CN113264991A - Preparation method and pharmaceutical composition of virus vaccine - Google Patents

Abstract

The invention provides a method for preparing a virus vaccine, which comprises the steps of providing a coding sequence of full-length or partial antigen peptide or protein of at least one virus, dividing the coding region of the full-length or partial antigen peptide or protein into one or more partial overlapping nucleic acid sequences, and connecting the partial overlapping nucleic acid sequences in a tandem mode to form a nucleic acid vaccine fragment. After the nucleic acid vaccine prepared by the preparation method of the virus vaccine is injected into animals and human bodies, the nucleic acid vaccine is processed by cells in vivo to form a small-segment antigen peptide vaccine, so that the serious side effect possibly caused by a full-length protein vaccine can be effectively avoided, and the immunogenicity of the antigen peptide or protein is kept, thereby effectively antagonizing virus infection. Meanwhile, the small fragment antigen peptide component can be quickly and correspondingly adjusted according to the mutation of the S protein of the new coronavirus, so that the nucleic acid vaccine can also be effective on the new coronavirus mutant strain. The invention also provides a pharmaceutical composition obtained by the preparation method of the virus vaccine.

Description

Preparation method and pharmaceutical composition of virus vaccine

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of a virus vaccine and a pharmaceutical composition.

Background

The first genome sequence data of the novel coronavirus was published at 10/1/2020. The researchers performed genome-wide alignment of the novel coronavirus with two kinds of coronaviruses known to definitely infect humans, namely, Severe Acute Respiratory Syndrome (SARS) coronavirus and Middle East Respiratory Syndrome (MERS) coronavirus, and found that the novel coronavirus has 70% and 40% sequence similarity respectively compared with SARS coronavirus and MERS coronavirus, wherein different coronaviruses have greater difference with a key gene acting on host cells, namely, a spike protein (spike protein) encoding gene.

No vaccine or specific treatment method aiming at the novel coronavirus exists at present, and patients diagnosed with the novel coronavirus infection only receive supportive treatment according to individual symptoms and clinical conditions, and the fatality rate is about 1-4%. Therefore, there is an urgent need for a safe and effective vaccine and pharmaceutical composition thereof against the infection with the novel coronavirus.

SARS is the first new infectious disease discovered since the twenty-first century, and the pathogen was found to be a novel coronavirus in month 4 of 2003, named SARS coronavirus. By 9 months in 2003, 8098 cases and 774 deaths were diagnosed in 29 countries in five continents, with a mortality rate of about 10%. The SARS coronavirus spike protein consists of two subunits, the S1 subunit contains a receptor binding domain that binds to the host cell receptor angiotensin converting enzyme 2(ACE2), and has a total of 666 amino acid residues. Whereas the S2 subunit mediates fusion of the virus with the host cell membrane and consists of 583 amino acid residues. The spike protein plays a key role in inducing neutralizing antibodies, T cell immune response and providing protective immunity during SARS coronavirus infection.

MERS coronavirus is also a newly discovered zoonotic pathogen, beginning with a fatal human case of saudi arabia in 2012, with nearly 1800 people in 25 countries becoming infected in succession. The fatality rate of the patients is about 30-40%. However, more than 60% of patients can recover, eliminate viruses and develop immunity, and the components such as spike protein of MERS coronavirus can be a suitable candidate vaccine. This was further confirmed by animal experiments. In these experiments, several anti-MERS coronavirus spike protein neutralizing antibodies isolated from patient sera were able to neutralize and prevent low concentrations of virus from entering cells and spreading, sometimes providing effective protection. While these experimental vaccines induce protective responses in animals, advances in vaccine development of SARS coronavirus and MERS coronavirus similar to the novel coronavirus indicate that the use of full-length spike protein and other viral proteins as vaccine candidates may present serious safety problems, including immunopathology and promotion of disease progression, possibly due to the induction of Th 2-biased immune responses and/or anti-spike protein non-neutralizing antibodies.

Therefore, there is a need to prepare novel viral vaccines and pharmaceutical compositions to avoid the above problems in the prior art.

Disclosure of Invention

The invention aims to provide a preparation method of a virus vaccine, which can effectively avoid serious side effects possibly caused by a full-length protein vaccine, and can keep the immunogenicity of the antigen peptide or protein, thereby effectively antagonizing virus infection. Meanwhile, the small fragment antigen peptide component can be quickly and correspondingly adjusted according to the mutation of the S protein of the new coronavirus, so that the nucleic acid vaccine can also be effective on the new coronavirus mutant strain. The invention also provides a virus vaccine obtained by the preparation method and application thereof. The invention can accurately, quickly and efficiently capture virus antigen peptide or protein which can stimulate the immune response of human bodies or other virus hosts and quickly prepare virus nucleic acid vaccines, and has wide application prospect in the fields of infectious diseases, tumors and autoimmune diseases.

In order to achieve the above object, the method for preparing the virus vaccine of the present invention comprises:

s1: providing a nucleic acid sequence encoding a full-length or partial antigenic peptide or protein of at least one virus;

s2: dividing the coding region of the full-length or partial antigenic peptide or protein into one or more partially overlapping nucleic acid sequences consisting of 60-150 nucleotides to encode 20-50 amino acid residues, each of said partially overlapping nucleic acid sequences being separated by a furin cleavage site coding sequence or a non-immunogenic glycine or serine linker coding sequence, overlapping 5-15 amino acid residue coding sequences, and ligating a plurality of said partially overlapping nucleic acid sequences in tandem to form a nucleic acid vaccine fragment;

s3: sequentially or randomly operably linking the linking elements in a5 'to 3' direction to form a nucleic acid sequence;

s4: the nucleic acid sequence is subjected to gene synthesis and subcloning to a conventional plasmid sequence by a molecular biological method, and a nucleic acid vaccine is prepared in a clean environment.

The preparation method of the virus vaccine has the beneficial effects that: dividing the coding region of the full-length or partial antigen peptide or protein into at least one partial overlapping nucleic acid sequence, and connecting a plurality of partial overlapping nucleic acid sequences in a tandem mode to form a nucleic acid vaccine fragment, so that the serious side effect possibly caused by a full-length protein vaccine can be effectively avoided, and the immunogenicity of the antigen peptide or protein is kept, thereby effectively antagonizing virus infection. Meanwhile, small fragment antigen peptide components can be quickly and correspondingly adjusted according to the mutation of the S protein of the new coronavirus, so that the nucleic acid vaccine can also be effective on the new coronavirus mutant strain.

