduction in mRNA immunogenicity by reducing the U (uridylic acid) content of the mRNA molecule by substitution of all uracils in the nucleic acid sequences SEQ ID NO:20-SEQ ID NO:22 with pseudouridine (ψ), the capping substituted sequences shown in SEQ ID NO:23-SEQ ID NO: 25.
In another embodiment, provided herein are pharmaceutical compositions for inducing a neutralizing antibody response in a subject to a novel coronal variant omicron (b.1.1.529) S glycoprotein, the pharmaceutical compositions described herein comprising mRNA molecules having the sequences shown in SEQ ID No. 20-SEQ ID No. 25, or any combination thereof, encapsulated in a lipid shell formulated in a lipid nanoparticle form, wherein the lipid nanoparticle generally comprises an ionizable cationic lipid, a non-cationic lipid, a sterol, and a PEG lipid component.
In another embodiment, provided herein are mRNAs as shown in SEQ ID NO. 20-SEQ ID NO. 25 useful for inducing a neutralizing antibody response in a subject to the S glycoprotein of the novel coronavariant omicron (B.1.1.529), which mRNAs provided herein can control, prevent or treat infectious diseases caused by coronaviruses in a subject to be administered.
In another embodiment, the subject of the invention is a human or non-human mammal. In another embodiment, the subject is an adult, a human child, or a human infant. In another embodiment, the subject is at risk of or is susceptible to a coronavirus infection. In another embodiment, the subject is an elderly person. In another embodiment, the subject has been diagnosed as positive for a coronavirus infection. In another embodiment, the subject is asymptomatic.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the effect of flow cytometry in example 2 of the present invention on the expression of different omacron (B.1.1.529) S glycoprotein mRNA transfected HEK293 cells;
FIG. 2 shows the effect of intermediate immunofluorescence detection of different omicron (B.1.1.529) S glycoprotein mRNA in HEK293 cells transfected according to example 3 of the present invention;
FIG. 3 shows the effect of Western blot detection on the expression of different omicron (B.1.1.529) S glycoprotein mRNA transfected HEK293 cells in example 4 of the present invention;
FIG. 4 shows serum antibodies after immunization of animals with different omacron (B.1.1.529) S glycoprotein mRNA preparations detected by IgG neutralizing antibodies of example 5 of the present invention;
FIG. 5 is a schematic diagram of mRNA cap structure.
Detailed Description
The present invention provides therapeutic nucleic acid molecules useful for the prevention, management and treatment of infectious diseases or conditions caused by the novel coronal variant virus omacron (b.1.1.529). By optimizing the nucleotide sequence of the S glycoprotein of encoding coronavirus omicron (B.1.1.529), the transcribed mRNA structure is more stable, and the translation efficiency of the target protein in mammals and human bodies is higher. Provided herein are pharmaceutical compositions for inducing a neutralizing antibody response in a subject to a novel coronal variant omicron (b.1.1.529) S glycoprotein, comprising mRNA molecules encoding the novel coronal variant omicron (b.1.1.529) S glycoprotein and related therapeutic methods and uses for preventing, managing and treating infectious diseases caused by the novel coronal variant omicron (b.1.1.529) to improve the problems of low antibody titer, low effective antigen amount, and high production cost of the novel coronal variant omicron in the prior art.
To make the invention easier to understand, certain terms are first defined. Additional definitions will be set forth throughout the detailed description.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art, which are fully explained in the technical literature and general textbooks of the art, such as Molecular Cloning: a Laboratory Manual (molecular cloning: laboratory Manual), etc.
Antigen (abbreviated Ag) refers to a substance that causes the production of antibodies, and any substance that induces an immune response. The foreign molecules can be passed through the recognition of immunoglobulins on B cells or through the treatment of antigen presenting cells and combined with the major histocompatibility complex to form a complex that reactivates T cells, eliciting a continuous immune response. In general, an antigen may be a protein capable of inducing an immune response (e.g., causing the immune system to produce antibodies to the antigen).
Nucleic acids, which are a class of biological macromolecules composed of nucleotides, are largely classified into two classes, deoxyribonucleic acid (Deoxyribonucleic acid, DNA) and Ribonucleic acid (RNA). In addition to prions, nucleic acids are biological macromolecules necessary for life, which act primarily as carriers of genetic information in the life system.
Open reading frame (open reading frame, ORF): it is meant that the normal nucleotide sequence of the structural gene, from the start codon to the stop codon, encodes the complete polypeptide chain without the stop codon interrupting translation.
CDS: is an abbreviation for Coding sequence. The CDS may be one ORF but may also include multiple ORFs.
Messenger ribonucleic acid (mRNA) is a template that directs the synthesis of proteins and is a messenger that transfers genetic information from DNA to proteins. The main sequence of mature mRNA is the coding region, with non-coding regions both 5' upstream and downstream. Eukaryotic mRNA molecules also have 5 'cap and 3' tail structures at both ends.
5' -cap structure: is a modified 5' end of messenger RNA (mRNA) in eukaryotes. It is linked to the 5' nucleotide of mRNA by pyrophosphorylation from methylated guanylic acid (m 7G) after mRNA transcription. The 5' end cap structure plays an important role in many biological functions of mRNA, including: shearing, stabilization, transport, and ribosome recruitment of mRNA. Due to the important biological functions of the 5 'end cap sub-structure, it is desirable to optimize the 5' end cap sub-structure during the development of mRNA vaccines and to "cap" the mRNA feed using a suitable process.
Untranslated region (UTR): refers to any fragment located at both ends of the mRNA strand coding sequence. If it is at the 5 'end, it is referred to as the 5' untranslated region (or "leader") and if it is at the 3 'end, it is referred to as the 3' untranslated region (or "trailer"). The untranslated region sequence is not a codon and cannot be translated into an amino acid, but can control the degradation and transcription efficiency of an mRNA product by an RNA-binding protein. Therefore, each mRNA vaccine needs to consider how to design the UTR module of the product, thereby improving the stability and transcription efficiency of the mRNA vaccine in the cells of interest.
Poly (A): the 3' -end of most eukaryotic mRNA has Poly (A) tail formed by polymerizing 100-200 adenylates, which can prevent exonuclease degradation and can be used as a marker of nuclear pore transport system, and is related to the transport of mature mRNA to cytoplasm through nuclear pores. The length of Poly (A) is also a very important optimization criterion in the design of the sequence of mRNA vaccine products.
Kozak sequence: eukaryotic translation initiation sequences are commonly referred to as "Kozak" consensus sequences. A Kozak sequence is a nucleic acid sequence located behind the 5' -UTR of eukaryotic mRNA and between the initiation codon AUG, which represents the translation initiation site of the methionine codon at position +1, typically ACCACCAUGG, GCCAUGAUGG, etc. The Kozak sequence on an mRNA molecule is recognized by the ribosome as a translation initiation site, which can bind to a translation initiation factor to mediate translation initiation of mRNA containing a 5' cap structure. The Kozak sequence may be used to enhance the translation efficiency of eukaryotic genes. The kozak sequence different from the present invention may be selected and replaced or deleted by conventional technical means, for example, the kozak sequence may be replaced with GCC, ACC, GCCACC or GCCANN etc., where N denotes A, T/U, C or G.
Transcription: is the process by which genetic information is copied from DNA to RNA (especially mRNA) under the catalysis of RNA polymerase. As a first step in protein biosynthesis, transcription is the pathway for the synthesis of mRNA as well as non-coding RNAs (tRNA, rRNA, etc.).
Polyadenylation: refers to the covalent linking of polyadenylation acids to messenger RNA (mRNA) molecules. During protein biosynthesis, this is part of the way in which mature mRNA is produced ready for translation.
Antibody titer: the minimum concentration (i.e., the maximum dilution) required for a certain antibody (anti) to recognize a particular epitope (epitope) is measured.
Neutralizing antibodies: is an antibody which is used for protecting cells from a certain antigen or infectious agent.
Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer found in their form in nature.
The term "subject" refers to any animal, including but not limited to humans, non-human primates, dogs, cats, rodents, etc., that is the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein with reference to a human subject of the present invention.
The term "effective amount" or "therapeutically effective amount" or "therapeutic effect" refers to a therapeutically effective amount to treat a disease or disorder in a subject. The therapeutically effective amount of the medicament has therapeutic effects, so that the development of diseases or symptoms can be prevented, slowed down or lightened, the morbidity and the mortality are reduced, and the quality of life is improved.
Nucleic acid molecules
The present invention provides nucleic acid molecules useful for the prevention, control and treatment of novel coronavariant viruses omicron (b.1.1.529) which, when administered to a subject in need thereof, are expressed by cells in the subject to produce a coded peptide or polypeptide. An optimized nucleic acid molecule comprises a coding region, wherein the coding region comprises one or more Open Reading Frames (ORFs), and wherein at least one ORF encodes an S glycoprotein of coronavirus omicron (b.1.1.529), said S glycoprotein having a protein sequence as set forth in SEQ ID NO: 1:
NLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDIFSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT*(SEQ ID NO:1)
in some embodiments, the nucleic acid molecule comprising a nucleic acid sequence encoding a coronavirus omicron (B.1.1.529) S glycoprotein has the nucleotide sequence shown in SEQ ID NO. 2.
AATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGTTATCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCATTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTATAGTGCGTGAGCCAGAAGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGATGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATCTCGCACCATTTTTCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAATATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAAGCTTGATTCTAAGGTTAGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAACAAACCTTGTAATGGTGTTGCAGGTTTTAATTGTTACTTTCCTTTACGATCATATAGTTTCCGACCCACTTATGGTGTTGGTCACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAAAAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAATATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAAGTCTCATCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAAACGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAATATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAAAGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCATAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAAATTTGGTGCAATTTCAAGTGTTTTAAATGATATCTTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGATTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATGA(SEQ ID NO:2)
In another embodiment, the nucleic acid molecule is a DNA molecule. The invention optimizes the codon of S glycoprotein (SEQ ID NO: 1) of encoding coronavirus omacron (B.1.1.529), and the sequence optimization comprises: optimizing GC content in human body, optimizing the use frequency of common codons, optimizing codon preference and the like to obtain corresponding DNA coding genes. In some embodiments, the invention provides an optimized nucleic acid molecule comprising an S glycoprotein encoding a coronavirus omicron (b.1.1.529), wherein the coding region comprises one or more Open Reading Frames (ORFs), and wherein at least one ORF encodes an S glycoprotein of coronavirus omicron (b.1.1.529) having a sequence as set forth in SEQ ID No. 1, but having a nucleotide sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID No. 2.
