CN113264991B - Preparation method of virus vaccine and pharmaceutical composition - Google Patents

Abstract

The invention provides a preparation method of a virus vaccine, which comprises the steps of providing a coding sequence of at least one full-length or partial antigen peptide or protein of a virus, dividing the coding region of the full-length or partial antigen peptide or protein into one or more partially overlapped nucleic acid sequences, and connecting the partially overlapped 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 small fragment antigen peptide vaccine is formed by in vivo cell treatment, so that serious side effects possibly caused by a full-length protein vaccine can be effectively avoided, the immunogenicity of the antigen peptide or protein is maintained, and the virus infection can be effectively antagonized. Meanwhile, the small fragment antigen peptide component can be correspondingly and rapidly adjusted according to the mutation of the novel coronavirus S protein, so that the nucleic acid vaccine can be also effective on the novel coronavirus mutant strain. The invention also provides a pharmaceutical composition obtained by the preparation method of the virus vaccine.

Description

Preparation method of virus vaccine and pharmaceutical composition

Technical Field

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

Background

The first genomic sequence data of the novel coronavirus was published on 1 month 10 2020. Researchers have performed whole genome alignment of the novel coronavirus with two coronaviruses known to specifically infect humans, namely severe acute respiratory syndrome (Severe Acute Respiratory Syndromes, SARS) coronavirus and middle east respiratory syndrome (Middle East Respiratory Syndromes, MERS) coronavirus, and found that the novel coronavirus has on average 70% and 40% sequence similarity compared to SARS coronavirus and MERS coronavirus, respectively, with greater variability in the key genes that different coronaviruses interact with host cells, namely spike protein (spike protein) encoding genes.

At present, no vaccine or specific treatment method aiming at the novel coronavirus exists, and patients diagnosed with the novel coronavirus infection are only treated in a supporting way according to the individual symptoms and clinical conditions, and the death rate is about 1-4%. Thus, there is an urgent need for a vaccine and pharmaceutical compositions thereof that are safe and effective against novel coronavirus infections.

SARS was the first new infectious disease discovered since the twenty-first century, and its pathogen was discovered as a novel coronavirus, designated SARS coronavirus, in month 4 of 2003. By 9 months of 2003, in 29 countries in five continents, 8098 cases were diagnosed altogether, 774 cases died, and the mortality rate was about 10%. SARS coronavirus spike protein consists of two subunits, the S1 subunit containing a receptor binding domain that binds to host cell receptor angiotensin converting enzyme 2 (ACE 2), totaling 666 amino acid residues. Whereas the S2 subunit mediates fusion of the virus with the host cell membrane, consisting of 583 amino acid residues. Spike proteins play a key role in inducing neutralizing antibodies, T cell immune responses and providing protective immunity upon infection by SARS coronavirus.

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

There is therefore a need for new viral vaccines and pharmaceutical compositions that avoid the above-mentioned problems of 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, but keep the immunogenicity of antigen peptide or protein, thereby effectively antagonizing virus infection. Meanwhile, the small fragment antigen peptide component can be correspondingly and rapidly adjusted according to the mutation of the novel coronavirus S protein, so that the nucleic acid vaccine can be also effective on the novel 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 capable of stimulating immune response of human body or other virus hosts and quickly prepare virus nucleic acid vaccine, and has wide application prospect in the fields of infectious diseases, tumors and autoimmune diseases.

In order to achieve the above object, the preparation method of 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 said full-length or partial antigen 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 such that the 5-15 amino acid residue coding sequences overlap, and ligating a plurality of said partially overlapping nucleic acid sequences in tandem to form a nucleic acid vaccine fragment;

s3: operably linking the linking elements sequentially or randomly from the 5 'end to the 3' end 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 the nucleic acid vaccine is prepared under a clean environment.

The preparation method of the virus vaccine has the beneficial effects that: 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 a plurality of the partial overlapping nucleic acid sequences are connected in series to form a nucleic acid vaccine fragment, so that serious side effects possibly caused by a full-length protein vaccine can be effectively avoided, the immunogenicity of the antigen peptide or protein is maintained, and the virus infection can be effectively antagonized. Meanwhile, the small fragment antigen peptide component can be correspondingly and rapidly adjusted according to the mutation of the novel coronavirus S protein, so that the nucleic acid vaccine can be also effective on the novel coronavirus mutant strain.