Preferably, the ligation element comprises a5 'untranslated fragment, a Kozak sequence, an initiation codon sequence, the nucleic acid vaccine fragment, a stop codon sequence, a poly (a) signal sequence and a 3' untranslated fragment, and the 5 'untranslated fragment, the Kozak sequence, the initiation codon sequence, the nucleic acid vaccine fragment, the stop codon sequence, the poly (a) signal sequence and the 3' untranslated fragment are operably ligated in sequence or randomly from the 5 'end to the 3' end in step S3 to prepare a DNA vaccine through step S4.

Further preferably, a signal peptide of an MHC class I molecule and a transport region coding sequence are linked between the initiation codon sequence and the nucleic acid vaccine fragment.

Preferably, the linking element comprises a5 'cap structure, a 5' untranslated fragment, a Kozak sequence, an initiation codon sequence, the nucleic acid vaccine fragment, a stop codon sequence, a 3 'untranslated fragment, and a poly (a) sequence, and the 5' cap structure, the 5 'untranslated fragment, the Kozak sequence, the initiation codon sequence, the nucleic acid vaccine fragment, the stop codon sequence, the 3' untranslated fragment, and the poly (a) sequence are sequentially or randomly operably linked in the 5 'to 3' direction in step S3 to prepare an mRNA vaccine through step S4 and in vitro transcription.

Further preferably, a signal peptide of an MHC class I molecule and a transport region coding sequence are linked between the initiation codon sequence and the nucleic acid vaccine fragment.

Preferably, the poly (a) sequence comprises 20-400 adenine nucleotides.

Further preferably, the poly (a) sequence comprises 60 to 250 adenine nucleotides.

Preferably, the partially overlapping nucleic acid sequence is derived from an artificially modified nucleic acid to enhance the stability of the nucleic acid sequence.

Further preferably, the coding region of the nucleic acid sequence has a codon adaptation index of 0.5 to 1 to fit human codon usage.

Preferably, the antigenic peptide or protein of the virus comprises a spike protein, the S1 subunit and the S2 subunit of the spike protein, a viral structural protein, or the spike protein, the S1 subunit and the S2 subunit of the spike protein, and a fragment, variant or derivative of any one or more of the viral structural proteins, the S1 subunit of the spike protein comprising a receptor binding domain, the viral structural protein comprising any one or more of hemagglutinin, neuraminidase, envelope, membrane and nucleocapsid proteins.

Further preferably, the viral antigenic peptide or protein comprises any one or more of the spike protein, or a fragment, variant or derivative of the spike protein.

Further preferably, the viral antigen peptide or protein comprises any one or more of the S1 subunit and the S2 subunit of the spike protein, or a variant or derivative thereof. The beneficial effects of the kit are that the kit can recognize host cell receptors and provide specific antigens.

Further preferably, the antigenic peptide or protein further comprises one or more of said hemagglutinin, neuraminidase, envelope protein, membrane protein or nucleocapsid protein, or a fragment, variant or derivative of these viral structural proteins, the hemagglutinin is the HA protein and the neuraminidase is the NA protein. The beneficial effects of the method are that the method plays an important role in recognizing host cell receptors, effectively assembling viruses and outputting cells, and can provide specific antigens.

Preferably, the antigenic peptide or protein of the virus is derived from any one or more of respiratory tract infection virus, digestive tract infection virus, hepatitis virus, encephalitis B virus, neurovirus and sexually transmitted virus, and the respiratory tract infection virus comprises any one or more of novel coronavirus, SARS virus, MERS virus, influenza virus, rhinovirus, adenovirus and respiratory syncytial virus.

The pharmaceutical composition of the present invention comprises a nucleic acid vaccine obtained by the method for preparing the virus vaccine.

The pharmaceutical composition has the beneficial effects that: the pharmaceutical composition is derived from a nucleic acid vaccine obtained by the preparation method of the viral vaccine, and in the preparation method of the viral vaccine, the coding region of the full-length or partial antigen peptide or protein is divided into at least one partial overlapping nucleic acid sequence, and then a plurality of partial overlapping nucleic acid sequences are connected in a tandem manner to form a nucleic acid vaccine fragment, so that the serious side effect possibly caused by the pharmaceutical composition can be effectively avoided, and the immunogenicity of the antigen peptide or protein is kept, thereby effectively antagonizing viral infection.

Preferably, the pharmaceutical composition further comprises a substance formed by complexing the nucleic acid vaccine with one or more liposomes.

Preferably, the pharmaceutical composition further comprises an adjuvant comprising poly-ICLC, TLR, 1018ISS, aluminum salts, immunomodulatory oligonucleotides, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFaimPt 321, IS Patch, ISS, ISOMATRIX, Juvlmmone, Lipovac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-MP-EC, ONTAK, PLGA microparticles, Requimod, SRL172, virosomes, virus-like particles, YF-17D, VEGF trap 848, R848, beta-glucan, Palam 3Cys, Adivan, Quickian 21, and Asmeza 59n to induce a specific immune responses in any one or more of the body, and/or organism, and reduces the production cost.

Drawings

FIG. 1 is a schematic diagram of plasmid DNA consisting of a partially overlapping nucleic acid sequence structure encoding the subunit S1 of the novel coronavirus spike protein including the receptor binding domain during the course of the experiment for injecting a mouse vaccine according to the present invention;

description of reference numerals: 11. a start codon; an MHC class I molecule Signal Peptide (SP) encoding nucleic acid sequence; 13. a nucleic acid sequence encoding a partial overlap of 36 amino acid residues; 14.7 amino acid residue overlapping coding nucleic acid sequence; 15. a nucleic acid sequence encoding a furin cleavage site; a nucleic acid sequence encoding amino acids (MITD) of the MHC class I molecule transport region; 17. a stop codon;

FIG. 2 is a plasmid map of pJM908 according to an embodiment of the present invention;

FIG. 3 is a plasmid map of pJM909 according to an embodiment of the present invention;

FIG. 4 is a graph showing the results of Western blotting of supernatant of 293T cells transfected with plasmid pJM908 and pJM909 DNA vaccines in example 1 of the present invention;

FIG. 5 is a graph comparing the ELISA titer levels of antibodies to the novel coronavirus S protein RBD in serum after vaccination of groups of experimental mice according to some embodiments of the present invention;

FIG. 6 is a graph comparing the percent competitive inhibition obtained after the detection of RBD neutralizing antibodies against the novel coronavirus S protein in different experimental group samples according to some embodiments of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without any inventive step, are within the scope of the present invention. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The use of "including" and "comprising" and similar words, as used herein, is intended to mean that the element or item preceding the word comprises the element or item listed after the word and its equivalent, but not the exclusion of other elements or items.