In another preferred embodiment, the invention provides an optimized nucleic acid molecule comprising an S glycoprotein encoding coronavirus omacron (B.1.1.529) having the sequence shown in SEQ ID NO:3-SEQ ID NO: 5. Increasing the GC content and optimizing the codons to obtain the nucleotide sequence enables the transcribed mRNA structure to be more stable and the translation efficiency of the target protein to be higher in mammals and human bodies. The DNA encoding gene is cloned into a vector and transformed into a host, which is a prokaryotic cell, eukaryotic cell or mammalian cell, preferably a prokaryotic cell, more preferably E.coli, etc.
AACCTAACCACCCGCACCCAACTACCGCCGGCGTACACCAACTCGTTCACCCGCGGGGTCTACTACCCGGACAAGGTCTTCCGCTCGTCGGTCCTACACTCGACCCAAGACCTATTCCTACCGTTCTTCTCGAACGTCACCTGGTTCCACGTCATATCGGGGACCAACGGGACCAAGCGCTTCGACAACCCGGTCCTACCGTTCAACGACGGGGTCTACTTCGCGTCGATAGAGAAGTCGAACATAATACGCGGCTGGATTTTCGGCACTACTCTGGACTCTAAGACTCAGTCTCTGCTGATTGTGAACAACGCTACTAACGTGGTGATTAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCTTTCCTGGACCACAAGAACAACAAGTCTTGGATGGAGTCTGAGTTCCGTGTGTACTCTTCTGCTAACAACTGCACTTTCGAGTACGTGTCTCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGTGAGTTCGTGTTCAAGAACATTGACGGCTACTTCAAGATTTACTCTAAGCACACTCCTATTATTGTGCGTGAGCCTGAGGACCTGCCTCAGGGCTTCTCTGCTCTGGAGCCTCTGGTGGACCTGCCTATTGGCATTAACATTACTCGTTTCCAGACTCTGCTGGCTCTGCACCGTTCTTACCTGACTCCTGGCGACTCTTCTTCTGGCTGGACTGCTGGCGCTGCTGCTTACTACGTGGGCTACCTGCAGCCTCGTACTTTCCTGCTGAAGTACAACGAGAACGGCACTATTACTGACGCTGTGGACTGCGCTCTGGACCCTCTGTCTGAGACTAAGTGCACTCTGAAGTCTTTCACTGTGGAGAAGGGCATTTACCAGACTTCTAACTTCCGTGTGCAGCCTACTGAGTCTATTGTGCGTTTCCCTAACATTACTAACCTGTGCCCTTTCGACGAGGTGTTCAACGCTACTCGTTTCGCTTCTGTGTACGCTTGGAACCGTAAGCGTATTTCTAACTGCGTGGCTGACTACTCTGTGCTGTACAACCTGGCTCCTTTCTTCACTTTCAAGTGCTACGGCGTGTCTCCTACTAAGCTGAACGACCTGTGCTTCACTAACGTGTACGCTGACTCTTTCGTGATTCGTGGCGACGAGGTGCGTCAGATTGCTCCTGGCCAGACTGGCAACATTGCTGACTACAACTACAAGCTGCCTGACGACTTCACTGGCTGCGTGATTGCTTGGAACTCTAACAAGCTGGACTCTAAGGTGTCTGGCAACTACAACTACCTGTACCGTCTGTTCCGTAAGTCTAACCTGAAGCCTTTCGAGCGTGACATTTCTACTGAGATTTACCAGGCTGGCAACAAGCCTTGCAACGGCGTGGCTGGCTTCAACTGCTACTTCCCTCTGCGTTCTTACTCTTTCCGTCCTACTTACGGCGTGGGCCACCAGCCTTACCGTGTGGTGGTGCTGTCTTTCGAGCTGCTGCACGCTCCTGCTACTGTGTGCGGCCCTAAGAAGTCTACTAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACTGGCGTGCTGACTGAGTCTAACAAGAAGTTCCTGCCTTTCCAGCAGTTCGGCCGTGACATTGCTGACACTACTGACGCTGTGCGTGACCCTCAGACTCTGGAGATTCTGGACATTACTCCTTGCTCTTTCGGCGGCGTGTCTGTGATTACTCCTGGCACTAACACTTCTAACCAGGTGGCTGTGCTGTACCAGGGCGTGAACTGCACTGAGGTGCCTGTGGCTATTCACGCTGACCAGCTGACTCCTACTTGGCGTGTGTACTCTACTGGCTCTAACGTGTTCCAGACTCGTGCTGGCTGCCTGATTGGCGCTGAGTACGTGAACAACTCTTACGAGTGCGACATTCCTATTGGCGCTGGCATTTGCGCTTCTTACCAGACTCAGACTAAGTCTCACCGTCGTGCTCGTTCTGTGGCTTCTCAGTCTATTATTGCTTACACTATGTCTCTGGGCGCTGAGAACTCTGTGGCTTACTCTAACAACTCTATTGCTATTCCTACTAACTTCACTATTTCTGTGACTACTGAGATTCTGCCTGTGTCTATGACTAAGACTTCTGTGGACTGCACTATGTACATTTGCGGCGACTCTACTGAGTGCTCTAACCTGCTGCTGCAGTACGGCTCTTTCTGCACTCAGCTGAAGCGTGCTCTGACTGGCATTGCTGTGGAGCAGGACAAGAACACTCAGGAGGTGTTCGCTCAGGTGAAGCAGATTTACAAGACTCCTCCTATTAAGTACTTCGGCGGCTTCAACTTCTCTCAGATTCTGCCTGACCCTTCTAAGCCTTCTAAGCGTTCTTTCATTGAGGACCTGCTGTTCAACAAGGTGACTCTGGCTGACGCTGGCTTCATTAAGCAGTACGGCGACTGCCTGGGCGACATTGCTGCTCGTGACCTGATTTGCGCTCAGAAGTTCAAGGGCCTGACTGTGCTGCCTCCTCTGCTGACTGACGAGATGATTGCTCAGTACACTTCTGCTCTGCTGGCTGGCACTATTACTTCTGGCTGGACTTTCGGCGCTGGCGCTGCTCTGCAGATTCCTTTCGCTATGCAGATGGCTTACCGTTTCAACGGCATTGGCGTGACTCAGAACGTGCTGTACGAGAACCAGAAGCTGATTGCTAACCAGTTCAACTCTGCTATTGGCAAGATTCAGGACTCTCTGTCTTCTACTGCTTCTGCTCTGGGCAAGCTGCAGGACGTGGTGAACCACAACGCTCAGGCTCTGAACACTCTGGTGAAGCAGCTGTCTTCTAAGTTCGGCGCTATTTCTTCTGTGCTGAACGACATTTTCTCTCGTCTGGACAAGGTGGAGGCTGAGGTGCAGATTGACCGTCTGATTACTGGCCGTCTGCAGTCTCTGCAGACTTACGTGACTCAGCAGCTGATTCGTGCTGCTGAGATTCGTGCTTCTGCTAACCTGGCTGCTACTAAGATGTCTGAGTGCGTGCTGGGCCAGTCTAAGCGTGTGGACTTCTGCGGCAAGGGCTACCACCTGATGTCTTTCCCTCAGTCTGCTCCTCACGGCGTGGTGTTCCTGCACGTGACTTACGTGCCTGCTCAGGAGAAGAACTTCACTACTGCTCCTGCTATTTGCCACGACGGCAAGGCTCACTTCCCTCGTGAGGGCGTGTTCGTGTCTAACGGCACTCACTGGTTCGTGACTCAGCGTAACTTCTACGAGCCTCAGATTATTACTACTGACAACACTTTCGTGTCTGGCAACTGCGACGTGGTGATTGGCATTGTGAACAACACTGTGTACGACCCTCTGCAGCCTGAGCTGGACTCTTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACTTCTCCTGACGTGGACCTGGGCGACATTTCTGGCATTAACGCTTCTGTGGTGAACATTCAGAAGGAGATTGACCGTCTGAACGAGGTGGCTAAGAACCTGAACGAGTCTCTGATTGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATTAAGTGGCCTTGGTACATTTGGCTGGGCTTCATTGCTGGCCTGATTGCTATTGTGATGGTGACTATTATGCTGTGCTGCATGACTTCTTGCTGCTCTTGCCTGAAGGGCTGCTGCTCTTGCGGCTCTTGCTGCAAGTTCGACGAGGACGACTCTGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACTTGA(SEQ ID NO:3)
AACCTGACTACTCGTACTCAGCTGCCTCCTGCTTACACTAACTCTTTCACTCGTGGCGTGTACTACCCTGACAAGGTGTTCCGTTCTTCTGTGCTGCACTCTACTCAGGACCTGTTCCTGCCTTTCTTCTCTAACGTGACTTGGTTCCACGTGATTTCTGGCACTAACGGCACTAAGCGTTTCGACAACCCTGTGCTGCCTTTCAACGACGGCGTGTACTTCGCTTCTATTGAGAAGTCTAACATTATTCGTGGCTGGATTTTCGGCACTACTCTGGACTCTAAGACTCAGTCTCTGCTGATTGTGAACAACGCTACTAACGTGGTGATTAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCTTTCCTGGACCACAAGAACAACAAGTCTTGGATGGAGTCTGAGTTCCGTGTGTACTCTTCTGCTAACAACTGCACTTTCGAGTACGTGTCTCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGTGAGTTCGTGTTCAAGAACATTGACGGCTACTTCAAGATTTACTCTAAGCACACTCCTATTATTGTGCGTGAGCCTGAGGACCTGCCTCAGGGCTTCTCTGCTCTGGAGCCTCTGGTGGACCTGCCTATTGGCATTAACATTACTCGTTTCCAGACTCTGCTGGCTCTGCACCGTTCTTACCTGACTCCTGGCGACTCTTCTTCTGGCTGGACTGCTGGCGCTGCTGCTTACTACGTGGGCTACCTGCAGCCTCGTACTTTCCTGCTGAAGTACAACGAGAACGGCACTATTACTGACGCTGTGGACTGCGCTCTGGACCCTCTGTCTGAGACTAAGTGCACTCTGAAGTCTTTCACTGTGGAGAAGGGCATTTACCAGACTTCTAACTTCCGTGTGCAGCCTACTGAGTCTATTGTGCGTTTCCCTAACATTACTAACCTGTGCCCTTTCGACGAGGTGTTCAACGCTACTCGTTTCGCTTCTGTGTACGCTTGGAACCGTAAGCGTATTTCTAACTGCGTGGCTGACTACTCTGTGCTGTACAACCTGGCTCCTTTCTTCACTTTCAAGTGCTACGGCGTGTCTCCTACTAAGCTGAACGACCTGTGCTTCACTAACGTGTACGCTGACTCTTTCGTGATTCGTGGCGACGAGGTGCGTCAGATTGCTCCTGGCCAGACTGGCAACATTGCTGACTACAACTACAAGCTGCCTGACGACTTCACTGGCTGCGTGATTGCTTGGAACTCTAACAAGCTGGACTCTAAGGTGTCTGGCAACTACAACTACCTGTACCGTCTGTTCCGTAAGTCTAACCTGAAGCCTTTCGAGCGTGACATTTCTACTGAGATTTACCAGGCTGGCAACAAGCCTTGCAACGGCGTGGCTGGCTTCAACTGCTACTTCCCTCTGCGTTCTTACTCTTTCCGTCCTACTTACGGCGTGGGCCACCAGCCTTACCGTGTGGTGGTGCTGTCTTTCGAGCTGCTGCACGCTCCTGCTACTGTGTGCGGCCCTAAGAAGTCTACTAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACTGGCGTGCTGACTGAGTCTAACAAGAAGTTCCTGCCTTTCCAGCAGTTCGGCCGTGACATTGCTGACACTACTGACGCTGTGCGTGACCCTCAGACTCTGGAGATTCTGGACATTACTCCTTGCTCTTTCGGCGGCGTGTCTGTGATTACTCCTGGCACTAACACTTCTAACCAGGTGGCTGTGCTGTACCAGGGCGTGAACTGCACTGAGGTGCCTGTGGCTATTCACGCTGACCAGCTGACTCCTACTTGGCGTGTGTACTCTACTGGCTCTAACGTGTTCCAGACTCGTGCTGGCTGCCTGATTGGCGCTGAGTACGTGAACAACTCTTACGAGTGCGACATTCCTATTGGCGCTGGCATTTGCGCTTCTTACCAGACTCAGACTAAGTCTCACCGTCGTGCTCGTTCTGTGGCTTCTCAGTCTATTATTGCTTACACTATGTCTCTGGGCGCTGAGAACTCTGTGGCTTACTCTAACAACTCTATTGCTATTCCTACTAACTTCACTATTTCTGTGACTACTGAGATTCTGCCTGTGTCTATGACTAAGACTTCTGTGGACTGCACTATGTACATTTGCGGCGACTCTACTGAGTGCTCTAACCTGCTGCTGCAGTACGGCTCTTTCTGCACTCAGCTGAAGCGTGCTCTGACTGGCATTGCTGTGGAGCAGGACAAGAACACTCAGGAGGTGTTCGCTCAGGTGAAGCAGATTTACAAGACTCCTCCTATTAAGTACTTCGGCGGCTTCAACTTCTCTCAGATTCTGCCTGACCCTTCTAAGCCTTCTAAGCGTTCTTTCATTGAGGACCTGCTGTTCAACAAGGTGACTCTGGCTGACGCTGGCTTCATTAAGCAGTACGGCGACTGCCTGGGCGACATTGCTGCTCGTGACCTGATTTGCGCTCAGAAGTTCAAGGGCCTGACTGTGCTGCCTCCTCTGCTGACTGACGAGATGATTGCTCAGTACACTTCTGCTCTGCTGGCTGGCACTATTACTTCTGGCTGGACTTTCGGCGCTGGCGCTGCTCTGCAGATTCCTTTCGCTATGCAGATGGCTTACCGTTTCAACGGCATTGGCGTGACTCAGAACGTGCTGTACGAGAACCAGAAGCTGATTGCTAACCAGTTCAACTCTGCTATTGGCAAGATTCAGGACTCTCTGTCTTCTACTGCTTCTGCTCTGGGCAAGCTGCAGGACGTGGTGAACCACAACGCTCAGGCTCTGAACACTCTGGTGAAGCAGCTGTCTTCTAAGTTCGGCGCTATTTCTTCTGTGCTGAACGACATTTTCTCTCGTCTGGACAAGGTGGAGGCTGAGGTGCAGATTGACCGTCTGATTACTGGCCGTCTGCAGTCTCTGCAGACTTACGTGACTCAGCAGCTGATTCGTGCTGCTGAGATTCGTGCTTCTGCTAACCTGGCTGCTACTAAGATGTCTGAGTGCGTGCTGGGCCAGTCTAAGCGTGTGGACTTCTGCGGCAAGGGCTACCACCTGATGTCTTTCCCTCAGTCTGCTCCTCACGGCGTGGTGTTCCTGCACGTGACTTACGTGCCTGCTCAGGAGAAGAACTTCACTACTGCTCCTGCTATTTGCCACGACGGCAAGGCTCACTTCCCTCGTGAGGGCGTGTTCGTGTCTAACGGCACTCACTGGTTCGTGACTCAGCGTAACTTCTACGAGCCTCAGATTATTACTACTGACAACACTTTCGTGTCTGGCAACTGCGACGTGGTGATTGGCATTGTGAACAACACTGTGTACGACCCTCTGCAGCCTGAGCTGGACTCTTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACTTCTCCTGACGTGGACCTGGGCGACATTTCTGGCATTAACGCTTCTGTGGTGAACATTCAGAAGGAGATTGACCGTCTGAACGAGGTGGCTAAGAACCTGAACGAGTCTCTGATTGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATTAAGTGGCCTTGGTACATTTGGCTGGGCTTCATTGCTGGCCTGATTGCTATTGTGATGGTGACTATTATGCTGTGCTGCATGACTTCTTGCTGCTCTTGCCTGAAGGGCTGCTGCTCTTGCGGCTCTTGCTGCAAGTTCGACGAGGACGACTCTGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACTTGA(SEQ ID NO:4)
AACCTGACGACGCGGACGCAGCTGCCCCCCGCCTACACGAACAGCTTCACGCGGGGCGTGTACTACCCCGACAAGGTGTTCCGGAGCAGCGTGCTGCACAGCACGCAGGACCTGTTCCTGCCCTTCTTCAGCAACGTGACGTGGTTCCACGTGATCAGCGGCACGAACGGCACGAAGCGGTTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCATCGAGAAGAGCAACATCATCCGGGGCTGGATCTTCGGCACGACGCTGGACAGCAAGACGCAGAGCCTGCTGATCGTGAACAACGCCACGAACGTGGTGATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACGTTCGAGTACGTGAGCCAGCCCTTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGGGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACGCCCATCATCGTGCGGGAGCCCGAGGACCTGCCCCAGGGCTTCAGCGCCCTGGAGCCCCTGGTGGACCTGCCCATCGGCATCAACATCACGCGGTTCCAGACGCTGCTGGCCCTGCACCGGAGCTACCTGACGCCCGGCGACAGCAGCAGCGGCTGGACGGCCGGCGCCGCCGCCTACTACGTGGGCTACCTGCAGCCCCGGACGTTCCTGCTGAAGTACAACGAGAACGGCACGATCACGGACGCCGTGGACTGCGCCCTGGACCCCCTGAGCGAGACGAAGTGCACGCTGAAGAGCTTCACGGTGGAGAAGGGCATCTACCAGACGAGCAACTTCCGGGTGCAGCCCACGGAGAGCATCGTGCGGTTCCCCAACATCACGAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACGCGGTTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACCTGGCCCCCTTCTTCACGTTCAAGTGCTACGGCGTGAGCCCCACGAAGCTGAACGACCTGTGCTTCACGAACGTGTACGCCGACAGCTTCGTGATCCGGGGCGACGAGGTGCGGCAGATCGCCCCCGGCCAGACGGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACGGGCTGCGTGATCGCCTGGAACAGCAACAAGCTGGACAGCAAGGTGAGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACGGAGATCTACCAGGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCTTCAACTGCTACTTCCCCCTGCGGAGCTACAGCTTCCGGCCCACGTACGGCGTGGGCCACCAGCCCTACCGGGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACGGTGTGCGGCCCCAAGAAGAGCACGAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACGGGCGTGCTGACGGAGAGCAACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCCGGGACATCGCCGACACGACGGACGCCGTGCGGGACCCCCAGACGCTGGAGATCCTGGACATCACGCCCTGCAGCTTCGGCGGCGTGAGCGTGATCACGCCCGGCACGAACACGAGCAACCAGGTGGCCGTGCTGTACCAGGGCGTGAACTGCACGGAGGTGCCCGTGGCCATCCACGCCGACCAGCTGACGCCCACGTGGCGGGTGTACAGCACGGGCAGCAACGTGTTCCAGACGCGGGCCGGCTGCCTGATCGGCGCCGAGTACGTGAACAACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATCTGCGCCAGCTACCAGACGCAGACGAAGAGCCACCGGCGGGCCCGGAGCGTGGCCAGCCAGAGCATCATCGCCTACACGATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGCATCGCCATCCCCACGAACTTCACGATCAGCGTGACGACGGAGATCCTGCCCGTGAGCATGACGAAGACGAGCGTGGACTGCACGATGTACATCTGCGGCGACAGCACGGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACGCAGCTGAAGCGGGCCCTGACGGGCATCGCCGTGGAGCAGGACAAGAACACGCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACGCCCCCCATCAAGTACTTCGGCGGCTTCAACTTCAGCCAGATCCTGCCCGACCCCAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAGGTGACGCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTGGGCGACATCGCCGCCCGGGACCTGATCTGCGCCCAGAAGTTCAAGGGCCTGACGGTGCTGCCCCCCCTGCTGACGGACGAGATGATCGCCCAGTACACGAGCGCCCTGCTGGCCGGCACGATCACGAGCGGCTGGACGTTCGGCGCCGGCGCCGCCCTGCAGATCCCCTTCGCCATGCAGATGGCCTACCGGTTCAACGGCATCGGCGTGACGCAGAACGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACGGCCAGCGCCCTGGGCAAGCTGCAGGACGTGGTGAACCACAACGCCCAGGCCCTGAACACGCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTGCTGAACGACATCTTCAGCCGGCTGGACAAGGTGGAGGCCGAGGTGCAGATCGACCGGCTGATCACGGGCCGGCTGCAGAGCCTGCAGACGTACGTGACGCAGCAGCTGATCCGGGCCGCCGAGATCCGGGCCAGCGCCAACCTGGCCGCCACGAAGATGAGCGAGTGCGTGCTGGGCCAGAGCAAGCGGGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAGCTTCCCCCAGAGCGCCCCCCACGGCGTGGTGTTCCTGCACGTGACGTACGTGCCCGCCCAGGAGAAGAACTTCACGACGGCCCCCGCCATCTGCCACGACGGCAAGGCCCACTTCCCCCGGGAGGGCGTGTTCGTGAGCAACGGCACGCACTGGTTCGTGACGCAGCGGAACTTCTACGAGCCCCAGATCATCACGACGGACAACACGTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTGAACAACACGGTGTACGACCCCCTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACGAGCCCCGACGTGGACCTGGGCGACATCAGCGGCATCAACGCCAGCGTGGTGAACATCCAGAAGGAGATCGACCGGCTGAACGAGGTGGCCAAGAACCTGAACGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACGATCATGCTGTGCTGCATGACGAGCTGCTGCAGCTGCCTGAAGGGCTGCTGCAGCTGCGGCAGCTGCTGCAAGTTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACGTGA(SEQ ID NO:5)