In view of the problems in the prior art, the embodiment of the invention provides a preparation method of a virus vaccine.

The preparation method of the virus vaccine comprises the following steps: providing a nucleic acid sequence encoding at least one full-length or partial antigenic peptide or protein of the virus; dividing the coding region of the full-length or partial antigenic peptide or protein into one or more overlapping nucleic acid sequences consisting of 60-150 nucleotides to encode 20-50 amino acid residues, each of said overlapping nucleic acid sequences separated by a furin cleavage site or a non-immunogenic glycine or serine linker coding sequence such that 5-15 amino acid residues overlap; and connecting a plurality of partially overlapped nucleic acids in a tandem mode to form the nucleic acid vaccine fragment.

In some embodiments of the invention, the coding region of the full-length or partial antigenic peptide or protein is divided into one or more partially overlapping nucleic acid sequences consisting of 90-120 nucleotides to encode 30-40 amino acid residues; each of said partially overlapping nucleic acid sequences is separated by a furin cleavage site coding sequence or a non-immunogenic glycine or serine linker coding sequence such that the 6-14 amino acid residue coding sequences overlap; and connecting a plurality of partially overlapped nucleic acids in a tandem mode to form the nucleic acid vaccine fragment.

In some embodiments of the invention, the coding region of the full-length or partial antigenic peptide or protein is divided into one or more partially overlapping nucleic acid sequences consisting of 99-108 nucleotides to encode 33-36 amino acid residues; each of said partially overlapping nucleic acid sequences is separated by a furin cleavage site coding sequence or a non-immunogenic glycine or serine linker coding sequence such that the 7-9 amino acid residue coding sequences overlap; and connecting a plurality of partially overlapped nucleic acids in a tandem mode to form the nucleic acid vaccine fragment.

Specifically, the non-immunogenic glycine or serine linker coding sequence is any one of a non-immunogenic glycine linker coding sequence and a non-immunogenic serine linker coding sequence.

In particular, furin is a precursor-protein converting enzyme similar to subtilisins. It is the major processing enzyme in the secretory pathway and is localized to the site of the network on the reverse side of the golgi apparatus. The substrates of the polypeptide comprise coagulation factors, serum proteins, growth factor receptors and the like, and the shortest cleavage recognition sites are as follows: Arg-X-X-Arg.

The preparation method of the virus vaccine further comprises the following steps: sequentially or randomly operably linking the linking elements in a5 'to 3' direction to form a nucleic acid sequence; the nucleic acid sequence is subcloned to a conventional plasmid sequence by a molecular biological method, and the nucleic acid vaccine is prepared in a clean environment.

In some embodiments of the invention, the linking element comprises a5 'untranslated fragment, a Kozak sequence, a start codon sequence, the nucleic acid vaccine fragment, a stop codon sequence, a poly (a) signal sequence, and a 3' untranslated fragment, wherein the 5 'untranslated fragment, the Kozak sequence, the start codon sequence, the nucleic acid vaccine fragment, the stop codon sequence, the poly (a) signal sequence, and the 3' untranslated fragment are operably linked sequentially or randomly from the 5 'end to the 3' end to form the nucleic acid sequence; the nucleic acid sequence is subcloned into a conventional plasmid sequence by a molecular biological method, and the DNA vaccine is prepared in a clean environment.

Specifically, between the above-mentioned initiation codon sequence and the described nucleic acid vaccine fragment, signal peptide of MHC class I molecule and transport region coding sequence also can be added.

In some embodiments of the present invention, the linking element comprises a5 'cap structure, a 5' untranslated fragment, a Kozak sequence, a start codon sequence, the nucleic acid vaccine fragment, a stop codon sequence, a 3 'untranslated fragment, and a poly (a) sequence, and the 5' cap structure, the 5 'untranslated fragment, the Kozak sequence, the start codon sequence, the nucleic acid vaccine fragment, the stop codon sequence, the 3' untranslated fragment, and the poly (a) sequence are operably linked sequentially or randomly from 5 'end to 3' end in step S3 to form the nucleic acid sequence; the nucleic acid sequence is subcloned to a conventional plasmid sequence by a molecular biological method, and the nucleic acid sequence is transcribed into an mRNA sequence in vitro under a clean environment to prepare the mRNA vaccine.

Specifically, between the above-mentioned initiation codon sequence and the described nucleic acid vaccine fragment, signal peptide of MHC class I molecule and transport region coding sequence also can be added.

In some embodiments of the invention, the signal peptide and transport region amino acid sequences of the MHC class I molecule are as follows: signal peptide, MRVTAPRTLILLLSGALALTETWAGS; a transit zone, IVGIVAGLAV LAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA; the signal peptide of the MHC class I molecule and the amino acid sequence of the transport region are used for optimizing MHC class I and II pathways and improving RNA stability and protein translation efficiency.

The coding region of the full-length antigenic peptide or protein is divided into one or more partially overlapping nucleic acid sequences, which are joined in tandem to form nucleic acid vaccine fragments. After the prepared nucleic acid vaccine is injected into animals and human bodies, the small-segment antigen peptide vaccine is formed by in vivo cell treatment, so that the serious side effect possibly caused by the full-length protein vaccine can be effectively avoided, and the immunogenicity of the antigen peptide or protein is kept, thereby effectively antagonizing virus infection.

In some embodiments of the invention, multiple partially overlapping nucleic acid sequences designed based on the nucleic acid sequence encoded by the S1 subunit of the novel coronavirus spike protein are joined in tandem to form the sequence of a nucleic acid vaccine fragment, see FIGS. 1-3 and SEQ ID NOS: 1-2, respectively.

Specifically, the Poly (A) sequence comprises 25-400 adenine nucleotides.

More specifically, the poly (a) sequence comprises 60-250 adenine nucleotides.

In some embodiments of the invention, the 5' CAP structure (capping) is a modified nucleotide, i.e., a metabolic gene activator protein (CAP) analog.

In some specific embodiments of the invention, guanine nucleotides are added to the 5 'end of the mRNA molecule to form the 5' cap structure.

In some embodiments of the invention, the 5 'cap structure is a methylguanosine cap, i.e., m7 gppppn is added to the 5' end.

In some specific embodiments of the invention, the 5 ' cap structure further comprises glycerol or 4 ', 5 ' -methylene nucleotides, and the like.

The fragments of various nucleic acid sequences described in the embodiments of the present invention belong to continuous or discontinuous nucleotide fragments in the corresponding full-length nucleic acid sequence. The nucleic acid sequence described in the variants or derivatives of each class of nucleic acid sequences is formed by deletion, insertion, addition or substitution of at least one nucleotide.

In some embodiments of the invention, the partially overlapping nucleic acid sequence is derived from an artificially modified nucleic acid to enhance the stability of the nucleic acid sequence. The coding region of the nucleic acid sequence has a codon adaptation index of 0.5 to 1 to fit human codon usage. The codons modified in such a way can optimize the expression of antigenic peptides or proteins and reduce the possibility of mutation due to recombination with endogenous viruses.

The antigenic peptides or proteins described in the embodiments of the present invention refer to substances that are recognized by the immune system of a mammal and are capable of triggering an antigen-specific immune response.

In some embodiments of the invention, the antigenic peptide or protein is a translation product of the nucleic acid sequence of the invention. In this case, the antigenic peptides or proteins are fragments, variants or derivatives of peptides and proteins comprising at least one epitope.

In particular, the epitopes are also referred to as antigenic determinants, and the T cell epitopes or protein portions of some embodiments of the invention have 8-11 amino acid residues, some of which contain identical or qualitatively similar amino acid residues, and others of which contain any amino acid residues, longer or shorter, that are still capable of binding effectively to MHC class I molecules. Wherein the fragments may be selected from any part of the full-length amino acid sequence of the antigenic peptide or protein, which fragments are normally recognized by T cells as a complex of peptide fragments and MHC class I molecules.

Some embodiments of the invention provide T-cell epitopes or protein moieties having from 10 to 25 amino acid residues, or from 13 to 18 amino acid residues, wherein certain positions contain identical or qualitatively similar amino acid residues, and others contain any amino acid residue, longer or shorter, but still capable of effective binding to MHC class II molecules. Wherein the fragments may be selected from any part of the full length amino acid sequence of the antigenic peptide or protein, such fragments are normally recognized by T cells as a complex of peptide fragments and MHC class II molecules.

In some embodiments of the invention, the antigenic peptide or protein is used as part of an adaptive immune response by forming any one or more of antibodies or antigen-specific T cells.

In some embodiments of the invention, the antigenic peptide or protein is presented to the T cell by MHC.

In some embodiments of the invention, the antigenic peptide or protein of the virus comprises a spike protein, or any one or more of a fragment, variant or derivative of said spike protein.

Specifically, the spike protein is a long rod-shaped envelope bulge to protrude on the surface of a coronavirus particle, and is used as a protective antigen of the virus to interact with a specific protein receptor of a host and mediate fusion of the virus and a protein membrane, so that the genome of the virus enters a cell.

In some embodiments of the invention, the antigenic peptide or protein of the virus further comprises a spike fragment derived from the virus, or any one or more of a variant or derivative of said spike fragment, said spike fragment comprising a receptor binding domain, or any one or more of a fragment, variant or derivative of said receptor binding domain.

Specifically, the spike fragment is the S1 subunit of the spike protein, and the S1 subunit comprises the receptor binding domain and receptor binding structure, and is responsible for recognizing a receptor of a cell.

In some embodiments of the invention, the antigenic peptide or protein of the virus further comprises any one or more of hemagglutinin (HA protein), neuraminidase (NA protein), envelope protein, membrane protein, nucleocapsid protein, and other viral structural proteins, or fragments, variants, or derivatives of these viral structural proteins. The beneficial effects are that the antigen plays an important role in recognizing host cell receptors, effectively assembling viruses and producing cells, and can provide specific antigens.

In some embodiments of the invention, the antigenic peptide or protein of the virus is derived from any one or more of respiratory infection virus, digestive tract infection virus, hepatitis virus, encephalitis b virus, neurovirus and sexually transmitted virus, including any one or more of novel coronavirus, SARS virus, MERS virus, influenza virus, rhinovirus, adenovirus and respiratory syncytial virus.

In some embodiments of the present invention, the pharmaceutical composition comprises the nucleic acid vaccine obtained by the method for preparing the virus vaccine, so as to be applied to antagonize virus infection.

In particular, the pharmaceutical composition can be used as a vaccine providing at least one antigen or antigenic function that stimulates the adaptive immune system of the body to provide an adaptive immune response. In particular, the nucleic acid sequence stimulates the immune system of the mammal to produce a specific immune response against the virus.

The immune system refers to a system that can protect an organism from infection. If a pathogen breaks the physical barrier of an organism into the organism, the innate immune system provides an immediate but non-specific response. If the pathogen escapes this innate response, the vertebrate possesses a second layer of protection, the adaptive immune system.

The adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic growth. Adaptive immune responses provide the vertebrate immune system with the ability to recognize and remember a particular pathogen, i.e., to generate an immune response, and to mount a stronger challenge each time a pathogen is encountered. The system is highly adaptable due to somatic mutations and MHC gene V (D) J recombination. This mechanism allows a small number of genes to produce a large number of different antigen receptors, which are then uniquely expressed on each lymphocyte. Since genetic recombination results in irreversible changes in the DNA of each cell, all progeny of the cell will inherit genes encoding the same receptor specificity, including memory B cells and memory T cells, which are critical for long-term specific immunity.

The immune response may typically be a specific response of the adaptive immune system to a specific antigen or a non-specific response of the innate immune system.

An adaptive immune response is generally understood to be antigen-specific. Antigen specificity allows for the generation of a response against a particular antigen, pathogen, or pathogen infected cell. The ability to maintain these customized responses in vivo through "memory cells". If the pathogen infects the human more than once, these specific memory cells are used to rapidly eliminate it.

Cellular or cellular immune responses are often associated with macrophages, natural killer cells (NK), activation of antigen-specific cytotoxic T lymphocytes, and release of various cytokines in an antigenic response. In general, cellular immunity is not associated with antibodies, but rather with activation of cells of the immune system. The cellular immune response is characterized by the ability to induce apoptosis of certain cells displaying antigenic epitopes on their surface, such as virus-infected cells, cells with intracellular bacteria and cancer cells displaying tumor antigens, by activating antigen-specific cytotoxic T lymphocytes; activating macrophages and natural killer cells, enabling them to destroy pathogens; it also stimulates cells to secrete various cytokines, affecting other cellular functions involved in adaptive and innate immune responses.

Humoral immunity generally refers to the production of antibodies and their attendant ancillary processes. The humoral immune response is typically characterized by the activation of Th2 and the production of cytokines, the formation of germinal centers and isotype switching, affinity maturation and the production of memory cells.