In another embodiment, the invention provides an mRNA molecule comprising an encoded coronavirus omacron (b.1.1.529) S glycoprotein. The DNA coding gene is transcribed by using a template to obtain an mRNA sequence for coding the novel coronary variant omacron (B.1.1.529) S glycoprotein, and the sequence is shown as SEQ ID NO. 6-SEQ ID NO. 8. The optimized DNA coding gene is used as a template for transcription, the nucleotide sequence enables the transcribed mRNA structure to be more stable, the translation efficiency of target proteins in mammals and human bodies to be higher, the technical problems that the translation efficiency of mRNA is poor, the stability is to be improved and the like in the prior art can be solved, and the immune response of organisms can be efficiently induced, so that the problems of low antibody titer, small effective antigen quantity and high production cost of novel crown variant omicron immune in the prior art can be improved. AACCUAACCACCCGCACCCAACUACCGCCGGCGUACACCAACUCGUUCACCCGCGGGGUCUACUACCCGGACAAGGUCUUCCGCUCGUCGGUCCUACACUCGACCCAAGACCUAUUCCUACCGUUCUUCUCGAACGUCACCUGGUUCCACGUCAUAUCGGGGACCAACGGGACCAAGCGCUUCGACAACCCGGUCCUACCGUUCAACGACGGGGUCUACUUCGCGUCGAUAGAGAAGUCGAACAUAAUACGCGGCUGGAUUUUCGGCACUACUCUGGACUCUAAGACUCAGUCUCUGCUGAUUGUGAACAACGCUACUAACGUGGUGAUUAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCUUUCCUGGACCACAAGAACAACAAGUCUUGGAUGGAGUCUGAGUUCCGUGUGUACUCUUCUGCUAACAACUGCACUUUCGAGUACGUGUCUCAGCCUUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGUGAGUUCGUGUUCAAGAACAUUGACGGCUACUUCAAGAUUUACUCUAAGCACACUCCUAUUAUUGUGCGUGAGCCUGAGGACCUGCCUCAGGGCUUCUCUGCUCUGGAGCCUCUGGUGGACCUGCCUAUUGGCAUUAACAUUACUCGUUUCCAGACUCUGCUGGCUCUGCACCGUUCUUACCUGACUCCUGGCGACUCUUCUUCUGGCUGGACUGCUGGCGCUGCUGCUUACUACGUGGGCUACCUGCAGCCUCGUACUUUCCUGCUGAAGUACAACGAGAACGGCACUAUUACUGACGCUGUGGACUGCGCUCUGGACCCUCUGUCUGAGACUAAGUGCACUCUGAAGUCUUUCACUGUGGAGAAGGGCAUUUACCAGACUUCUAACUUCCGUGUGCAGCCUACUGAGUCUAUUGUGCGUUUCCCUAACAUUACUAACCUGUGCCCUUUCGACGAGGUGUUCAACGCUACUCGUUUCGCUUCUGUGUACGCUUGGAACCGUAAGCGUAUUUCUAACUGCGUGGCUGACUACUCUGUGCUGUACAACCUGGCUCCUUUCUUCACUUUCAAGUGCUACGGCGUGUCUCCUACUAAGCUGAACGACCUGUGCUUCACUAACGUGUACGCUGACUCUUUCGUGAUUCGUGGCGACGAGGUGCGUCAGAUUGCUCCUGGCCAGACUGGCAACAUUGCUGACUACAACUACAAGCUGCCUGACGACUUCACUGGCUGCGUGAUUGCUUGGAACUCUAACAAGCUGGACUCUAAGGUGUCUGGCAACUACAACUACCUGUACCGUCUGUUCCGUAAGUCUAACCUGAAGCCUUUCGAGCGUGACAUUUCUACUGAGAUUUACCAGGCUGGCAACAAGCCUUGCAACGGCGUGGCUGGCUUCAACUGCUACUUCCCUCUGCGUUCUUACUCUUUCCGUCCUACUUACGGCGUGGGCCACCAGCCUUACCGUGUGGUGGUGCUGUCUUUCGAGCUGCUGCACGCUCCUGCUACUGUGUGCGGCCCUAAGAAGUCUACUAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACUGGCGUGCUGACUGAGUCUAACAAGAAGUUCCUGCCUUUCCAGCAGUUCGGCCGUGACAUUGCUGACACUACUGACGCUGUGCGUGACCCUCAGACUCUGGAGAUUCUGGACAUUACUCCUUGCUCUUUCGGCGGCGUGUCUGUGAUUACUCCUGGCACUAACACUUCUAACCAGGUGGCUGUGCUGUACCAGGGCGUGAACUGCACUGAGGUGCCUGUGGCUAUUCACGCUGACCAGCUGACUCCUACUUGGCGUGUGUACUCUACUGGCUCUAACGUGUUCCAGACUCGUGCUGGCUGCCUGAUUGGCGCUGAGUACGUGAACAACUCUUACGAGUGCGACAUUCCUAUUGGCGCUGGCAUUUGCGCUUCUUACCAGACUCAGACUAAGUCUCACCGUCGUGCUCGUUCUGUGGCUUCUCAGUCUAUUAUUGCUUACACUAUGUCUCUGGGCGCUGAGAACUCUGUGGCUUACUCUAACAACUCUAUUGCUAUUCCUACUAACUUCACUAUUUCUGUGACUACUGAGAUUCUGCCUGUGUCUAUGACUAAGACUUCUGUGGACUGCACUAUGUACAUUUGCGGCGACUCUACUGAGUGCUCUAACCUGCUGCUGCAGUACGGCUCUUUCUGCACUCAGCUGAAGCGUGCUCUGACUGGCAUUGCUGUGGAGCAGGACAAGAACACUCAGGAGGUGUUCGCUCAGGUGAAGCAGAUUUACAAGACUCCUCCUAUUAAGUACUUCGGCGGCUUCAACUUCUCUCAGAUUCUGCCUGACCCUUCUAAGCCUUCUAAGCGUUCUUUCAUUGAGGACCUGCUGUUCAACAAGGUGACUCUGGCUGACGCUGGCUUCAUUAAGCAGUACGGCGACUGCCUGGGCGACAUUGCUGCUCGUGACCUGAUUUGCGCUCAGAAGUUCAAGGGCCUGACUGUGCUGCCUCCUCUGCUGACUGACGAGAUGAUUGCUCAGUACACUUCUGCUCUGCUGGCUGGCACUAUUACUUCUGGCUGGACUUUCGGCGCUGGCGCUGCUCUGCAGAUUCCUUUCGCUAUGCAGAUGGCUUACCGUUUCAACGGCAUUGGCGUGACUCAGAACGUGCUGUACGAGAACCAGAAGCUGAUUGCUAACCAGUUCAACUCUGCUAUUGGCAAGAUUCAGGACUCUCUGUCUUCUACUGCUUCUGCUCUGGGCAAGCUGCAGGACGUGGUGAACCACAACGCUCAGGCUCUGAACACUCUGGUGAAGCAGCUGUCUUCUAAGUUCGGCGCUAUUUCUUCUGUGCUGAACGACAUUUUCUCUCGUCUGGACAAGGUGGAGGCUGAGGUGCAGAUUGACCGUCUGAUUACUGGCCGUCUGCAGUCUCUGCAGACUUACGUGACUCAGCAGCUGAUUCGUGCUGCUGAGAUUCGUGCUUCUGCUAACCUGGCUGCUACUAAGAUGUCUGAGUGCGUGCUGGGCCAGUCUAAGCGUGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGUCUUUCCCUCAGUCUGCUCCUCACGGCGUGGUGUUCCUGCACGUGACUUACGUGCCUGCUCAGGAGAAGAACUUCACUACUGCUCCUGCUAUUUGCCACGACGGCAAGGCUCACUUCCCUCGUGAGGGCGUGUUCGUGUCUAACGGCACUCACUGGUUCGUGACUCAGCGUAACUUCUACGAGCCUCAGAUUAUUACUACUGACAACACUUUCGUGUCUGGCAACUGCGACGUGGUGAUUGGCAUUGUGAACAACACUGUGUACGACCCUCUGCAGCCUGAGCUGGACUCUUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACUUCUCCUGACGUGGACCUGGGCGACAUUUCUGGCAUUAACGCUUCUGUGGUGAACAUUCAGAAGGAGAUUGACCGUCUGAACGAGGUGGCUAAGAACCUGAACGAGUCUCUGAUUGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUUAAGUGGCCUUGGUACAUUUGGCUGGGCUUCAUUGCUGGCCUGAUUGCUAUUGUGAUGGUGACUAUUAUGCUGUGCUGCAUGACUUCUUGCUGCUCUUGCCUGAAGGGCUGCUGCUCUUGCGGCUCUUGCUGCAAGUUCGACGAGGACGACUCUGAGCCUGUGCUGAAGGGCGUGAAGCUGCACUACACUUGA (SEQ ID NO: 6)
AACCUGACUACUCGUACUCAGCUGCCUCCUGCUUACACUAACUCUUUCACUCGUGGCGUGUACUACCCUGACAAGGUGUUCCGUUCUUCUGUGCUGCACUCUACUCAGGACCUGUUCCUGCCUUUCUUCUCUAACGUGACUUGGUUCCACGUGAUUUCUGGCACUAACGGCACUAAGCGUUUCGACAACCCUGUGCUGCCUUUCAACGACGGCGUGUACUUCGCUUCUAUUGAGAAGUCUAACAUUAUUCGUGGCUGGAUUUUCGGCACUACUCUGGACUCUAAGACUCAGUCUCUGCUGAUUGUGAACAACGCUACUAACGUGGUGAUUAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCUUUCCUGGACCACAAGAACAACAAGUCUUGGAUGGAGUCUGAGUUCCGUGUGUACUCUUCUGCUAACAACUGCACUUUCGAGUACGUGUCUCAGCCUUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGUGAGUUCGUGUUCAAGAACAUUGACGGCUACUUCAAGAUUUACUCUAAGCACACUCCUAUUAUUGUGCGUGAGCCUGAGGACCUGCCUCAGGGCUUCUCUGCUCUGGAGCCUCUGGUGGACCUGCCUAUUGGCAUUAACAUUACUCGUUUCCAGACUCUGCUGGCUCUGCACCGUUCUUACCUGACUCCUGGCGACUCUUCUUCUGGCUGGACUGCUGGCGCUGCUGCUUACUACGUGGGCUACCUGCAGCCUCGUACUUUCCUGCUGAAGUACAACGAGAACGGCACUAUUACUGACGCUGUGGACUGCGCUCUGGACCCUCUGUCUGAGACUAAGUGCACUCUGAAGUCUUUCACUGUGGAGAAGGGCAUUUACCAGACUUCUAACUUCCGUGUGCAGCCUACUGAGUCUAUUGUGCGUUUCCCUAACAUUACUAACCUGUGCCCUUUCGACGAGGUGUUCAACGCUACUCGUUUCGCUUCUGUGUACGCUUGGAACCGUAAGCGUAUUUCUAACUGCGUGGCUGACUACUCUGUGCUGUACAACCUGGCUCCUUUCUUCACUUUCAAGUGCUACGGCGUGUCUCCUACUAAGCUGAACGACCUGUGCUUCACUAACGUGUACGCUGACUCUUUCGUGAUUCGUGGCGACGAGGUGCGUCAGAUUGCUCCUGGCCAGACUGGCAACAUUGCUGACUACAACUACAAGCUGCCUGACGACUUCACUGGCUGCGUGAUUGCUUGGAACUCUAACAAGCUGGACUCUAAGGUGUCUGGCAACUACAACUACCUGUACCGUCUGUUCCGUAAGUCUAACCUGAAGCCUUUCGAGCGUGACAUUUCUACUGAGAUUUACCAGGCUGGCAACAAGCCUUGCAACGGCGUGGCUGGCUUCAACUGCUACUUCCCUCUGCGUUCUUACUCUUUCCGUCCUACUUACGGCGUGGGCCACCAGCCUUACCGUGUGGUGGUGCUGUCUUUCGAGCUGCUGCACGCUCCUGCUACUGUGUGCGGCCCUAAGAAGUCUACUAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACUGGCGUGCUGACUGAGUCUAACAAGAAGUUCCUGCCUUUCCAGCAGUUCGGCCGUGACAUUGCUGACACUACUGACGCUGUGCGUGACCCUCAGACUCUGGAGAUUCUGGACAUUACUCCUUGCUCUUUCGGCGGCGUGUCUGUGAUUACUCCUGGCACUAACACUUCUAACCAGGUGGCUGUGCUGUACCAGGGCGUGAACUGCACUGAGGUGCCUGUGGCUAUUCACGCUGACCAGCUGACUCCUACUUGGCGUGUGUACUCUACUGGCUCUAACGUGUUCCAGACUCGUGCUGGCUGCCUGAUUGGCGCUGAGUACGUGAACAACUCUUACGAGUGCGACAUUCCUAUUGGCGCUGGCAUUUGCGCUUCUUACCAGACUCAGACUAAGUCUCACCGUCGUGCUCGUUCUGUGGCUUCUCAGUCUAUUAUUGCUUACACUAUGUCUCUGGGCGCUGAGAACUCUGUGGCUUACUCUAACAACUCUAUUGCUAUUCCUACUAACUUCACUAUUUCUGUGACUACUGAGAUUCUGCCUGUGUCUAUGACUAAGACUUCUGUGGACUGCACUAUGUACAUUUGCGGCGACUCUACUGAGUGCUCUAACCUGCUGCUGCAGUACGGCUCUUUCUGCACUCAGCUGAAGCGUGCUCUGACUGGCAUUGCUGUGGAGCAGGACAAGAACACUCAGGAGGUGUUCGCUCAGGUGAAGCAGAUUUACAAGACUCCUCCUAUUAAGUACUUCGGCGGCUUCAACUUCUCUCAGAUUCUGCCUGACCCUUCUAAGCCUUCUAAGCGUUCUUUCAUUGAGGACCUGCUGUUCAACAAGGUGACUCUGGCUGACGCUGGCUUCAUUAAGCAGUACGGCGACUGCCUGGGCGACAUUGCUGCUCGUGACCUGAUUUGCGCUCAGAAGUUCAAGGGCCUGACUGUGCUGCCUCCUCUGCUGACUGACGAGAUGAUUGCUCAGUACACUUCUGCUCUGCUGGCUGGCACUAUUACUUCUGGCUGGACUUUCGGCGCUGGCGCUGCUCUGCAGAUUCCUUUCGCUAUGCAGAUGGCUUACCGUUUCAACGGCAUUGGCGUGACUCAGAACGUGCUGUACGAGAACCAGAAGCUGAUUGCUAACCAGUUCAACUCUGCUAUUGGCAAGAUUCAGGACUCUCUGUCUUCUACUGCUUCUGCUCUGGGCAAGCUGCAGGACGUGGUGAACCACAACGCUCAGGCUCUGAACACUCUGGUGAAGCAGCUGUCUUCUAAGUUCGGCGCUAUUUCUUCUGUGCUGAACGACAUUUUCUCUCGUCUGGACAAGGUGGAGGCUGAGGUGCAGAUUGACCGUCUGAUUACUGGCCGUCUGCAGUCUCUGCAGACUUACGUGACUCAGCAGCUGAUUCGUGCUGCUGAGAUUCGUGCUUCUGCUAACCUGGCUGCUACUAAGAUGUCUGAGUGCGUGCUGGGCCAGUCUAAGCGUGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGUCUUUCCCUCAGUCUGCUCCUCACGGCGUGGUGUUCCUGCACGUGACUUACGUGCCUGCUCAGGAGAAGAACUUCACUACUGCUCCUGCUAUUUGCCACGACGGCAAGGCUCACUUCCCUCGUGAGGGCGUGUUCGUGUCUAACGGCACUCACUGGUUCGUGACUCAGCGUAACUUCUACGAGCCUCAGAUUAUUACUACUGACAACACUUUCGUGUCUGGCAACUGCGACGUGGUGAUUGGCAUUGUGAACAACACUGUGUACGACCCUCUGCAGCCUGAGCUGGACUCUUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACUUCUCCUGACGUGGACCUGGGCGACAUUUCUGGCAUUAACGCUUCUGUGGUGAACAUUCAGAAGGAGAUUGACCGUCUGAACGAGGUGGCUAAGAACCUGAACGAGUCUCUGAUUGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUUAAGUGGCCUUGGUACAUUUGGCUGGGCUUCAUUGCUGGCCUGAUUGCUAUUGUGAUGGUGACUAUUAUGCUGUGCUGCAUGACUUCUUGCUGCUCUUGCCUGAAGGGCUGCUGCUCUUGCGGCUCUUGCUGCAAGUUCGACGAGGACGACUCUGAGCCUGUGCUGAAGGGCGUGAAGCUGCACUACACUUGA(SEQ ID NO:7)
AACCUGACGACGCGGACGCAGCUGCCCCCCGCCUACACGAACAGCUUCACGCGGGGCGUGUACUACCCCGACAAGGUGUUCCGGAGCAGCGUGCUGCACAGCACGCAGGACCUGUUCCUGCCCUUCUUCAGCAACGUGACGUGGUUCCACGUGAUCAGCGGCACGAACGGCACGAAGCGGUUCGACAACCCCGUGCUGCCCUUCAACGACGGCGUGUACUUCGCCAGCAUCGAGAAGAGCAACAUCAUCCGGGGCUGGAUCUUCGGCACGACGCUGGACAGCAAGACGCAGAGCCUGCUGAUCGUGAACAACGCCACGAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACCACAAGAACAACAAGAGCUGGAUGGAGAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACGUUCGAGUACGUGAGCCAGCCCUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGGGAGUUCGUGUUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACGCCCAUCAUCGUGCGGGAGCCCGAGGACCUGCCCCAGGGCUUCAGCGCCCUGGAGCCCCUGGUGGACCUGCCCAUCGGCAUCAACAUCACGCGGUUCCAGACGCUGCUGGCCCUGCACCGGAGCUACCUGACGCCCGGCGACAGCAGCAGCGGCUGGACGGCCGGCGCCGCCGCCUACUACGUGGGCUACCUGCAGCCCCGGACGUUCCUGCUGAAGUACAACGAGAACGGCACGAUCACGGACGCCGUGGACUGCGCCCUGGACCCCCUGAGCGAGACGAAGUGCACGCUGAAGAGCUUCACGGUGGAGAAGGGCAUCUACCAGACGAGCAACUUCCGGGUGCAGCCCACGGAGAGCAUCGUGCGGUUCCCCAACAUCACGAACCUGUGCCCCUUCGACGAGGUGUUCAACGCCACGCGGUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACCUGGCCCCCUUCUUCACGUUCAAGUGCUACGGCGUGAGCCCCACGAAGCUGAACGACCUGUGCUUCACGAACGUGUACGCCGACAGCUUCGUGAUCCGGGGCGACGAGGUGCGGCAGAUCGCCCCCGGCCAGACGGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACGGGCUGCGUGAUCGCCUGGAACAGCAACAAGCUGGACAGCAAGGUGAGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACGGAGAUCUACCAGGCCGGCAACAAGCCCUGCAACGGCGUGGCCGGCUUCAACUGCUACUUCCCCCUGCGGAGCUACAGCUUCCGGCCCACGUACGGCGUGGGCCACCAGCCCUACCGGGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACGGUGUGCGGCCCCAAGAAGAGCACGAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACGGGCGUGCUGACGGAGAGCAACAAGAAGUUCCUGCCCUUCCAGCAGUUCGGCCGGGACAUCGCCGACACGACGGACGCCGUGCGGGACCCCCAGACGCUGGAGAUCCUGGACAUCACGCCCUGCAGCUUCGGCGGCGUGAGCGUGAUCACGCCCGGCACGAACACGAGCAACCAGGUGGCCGUGCUGUACCAGGGCGUGAACUGCACGGAGGUGCCCGUGGCCAUCCACGCCGACCAGCUGACGCCCACGUGGCGGGUGUACAGCACGGGCAGCAACGUGUUCCAGACGCGGGCCGGCUGCCUGAUCGGCGCCGAGUACGUGAACAACAGCUACGAGUGCGACAUCCCCAUCGGCGCCGGCAUCUGCGCCAGCUACCAGACGCAGACGAAGAGCCACCGGCGGGCCCGGAGCGUGGCCAGCCAGAGCAUCAUCGCCUACACGAUGAGCCUGGGCGCCGAGAACAGCGUGGCCUACAGCAACAACAGCAUCGCCAUCCCCACGAACUUCACGAUCAGCGUGACGACGGAGAUCCUGCCCGUGAGCAUGACGAAGACGAGCGUGGACUGCACGAUGUACAUCUGCGGCGACAGCACGGAGUGCAGCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACGCAGCUGAAGCGGGCCCUGACGGGCAUCGCCGUGGAGCAGGACAAGAACACGCAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACGCCCCCCAUCAAGUACUUCGGCGGCUUCAACUUCAGCCAGAUCCUGCCCGACCCCAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAGGUGACGCUGGCCGACGCCGGCUUCAUCAAGCAGUACGGCGACUGCCUGGGCGACAUCGCCGCCCGGGACCUGAUCUGCGCCCAGAAGUUCAAGGGCCUGACGGUGCUGCCCCCCCUGCUGACGGACGAGAUGAUCGCCCAGUACACGAGCGCCCUGCUGGCCGGCACGAUCACGAGCGGCUGGACGUUCGGCGCCGGCGCCGCCCUGCAGAUCCCCUUCGCCAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGCGUGACGCAGAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACGGCCAGCGCCCUGGGCAAGCUGCAGGACGUGGUGAACCACAACGCCCAGGCCCUGAACACGCUGGUGAAGCAGCUGAGCAGCAAGUUCGGCGCCAUCAGCAGCGUGCUGAACGACAUCUUCAGCCGGCUGGACAAGGUGGAGGCCGAGGUGCAGAUCGACCGGCUGAUCACGGGCCGGCUGCAGAGCCUGCAGACGUACGUGACGCAGCAGCUGAUCCGGGCCGCCGAGAUCCGGGCCAGCGCCAACCUGGCCGCCACGAAGAUGAGCGAGUGCGUGCUGGGCCAGAGCAAGCGGGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCCCAGAGCGCCCCCCACGGCGUGGUGUUCCUGCACGUGACGUACGUGCCCGCCCAGGAGAAGAACUUCACGACGGCCCCCGCCAUCUGCCACGACGGCAAGGCCCACUUCCCCCGGGAGGGCGUGUUCGUGAGCAACGGCACGCACUGGUUCGUGACGCAGCGGAACUUCUACGAGCCCCAGAUCAUCACGACGGACAACACGUUCGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAACACGGUGUACGACCCCCUGCAGCCCGAGCUGGACAGCUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACGAGCCCCGACGUGGACCUGGGCGACAUCAGCGGCAUCAACGCCAGCGUGGUGAACAUCCAGAAGGAGAUCGACCGGCUGAACGAGGUGGCCAAGAACCUGAACGAGAGCCUGAUCGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACGAUCAUGCUGUGCUGCAUGACGAGCUGCUGCAGCUGCCUGAAGGGCUGCUGCAGCUGCGGCAGCUGCUGCAAGUUCGACGAGGACGACAGCGAGCCCGUGCUGAAGGGCGUGAAGCUGCACUACACGUGA(SEQ ID NO:8)
In another embodiment, the present disclosure provides an mRNA nucleic acid molecule comprising:
(i) A 5 'untranslated region (5' -UTR);
(ii) A CDS, wherein the CDS comprises an Open Reading Frame (ORF) encoding a coronavirus omicron (b.1.1.529) antigen S glycoprotein capable of inducing an immune response comprising a nucleotide sequence as set forth in SEQ ID No. 6-SEQ ID No. 8;
(iii) 3 '-untranslated region (3' -UTR).