In some embodiments of the invention, the pharmaceutical composition further comprises a substance formed by complexing the nucleic acid vaccine with one or more liposomes. Specifically, the substance is one or more of novel liposome, lipid nanoparticle or liposome-vaccine pharmaceutical composition, and has the characteristics of targeting property, long-acting property, cell affinity and histocompatibility, improvement of stability of vaccine components, reduction of side effects of the vaccine and the like.

In some embodiments of the invention, the pharmaceutical composition further comprises an adjuvant to induce the body to generate long-term and efficient specific immune response, improve the protective capacity of the body, reduce the dosage of immune substances and reduce the production cost of the vaccine.

Specifically, the adjuvant comprises one or more of poly-ICLC, TLR, 1018ISS, aluminum salts, immunomodulatory oligonucleotides, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISOMATRIX, Juvlmmue, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap 848, R, β -glycan, Pam3Cys, Quila QS _ Vadidaxan 21, XA A or any exciton 404 (AsmeA). Specifically, the immune regulatory oligonucleotide is Amplivax.

The pharmaceutical compositions of the embodiments of the present invention are capable of being stored and transported without a cold chain and can be produced quickly and scalable.

The beneficial effects of the present invention are illustrated below by the experiments of example 1DNA vaccine plasmid transfection into 293T cells in vitro, example 2 mouse vaccine injection immunization and example 3 rhesus monkey vaccine injection.

FIG. 1 is a schematic representation of the overlapping nucleic acid sequence of the fragment encoding the S1 subunit of the novel coronavirus spike protein including the receptor binding domain during the course of the experiments described in some of the examples. Although the S1 subunit has good immunogenicity, it contains more than 650 amino acid residues and may cause serious side effects after entering human body. Referring to FIG. 1, the pJM908 circular DNA plasmid has an S1 subunit NTD encoding nucleic acid fragment comprising 11 partially overlapping nucleic acid sequences each encoding 36 amino acids forming a partially overlapping 36 amino acid residue encoding nucleic acid sequence 13, which is downstream repeated with adjacent partially overlapping regions encoding 7 amino acids forming a 7 amino acid residue overlapping encoding nucleic acid sequence 14. The 1 st sequence at the 5 'end contains an initiation codon 11 and an MHC class I molecule signal peptide coding nucleic acid sequence 12, the tail end of each sequence contains a furin enzyme cutting site coding nucleic acid sequence 15, the sequence at the 3' end contains an MHC class I molecule transport region amino acid coding sequence 16 and a stop codon 17, a plurality of sequences are connected in series to form a complete nucleic acid vaccine fragment, and a circular DNA plasmid is formed through gene synthesis and subcloning. The pJM909 circular DNA plasmid has an RBD-encoding nucleic acid fragment of the S1 subunit and a small portion of an RBD-encoding nucleic acid fragment of the S2 subunit, contains 13 partially overlapping nucleic acid sequences, has the same structure as that of pJM908, and is genetically synthesized and subcloned to form a circular DNA plasmid. Two candidate DNA vaccines were prepared in a clean environment. After the prepared candidate DNA vaccine is injected, electrified and introduced into animals and human bodies, a plurality of partial overlapping nucleic acid sequences and nucleic acid segments encoding the downstream furin enzyme cutting sites of the nucleic acid vaccine can be identified and translated through in vivo cell treatment, so that the nucleic acid vaccine segments are treated by furin enzyme to further form a plurality of small-segment antigen peptides which are directly combined with histocompatibility complex MHCI or II molecules, thereby effectively avoiding the serious side effect possibly caused by S1 subunit large-segment or full-length S protein vaccines, but keeping the immunogenicity of the antigen peptides or proteins, and further effectively antagonizing virus infection. Meanwhile, the small fragment antigen peptide component can be quickly and correspondingly adjusted according to the mutation of the S protein of the new coronavirus, so that the nucleic acid vaccine can also be effective on the new coronavirus mutant strain.

Examples 1-3 of the present invention provide DNA vaccines numbered pJM908 and pJM909, specifically:

said step S1 uses the DNA coding sequence of the spike protein S1 subunit of the novel coronavirus as the coding nucleic acid sequence;

in step S2, each of the dozens of partial overlapping nucleic acid sequences consists of 108 nucleotides to encode 36 amino acid residues, and each partial overlapping nucleic acid sequence is separated by furin cleavage site coding sequence to overlap the 7 amino acid residue coding sequences, and then connected in tandem to form the nucleic acid vaccine fragment.

The plasmids used in step S4 are pJM908 and pJM909, and their plasmid maps are shown in FIGS. 2-3, respectively, and their sequences are shown in SEQ ID NO. 1-2, respectively.

In the embodiment 1 of the invention, the experiment of in vitro transfection of 293T cells with DNA vaccine plasmids comprises the following specific steps: the frozen 293T cells are recovered, centrifuged to take the supernatant, inoculated in a culture dish and placed in an incubator for culture. The 293T cells were observed to grow to around 80% for electroporation. Collecting cells, diluting the cell pellet with the counting electrotransfer solution R to a cell density of 1X 10⁶-5×10⁶. 5ug of pJM908 and pJM909 plasmids were added to each well of cells, respectively, to finally transfect a volume of 120 ul. Electrotransformation was carried out using a Neon electrotransfer instrument (Invitrogen). Injecting the shocked cells into a new culture dish, adding a serum-free culture medium for culturing for 4 hours, changing into a complete culture medium, changing the culture medium for 24 hours and 48 hours, respectively, and collecting cell supernatantAnd collecting the body for later use.

FIG. 4 shows the results of Western blot experiments on cell supernatant collected after transfection of 293T cells with pJM908 and pJM909 DNA vaccine plasmids. Wherein the primary antibody used is a mouse anti-neocoronal S protein RBD monoclonal antibody from Xinbo Sheng Biotechnology Limited; the secondary antibody is goat anti-mouse-HRP; JP-396 polypeptide covering the RBD region served as a positive control. The results of this experiment show that the S protein fragment, particularly the small fragment antigenic peptide of pJM909 plasmid, has been expressed in transfected cells.