In another embodiment, the mRNA nucleic acid molecules of the invention comprise a 5 'untranslated region (5' -UTR). Optionally, wherein the 5' UTR comprises the sequence of SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO. 11. Specifically, the 5' -UTR sequence may or may not comprise a Kozak sequence. In another preferred embodiment, wherein the 5' UTR may comprise a kozak sequence, such as GCCACC or GCCANN, etc., N refers to A, T/U, C or G.
AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC(SEQ ID NO:9)
AGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC(SEQ ID NO:10)
AGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC(SEQ ID NO:11)
In another embodiment, the nucleic acid molecules of the invention further comprise a 3 'untranslated region (3' -UTR). Optionally, wherein the 3' UTR comprises the sequence of SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14.
UAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(SEQ ID NO:12)
UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(SEQ ID NO:13)
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(SEQ ID NO:14)
In another embodiment, the mRNA nucleic acid molecules of the present invention further comprise a native 5' -cap structure or analog thereof produced by an endogenous process. Modification of the 5' -cap can increase the stability of the nucleic acid molecule, increase its half-life and can increase translation efficiency. Modifications to the native 5' -cap structure include 2' -O-methylation at the 5' -end of the polynucleotide and/or at the ribose 2' -hydroxy group of the 5' -end nucleic acid. The 5' -cap analogue may optionally be selected from m7G (5 ') ppp (5 ') (2 ' OMeA) pG, 3' -O-Me-m7G (5 ') ppp (5 ') G; g (5 ') ppp (5') A; g (5 ') ppp (5') G; m7G (5 ') ppp (5') A; m7G (5 ') ppp (5') G, etc. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or attached to the 5' end of an RNA molecule. The mRNA molecules produced after transcription can be modified by vaccinia virus capping enzymes to produce a 7-methylguanosine (m 7G) Cap bridged by triphosphate (m 7G (5 ' ') PPP (5 ') G), a structure known as Cap 0. Cap 1 structures (m 7 GpppmN) and Cap 2 structures (m 7 GpppmNmN) can be produced using vaccinia virus capping enzyme and 2' -O methyltransferase. The 5' -cap structures that may be used in connection with the present disclosure may be referred to those described in international patent publication nos. WO2008127688, WO2008016473 and WO2011015347, the entire contents of which are incorporated herein by reference. In a preferred embodiment, the 5' -cap selected for mRNA co-transcription capping according to the invention is similarly selected from commercially available Reagent AG- (N-7113), which is m7G (5 ') ppp (5 ') (2 ' OMeA) pG, can generate Cap 1 structure when mRNA is capped by in vitro transcription. Clearcap has capping efficiency as high as 98%. Cap 1mRNA has higher in vivo activity than Cap 0mRNA produced by conventional capping methods such as mCap or anti-reverse Cap analog (ARCA).
In another embodiment, the mRNA nucleic acid molecules of the invention further comprise a poly-A tail or polyadenylation signal, optionally having a length of 80 to 180 nucleotides. In a preferred embodiment, the 3' -polyadenylation sequence (polyA) is preferably 80-180A with a linker sequence in between, as shown in SEQ ID NO. 15; more preferably 80-160A's, still more preferably 130A's, as shown in SEQ ID NO. 16: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 15)
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:16)
In another preferred embodiment, the CDS may further comprise a signal peptide sequence. The purpose of adding the signal peptide is to enhance the translation of the protein, improve the translation stability and promote better folding of the protein. The preferred signal peptide sequence is shown in SEQ ID NO. 17-SEQ ID NO. 19. The signal peptide is positioned in front of the mature peptide, and the sequence of the mature peptide is shown as SEQ ID NO. 1.
AUGUUCGUCUUCCUAGUCCUACUACCGCUAGUCUCGUCGCAAUGCGUC(SEQ ID NO:17)
AUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCUUCUCAGUGCGUG(SEQ ID NO:18)
AUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCCAGUGCGUG(SEQ ID NO:19)
In another embodiment, the mRNA nucleic acid molecules of the present invention further comprise one or more functional nucleotide analog modifications selected from the group consisting of pseudouridine (ψ), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (e 1 ψ), 5-methoxy-uridine (mo 5U) and 5-methylcytosine (m 5C). Human immune response to mRNA is mainly related to uridine (partially consisting of uracil), whereas replacement of uracil with pseudouracil reduces mRNA recognition by the immune system. Methods for replacing uracil with pseudo-uracil refer to U.S. patent nos. 8278036B2, US9750824B2, US8835108B2, US8748089B2, US8691966B2, etc.; reference is made to US9428535B2 using the 1-methyl pseudouracil modification method; the effect of reducing the immunogenicity of mRNA by reducing the amount of U (uridylic acid) in the mRNA molecule is described in WO2017036889A1; the relevant technical content is incorporated herein by reference in its entirety. In some embodiments, the presently disclosed mRNA includes pseudo-uridine (ψ) substitutions at one or more or all uracil positions of the nucleic acid. In a particularly preferred embodiment, the uracil (U) sequences shown in SEQ ID NO. 6-SEQ ID NO. 25 are each replaced by a pseudouridine (ψ).
In another embodiment, the invention provides an mRNA nucleic acid molecule comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA), wherein CDS comprises an Open Reading Frame (ORF) encoding coronavirus omicron (B.1.1.529) antigen S glycoprotein capable of inducing immune response, the nucleotide sequence is shown as SEQ ID NO:6-SEQ ID NO: 8. The inventor optimizes the novel coronal variant omacron (B.1.1.529) S glycoprotein coding region (CDS), and the sequence optimization comprises adjusting GC content when expressing in human body, improving the GC content of the sequence, so that the transcribed mRNA structure is more stable, and the translation efficiency of target proteins in mammals and human bodies is higher. Preferably, the 5' -UTR comprises a Kozak sequence which may be used to enhance translation efficiency of mRNA. The addition of 5 '-cap structure, 5' -UTR, kozak, 3 '-poly A sequence (polyA) and 3' -UTR sequence elements can further improve the stability of the sequence and avoid degradation.
In another preferred embodiment, the invention provides an mRNA molecule comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA);
Wherein said 5' -cap structure is optionally selected from m7G (5 ') ppp (5 ') (2 ' OMeA) pG, 3' -O-Me-m7G (5 ') ppp (5 ') G; g (5 ') ppp (5') A; g (5 ') ppp (5') G; m7G (5 ') ppp (5') A; m7G (5 ') ppp (5') G, etc.;
wherein the 5' -UTR comprises a sequence shown as SEQ ID NO. 9-SEQ ID NO. 11;
wherein the CDS comprises an Open Reading Frame (ORF) which encodes a signal peptide and a coronavirus omacron (B.1.1.529) antigen S glycoprotein capable of inducing an immune response, wherein the sequence of the signal peptide is shown as SEQ ID NO. 17-SEQ ID NO. 19, and the sequence of the coronavirus omacron (B.1.1.529) antigen S glycoprotein is shown as SEQ ID NO. 6-SEQ ID NO. 8;
wherein the 3' -UTR comprises a nucleotide sequence shown as SEQ ID NO. 12-SEQ ID NO. 14;
wherein the 3' -polyadenylation sequence (polyA) optionally has a length of 80 to 180 adenylates.
In another preferred embodiment, the invention provides an mRNA molecule comprising the following elements: 5' -cap structure, 5' -UTR, CDS, 3' -UTR and 3' -polyadenylation sequence (polyA), wherein 5' -UTR comprises Kozak sequence, CDS comprises signal peptide and mature peptide, and the sequence is shown as SEQ ID NO:20-SEQ ID NO: 22.
GAGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACCAUGUUCGUCUUCCUAGUCCUACUACCGCUAGUCUCGUCGCAAUGCGUCAACCUAACCACCCGCACCCAACUACCGCCGGCGUACACCAACUCGUUCACCCGCGGGGUCUACUACCCGGACAAGGUCUUCCGCUCGUCGGUCCUACACUCGACCCAAGACCUAUUCCUACCGUUCUUCUCGAACGUCACCUGGUUCCACGUCAUAUCGGGGACCAACGGGACCAAGCGCUUCGACAACCCGGUCCUACCGUUCAACGACGGGGUCUACUUCGCGUCGAUAGAGAAGUCGAACAUAAUACGCGGCUGGAUUUUCGGCACUACUCUGGACUCUAAGACUCAGUCUCUGCUGAUUGUGAACAACGCUACUAACGUGGUGAUUAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCUUUCCUGGACCACAAGAACAACAAGUCUUGGAUGGAGUCUGAGUUCCGUGUGUACUCUUCUGCUAACAACUGCACUUUCGAGUACGUGUCUCAGCCUUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGUGAGUUCGUGUUCAAGAACAUUGACGGCUACUUCAAGAUUUACUCUAAGCACACUCCUAUUAUUGUGCGUGAGCCUGAGGACCUGCCUCAGGGCUUCUCUGCUCUGGAGCCUCUGGUGGACCUGCCUAUUGGCAUUAACAUUACUCGUUUCCAGACUCUGCUGGCUCUGCACCGUUCUUACCUGACUCCUGGCGACUCUUCUUCUGGCUGGACUGCUGGCGCUGCUGCUUACUACGUGGGCUACCUGCAGCCUCGUACUUUCCUGCUGAAGUACAACGAGAACGGCACUAUUACUGACGCUGUGGACUGCGCUCUGGACCCUCUGUCUGAGACUAAGUGCACUCUGAAGUCUUUCACUGUGGAGAAGGGCAUUUACCAGACUUCUAACUUCCGUGUGCAGCCUACUGAGUCUAUUGUGCGUUUCCCUAACAUUACUAACCUGUGCCCUUUCGACGAGGUGUUCAACGCUACUCGUUUCGCUUCUGUGUACGCUUGGAACCGUAAGCGUAUUUCUAACUGCGUGGCUGACUACUCUGUGCUGUACAACCUGGCUCCUUUCUUCACUUUCAAGUGCUACGGCGUGUCUCCUACUAAGCUGAACGACCUGUGCUUCACUAACGUGUACGCUGACUCUUUCGUGAUUCGUGGCGACGAGGUGCGUCAGAUUGCUCCUGGCCAGACUGGCAACAUUGCUGACUACAACUACAAGCUGCCUGACGACUUCACUGGCUGCGUGAUUGCUUGGAACUCUAACAAGCUGGACUCUAAGGUGUCUGGCAACUACAACUACCUGUACCGUCUGUUCCGUAAGUCUAACCUGAAGCCUUUCGAGCGUGACAUUUCUACUGAGAUUUACCAGGCUGGCAACAAGCCUUGCAACGGCGUGGCUGGCUUCAACUGCUACUUCCCUCUGCGUUCUUACUCUUUCCGUCCUACUUACGGCGUGGGCCACCAGCCUUACCGUGUGGUGGUGCUGUCUUUCGAGCUGCUGCACGCUCCUGCUACUGUGUGCGGCCCUAAGAAGUCUACUAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACUGGCGUGCUGACUGAGUCUAACAAGAAGUUCCUGCCUUUCCAGCAGUUCGGCCGUGACAUUGCUGACACUACUGACGCUGUGCGUGACCCUCAGACUCUGGAGAUUCUGGACAUUACUCCUUGCUCUUUCGGCGGCGUGUCUGUGAUUACUCCUGGCACUAACACUUCUAACCAGGUGGCUGUGCUGUACCAGGGCGUGAACUGCACUGAGGUGCCUGUGGCUAUUCACGCUGACCAGCUGACUCCUACUUGGCGUGUGUACUCUACUGGCUCUAACGUGUUCCAGACUCGUGCUGGCUGCCUGAUUGGCGCUGAGUACGUGAACAACUCUUACGAGUGCGACAUUCCUAUUGGCGCUGGCAUUUGCGCUUCUUACCAGACUCAGACUAAGUCUCACCGUCGUGCUCGUUCUGUGGCUUCUCAGUCUAUUAUUGCUUACACUAUGUCUCUGGGCGCUGAGAACUCUGUGGCUUACUCUAACAACUCUAUUGCUAUUCCUACUAACUUCACUAUUUCUGUGACUACUGAGAUUCUGCCUGUGUCUAUGACUAAGACUUCUGUGGACUGCACUAUGUACAUUUGCGGCGACUCUACUGAGUGCUCUAACCUGCUGCUGCAGUACGGCUCUUUCUGCACUCAGCUGAAGCGUGCUCUGACUGGCAUUGCUGUGGAGCAGGACAAGAACACUCAGGAGGUGUUCGCUCAGGUGAAGCAGAUUUACAAGACUCCUCCUAUUAAGUACUUCGGCGGCUUCAACUUCUCUCAGAUUCUGCCUGACCCUUCUAAGCCUUCUAAGCGUUCUUUCAUUGAGGACCUGCUGUUCAACAAGGUGACUCUGGCUGACGCUGGCUUCAUUAAGCAGUACGGCGACUGCCUGGGCGACAUUGCUGCUCGUGACCUGAUUUGCGCUCAGAAGUUCAAGGGCCUGACUGUGCUGCCUCCUCUGCUGACUGACGAGAUGAUUGCUCAGUACACUUCUGCUCUGCUGGCUGGCACUAUUACUUCUGGCUGGACUUUCGGCGCUGGCGCUGCUCUGCAGAUUCCUUUCGCUAUGCAGAUGGCUUACCGUUUCAACGGCAUUGGCGUGACUCAGAACGUGCUGUACGAGAACCAGAAGCUGAUUGCUAACCAGUUCAACUCUGCUAUUGGCAAGAUUCAGGACUCUCUGUCUUCUACUGCUUCUGCUCUGGGCAAGCUGCAGGACGUGGUGAACCACAACGCUCAGGCUCUGAACACUCUGGUGAAGCAGCUGUCUUCUAAGUUCGGCGCUAUUUCUUCUGUGCUGAACGACAUUUUCUCUCGUCUGGACAAGGUGGAGGCUGAGGUGCAGAUUGACCGUCUGAUUACUGGCCGUCUGCAGUCUCUGCAGACUUACGUGACUCAGCAGCUGAUUCGUGCUGCUGAGAUUCGUGCUUCUGCUAACCUGGCUGCUACUAAGAUGUCUGAGUGCGUGCUGGGCCAGUCUAAGCGUGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGUCUUUCCCUCAGUCUGCUCCUCACGGCGUGGUGUUCCUGCACGUGACUUACGUGCCUGCUCAGGAGAAGAACUUCACUACUGCUCCUGCUAUUUGCCACGACGGCAAGGCUCACUUCCCUCGUGAGGGCGUGUUCGUGUCUAACGGCACUCACUGGUUCGUGACUCAGCGUAACUUCUACGAGCCUCAGAUUAUUACUACUGACAACACUUUCGUGUCUGGCAACUGCGACGUGGUGAUUGGCAUUGUGAACAACACUGUGUACGACCCUCUGCAGCCUGAGCUGGACUCUUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACUUCUCCUGACGUGGACCUGGGCGACAUUUCUGGCAUUAACGCUUCUGUGGUGAACAUUCAGAAGGAGAUUGACCGUCUGAACGAGGUGGCUAAGAACCUGAACGAGUCUCUGAUUGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUUAAGUGGCCUUGGUACAUUUGGCUGGGCUUCAUUGCUGGCCUGAUUGCUAUUGUGAUGGUGACUAUUAUGCUGUGCUGCAUGACUUCUUGCUGCUCUUGCCUGAAGGGCUGCUGCUCUUGCGGCUCUUGCUGCAAGUUCGACGAGGACGACUCUGAGCCUGUGCUGAAGGGCGUGAAGCUGCACUACACUUGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:20)
GAGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCUUCUCAGUGCGUGAACCUGACUACUCGUACUCAGCUGCCUCCUGCUUACACUAACUCUUUCACUCGUGGCGUGUACUACCCUGACAAGGUGUUCCGUUCUUCUGUGCUGCACUCUACUCAGGACCUGUUCCUGCCUUUCUUCUCUAACGUGACUUGGUUCCACGUGAUUUCUGGCACUAACGGCACUAAGCGUUUCGACAACCCUGUGCUGCCUUUCAACGACGGCGUGUACUUCGCUUCUAUUGAGAAGUCUAACAUUAUUCGUGGCUGGAUUUUCGGCACUACUCUGGACUCUAAGACUCAGUCUCUGCUGAUUGUGAACAACGCUACUAACGUGGUGAUUAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCUUUCCUGGACCACAAGAACAACAAGUCUUGGAUGGAGUCUGAGUUCCGUGUGUACUCUUCUGCUAACAACUGCACUUUCGAGUACGUGUCUCAGCCUUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGUGAGUUCGUGUUCAAGAACAUUGACGGCUACUUCAAGAUUUACUCUAAGCACACUCCUAUUAUUGUGCGUGAGCCUGAGGACCUGCCUCAGGGCUUCUCUGCUCUGGAGCCUCUGGUGGACCUGCCUAUUGGCAUUAACAUUACUCGUUUCCAGACUCUGCUGGCUCUGCACCGUUCUUACCUGACUCCUGGCGACUCUUCUUCUGGCUGGACUGCUGGCGCUGCUGCUUACUACGUGGGCUACCUGCAGCCUCGUACUUUCCUGCUGAAGUACAACGAGAACGGCACUAUUACUGACGCUGUGGACUGCGCUCUGGACCCUCUGUCUGAGACUAAGUGCACUCUGAAGUCUUUCACUGUGGAGAAGGGCAUUUACCAGACUUCUAACUUCCGUGUGCAGCCUACUGAGUCUAUUGUGCGUUUCCCUAACAUUACUAACCUGUGCCCUUUCGACGAGGUGUUCAACGCUACUCGUUUCGCUUCUGUGUACGCUUGGAACCGUAAGCGUAUUUCUAACUGCGUGGCUGACUACUCUGUGCUGUACAACCUGGCUCCUUUCUUCACUUUCAAGUGCUACGGCGUGUCUCCUACUAAGCUGAACGACCUGUGCUUCACUAACGUGUACGCUGACUCUUUCGUGAUUCGUGGCGACGAGGUGCGUCAGAUUGCUCCUGGCCAGACUGGCAACAUUGCUGACUACAACUACAAGCUGCCUGACGACUUCACUGGCUGCGUGAUUGCUUGGAACUCUAACAAGCUGGACUCUAAGGUGUCUGGCAACUACAACUACCUGUACCGUCUGUUCCGUAAGUCUAACCUGAAGCCUUUCGAGCGUGACAUUUCUACUGAGAUUUACCAGGCUGGCAACAAGCCUUGCAACGGCGUGGCUGGCUUCAACUGCUACUUCCCUCUGCGUUCUUACUCUUUCCGUCCUACUUACGGCGUGGGCCACCAGCCUUACCGUGUGGUGGUGCUGUCUUUCGAGCUGCUGCACGCUCCUGCUACUGUGUGCGGCCCUAAGAAGUCUACUAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACUGGCGUGCUGACUGAGUCUAACAAGAAGUUCCUGCCUUUCCAGCAGUUCGGCCGUGACAUUGCUGACACUACUGACGCUGUGCGUGACCCUCAGACUCUGGAGAUUCUGGACAUUACUCCUUGCUCUUUCGGCGGCGUGUCUGUGAUUACUCCUGGCACUAACACUUCUAACCAGGUGGCUGUGCUGUACCAGGGCGUGAACUGCACUGAGGUGCCUGUGGCUAUUCACGCUGACCAGCUGACUCCUACUUGGCGUGUGUACUCUACUGGCUCUAACGUGUUCCAGACUCGUGCUGGCUGCCUGAUUGGCGCUGAGUACGUGAACAACUCUUACGAGUGCGACAUUCCUAUUGGCGCUGGCAUUUGCGCUUCUUACCAGACUCAGACUAAGUCUCACCGUCGUGCUCGUUCUGUGGCUUCUCAGUCUAUUAUUGCUUACACUAUGUCUCUGGGCGCUGAGAACUCUGUGGCUUACUCUAACAACUCUAUUGCUAUUCCUACUAACUUCACUAUUUCUGUGACUACUGAGAUUCUGCCUGUGUCUAUGACUAAGACUUCUGUGGACUGCACUAUGUACAUUUGCGGCGACUCUACUGAGUGCUCUAACCUGCUGCUGCAGUACGGCUCUUUCUGCACUCAGCUGAAGCGUGCUCUGACUGGCAUUGCUGUGGAGCAGGACAAGAACACUCAGGAGGUGUUCGCUCAGGUGAAGCAGAUUUACAAGACUCCUCCUAUUAAGUACUUCGGCGGCUUCAACUUCUCUCAGAUUCUGCCUGACCCUUCUAAGCCUUCUAAGCGUUCUUUCAUUGAGGACCUGCUGUUCAACAAGGUGACUCUGGCUGACGCUGGCUUCAUUAAGCAGUACGGCGACUGCCUGGGCGACAUUGCUGCUCGUGACCUGAUUUGCGCUCAGAAGUUCAAGGGCCUGACUGUGCUGCCUCCUCUGCUGACUGACGAGAUGAUUGCUCAGUACACUUCUGCUCUGCUGGCUGGCACUAUUACUUCUGGCUGGACUUUCGGCGCUGGCGCUGCUCUGCAGAUUCCUUUCGCUAUGCAGAUGGCUUACCGUUUCAACGGCAUUGGCGUGACUCAGAACGUGCUGUACGAGAACCAGAAGCUGAUUGCUAACCAGUUCAACUCUGCUAUUGGCAAGAUUCAGGACUCUCUGUCUUCUACUGCUUCUGCUCUGGGCAAGCUGCAGGACGUGGUGAACCACAACGCUCAGGCUCUGAACACUCUGGUGAAGCAGCUGUCUUCUAAGUUCGGCGCUAUUUCUUCUGUGCUGAACGACAUUUUCUCUCGUCUGGACAAGGUGGAGGCUGAGGUGCAGAUUGACCGUCUGAUUACUGGCCGUCUGCAGUCUCUGCAGACUUACGUGACUCAGCAGCUGAUUCGUGCUGCUGAGAUUCGUGCUUCUGCUAACCUGGCUGCUACUAAGAUGUCUGAGUGCGUGCUGGGCCAGUCUAAGCGUGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGUCUUUCCCUCAGUCUGCUCCUCACGGCGUGGUGUUCCUGCACGUGACUUACGUGCCUGCUCAGGAGAAGAACUUCACUACUGCUCCUGCUAUUUGCCACGACGGCAAGGCUCACUUCCCUCGUGAGGGCGUGUUCGUGUCUAACGGCACUCACUGGUUCGUGACUCAGCGUAACUUCUACGAGCCUCAGAUUAUUACUACUGACAACACUUUCGUGUCUGGCAACUGCGACGUGGUGAUUGGCAUUGUGAACAACACUGUGUACGACCCUCUGCAGCCUGAGCUGGACUCUUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACUUCUCCUGACGUGGACCUGGGCGACAUUUCUGGCAUUAACGCUUCUGUGGUGAACAUUCAGAAGGAGAUUGACCGUCUGAACGAGGUGGCUAAGAACCUGAACGAGUCUCUGAUUGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUUAAGUGGCCUUGGUACAUUUGGCUGGGCUUCAUUGCUGGCCUGAUUGCUAUUGUGAUGGUGACUAUUAUGCUGUGCUGCAUGACUUCUUGCUGCUCUUGCCUGAAGGGCUGCUGCUCUUGCGGCUCUUGCUGCAAGUUCGACGAGGACGACUCUGAGCCUGUGCUGAAGGGCGUGAAGCUGCACUACACUUGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:21)
GAGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCCAGUGCGUGAACCUGACGACGCGGACGCAGCUGCCCCCCGCCUACACGAACAGCUUCACGCGGGGCGUGUACUACCCCGACAAGGUGUUCCGGAGCAGCGUGCUGCACAGCACGCAGGACCUGUUCCUGCCCUUCUUCAGCAACGUGACGUGGUUCCACGUGAUCAGCGGCACGAACGGCACGAAGCGGUUCGACAACCCCGUGCUGCCCUUCAACGACGGCGUGUACUUCGCCAGCAUCGAGAAGAGCAACAUCAUCCGGGGCUGGAUCUUCGGCACGACGCUGGACAGCAAGACGCAGAGCCUGCUGAUCGUGAACAACGCCACGAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACCACAAGAACAACAAGAGCUGGAUGGAGAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACGUUCGAGUACGUGAGCCAGCCCUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGGGAGUUCGUGUUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACGCCCAUCAUCGUGCGGGAGCCCGAGGACCUGCCCCAGGGCUUCAGCGCCCUGGAGCCCCUGGUGGACCUGCCCAUCGGCAUCAACAUCACGCGGUUCCAGACGCUGCUGGCCCUGCACCGGAGCUACCUGACGCCCGGCGACAGCAGCAGCGGCUGGACGGCCGGCGCCGCCGCCUACUACGUGGGCUACCUGCAGCCCCGGACGUUCCUGCUGAAGUACAACGAGAACGGCACGAUCACGGACGCCGUGGACUGCGCCCUGGACCCCCUGAGCGAGACGAAGUGCACGCUGAAGAGCUUCACGGUGGAGAAGGGCAUCUACCAGACGAGCAACUUCCGGGUGCAGCCCACGGAGAGCAUCGUGCGGUUCCCCAACAUCACGAACCUGUGCCCCUUCGACGAGGUGUUCAACGCCACGCGGUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACCUGGCCCCCUUCUUCACGUUCAAGUGCUACGGCGUGAGCCCCACGAAGCUGAACGACCUGUGCUUCACGAACGUGUACGCCGACAGCUUCGUGAUCCGGGGCGACGAGGUGCGGCAGAUCGCCCCCGGCCAGACGGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACGGGCUGCGUGAUCGCCUGGAACAGCAACAAGCUGGACAGCAAGGUGAGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACGGAGAUCUACCAGGCCGGCAACAAGCCCUGCAACGGCGUGGCCGGCUUCAACUGCUACUUCCCCCUGCGGAGCUACAGCUUCCGGCCCACGUACGGCGUGGGCCACCAGCCCUACCGGGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACGGUGUGCGGCCCCAAGAAGAGCACGAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACGGGCGUGCUGACGGAGAGCAACAAGAAGUUCCUGCCCUUCCAGCAGUUCGGCCGGGACAUCGCCGACACGACGGACGCCGUGCGGGACCCCCAGACGCUGGAGAUCCUGGACAUCACGCCCUGCAGCUUCGGCGGCGUGAGCGUGAUCACGCCCGGCACGAACACGAGCAACCAGGUGGCCGUGCUGUACCAGGGCGUGAACUGCACGGAGGUGCCCGUGGCCAUCCACGCCGACCAGCUGACGCCCACGUGGCGGGUGUACAGCACGGGCAGCAACGUGUUCCAGACGCGGGCCGGCUGCCUGAUCGGCGCCGAGUACGUGAACAACAGCUACGAGUGCGACAUCCCCAUCGGCGCCGGCAUCUGCGCCAGCUACCAGACGCAGACGAAGAGCCACCGGCGGGCCCGGAGCGUGGCCAGCCAGAGCAUCAUCGCCUACACGAUGAGCCUGGGCGCCGAGAACAGCGUGGCCUACAGCAACAACAGCAUCGCCAUCCCCACGAACUUCACGAUCAGCGUGACGACGGAGAUCCUGCCCGUGAGCAUGACGAAGACGAGCGUGGACUGCACGAUGUACAUCUGCGGCGACAGCACGGAGUGCAGCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACGCAGCUGAAGCGGGCCCUGACGGGCAUCGCCGUGGAGCAGGACAAGAACACGCAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACGCCCCCCAUCAAGUACUUCGGCGGCUUCAACUUCAGCCAGAUCCUGCCCGACCCCAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAGGUGACGCUGGCCGACGCCGGCUUCAUCAAGCAGUACGGCGACUGCCUGGGCGACAUCGCCGCCCGGGACCUGAUCUGCGCCCAGAAGUUCAAGGGCCUGACGGUGCUGCCCCCCCUGCUGACGGACGAGAUGAUCGCCCAGUACACGAGCGCCCUGCUGGCCGGCACGAUCACGAGCGGCUGGACGUUCGGCGCCGGCGCCGCCCUGCAGAUCCCCUUCGCCAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGCGUGACGCAGAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACGGCCAGCGCCCUGGGCAAGCUGCAGGACGUGGUGAACCACAACGCCCAGGCCCUGAACACGCUGGUGAAGCAGCUGAGCAGCAAGUUCGGCGCCAUCAGCAGCGUGCUGAACGACAUCUUCAGCCGGCUGGACAAGGUGGAGGCCGAGGUGCAGAUCGACCGGCUGAUCACGGGCCGGCUGCAGAGCCUGCAGACGUACGUGACGCAGCAGCUGAUCCGGGCCGCCGAGAUCCGGGCCAGCGCCAACCUGGCCGCCACGAAGAUGAGCGAGUGCGUGCUGGGCCAGAGCAAGCGGGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCCCAGAGCGCCCCCCACGGCGUGGUGUUCCUGCACGUGACGUACGUGCCCGCCCAGGAGAAGAACUUCACGACGGCCCCCGCCAUCUGCCACGACGGCAAGGCCCACUUCCCCCGGGAGGGCGUGUUCGUGAGCAACGGCACGCACUGGUUCGUGACGCAGCGGAACUUCUACGAGCCCCAGAUCAUCACGACGGACAACACGUUCGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAACACGGUGUACGACCCCCUGCAGCCCGAGCUGGACAGCUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACGAGCCCCGACGUGGACCUGGGCGACAUCAGCGGCAUCAACGCCAGCGUGGUGAACAUCCAGAAGGAGAUCGACCGGCUGAACGAGGUGGCCAAGAACCUGAACGAGAGCCUGAUCGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACGAUCAUGCUGUGCUGCAUGACGAGCUGCUGCAGCUGCCUGAAGGGCUGCUGCAGCUGCGGCAGCUGCUGCAAGUUCGACGAGGACGACAGCGAGCCCGUGCUGAAGGGCGUGAAGCUGCACUACACGUGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:22)
In another preferred embodiment, the mRNA nucleic acid molecule further comprises one or more functional nucleotide analogue modifications selected from the group consisting of pseudouridine (ψ), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e 1 ψ), 5-methoxy-uridine (mo 5U) and 5-methylcytosine (m 5C), preferably one or more or all uracils of the mRNA nucleic acid molecule are substituted with pseudouridine (ψ). In a particularly preferred embodiment, the inventors have achieved a reduction in mRNA immunogenicity by reducing the U (uridylic acid) content of the mRNA molecule by substitution of all uracils in the nucleic acid sequences SEQ ID NO:20-SEQ ID NO:22 with pseudouridine (ψ), the capping substituted sequences shown in SEQ ID NO:23-SEQ ID NO: 25. All uracils in the nucleic acid sequences SEQ ID NO. 6-SEQ ID NO. 8 are replaced by pseudouridine (psi), and the sequence after capping replacement is shown as SEQ ID NO. 26-SEQ ID NO. 28.
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Pharmaceutical composition
In another embodiment, provided herein is a pharmaceutical composition for inducing a neutralizing antibody response in a subject to a novel coronal variant omicron (b.1.1.529) S glycoprotein comprising an mRNA nucleic acid molecule as described above and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical compositions described herein comprise an mRNA molecule of the sequence shown in any one of SEQ ID NOs 20 to 25 or a combination thereof, and a pharmaceutically acceptable carrier.
In some embodiments, the mRNA of the present disclosure is formulated in lipid nanoparticles (Lipid nanoparticle, LNP) to form a lipid nanoparticle form. Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising mRNA comprising the following components:
(1) Any one of the mRNA molecules with the sequences shown in SEQ ID NO. 20-SEQ ID NO. 25 or the combination of the mRNA molecules;
(2) 20-60% by mole of ionizable cationic lipids (ionizable lipids);
(3) 5-25% by mole of a non-cationic lipid;
(4) 25-55% by mole of sterols; and
(5) 0.5-15% by mole of PEG modified lipid.
In another embodiment, the ionizable cationic lipid whole structure can be divided into three parts, a head part, a connecting fragment and a tail part. In LNP formulations, the ionizable lipids appear neutral at physiological pH, while being positively charged in the acidic environment of the endosome. The pH-dependent ionization capacity makes ionizable lipids suitable materials for nucleic acid delivery due to the substantial improvement in effectiveness and toxicity characteristics. Preferably, the ionizable lipid is present in the formulation in a molar ratio of typically 30% to 50% of the total lipid. Preferred ionizable cationic lipids that have been used clinically include DLin-MC3-DMA, SM-102, ALC-0315, and the like. The head groups such as ALC-0315 and SM-102 contain a terminal hydroxyl group, which can reduce hydration of the head groups and enhance hydrogen bonding interactions with nucleic acids, thereby possibly enhancing transfection ability.
In another embodiment, the non-cationic lipid is generally a neutral helper phospholipid. Phospholipids are often used as structural lipids for LNP formulations because they can spontaneously organize into lipid bilayers and higher phase transition temperatures enhance the membrane stability of LNP. Preferably, the phospholipid is present in the formulation in a molar ratio of 10% to 20% of the total lipid. Preferred phospholipids that have been used clinically may be DSPC, DOPE and DGTS. Wherein the 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) structure consists of a phosphatidylcholine head group and two saturated 18 carbon tails, both tails forming a tightly packed lipid bilayer. DSPC is a preferred structural lipid in the clinically validated mRNA vaccine LNP.
In another embodiment, the sterols may be selected from cholesterol, beta-sitosterol, oxidized cholesterol derivatives, and the like. Cholesterol is a naturally abundant component of cell membranes and is often used as a structural lipid in LNP formulations. The cholesterol ratio in the LNP formulation may preferably be 30-50 mole percent.
In another embodiment, PEG-modified lipids (PEG-modified lipids) are an important component in LNP that regulates half-life and cellular uptake. Preferably, the PEG-modified lipids are present in the formulation at a molar ratio of 0.5-2.5% of the total lipid. PEG provides an external polymeric layer for LNP to prevent serum protein adsorption and uptake by the mononuclear phagocyte system, prolonging circulation time in vivo. PEG can also prevent aggregation of nanoparticles during storage and in blood. In some embodiments, the PEG-modified lipids of the present disclosure include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some examples, the PEG-modified lipid is DMG-PEG, PEG-DOMG, PEG-DSG, and/or PEG-DPG, or the like. More preferred PEG modified lipid ALC0159.
In another embodiment, the LNP of the present disclosure comprises a mass ratio of ionizable cationic lipid component to mRNA of from about 10:1 to about 100:1. In another embodiment, the LNP of the present disclosure comprises a mass ratio of ionizable cationic lipid component to mRNA of from about 20:1 to about 40:1.
In another embodiment, the disclosed pharmaceutical composition, the mRNA molecules are encapsulated in a lipid shell and formulated into a lipid nanoparticle form, wherein the lipid nanoparticle generally comprises four lipid components of ALC0315, DSPC, cholesterol, and ALC 0159.
In another preferred embodiment, the present invention provides a pharmaceutical composition comprising mRNA comprising the following components:
(1) mRNA molecules with the sequence shown in any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof;
(2) 20-60% by mole of ionizable cationic lipid ALC0315;
(3) 5-25% by mole of DSPC;
(4) Cholesterol in 25-55% molar ratio; and
(5) 0.5-15% by mole of PEG modified lipid ALC0159;
wherein the mass ratio of the ionizable cationic lipid component to the mRNA is from about 10:1 to about 100:1.
In another preferred embodiment, the present invention provides a pharmaceutical composition comprising mRNA comprising the following components:
(1) mRNA molecules with the sequence shown in any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof;
(2) 30-50% by mole of ionizable cationic lipid ALC0315;
(3) 10-20% by mole of DSPC;
(4) 23-50% cholesterol by mole; and
(5) 0.5-2.5% by mole of PEG modified lipid ALC0159;
wherein the mass ratio of the ionizable cationic lipid component to the mRNA is from about 20:1 to about 40:1.
The mean particle size and particle size distribution of the LNP are important initial determinants of LNP quality and suitability for various applications. These features are typically characterized by Dynamic Light Scattering (DLS). In some embodiments, the presently disclosed LNPs have an average diameter of about 20-200 nm. In other embodiments, the LNPs of the disclosure have an average diameter of about 50nm to 150 nm. In other embodiments, the LNPs of the disclosure have an average diameter of about 70nm to 120 nm.
The surface charge of LNP is responsible for interactions with cell membranes and biological environments, and is typically assessed by Zeta potential measurements. One common method of adjusting the total charge on the surface of the LNP is to adjust the N/P ratio, i.e., the ratio of ionizable lipids (N, representing cationic amines) to nucleic acids (P, representing anionic phosphates). In some embodiments, the LNPs disclosed herein comprise an N to P ratio of about 2:1 to about 30:1. In other embodiments, the LNPs of the present disclosure include an N to P ratio of about 6:1. In other embodiments, the LNPs of the present disclosure include an N to P ratio of about 3:1.