In embodiment 2 of the present invention, the specific steps of the mouse vaccine injection immunization experiment include:

female BALB/C mice, 6-8 weeks old, were divided into several experimental groups of 24 mice each for intramuscular injection treatment, specifically: group 1 was injected with pJM909 vaccine at 30. mu.g/vaccine, group 2 was injected with pJM909 vaccine alone at 100. mu.g/vaccine, group 3 was injected with pJM908 and pJM909 vaccines at the same content at 60. mu.g/vaccine, group 4 was injected with pJM908 and pJM909 vaccines at the same content at 200. mu.g/vaccine; group 5 was injected with the empty pVax1 plasmid at 100. mu.g/mouse.

Specifically, muscle cells were electroporated immediately after injection using a tassaka electroporation device (from shanghatassaka health technology limited).

Specifically, it was observed that after BALB/c mice were repeatedly injected intramuscularly with pJM909 and the mixed plasmid for 9 weeks (1 administration per 2 weeks, 5 total administrations), the body had no significant toxic reaction and no significant irritation to the administration site.

All experimental mice survive for the planned dissection time, and through observation and dissection, compared with control mice, macroscopic lesions are not seen, no change of the weight of organs and histopathology is observed, and the visible vaccine is safe and reliable. The changes refer to inflammation, mineralization and degeneration phenomena to a greater than slight degree.

In the embodiment 2 of the invention, the ELISA titer level of the RBD antibody of the new coronavirus S protein in the serum of each group of experimental mice after inoculation is detected 5-7 days after the 3 rd intramuscular injection is completed, the number of the detected animals in each group is 10, and the samples to be detected in other groups are subjected to 1: the dilution of 100, the specific titer level is characterized by absorbance at a wavelength of 450 nm. The specific detection method is a conventional technical means for those skilled in the art.

Specifically, FIG. 5 is a graph comparing the ELISA titer levels of the serum RBD antibody against the novel coronavirus S protein of each group of experimental mice after inoculation. Referring to FIG. 5, compared with group 5, the titer level in group 1 was significantly higher than that in group 5, and the titer levels in groups 2 to 4 were comparable to that in group 5, but since the test specimens of the groups other than the blank group of group 5 were subjected to 1: 100 dilution, the vaccine of the present application was identified as being effective in inducing the production of antibodies to the novel coronavirus S protein RBD in mice.

In the embodiment 3 of the invention, the following experimental groups are formed by intramuscular injection of male rhesus monkeys of 3-6 years old and 3.5-8 kg body weight by referring to the mouse vaccine injection immunization experiment:

group 6 and group 7: 2 rhesus monkeys were injected with the empty plasmid pVax1 in an amount of 2 mg/monkey;

group 8 and group 9: 2 rhesus monkeys were injected individually with the pJM909 vaccine at 2 mg/monkey;

group 10 and group 11: 2 rhesus monkeys were injected with the same amount of pJM908 vaccine and pJM909 vaccine in a mixed manner, and the injection amount was 4 mg/monkey.

Specifically, muscle cells were electroporated immediately after injection using a tassaka electroporation device (from shanghatassaka health technology limited).

Specifically, it was observed that the body had no significant toxic reaction and no significant irritation to the administered site after repeated intramuscular injections of pJM909 and the mixed plasmid into rhesus monkeys for 8 weeks (1 administration per 4 weeks, 4 total administrations).

On days 5-7 after the end of the 3 rd injection, serum samples from each animal of the above experimental groups were subjected to 1: 100, serum samples from non-injected animals were subjected to a 1: 100 as reference, the neutralizing antibody of new coronavirus S protein RBD was detected. The specific process is as follows:

respectively mixing the test substance and the reference substance with an HRP-RBD complex according to the proportion of 1: 1 volume of the resulting mixture was incubated at room temperature for 20-30 minutes to allow circulating neutralizing antibodies to bind to the HRP-RBD.

The mixtures were then added separately to capture plates pre-coated with human ACE2 polypeptide, incubated for 10-15 minutes at room temperature, and the capture plates were washed 2-4 times. During the washing process, unbound HRP-RBD and any HRP-RBD bound to non-neutralizing antibodies will be captured on the surface of the capture plate, while the material that is bound to HRP-RBD by circulating neutralizing antibodies will be removed from the capture plate with the washing process.

Specifically, the washing was carried out using 0.15 mol/l PBS dialysate having a pH of 7.4 as a washing solution.

The substrate TMB solution was added to the capture plate sequentially as a chromogenic solution and a stop solution was added to quench the reaction to give a yellow final solution.

Finally, the absorbance of the final solution was measured at a wavelength of 450 nm using a microtiter plate reader in combination with a spectrophotometer to examine the virus-neutralizing antibody titer, i.e., the OD value, in the test sample. The absorbance of the sample is inversely proportional to the titer.

The percentage of competitive inhibition was calculated by the following formula:

percent competitive inhibition was 100% × (1-OD value of test sample at 450 nm)/OD value of control sample at 450 nm.

FIG. 6 is a graph comparing the percent competitive inhibition obtained after the detection of the RBD neutralizing antibodies of the new coronavirus S protein for different experimental group samples.

Referring to fig. 6, based on the percentage of the average competitive inhibition in groups 6 and 7, the antibody levels in the serum samples of groups 11 and 3 in example 2 were significantly higher than the baseline, indicating that the vaccine of the present application was able to effectively induce not only the production of antibodies to the new coronavirus S protein RBD in mice, but also the production of antibodies to the new coronavirus S protein RBD in rhesus monkeys.

Although the embodiments of the present invention have been described in detail hereinabove, it is apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention as described herein is capable of other embodiments and of being practiced or of being carried out in various ways.

Sequence listing

<110> Shanghai name of the last ruler of the Xia Dynasty Mongolian Biotechnology Co., Ltd