Use of the same
In another embodiment, provided herein is the use of an mRNA nucleic acid molecule or pharmaceutical composition thereof for inducing a neutralizing antibody response in a subject to the novel coronavirus variant omacron (b.1.1.529) S glycoprotein in the manufacture of a medicament or vaccine for treating or preventing a novel coronavirus infection. Preferably, provided herein are mRNAs as shown in SEQ ID NO:20-SEQ ID NO:25 useful for inducing a neutralizing antibody response in a subject against the S glycoprotein of the novel coronavariant strain omacron (B.1.1.529), which mRNAs provided herein can control, prevent or treat infectious diseases caused by coronaviruses in a subject to be administered.
In some embodiments, the invention discloses the use of mRNA as shown in SEQ ID NO. 20-SEQ ID NO. 25 for the preparation of a medicament or vaccine for the treatment or prevention of novel coronavirus infections. In some embodiments, compositions according to the present disclosure, including mRNA and polypeptides encoded thereby, are useful for treating or preventing coronavirus infections, particularly novel coronavariant omicron (b.1.1.529) virus infections. The pharmaceutical composition may be administered to a subject as part of an active immunization regimen or as a therapeutic pharmaceutical composition. In some embodiments, the amount of mRNA provided to the subject may be a dose effective for immunoprophylaxis.
In another embodiment, the subject of the invention is a human or non-human mammal. In another embodiment, the subject is an adult, a human child, or a human infant. In another embodiment, the subject is at risk of or is susceptible to a coronavirus infection. In another embodiment, the subject is an elderly person. In another embodiment, the subject has been diagnosed as positive for a coronavirus infection. In another embodiment, the subject is asymptomatic.
The pharmaceutical composition may be administered with other prophylactic or therapeutic treatments. As used herein, when referring to a prophylactic composition (e.g., vaccine), the term "enhancer" refers to the additional administration of the prophylactic (vaccine) composition. After administration of the prophylactic pharmaceutical composition, an enhancer (or an enhanced vaccine) may be administered. The time of intermittent administration of the prophylactic pharmaceutical composition and the enhancer may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year. The mRNA described herein can be formulated into the dosage forms described herein, for example, intranasal, intraperitoneal, intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac intraperitoneal, or subcutaneous administration, preferably subcutaneous or intramuscular administration.
The mRNA provided herein can control, prevent or treat infectious diseases caused by coronaviruses in a subject to be administered, and the effective dose of mRNA can range from 10 μg to 5mg, more preferably from 10 μg to 1mg, from 10 μg to 500 μg, from 20 μg to 300 μg or from 40 μg to 200 μg, etc. The amount of therapeutic nucleic acid or composition thereof effective in treating, preventing and/or treating an infectious disease will depend on the nature of the disease being treated, the route of administration, the general health of the subject being administered, etc., and will generally be at the discretion of the physician. Standard clinical techniques, such as in vitro assays, may optionally be employed to assist in determining the optimal dosage range.
The present invention provides mRNA nucleic acid molecules, pharmaceutical compositions comprising the mRNA nucleic acid molecules, and uses thereof for controlling, preventing, and treating coronavirus infections. The technical scheme of the invention is summarized as follows:
1. a nucleic acid molecule comprising an S glycoprotein encoding coronavirus omicron (b.1.1.529), wherein said coding region comprises one or more open reading frames (open reading frame, ORFs), and wherein at least one ORF encodes an S glycoprotein of coronavirus omicron (b.1.1.529) having a protein sequence as set forth in SEQ ID No. 1; and having a nucleotide sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence shown in SEQ ID NO. 2.
2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule has a sequence shown in SEQ ID NO. 3-SEQ ID NO. 5.
3. Comprises mRNA molecules encoding coronavirus omacron (B.1.1.529) S glycoprotein, and the sequence of the mRNA molecules is shown as SEQ ID NO. 6-SEQ ID NO. 8.
4. An mRNA nucleic acid molecule comprising:
(i) A 5 'untranslated region (5' -UTR);
(ii) CDS, wherein the CDS comprises an Open Reading Frame (ORF) encoding a coronavirus omicron (B.1.1.529) antigen S glycoprotein capable of inducing an immune response comprising a polypeptide as set forth in SEQ ID NO:6-SEQ ID NO:8
The nucleotide sequence shown;
(iii) 3 '-untranslated region (3' -UTR).
5. The mRNA nucleic acid molecule of claim 4, wherein: wherein the 5' UTR comprises the sequence of SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO. 11.
6. The mRNA nucleic acid molecule of claim 4, wherein: the 5' -UTR sequence may or may not comprise a Kozak sequence.
7. The mRNA nucleic acid molecule of claim 6, wherein: wherein the 5' UTR may comprise the kozak sequence GCCACC or GCCANN et al, wherein N refers to A, T/U, C or G.
8. The mRNA nucleic acid molecule of claim 4, wherein: wherein the 3' UTR comprises the sequence of SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14.
9. The mRNA nucleic acid molecule of claim 4, wherein: the mRNA nucleic acid molecules also contain native 5' -cap structures or analogs thereof produced by endogenous processes.
10. The mRNA nucleic acid molecule of claim 9, wherein: the 5' -cap analogue may optionally be selected from m7G (5 ') ppp (5 ') (2 ' OMeA) pG, 3' -O-Me-m7G (5 ') ppp (5 ') G; g (5 ') ppp (5') A; g (5 ') ppp (5') G; m7G (5 ') ppp (5') A; m7G (5 ') ppp (5') G, etc.
11. The mRNA nucleic acid molecule of claim 4, wherein: the mRNA nucleic acid molecule also comprises a poly-a tail or polyadenylation signal, optionally having a length of 80 to 180 nucleotides.
12. The mRNA nucleic acid molecule of claim 11, wherein: the poly A sequence (polyA) is 80-180A, and a linker sequence is arranged in the middle.
13. The mRNA nucleic acid molecule of claim 12, wherein: the poly A sequence (polyA) is 80-160A.
14. The mRNA nucleic acid molecule of claim 11, wherein: the poly A sequence is shown as SEQ ID NO. 15 or SEQ ID NO. 16.
15. The mRNA nucleic acid molecule according to any one of claims 3-11, characterized in that: the mRNA nucleic acid molecule further comprises one or more functional nucleotide analog modifications selected from the group consisting of pseudouridine (ψ), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (e 1 ψ), 5-methoxy-uridine (mo 5U) and 5-methylcytosine (m 5C).
16. The mRNA nucleic acid molecule of claim 15, wherein: the mRNA includes pseudouridine (ψ) substitutions at one or more or all uracil positions of the nucleic acid.
17. The mRNA nucleic acid molecule of claim 16, wherein: uracil (U) as shown in any one of SEQ ID NO. 6-SEQ ID NO. 25 is replaced by pseudouridine (ψ).
18. An mRNA nucleic acid molecule, said mRNA molecule comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA), wherein CDS comprises an Open Reading Frame (ORF) encoding coronavirus omicron (B.1.1.529) antigen S glycoprotein capable of inducing immune response, the nucleotide sequence is shown as SEQ ID NO:6-SEQ ID NO: 8.
19. The mRNA nucleic acid molecule of claim 18, wherein: wherein the 5' -UTR comprises a Kozak sequence.
20. The mRNA nucleic acid molecule of claim 18, wherein: the CDS may further comprise a signal peptide sequence.
21. The mRNA nucleic acid molecule of claim 20, wherein: the signal peptide sequence is shown as SEQ ID NO. 17-SEQ ID NO. 19.
22. An mRNA molecule, said mRNA comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA);
wherein said 5' -cap structure is optionally selected from m7G (5 ') ppp (5 ') (2 ' OMeA) pG, 3' -O-Me-m7G (5 ') ppp (5 ') G; g (5 ') ppp (5') A; g (5 ') ppp (5') G; m7G (5 ') ppp (5') A; m7G (5 ') ppp (5') G, etc.;
wherein the 5' -UTR comprises a sequence shown as SEQ ID NO. 9-SEQ ID NO. 11;
wherein the CDS comprises an Open Reading Frame (ORF) which encodes a signal peptide and a coronavirus omacron (B.1.1.529) antigen S glycoprotein capable of inducing an immune response, wherein the sequence of the signal peptide is shown as SEQ ID NO. 17-SEQ ID NO. 19, and the sequence of the coronavirus omacron (B.1.1.529) antigen S glycoprotein is shown as SEQ ID NO. 6-SEQ ID NO. 8;
wherein the 3' -UTR comprises a nucleotide sequence shown as SEQ ID NO. 12-SEQ ID NO. 14;
Wherein the 3' -polyadenylation sequence (polyA) optionally has a length of 80 to 180 adenylates.
23. The mRNA nucleic acid molecule of claim 22, wherein: the sequence is shown as SEQ ID NO. 20-SEQ ID NO. 22.
24. The mRNA nucleic acid molecule of any one of claims 18-23, wherein: the mRNA nucleic acid molecule further comprises one or more functional nucleotide analog modifications selected from the group consisting of pseudouridine (ψ), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (e 1 ψ), 5-methoxy-uridine (mo 5U) and 5-methylcytosine (m 5C).
25. The mRNA nucleic acid molecule of claim 24, wherein: one or more or all uracils of the mRNA nucleic acid molecule are replaced with pseudouridine (ψ).
26. The mRNA nucleic acid molecule of claim 24, wherein: all uracils in the nucleic acid sequences SEQ ID NO. 20-SEQ ID NO. 22 are replaced by pseudouridine (psi), and the replaced sequences are shown as SEQ ID NO. 23-SEQ ID NO. 25.
27. A pharmaceutical composition for inducing a neutralizing antibody response in a subject to a novel coronal variant omacron (b.1.1.529) S glycoprotein comprising the mRNA nucleic acid molecule of any of claims 4-26 and a pharmaceutically acceptable carrier.
28. The pharmaceutical composition according to claim 27, wherein: the pharmaceutical composition comprises an mRNA molecule shown as any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof and a pharmaceutically acceptable carrier.
29. A pharmaceutical composition comprising mRNA comprising the following components:
(1) mRNA molecules with the sequence shown in any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof;
(2) 20-60% by mole of ionizable cationic lipids (ionizable lipids);
(3) 5-25% by mole of a non-cationic lipid;
(4) 25-55% by mole of sterols; and
(5) 0.5-15% by mole of PEG modified lipid.
30. The pharmaceutical composition according to claim 29, wherein: the mole ratio of the ionizable cationic lipid is 30% -50%.
31. The pharmaceutical composition according to claim 29, wherein: the ionizable cationic lipid comprises DLin-MC3-DMA, SM-102, ALC-0315, etc.
32. The pharmaceutical composition according to claim 29, wherein: the molar ratio of the non-cationic lipid is 10% -20%.
33. The pharmaceutical composition according to claim 29, wherein: the non-cationic lipid is DSPC, DOPE or DGTS.
34. The pharmaceutical composition according to claim 29, wherein: the sterol is selected from cholesterol, beta-sitosterol, oxidized cholesterol derivatives and the like.
35. The pharmaceutical composition according to claim 29, wherein: the mole ratio of the sterol is 30-50%.
35. The pharmaceutical composition according to claim 29, wherein: the molar ratio of the PEG modified lipid is 0.5-2.5%.
36. The pharmaceutical composition according to claim 29, wherein: the PEG modified lipid comprises PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide, PEG modified dialkylamine, PEG modified diacylglycerol, PEG modified dialkylglycerol and mixture thereof.
37. A pharmaceutical composition according to claim 36, wherein: the PEG modified lipid is DMG-PEG, PEG-DOMG, PEG-DSG and/or PEG-DPG.
38. The pharmaceutical composition according to claim 37, wherein: the PEG modified lipid is ALC0159.
39. The pharmaceutical composition according to claim 29, wherein: the pharmaceutical composition has a mass ratio of ionizable cationic lipid component to mRNA of from about 10:1 to about 100:1.
40. The pharmaceutical composition according to claim 39, wherein: the pharmaceutical composition has a mass ratio of ionizable cationic lipid component to mRNA of from about 20:1 to about 40:1.
41. A pharmaceutical composition comprising mRNA comprising the following components:
(1) mRNA molecules with the sequence shown in any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof;
(2) 20-60% by mole of ionizable cationic lipid ALC0315;
(3) 5-25% by mole of DSPC;
(4) Cholesterol in 25-55% molar ratio; and
(5) 0.5-15% by mole of PEG modified lipid ALC0159;
wherein the mass ratio of the ionizable cationic lipid component to the mRNA is from about 10:1 to about 100:1.
42. A pharmaceutical composition comprising mRNA comprising the following components:
(1) mRNA molecules with the sequence shown in any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof;
(2) 30-50% by mole of ionizable cationic lipid ALC0315;
(3) 10-20% by mole of DSPC;
(4) 23-50% cholesterol by mole; and
(5) 0.5-2.5% by mole of PEG modified lipid ALC0159;
wherein the mass ratio of the ionizable cationic lipid component to the mRNA is from about 20:1 to about 40:1.
43. The pharmaceutical composition according to any one of claims 29-42, wherein: the pharmaceutical composition forms Lipid Nanoparticles (LNPs), the LNPs having an average diameter of about 20-200 nm.
44. The pharmaceutical composition according to claim 43, wherein: the LNP has an average diameter of about 50nm to 150 nm.
45. The pharmaceutical composition of claim 44, wherein: the LNP has an average diameter of about 70nm to 120 nm.
46. The pharmaceutical composition according to claim 43, wherein: the LNP comprises an N to P ratio of about 2:1 to about 30:1, wherein the N/P ratio is the ratio of ionizable lipids (N, representing cationic amines) to nucleic acids (P, representing anionic phosphates).
47. The pharmaceutical composition according to claim 46, wherein: the LNP includes an N to P ratio of about 6:1.
48. The pharmaceutical composition according to claim 46, wherein: the LNP includes an N to P ratio of about 3:1.
49. Use of an mRNA nucleic acid molecule according to any one of claims 4-26 or a pharmaceutical composition according to any one of claims 27-48 for the preparation of a medicament or vaccine for the treatment or prevention of a novel coronavirus infection.
50. The use according to claim 49, wherein: the mRNA has a sequence shown in any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof.
51. The use according to claim 49, wherein: the mRNA drug or vaccine is administered to a subject that is a human or non-human mammal.
52. The use according to claim 51, characterized in that: the mRNA drug or vaccine is administered to a subject who is an adult, a human child, or a human young child.
53. The use according to claim 52, characterized in that: the subject to which the mRNA drug or vaccine is administered is at risk of, or is susceptible to, a coronavirus infection.
54. The use according to claim 52, characterized in that: the administration subject of the mRNA drug or vaccine is elderly.
55. The use according to claim 52, characterized in that: the subject to whom the mRNA drug or vaccine is administered has been diagnosed as positive for coronavirus infection.
56. The use according to claim 52, characterized in that: the administration subject of the mRNA drug or vaccine is asymptomatic.
57. The use according to claim 49, wherein: the mRNA drug or vaccine may be administered with the enhancer (or enhanced vaccine) after administration of the prophylactic pharmaceutical composition, and the time of intermittent administration of the prophylactic pharmaceutical composition and enhancer may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.
58. The use according to claim 49, wherein: the mRNA drug or vaccine is formulated for intranasal, intraperitoneal, intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac intraperitoneal, or subcutaneous administration.
59. The use according to claim 58, wherein: the mRNA drug or vaccine is administered subcutaneously or intramuscularly.
60. The use according to claim 49, wherein: the effective dosage range of the mRNA drug or vaccine is 10 mug-5 mg.
61. The use according to claim 60, characterized in that: the effective dosage range of the mRNA medicine or vaccine is 10 mug-1 mg,10 mug-500 mug, 20 mug-300 mug or 40 mug-200 mug, etc.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.
Example 1 preparation of mRNA
The inventors optimized the novel coronal variant omacron (B.1.1.529) S glycoprotein coding region (SEQ ID NO: 2), and sequence optimization included: the GC content is regulated aiming at the expression in human body, the GC content of the sequence is improved, and the transcription optimization omicron (B.1.1.529) S glycoprotein DNA sequence with the nucleotide sequence shown as SEQ ID NO. 3 is obtained through optimization; optimizing the codon to obtain a transcription optimized omicron (B.1.1.529) S glycoprotein DNA sequence with a nucleotide sequence shown as SEQ ID NO. 4; increasing GC content and optimizing codon to obtain transcription optimized omicron (B.1.1.529) S glycoprotein DNA sequence with nucleotide sequence shown as SEQ ID N0.5. The nucleotide sequence makes the transcribed mRNA structure more stable and the translation efficiency of target protein in mammal and human body higher. mRNA sequences obtained through transcription according to the sequences of SEQ ID NO. 3-SEQ ID NO. 5 are respectively shown as SEQ ID NO. 6-SEQ ID NO. 8. The inventor realizes reducing mRNA immunogenicity by reducing the content of U (uridylic acid) in mRNA molecules, and substitutes all uracils in nucleic acid sequences SEQ ID NO. 6-SEQ ID NO. 8 with pseudo-uridine (psi), and the substituted sequences are shown as SEQ ID NO. 26-SEQ ID NO. 28.
The nucleotide sequence of the optimized omicron (B.1.1.529) S glycoprotein mRNA is shown as SEQ ID NO. 26: 5' -AACC psi AACCACCCGCACCCAAC psi ACCGCCGGCG psi ACACACC psi CG psi CACCCGCGGGG psi C psi AC psi ACCCGGACAAGG psi C psi CCGC psi CG psi CGG psi CC psi ACC psi CGACCCAAGACC psi A psi CC psi ACG psi C psi CGA CAC psi C psi CAC psi GG psi CCACpsi A psi CGGGGACCAACGGGACCAAGCGC psi A kind of 13-CGACAACCCGG-13-3-13-9-13-3-13-9-13-3-13-to-13-to-13 psi CGACAACCCGG psi CC psi ACCG psi CAACGACGGGG psi C psi AC psi CGCG psi CGA psi AGAGAAG psi CGAAG psi AA psi ACGCGGC psi GGA psi A kind of psi CGGCAC psi AC psi C psi GGAC psi C psi AAGAC psi CAG psi C psi GC psi GA psi G psi GAACAACGC psi AC psi AAG psi GG A kind of 13C, 3G, C, 3G, C, G, C, G, 3, C, 3, C, of; GC-PSGGCGC-PSI-PSG-GAG-PSG-G-PSG-G-carrier GC ψGGCGC ψGC ψ GC ψ GC ψ AC ψ ACG ψ GGGC ψ ACC ψ GCAGCC ψ CG ψ AC ψ CC ψ GC ψ GAAG ψ ACAACGAGAACGGCAC A.psigargin AC psi GACGC psi G psi GGAC psi GCGC psi GGACCC psi C psi G psi C psi GAGAGAC psi AAG psi GCAC psi C psi GAAG GAGA psi-psi ACCAGGC-psi GGCAACAAGCC psi-psi GCAACGGCG psi GGC-psi CAAC-psi GC-psi CCC-psi GCG-psi C-psi AC ψ C ψ cci CCG ψ CC ψ AC ψ acggg G psi GGGCCACCAGCC ψ cpcg CG psi G dg GG GC ψ C ψ CGAGC GAGA psi ACCAGGC GGCAACAAGCC psi ACGGC psi GCAACGGCG psi GGC psi CAAC psi GC psi CCC psi GCG psi C psi AC psi CCC psi ACpsi psi GGGCCACCAGCC psi ACCG psi G psi GG GGC psi G psi CGpsi C psi CGpsi CGstep CGAGC CGstep A kind of 13-GC-13-GCACGC-13-CC-13-GC-13-C-13-G-C-13-G-13-C-13-G-13-G and G and a G G G and a G G and G and G the level of the ground plane is the same as the level of the ground plane, the level of the ground plane is the same as the level of the ground plane, and the level of the ground plane The method comprises the steps of determining a total of a total and a total of psi AAGCAG psi ACGGCGAC psi GCC psi GGGCGACA psi GC psi CG psi GACC psi GA psi GG psi GCGAAG GGCC A kind of PSAAGCG PSPSPSPSPSPSGAGGACACGAPSGAPSGAPSGAPSGAGAGGCGCAPSGAGGCCs of PSACPSGAPSGAPSGAPSGAGGCC of PSACGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAGGCGACPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSGAPSPSPSPSGAPSPSGAPSPSPSPSGAPSGAPSPSGAPSPSPSPSPSPSPSPSGAPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPSPScarrier to a-type-of three-stage dual-purpose antenna, common antenna, with high-core, common antenna, common mode, common mode,), common mode,), or method,) to), A method for preparing a composite material of a plurality of components, wherein the composite material comprises the steps of a plurality of components, a grade, a one to be one G the level of the ground plane is the same as the level of the ground plane, the level of the ground plane is the same as the level of the ground plane Psi CAGAAGGAGA psi GACCG psi C psi GAACGAGG psi GGC psi AAGAACGAACGAACGAG psi C psi GA psi GACC psi GCAGGAGC psi GGGCAAG psi ACGAGCAG either of the above-mentioned modes A kind of 13-C GACCG-3-C GAACGAGG-C-3-G-C-G-3-G-C-G GG-GAC-GAG-GAC-GAG-GAAGC-GAC-ACC-GAC-3' (SEQ ID NO: 26).
An optimized omicron (B.1.1.529) S glycoprotein mRNA (opt) nucleotide sequence is shown in SEQ ID NO. 27.
5’-AACCΨGACΨACΨCGΨACΨCAGCΨGCCΨCCΨGCΨΨACACΨAACΨCΨΨΨCACΨCGΨGGCGΨGΨACΨACCCΨGACAAGGΨGΨΨCCGΨΨCΨΨCΨGΨGCΨGCACΨCΨACΨCAGGACCΨGΨΨCCΨGCCΨΨΨCΨΨCΨCΨAACGΨGACΨΨGGΨΨCCACGΨGAΨΨΨCΨGGCACΨAACGGCACΨAAGCGΨΨΨCGACAACCCΨGΨGCΨGCCΨΨΨCAACGACGGCGΨGΨACΨΨCGCΨΨCΨAΨΨGAGAAGΨCΨAACAΨΨAΨΨCGΨGGCΨGGAΨΨΨΨCGGCACΨACΨCΨGGACΨCΨAAGACΨCAGΨCΨCΨGCΨGAΨΨGΨGAACAACGCΨACΨAACGΨGGΨGAΨΨAAGGΨGΨGCGAGΨΨCCAGΨΨCΨGCAACGACCCΨΨΨCCΨGGACCACAAGAACAACAAGΨCΨΨGGAΨGGAGΨCΨGAGΨΨCCGΨGΨGΨACΨCΨΨCΨGCΨAACAACΨGCACΨΨΨCGAGΨACGΨGΨCΨCAGCCΨΨΨCCΨGAΨGGACCΨGGAGGGCAAGCAGGGCAACΨΨCAAGAACCΨGCGΨGAGΨΨCGΨGΨΨCAAGAACAΨΨGACGGCΨACΨΨCAAGAΨΨΨACΨCΨAAGCACACΨCCΨAΨΨAΨΨGΨGCGΨGAGCCΨGAGGACCΨGCCΨCAGGGCΨΨCΨCΨGCΨCΨGGAGCCΨCΨGGΨGGACCΨGCCΨAΨΨGGCAΨΨAACAΨΨACΨCGΨΨΨCCAGACΨCΨGCΨGGCΨCΨGCACCGΨΨCΨΨACCΨGACΨCCΨGGCGACΨCΨΨCΨΨCΨGGCΨGGACΨGCΨGGCGCΨGCΨGCΨΨACΨACGΨGGGCΨACCΨGCAGCCΨCGΨACΨΨΨCCΨGCΨGAAGΨACAACGAGAACGGCACΨAΨΨACΨGACGCΨGΨGGACΨGCGCΨCΨGGACCCΨCΨGΨCΨGAGACΨAAGΨGCACΨCΨGAAGΨCΨΨΨCACΨGΨGGAGAAGGGCAΨΨΨACCAGACΨΨCΨAACΨΨCCGΨGΨGCAGCCΨACΨGAGΨCΨAΨΨGΨGCGΨΨΨCCCΨAACAΨΨACΨAACCΨGΨGCCCΨΨΨCGACGAGGΨGΨΨCAACGCΨACΨCGΨΨΨCGCΨΨCΨGΨGΨACGCΨΨGGAACCGΨAAGCGΨAΨΨΨCΨAACΨGCGΨGGCΨGACΨACΨCΨGΨGCΨGΨACAACCΨGGCΨCCΨΨΨCΨΨCACΨΨΨCAAGΨGCΨACGGCGΨGΨCΨCCΨACΨAAGCΨGAACGACCΨGΨGCΨΨCACΨAACGΨGΨACGCΨGACΨCΨΨΨCGΨGAΨΨCGΨGGCGACGAGGΨGCGΨCAGAΨΨGCΨCCΨGGCCAGACΨGGCAACAΨΨGCΨGACΨACAACΨACAAGCΨGCCΨGACGACΨΨCACΨGGCΨGCGΨGAΨΨGCΨΨGGAACΨCΨAACAAGCΨGGACΨCΨAAGGΨGΨCΨGGCAACΨACAACΨACCΨGΨACCGΨCΨGΨΨCCGΨAAGΨCΨAACCΨGAAGCCΨΨΨCGAGCGΨGACAΨΨΨCΨACΨGAGAΨΨΨACCAGGCΨGGCAACAAGCCΨΨGCAACGGCGΨGGCΨGGCΨΨCAACΨGCΨACΨΨCCCΨCΨGCGΨΨCΨΨACΨCΨΨΨCCGΨCCΨACΨΨACGGCGΨGGGCCACCAGCCΨΨACCGΨGΨGGΨGGΨGCΨGΨCΨΨΨCGAGCΨGCΨGCACGCΨCCΨGCΨACΨGΨGΨGCGGCCCΨAAGAAGΨCΨACΨAACCΨGGΨGAAGAACAAGΨGCGΨGAACΨΨCAACΨΨCAACGGCCΨGAAGGGCACΨGGCGΨGCΨGACΨGAGΨCΨAACAAGAAGΨΨCCΨGCCΨΨΨCCAGCAGΨΨCGGCCGΨGACAΨΨGCΨGACACΨACΨGACGCΨGΨGCGΨGACCCΨCAGACΨCΨGGAGAΨΨCΨGGACAΨΨACΨCCΨΨGCΨCΨΨΨCGGCGGCGΨGΨCΨGΨGAΨΨACΨCCΨGGCACΨAACACΨΨCΨAACCAGGΨGGCΨGΨGCΨGΨACCAGGGCGΨGAACΨGCACΨGAGGΨGCCΨGΨGGCΨAΨΨCACGCΨGACCAGCΨGACΨCCΨACΨΨGGCGΨGΨGΨACΨCΨACΨGGCΨCΨAACGΨGΨΨCCAGACΨCGΨGCΨGGCΨGCCΨGAΨΨGGCGCΨGAGΨACGΨGAACAACΨCΨΨACGAGΨGCGACAΨΨCCΨAΨΨGGCGCΨGGCAΨΨΨGCGCΨΨCΨΨACCAGACΨCAGACΨAAGΨCΨCACCGΨCGΨGCΨCGΨΨCΨGΨGGCΨΨCΨCAGΨCΨAΨΨAΨΨGCΨΨACACΨAΨGΨCΨCΨGGGCGCΨGAGAACΨCΨGΨGGCΨΨACΨCΨAACAACΨCΨAΨΨGCΨAΨΨCCΨACΨAACΨΨCACΨAΨΨΨCΨGΨGACΨACΨGAGAΨΨCΨGCCΨGΨGΨCΨAΨGACΨAAGACΨΨCΨGΨGGACΨGCACΨAΨGΨACAΨΨΨGCGGCGACΨCΨACΨGAGΨGCΨCΨAACCΨGCΨGCΨGCAGΨACGGCΨCΨΨΨCΨGCACΨCAGCΨGAAGCGΨGCΨCΨGACΨGGCAΨΨGCΨGΨGGAGCAGGACAAGAACACΨCAGGAGGΨGΨΨCGCΨCAGGΨGAAGCAGAΨΨΨACAAGACΨCCΨCCΨAΨΨAAGΨACΨΨCGGCGGCΨΨCAACΨΨCΨCΨCAGAΨΨCΨGCCΨGACCCΨΨCΨAAGCCΨΨCΨAAGCGΨΨCΨΨΨCAΨΨGAGGACCΨGCΨGΨΨCAACAAGGΨGACΨCΨGGCΨGACGCΨGGCΨΨCAΨΨAAGCAGΨACGGCGACΨGCCΨGGGCGACAΨΨGCΨGCΨCGΨGACCΨGAΨΨΨGCGCΨCAGAAGΨΨCAAGGGCCΨGACΨGΨGCΨGCCΨCCΨCΨGCΨGACΨGACGAGAΨGAΨΨGCΨCAGΨACACΨΨCΨGCΨCΨGCΨGGCΨGGCACΨAΨΨACΨΨCΨGGCΨGGACΨΨΨCGGCGCΨGGCGCΨGCΨCΨGCAGAΨΨCCΨΨΨCGCΨAΨGCAGAΨGGCΨΨACCGΨΨΨCAACGGCAΨΨGGCGΨGACΨCAGAACGΨGCΨGΨACGAGAACCAGAAGCΨGAΨΨGCΨAACCAGΨΨCAACΨCΨGCΨAΨΨGGCAAGAΨΨCAGGACΨCΨCΨGΨCΨΨCΨACΨGCΨΨCΨGCΨCΨGGGCAAGCΨGCAGGACGΨGGΨGAACCACAACGCΨCAGGCΨCΨGAACACΨCΨGGΨGAAGCAGCΨGΨCΨΨCΨAAGΨΨCGGCGCΨAΨΨΨCΨΨCΨGΨGCΨGAACGACAΨΨΨΨCΨCΨCGΨCΨGGACAAGGΨGGAGGCΨGAGGΨGCAGAΨΨGACCGΨCΨGAΨΨACΨGGCCGΨCΨGCAGΨCΨCΨGCAGACΨΨACGΨGACΨCAGCAGCΨGAΨΨCGΨGCΨGCΨGAGAΨΨCGΨGCΨΨCΨGCΨAACCΨGGCΨGCΨACΨAAGAΨGΨCΨGAGΨGCGΨGCΨGGGCCAGΨCΨAAGCGΨGΨGGACΨΨCΨGCGGCAAGGGCΨACCACCΨGAΨGΨCΨΨΨCCCΨCAGΨCΨGCΨCCΨCACGGCGΨGGΨGΨΨCCΨGCACGΨGACΨΨACGΨGCCΨGCΨCAGGAGAAGAACΨΨCACΨACΨGCΨCCΨGCΨAΨΨΨGCCACGACGGCAAGGCΨCACΨΨCCCΨCGΨGAGGGCGΨGΨΨCGΨGΨCΨAACGGCACΨCACΨGGΨΨCGΨGACΨCAGCGΨAACΨΨCΨACGAGCCΨCAGAΨΨAΨΨACΨACΨGACAACACΨΨΨCGΨGΨCΨGGCAACΨGCGACGΨGGΨGAΨΨGGCAΨΨGΨGAACAACACΨGΨGΨACGACCCΨCΨGCAGCCΨGAGCΨGGACΨCΨΨΨCAAGGAGGAGCΨGGACAAGΨACΨΨCAAGAACCACACΨΨCΨCCΨGACGΨGGACCΨGGGCGACAΨΨΨCΨGGCAΨΨAACGCΨΨCΨGΨGGΨGAACAΨΨCAGAAGGAGAΨΨGACCGΨCΨGAACGAGGΨGGCΨAAGAACCΨGAACGAGΨCΨCΨGAΨΨGACCΨGCAGGAGCΨGGGCAAGΨACGAGCAGΨACAΨΨAAGΨGGCCΨΨGGΨACAΨΨΨGGCΨGGGCΨΨCAΨΨGCΨGGCCΨGAΨΨGCΨAΨΨGΨGAΨGGΨGACΨAΨΨAΨGCΨGΨGCΨGCAΨGACΨΨCΨΨGCΨGCΨCΨΨGCCΨGAAGGGCΨGCΨGCΨCΨΨGCGGCΨCΨΨGCΨGCAAGΨΨCGACGAGGACGACΨCΨGAGCCΨGΨGCΨGAAGGGCGΨGAAGCΨGCACΨACACΨΨGA-3’(SEQ ID NO:27)。
An optimized omicron (B.1.1.529) S glycoprotein mRNA (gcH global opt) has the nucleotide sequence shown in SEQ ID NO. 28.