Preparation method and pharmaceutical composition of <120> virus vaccine

<130> 2021.01.28

<150> HRCN20L31002-YX

<151> 2021-01-28

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4935

<212> DNA

<213> Artificial Sequence

<400> 1

gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc 780

ggccgcccac catgcgggtc acggcgcccc gaaccctcat cctgctgctc agcggggcac 840

tggccctgac cgagacctgg gctggctcct cacaatgcgt gaaccttaca acacgcacgc 900

agttaccacc ggcatacaca aactcattta caagaggcgt ctattatcca gacaaggtat 960

tccgtagttc tgtgagaggt cgtaagcgga ggagtaaagt atttcgatcc agcgtactac 1020

attcgaccca ggacctgttc ctgccattct tctctaatgt gacatggttt cacgcaatcc 1080

acgtaagcgg gaccaatggg actcgcggtc gcaaacgacg ttccgtatcg ggtacaaacg 1140

gcacgaaacg attcgataac cctgtgcttc ctttcaatga cggagtgtac tttgcatcaa 1200

cagagaagag taacataatt cgtggttgga tacgtggtag gaagcgtagg agtaatatta 1260

tacgaggttg gatatttggt acaacacttg attcaaagac ccagtcactt cttatcgtga 1320

acaacgcaac aaacgtggtg attaaggttt gtgagttcca gcggggacgg aagcgtcgat 1380

caatcaaagt ctgtgagttc cagttttgca atgatccttt cctgggtgtt tactatcata 1440

agaacaacaa atcatggatg gaatcagaat tccgggtcta ttcgtccgcg cgaggacgga 1500

agcgaagatc cttccgagtg tattcttccg cgaataattg cacatttgaa tacgtgtcac 1560

aacctttcct catggactta gagggaaagc agggcaattt caagaatctc agagaatttc 1620

gaggtcgcaa gcggcgctcc tttaagaacc tgcgggaatt tgtcttcaag aatatcgatg 1680

gatactttaa gatatattca aagcatactc ccataaacct tgtgagagac ctaccgcaag 1740

gtttctctcg cggacgtaag cgcagaagcg acctgccgca gggtttctct gccttggaac 1800

ctcttgtgga tcttcctatc ggaatcaaca tcacaagatt tcaaacactt cttgctctcc 1860

atcgctctta tctaacccgc ggacggaaga gaaggtcact acataggtct tatttgacac 1920

caggcgattc ctcatcagga tggacagcag gagcagcagc atactacgtg ggatacctcc 1980

agccaaggac atttctccta aagtaccgcg gaagaaagcg tcggagtaga acattcttac 2040

tcaagtataa tgagaacgga acaatcacag atgcagtgga ttgcgcactt gatcctcttt 2100

cagaaacaaa gtgtactctg aagagcttca cggttagagg taggaaacgc cgaagcacac 2160

taaagtcgtt cacggtagag aagggtatct accaaacatc aaactttaga gtgcaaccta 2220

cagaatcaat cgtgagattt cctaacatca caaacctttg ccctagagga cgtaaaagac 2280

gtagtatcgt gggcattgtt gctggcctgg ctgtcctagc agttgtggtc atcggagctg 2340

tggtcgctac tgtgatgtgt aggaggaaga gctcaggtgg aaaaggaggg agctactctc 2400

aggctgcgtc cagcgacagt gcccagggct ctgatgtgtc tctcacagct taattactcg 2460

agagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac 2520

tactaaactg ggggatatta tgaagggcct tgagcatctg gattctgcct aataaaaaac 2580

atttattttc attgcagctc gctttcttgc tgtccaattt ctattaaagg ttcctttgtt 2640

ccctaagtcc aactactaaa ctgggggata ttatgaaggg ccttgagcat ctggattctg 2700

cctaataaaa aacatttatt ttcattgctc tagagggccc gtttaaaccc gctgatcagc 2760

ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 2820

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 2880

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 2940

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctactgg 3000

gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt 3060

gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg 3120

ggatcaagct ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga 3180

ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 3240

cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 3300

ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg 3360

ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 3420

gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 3480

cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 3540

gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 3600

cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 3660

ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg 3720

acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 3780

atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 3840

gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 3900

gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt 3960

attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4020

caccgcatca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 4080

ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 4140

atagcacgtg ctaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 4200

taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 4260

agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 4320

aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 4380

ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 4440

gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 4500

aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 4560

aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 4620

gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 4680

aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 4740

aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 4800

cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 4860

cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 4920

tgctcacatg ttctt 4935

<210> 2

<211> 5193

<212> DNA

<213> Artificial Sequence

<400> 2

gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc 780

ggccgcccac catgcgggtc acggcgcccc gaaccctcat cctgctgctc agcggggcac 840

tggccctgac cgagacctgg gctggctcca gagtgcaacc tacagaatca atcgtgagat 900

ttcctaacat cacaaacctt tgccctttcg gagaagtatt taacgcaaca agattcgcct 960

ctgtctatgc ttggcgcgga agaaagaggc gatccttcgc cagtgtttat gcctggaatc 1020

gaaagaggat ttcaaactgc gtggcagatt actcagtgct ttacaactca gcatcattca 1080

gtacgttcaa gtgttatggc gtgagaggta ggaaacgacg atctactttt aaatgctatg 1140

gagtatctcc gacgaagttg aatgatcttt gctttacaaa cgtgtacgca gattcatttg 1200

tgatcagagg agacgaggtc cggcagattg cacgaggtag gaagaggcgg agcgatgaag 1260

tccgacagat agcacctggc cagactggaa agatagctga ttacaactac aaacttcctg 1320

atgatttcac cggttgcgtg attgcttgga acagtaacaa tcgagggagg aagcgtaggt 1380

caatcgcatg gaactcaaac aaccttgact caaaggttgg tggaaactac aactaccttt 1440

acagactatt ccggaaatcc aatcttaaac cgtttgaaag agacataagt cgcggtagaa 1500

agagaaggtc gcctttcgag cgggacatta gtacggagat ctaccaagca ggatcaacac 1560

cttgcaacgg agtggaagga tttaactgct actttcctct ccagtcctat ggtttccagc 1620

gtggacgcaa gcgcagatcg ctccaatcgt atgggttcca gcccaccaac ggagtgggat 1680

accaacctta cagagtggtg gtgctttcat ttgaacttct tcacgctcca gcgactgtct 1740

gtgggccacg ggggagaaag agacggagtc cggctacggt ctgtggccca aagaagagca 1800

caaaccttgt gaagaataag tgcgtgaact ttaactttaa cggacttaca ggaactggtg 1860

tacttaccga gtcgaaccgt ggaaggaaac ggcgttctgg cgtgctgacc gagagtaata 1920

agaagtttct acctttccag cagtttggaa gagatatcgc agatacaaca gatgcagtga 1980

gagatccgca aaccttggag atactccgtg gccggaaacg gagaagtcca cagaccctag 2040

agatactaga tataacacct tgctcatttg gaggagtgtc agtgatcaca cctggaacaa 2100

acacatcaaa ccaagttgcc gtactgtatc aggaccgcgg gagaaagcgg cgctctgtgg 2160

cggtattgta tcaggacgtt aattgcacag aagtgcctgt ggcaatccac gcagatcaac 2220

ttacacctac atggagagtg tactcgaccg gctctaatgt ctttcgagga aggaagcgtc 2280

gcagttcaac cgggtcgaat gtattccaga cgagagcagg atgccttatc ggagcagaac 2340

acgtgaacaa ctcatacgaa tgcgatatcc ctattggtgc cgggatatgt gcacgcggtc 2400

gcaagcgccg gtctattggt gcaggtatat gtgcgagcta tcaaacacaa acaaactcac 2460

ctagaagagc acgctctgtc gccagccagt caatcatcgc atacacaatg tcacttggag 2520

caagaggacg taaaagacgt agtatcgtgg gcattgttgc tggcctggct gtcctagcag 2580

ttgtggtcat cggagctgtg gtcgctactg tgatgtgtag gaggaagagc tcaggtggaa 2640

aaggagggag ctactctcag gctgcgtcca gcgacagtgc ccagggctct gatgtgtctc 2700