5’-AACCΨGACGACGCGGACGCAGCΨGCCCCCCGCCΨACACGAACAGCΨΨCACGCGGGGCGΨGΨACΨACCCCGACAAGGΨGΨΨCCGGAGCAGCGΨGCΨGCACAGCACGCAGGACCΨGΨΨCCΨGCCCΨΨCΨΨCAGCAACGΨGACGΨGGΨΨCCACGΨGAΨCAGCGGCACGAACGGCACGAAGCGGΨΨCGACAACCCCGΨGCΨGCCCΨΨCAACGACGGCGΨGΨACΨΨCGCCAGCAΨCGAGAAGAGCAACAΨCAΨCCGGGGCΨGGAΨCΨΨCGGCACGACGCΨGGACAGCAAGACGCAGAGCCΨGCΨGAΨCGΨGAACAACGCCACGAACGΨGGΨGAΨCAAGGΨGΨGCGAGΨΨCCAGΨΨCΨGCAACGACCCCΨΨCCΨGGACCACAAGAACAACAAGAGCΨGGAΨGGAGAGCGAGΨΨCCGGGΨGΨACAGCAGCGCCAACAACΨGCACGΨΨCGAGΨACGΨGAGCCAGCCCΨΨCCΨGAΨGGACCΨGGAGGGCAAGCAGGGCAACΨΨCAAGAACCΨGCGGGAGΨΨCGΨGΨΨCAAGAACAΨCGACGGCΨACΨΨCAAGAΨCΨACAGCAAGCACACGCCCAΨCAΨCGΨGCGGGAGCCCGAGGACCΨGCCCCAGGGCΨΨCAGCGCCCΨGGAGCCCCΨGGΨGGACCΨGCCCAΨCGGCAΨCAACAΨCACGCGGΨΨCCAGACGCΨGCΨGGCCCΨGCACCGGAGCΨACCΨGACGCCCGGCGACAGCAGCAGCGGCΨGGACGGCCGGCGCCGCCGCCΨACΨACGΨGGGCΨACCΨGCAGCCCCGGACGΨΨCCΨGCΨGAAGΨACAACGAGAACGGCACGAΨCACGGACGCCGΨGGACΨGCGCCCΨGGACCCCCΨGAGCGAGACGAAGΨGCACGCΨGAAGAGCΨΨCACGGΨGGAGAAGGGCAΨCΨACCAGACGAGCAACΨΨCCGGGΨGCAGCCCACGGAGAGCAΨCGΨGCGGΨΨCCCCAACAΨCACGAACCΨGΨGCCCCΨΨCGACGAGGΨGΨΨCAACGCCACGCGGΨΨCGCCAGCGΨGΨACGCCΨGGAACCGGAAGCGGAΨCAGCAACΨGCGΨGGCCGACΨACAGCGΨGCΨGΨACAACCΨGGCCCCCΨΨCΨΨCACGΨΨCAAGΨGCΨACGGCGΨGAGCCCCACGAAGCΨGAACGACCΨGΨGCΨΨCACGAACGΨGΨACGCCGACAGCΨΨCGΨGAΨCCGGGGCGACGAGGΨGCGGCAGAΨCGCCCCCGGCCAGACGGGCAACAΨCGCCGACΨACAACΨACAAGCΨGCCCGACGACΨΨCACGGGCΨGCGΨGAΨCGCCΨGGAACAGCAACAAGCΨGGACAGCAAGGΨGAGCGGCAACΨACAACΨACCΨGΨACCGGCΨGΨΨCCGGAAGAGCAACCΨGAAGCCCΨΨCGAGCGGGACAΨCAGCACGGAGAΨCΨACCAGGCCGGCAACAAGCCCΨGCAACGGCGΨGGCCGGCΨΨCAACΨGCΨACΨΨCCCCCΨGCGGAGCΨACAGCΨΨCCGGCCCACGΨACGGCGΨGGGCCACCAGCCCΨACCGGGΨGGΨGGΨGCΨGAGCΨΨCGAGCΨGCΨGCACGCCCCCGCCACGGΨGΨGCGGCCCCAAGAAGAGCACGAACCΨGGΨGAAGAACAAGΨGCGΨGAACΨΨCAACΨΨCAACGGCCΨGAAGGGCACGGGCGΨGCΨGACGGAGAGCAACAAGAAGΨΨCCΨGCCCΨΨCCAGCAGΨΨCGGCCGGGACAΨCGCCGACACGACGGACGCCGΨGCGGGACCCCCAGACGCΨGGAGAΨCCΨGGACAΨCACGCCCΨGCAGCΨΨCGGCGGCGΨGAGCGΨGAΨCACGCCCGGCACGAACACGAGCAACCAGGΨGGCCGΨGCΨGΨACCAGGGCGΨGAACΨGCACGGAGGΨGCCCGΨGGCCAΨCCACGCCGACCAGCΨGACGCCCACGΨGGCGGGΨGΨACAGCACGGGCAGCAACGΨGΨΨCCAGACGCGGGCCGGCΨGCCΨGAΨCGGCGCCGAGΨACGΨGAACAACAGCΨACGAGΨGCGACAΨCCCCAΨCGGCGCCGGCAΨCΨGCGCCAGCΨACCAGACGCAGACGAAGAGCCACCGGCGGGCCCGGAGCGΨGGCCAGCCAGAGCAΨCAΨCGCCΨACACGAΨGAGCCΨGGGCGCCGAGAACAGCGΨGGCCΨACAGCAACAACAGCAΨCGCCAΨCCCCACGAACΨΨCACGAΨCAGCGΨGACGACGGAGAΨCCΨGCCCGΨGAGCAΨGACGAAGACGAGCGΨGGACΨGCACGAΨGΨACAΨCΨGCGGCGACAGCACGGAGΨGCAGCAACCΨGCΨGCΨGCAGΨACGGCAGCΨΨCΨGCACGCAGCΨGAAGCGGGCCCΨGACGGGCAΨCGCCGΨGGAGCAGGACAAGAACACGCAGGAGGΨGΨΨCGCCCAGGΨGAAGCAGAΨCΨACAAGACGCCCCCCAΨCAAGΨACΨΨCGGCGGCΨΨCAACΨΨCAGCCAGAΨCCΨGCCCGACCCCAGCAAGCCCAGCAAGCGGAGCΨΨCAΨCGAGGACCΨGCΨGΨΨCAACAAGGΨGACGCΨGGCCGACGCCGGCΨΨCAΨCAAGCAGΨACGGCGACΨGCCΨGGGCGACAΨCGCCGCCCGGGACCΨGAΨCΨGCGCCCAGAAGΨΨCAAGGGCCΨGACGGΨGCΨGCCCCCCCΨGCΨGACGGACGAGAΨGAΨCGCCCAGΨACACGAGCGCCCΨGCΨGGCCGGCACGAΨCACGAGCGGCΨGGACGΨΨCGGCGCCGGCGCCGCCCΨGCAGAΨCCCCΨΨCGCCAΨGCAGAΨGGCCΨACCGGΨΨCAACGGCAΨCGGCGΨGACGCAGAACGΨGCΨGΨACGAGAACCAGAAGCΨGAΨCGCCAACCAGΨΨCAACAGCGCCAΨCGGCAAGAΨCCAGGACAGCCΨGAGCAGCACGGCCAGCGCCCΨGGGCAAGCΨGCAGGACGΨGGΨGAACCACAACGCCCAGGCCCΨGAACACGCΨGGΨGAAGCAGCΨGAGCAGCAAGΨΨCGGCGCCAΨCAGCAGCGΨGCΨGAACGACAΨCΨΨCAGCCGGCΨGGACAAGGΨGGAGGCCGAGGΨGCAGAΨCGACCGGCΨGAΨCACGGGCCGGCΨGCAGAGCCΨGCAGACGΨACGΨGACGCAGCAGCΨGAΨCCGGGCCGCCGAGAΨCCGGGCCAGCGCCAACCΨGGCCGCCACGAAGAΨGAGCGAGΨGCGΨGCΨGGGCCAGAGCAAGCGGGΨGGACΨΨCΨGCGGCAAGGGCΨACCACCΨGAΨGAGCΨΨCCCCCAGAGCGCCCCCCACGGCGΨGGΨGΨΨCCΨGCACGΨGACGΨACGΨGCCCGCCCAGGAGAAGAACΨΨCACGACGGCCCCCGCCAΨCΨGCCACGACGGCAAGGCCCACΨΨCCCCCGGGAGGGCGΨGΨΨCGΨGAGCAACGGCACGCACΨGGΨΨCGΨGACGCAGCGGAACΨΨCΨACGAGCCCCAGAΨCAΨCACGACGGACAACACGΨΨCGΨGAGCGGCAACΨGCGACGΨGGΨGAΨCGGCAΨCGΨGAACAACACGGΨGΨACGACCCCCΨGCAGCCCGAGCΨGGACAGCΨΨCAAGGAGGAGCΨGGACAAGΨACΨΨCAAGAACCACACGAGCCCCGACGΨGGACCΨGGGCGACAΨCAGCGGCAΨCAACGCCAGCGΨGGΨGAACAΨCCAGAAGGAGAΨCGACCGGCΨGAACGAGGΨGGCCAAGAACCΨGAACGAGAGCCΨGAΨCGACCΨGCAGGAGCΨGGGCAAGΨACGAGCAGΨACAΨCAAGΨGGCCCΨGGΨACAΨCΨGGCΨGGGCΨΨCAΨCGCCGGCCΨGAΨCGCCAΨCGΨGAΨGGΨGACGAΨCAΨGCΨGΨGCΨGCAΨGACGAGCΨGCΨGCAGCΨGCCΨGAAGGGCΨGCΨGCAGCΨGCGGCAGCΨGCΨGCAAGΨΨCGACGAGGACGACAGCGAGCCCGΨGCΨGAAGGGCGΨGAAGCΨGCACΨACACGΨGA-3’(SEQ ID NO:28)。
The sequence of the transcription optimized omicron (b.1.1.529) S glycoprotein mRNA also includes the following elements: 5 '-cap structure, 5' -UTR (comprising Kozak sequence), CDS, 3'-UTR and 3' -polyadenylation sequence (polyA). The addition of 5 '-cap structure, 5' -UTR, kozak, 3 '-poly A sequence (polyA) and 3' -UTR sequence elements can further improve the stability of the sequence and avoid degradation. The CDS may further comprise a signal peptide sequence, a preferred signal peptide sequence being shown in SEQ ID NO 17-SEQ ID NO 19, for the purpose of enhancing translation of the protein, enhancing translation stability of the block and better folding of subsequent proteins. Key elements of omicron (b.1.1.529) S glycoprotein mRNA sequences are the coding region nucleotides, HBA1 'utr and polyA sequences using HBA 1' utr, kozak, novel coronal variants Omcron virus S glycoprotein. The sequence of the complete mRNA molecule is shown as SEQ ID NO. 20-SEQ ID NO. 22.
TABLE 1 mRNA sequence key elements
The sequence optimization scheme of omicron (b.1.1.529) S glycoprotein mRNA in this example is shown in table 1, and the specific preparation process is as follows:
pUC57-Kan plasmid containing Omicron mRNA (gcH global nonopt), omicron mRNA (opt) and Omicron mRNA (gcH global opt) DNA sequences in example 1 was transcribed in vitro to prepare Omicron mRNA (gcH global nonopt) mRNA, omicron mRNA (opt) mRNA and Omicron mRNA (gcH global opt) mRNA stock. The preparation of the original liquid by in vitro transcription of the plasmid is completed by entrusting the preparation of the Norflua company, and the preparation method comprises four steps:
in the first step, three different pUC57-Kan mRNA plasmids, omicron mRNA (gcH global nonopt), omicron mRNA (opt) and Omicron mRNA (gcH global opt), were digested with linearizing enzyme and purified to give linearized plasmids;
secondly, the obtained linearized plasmid was added to the corresponding T7 RNA Polymerase Mix, 10 Xreaction buffer, N1-Me-Pseudo UTP Solution (100 mM), ATP (100 mM), GTP (100 mM), CTP (100 mM) and N1-Me-pseudoUTP according to Kit instructions using a commercial mRNA IVT Kit T7 High Yield RNA Transcription Kit (N1-Me-pseudoUTP) -DD4202, the Reaction system was prepared, reacted at 37℃for 16 hours using RNase-free H2O, DNase I was added to the Reaction system, and the transcribed DNA template was digested at 37℃for 15 minutes (in a PCR apparatus). The reaction system is as follows:
T7 RNA Polymerase Mix may be added last when the system is configured.
Thirdly, purifying the mRNA stock solution synthesized in the second step by using magnetic beads according to an Oligo (dT) magnetic bead purification kit to obtain a purified product mRNA.
And fourthly, adding 10 multiplied by the buffer, GTP (10 mM), SAM (thioadenosylmethionine), vaccinia Capping Enzyme and mRNA Cap 2' -O-methyl-transfer ferrose into the obtained mRNA stock solution, supplementing the mRNA stock solution into a corresponding reaction system by using RNase-free H2O, reacting for 60-100min at 37 ℃, and purifying the mRNA stock solution according to the operation of the third step.
Reaction conditions: reacting at 37 ℃ for 60min
The inventor realizes reducing mRNA immunogenicity by reducing the content of U (uridylic acid) in mRNA molecules, and substitutes all uracils in the nucleic acid sequences SEQ ID NO. 20-SEQ ID NO. 22 by pseudo uridine (psi), and the sequence after capping substitution is finally shown as SEQ ID NO. 23-SEQ ID NO. 25.
Example 2 flow cytometry detection of expression effects after transfection of HEK293 cells with different omacron (B.1.1.529) S glycoprotein mRNA
HEK293 cells were transfected with the above-mentioned Omacron mRNA (gcH global nonopt), omacron mRNA (opt) and Omacron mRNA (gcH global opt) prepared by the in vitro transcription process, respectively. First, 4×10 is used 5 Plating of HEK293 cells at individual cell/mlCell transfection was performed at about 80% of cell fusion after 24 hours. Transfection System in 1 well HEK293 cells added to 6 well plates included 1. Mu.g mRNA and transfection reagent Lipofectamine TM 3000 Transfection Reagent, the specific transfection procedure was performed according to the transfection reagent product instructions. HEK293 cells 10, 24 and 72 hours post-transfection were immunolabeled with omacron virus S glycoprotein antibody (fig. 1A) and then the S glycoprotein expression positive cell proportion was detected using a flow cytometer (fig. 1B). Empty liposome wells served as negative controls. The results are shown in FIG. 1, and it can be seen from the results in the figure that there was no significant difference in the proportion of S glycoprotein expression-positive cells compared to the Omicron mRNA (gcH global nonopt) group after cells were transfected with Omicron mRNA (gcH global nonopt) mRNA, omicron mRNA (opt) mRNA and Omicron mRNA (gcH global opt).
Example 3 Indirect immunofluorescence detection of expression Effect of different omacron (B.1.1.529) S glycoprotein mRNA transfected HEK293 cells
HEK293 cells were transfected with the above-mentioned Omacron mRNA (gcH global nonopt), omacron mRNA (opt) and Omacron mRNA (gcH global opt) prepared by the in vitro transcription process, respectively. First, 4×10 is used 5 Cell transfection was performed at a density of individual cells/ml by plating HEK293 cells at a cell fusion state of approximately 80% after 24 hours. Transfection System in 1 well HEK293 cells added to 6 well plates included 1. Mu.g mRNA and transfection reagent Lipofectamine TM 3000 Transfection Reagent, the specific transfection procedure was performed according to the transfection reagent product instructions. HEK293 cells 24 hours after transfection were immunolabeled with omicron virus S glycoprotein antibody and then the proportion of S glycoprotein expressing positive cells was detected using fluorescence microscopy. Empty liposome wells served as negative controls. The results are shown in FIG. 2, from which it can be seen that there was no significant difference in the proportion of S glycoprotein expression-positive cells compared to the Omicron mRNA (gcH global nonopt) group after cells were transfected with Omicron mRNA (gcH global nonopt) mRNA, omicron mRNA (opt) mRNA and Omicron mRNA (gcH global opt).
Example 4Western blot detection of expression Effect of different omacron (B.1.1.529) S glycoprotein mRNA transfected HEK293 cells
HEK293 cells were transfected with the above-mentioned Omacron mRNA (opt), and Omacron mRNA (gcH global opt) prepared by the in vitro transcription process, respectively. First, 4×10 is used 5 Cell transfection was performed at a density of individual cells/ml by plating HEK293 cells at a cell fusion state of approximately 80% after 24 hours. Transfection System in 1 well HEK293 cells added to 6 well plates included 1. Mu.g mRNA and transfection reagent Lipofectamine TM 3000 Transfection Reagent, the specific transfection procedure was performed according to the transfection reagent product instructions. HEK293 cells 24, 48 and 72 hours after transfection were lysed with lysate, and then the amount of S glycoprotein expression was detected using Western blot. Empty liposome wells served as negative controls.
As shown in FIG. 3, it was found from the results of the figures that the expression level of S glycoprotein was higher in the Omicron mRNA (opt) and Omicron mRNA (gcH global opt) transfected groups than in the Omicron mRNA (gcH global nonopt) transfected groups, and that optimizing the ORF codons and increasing the GC content could increase the protein expression level. Omicron mRNA (opt) and Omicron mRNA (gcH global opt) are the mRNA with the sequences of SEQ ID NO. 23 and SEQ ID NO. 24 provided by the invention.
Example 5IgG neutralizing antibodies detection of serum antibodies after immunization of animals with different preparations of omacron (B.1.1.529) S glycoprotein mRNA
mRNA the mice immunization experiments were performed using Omacron mRNA (opt) and Omacron mRNA (gcH global opt) from example 4, and the vaccine delivery system formulation was: mRNA molecule 1mg in quantity, ionizable cationic lipid ALC0315 14.00mg,DSPC 2.93mg, cholesterol 6.52mg and PEG modified lipid ALC0159 1.55mg. The prepared mRNA-LNP (Lipid nanoparticles) was used in a mouse administration experiment in which each mouse of the Omacron mRNA (opt) mRNA test group was injected at an administration amount of 10. Mu.g, the injection volume was 100. Mu.l/mouse, and the Omacron mRNA (gcH global opt) mRNA test group was divided into 3 dose groups of 5. Mu.g, 10. Mu.g and 20. Mu.g, respectively, and each mouse was injected at an injection volume of 100. Mu.l/mouse, respectively. Spike positive control test groups were prepared using omacron recombinant S glycoprotein according to the formulation method described in Thermo Scientific Imject alum adjuvant instructions (2. Mu.g dose per mouse, 100. Mu.l/mouse injection volume) and negative control as an equal volume saline injection. The results of measuring the neutralizing antibody titer of the serum Omicron virus of the mice 12 days after one administration of the above various vaccine preparations are shown in FIG. 4, from which it can be seen that Omicron S specific IgG neutralizing antibody titers were significantly higher in the group consisting of 10. Mu.g of Omicron mRNA (opt) mRNA, 5. Mu.g of Omicron mRNA (gcH global opt), 10. Mu.g of Omicron mRNA (gcH global opt) and 20. Mu.g of Omicron mRNA (gcH global opt) than in the Spike positive control test group, and the group consisting of 10. Mu.g of Omicron mRNA (gcH global opt) and 20. Mu.g of Omicron mRNA (gcH global opt) from the results.
Claims (10)
1. A nucleic acid molecule comprising an S glycoprotein encoding coronavirus omicron (b.1.1.529), wherein said coding region comprises one or more open reading frames (open reading frame, ORFs), and wherein at least one ORF encodes an S glycoprotein of coronavirus omicron (b.1.1.529) having a protein sequence as set forth in SEQ ID No. 1; and having a nucleotide sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence shown in SEQ ID NO. 2.
2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule has a sequence as set forth in SEQ ID NO. 3-SEQ ID NO. 5.
3. Comprises mRNA molecules encoding coronavirus omacron (B.1.1.529) S glycoprotein, and the sequence of the mRNA molecules is shown as SEQ ID NO. 6-SEQ ID NO. 8.
An mrna nucleic acid molecule comprising:
(i) A 5 'untranslated region (5' -UTR);
(ii) A CDS, wherein the CDS comprises an Open Reading Frame (ORF) encoding a coronavirus omicron (b.1.1.529) antigen S glycoprotein capable of inducing an immune response comprising a nucleotide sequence as set forth in SEQ ID No. 6-SEQ ID No. 8;
(iii) 3 '-untranslated region (3' -UTR).
5. An mRNA nucleic acid molecule, said mRNA molecule comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA), wherein CDS comprises an Open Reading Frame (ORF) encoding coronavirus omicron (B.1.1.529) antigen S glycoprotein capable of inducing immune response, the nucleotide sequence is shown as SEQ ID NO:6-SEQ ID NO: 8.
6. An mRNA molecule, said mRNA comprising the following elements: 5 '-cap structure, 5' -UTR, CDS, 3'-UTR and 3' -polyadenylation sequence (polyA);
wherein said 5' -cap structure is optionally selected from m7G (5 ') ppp (5 ') (2 ' OMeA) pG, 3' -O-Me-m7G (5 ') ppp (5 ') G; g (5 ') ppp (5') A; g (5 ') ppp (5') G;
m7G (5 ') ppp (5') A; m7G (5 ') ppp (5') G, etc.;
wherein the 5' -UTR comprises a sequence shown as SEQ ID NO. 9-SEQ ID NO. 11;
wherein the CDS comprises an Open Reading Frame (ORF) which encodes a signal peptide and a coronavirus omacron (B.1.1.529) antigen S glycoprotein capable of inducing an immune response, wherein the sequence of the signal peptide is shown as SEQ ID NO. 17-SEQ ID NO. 19, and the sequence of the coronavirus omacron (B.1.1.529) antigen S glycoprotein is shown as SEQ ID NO. 6-SEQ ID NO. 8;
Wherein the 3' -UTR comprises a nucleotide sequence shown as SEQ ID NO. 12-SEQ ID NO. 14;
wherein the 3' -polyadenylation sequence (polyA) optionally has a length of 80 to 180 adenylates.
7. The mRNA nucleic acid molecule of any one of claims 3-6, wherein: the mRNA nucleic acid molecule further comprises one or more functional nucleotide analog modifications selected from the group consisting of pseudouridine (ψ), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (e 1 ψ), 5-methoxy-uridine (mo 5U) and 5-methylcytosine (m 5C).
8. A pharmaceutical composition for inducing a neutralizing antibody response in a subject to a novel coronal variant omacron (b.1.1.529) S glycoprotein comprising the mRNA nucleic acid molecule of any of claims 3-7 and a pharmaceutically acceptable carrier.
9. A pharmaceutical composition comprising mRNA comprising the following components:
(1) mRNA molecules with the sequence shown in any one of SEQ ID NO. 20-SEQ ID NO. 25 or a combination thereof;
(2) 20-60% by mole of ionizable cationic lipids (ionizable lipids);
(3) 5-25% by mole of a non-cationic lipid;
(4) 25-55% by mole of sterols; and
(5) 0.5-15% by mole of PEG modified lipid.
10. Use of an mRNA nucleic acid molecule according to any one of claims 3-7 or a pharmaceutical composition according to any one of claims 8-9 for the preparation of a medicament or vaccine for the treatment or prevention of a novel coronavirus infection.
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