tcacagctta attactcgag agctcgcttt cttgctgtcc aatttctatt aaaggttcct 2760

ttgttcccta agtccaacta ctaaactggg ggatattatg aagggccttg agcatctgga 2820

ttctgcctaa taaaaaacat ttattttcat tgcagctcgc tttcttgctg tccaatttct 2880

attaaaggtt cctttgttcc ctaagtccaa ctactaaact gggggatatt atgaagggcc 2940

ttgagcatct ggattctgcc taataaaaaa catttatttt cattgctcta gagggcccgt 3000

ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 3060

ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 3120

tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3180

gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3240

ctctatggct tctactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg 3300

gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca 3360

aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc 3420

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 3480

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 3540

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 3600

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 3660

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 3720

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 3780

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 3840

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 3900

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 3960

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 4020

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 4080

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 4140

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 4200

gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt tctccttacg 4260

catctgtgcg gtatttcaca ccgcatcagg tggcactttt cggggaaatg tgcgcggaac 4320

ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 4380

ctgataaatg cttcaataat agcacgtgct aaaacttcat ttttaattta aaaggatcta 4440

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 4500

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 4560

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 4620

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 4680

tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 4740

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 4800

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 4860

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 4920

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 4980

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 5040

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 5100

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 5160

ggccttttgc tggccttttg ctcacatgtt ctt 5193

Claims (10)

1. A method of preparing a viral vaccine, comprising:

s1: providing a nucleic acid sequence encoding a full-length or partial antigenic peptide or protein of at least one virus;

s2: dividing the coding region of the full-length or partial antigenic peptide or protein into one or more overlapping nucleic acid sequences of 60-150 nucleotides to encode 20-50 amino acid residues, each of the overlapping nucleic acid sequences separated by a furin cleavage site coding sequence or a non-immunogenic glycine or serine linker coding sequence, overlapping the 5-15 amino acid residue coding sequences, and ligating the overlapping nucleic acid sequences in tandem to form a nucleic acid vaccine fragment;

s3: sequentially or randomly operably linking the linking elements in a5 'to 3' direction to form a nucleic acid sequence;

s4: the nucleic acid sequence is subjected to gene synthesis and subcloning to conventional plasmids by a molecular biological method, and the nucleic acid vaccine is prepared in a clean environment.

2. The method for preparing a viral vaccine according to claim 1, wherein the linking element comprises a5 'untranslated fragment, a Kozak sequence, a start codon sequence, the nucleic acid vaccine fragment, a stop codon sequence, a Poly (A) signal sequence and a 3' untranslated fragment, and in step S3, the 5 'untranslated fragment, the Kozak sequence, the start codon sequence, the nucleic acid vaccine fragment, the stop codon sequence, the Poly (A) signal sequence and the 3' untranslated fragment are sequentially or randomly operably linked in a5 'to 3' direction to prepare a DNA vaccine through step S4.

3. The method for preparing a viral vaccine according to claim 1, wherein the linking element comprises a5 'cap structure, a 5' untranslated fragment, a Kozak sequence, a start codon sequence, the nucleic acid vaccine fragment, a stop codon sequence, a 3 'untranslated fragment, and a Poly (A) sequence, and the 5' cap structure, the 5 'untranslated fragment, the Kozak sequence, the start codon sequence, the nucleic acid vaccine fragment, the stop codon sequence, the 3' untranslated fragment, and the Poly (A) sequence are sequentially or randomly operably linked from the 5 'end to the 3' end in step S3 to prepare an mRNA vaccine through step S4 and in vitro transcription.

4. The method of claim 1, wherein the partially overlapping nucleic acid sequence is derived from an artificially modified nucleic acid to enhance the stability of the nucleic acid sequence.

5. The method of claim 4, wherein the coding region of the nucleic acid sequence has a codon adaptation index of 0.5 to 1 to adapt to human codon usage.

6. The method of claim 1, wherein the antigenic peptide or protein of the virus comprises a spike protein, the S1 and S2 subunits of the spike protein, a viral structural protein, or a fragment, variant or derivative of any one or more of the spike protein, the S1 and S2 subunits of the spike protein, and the viral structural protein, wherein the S1 subunit of the spike protein comprises a receptor binding domain, and wherein the viral structural protein comprises any one or more of a hemagglutinin, a neuraminidase, an envelope protein, a membrane protein, and a nucleocapsid protein.

7. The method for preparing a viral vaccine according to claim 6, wherein the antigenic peptide or protein of the virus is derived from any one or more of respiratory infection virus, digestive tract infection virus, hepatitis virus, encephalitis B virus, neurovirus and sexually transmitted virus, and the respiratory infection virus comprises any one or more of novel coronavirus, SARS virus, MERS virus, influenza virus, rhinovirus, adenovirus and respiratory syncytial virus.

8. A pharmaceutical composition comprising the nucleic acid vaccine obtained by the method for producing a viral vaccine according to claim 1, for use in antagonizing viral infection.

9. The pharmaceutical composition of claim 8, further comprising a substance formed by complexing the nucleic acid vaccine with one or more liposomes.

10. The pharmaceutical composition of claim 8, further comprising an adjuvant comprising one or more of poly-ICLC, TLR, 1018ISS, aluminum salts, immunomodulatory oligonucleotides, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, dsim, GM-CSF, IC30, IC31, imiquimod, imufacp 321, IS Patch, ISS, ISCOMATRIX, juvlmmone, LipoVac, MF59, monophosphoryl lipid a, montanide IMS 1312, montanide ISA206, montanide ISA50V, tananide ISA-51, OK-432, OM-174, ont-197-MP-EC, ont, PLGA microparticles, resiquimod, SRL172, virus microbodies, virus-like particles, YF-17D, VEGF, R848, β -glucan, palam 3Cys, acandin 21, quinacr 404 a, and any of the excitons aszaa.

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