Preferably, the linking element comprises an untranslated fragment at the 5 'end, a Kozak sequence, an initiation codon sequence, the nucleic acid vaccine fragment, a termination codon sequence, a Poly (a) signal sequence, and an untranslated fragment at the 3' end, and in step S3, the untranslated fragment at the 5 'end, the Kozak sequence, the initiation codon sequence, the nucleic acid vaccine fragment, the termination codon sequence, the Poly (a) signal sequence, and the untranslated fragment at the 3' end are sequentially or randomly operatively linked in the direction from the 5 'end to the 3' end to prepare the DNA vaccine through step S4.

It is further preferred that a signal peptide of an MHC class I molecule and a transport region coding sequence are linked between said initiation codon sequence and said nucleic acid vaccine fragment.

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

It is further preferred that a signal peptide of an MHC class I molecule and a transport region coding sequence are linked between said initiation codon sequence and said nucleic acid vaccine fragment.

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

Further preferably, the Poly (A) sequence comprises 60-250 adenine nucleotides.

Preferably, the partially overlapping nucleic acid sequences are derived from artificially modified nucleic acids to enhance the stability of the nucleic acid sequences.

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

Preferably, 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 comprising any one or more of a hemagglutinin, a neuraminidase, an envelope protein, a membrane protein and a nucleocapsid 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.

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

Further preferred, the viral antigen peptide or protein comprises any one or more of the S1 subunit and the S2 subunit of the spike protein, or variants or derivatives thereof. The beneficial effects are that it can recognize host cell receptor and provide specific antigen.

Further preferred, the antigenic peptide or protein further comprises any one or more of the hemagglutinin, neuraminidase, envelope protein, membrane protein or nucleocapsid protein, or fragments, variants or derivatives of these viral structural proteins, wherein the hemagglutinin is an HA protein and the neuraminidase is an NA protein. The beneficial effects are that the effective assembly and the cell outlet of the virus play an important role in recognizing host cell receptors, 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 virus, nerve virus 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 invention comprises a nucleic acid vaccine obtained by the preparation method of the virus vaccine.

The pharmaceutical composition of the invention has the beneficial effects that: the pharmaceutical composition is derived from a nucleic acid vaccine obtained by the preparation method of the virus vaccine, wherein in the preparation method of the virus 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 a plurality of the partial overlapping nucleic acid sequences are connected in series to form a nucleic acid vaccine fragment, so that serious side effects possibly caused by the pharmaceutical composition can be effectively avoided, and the immunogenicity of the antigen peptide or protein is maintained, thereby effectively antagonizing antiviral infection.

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

Preferably, the pharmaceutical composition further has an adjuvant comprising any one or more of poly-ICLC, TLR,1018ISS, aluminum salt, immunomodulatory oligonucleotide, AS15, BCG, CP-870, CP-893, cpG7909, cyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, imuFact IMP321, IS Patch, ISS, ISCOMATRIX, juvlmmune, lipoVac, MF59, monophosphoryl lipid a, meng Dani dema 1312, meng Dani dema 206, meng Dani dema 50V, meng Dani dema-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, requimod, SRL172, viral microsomes, virus-like particles, YF-17D, VEGF trap, R848, β -glucan, pam3Cys, nystatin QS21, vadimezan and AsA404, facilitating the induction of a long-term, efficient specific immune response and reducing the production costs of the organism.

Drawings

FIG. 1 is a schematic diagram of plasmid DNA comprising a partially overlapping nucleic acid sequence structure encoding a novel coronavirus spike protein S1 subunit including a receptor binding domain in a pestivirus injection immunization experiment according to the present invention;

reference numerals illustrate: 11. a start codon; mhc class I molecule Signal Peptide (SP) encoding nucleic acid sequences; 13. a nucleic acid sequence encoding a sequence of 36 amino acid residues partially overlapping; 14.7 amino acid residues; 15. a furin cleavage site encoding nucleic acid sequence; an mhc class I molecule transport region amino acid (MITD) encoding nucleic acid sequence; 17. a stop codon;

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

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

FIG. 4 is a graph showing the Western blot of the supernatant of 293T cells transfected with pJM908 and pJM909 DNA vaccine plasmids according to example 1 of the present invention;

FIG. 5 is a graph showing comparison of ELISA titer levels of novel coronavirus S protein RBD antibodies in serum after inoculation 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 detection of novel coronavirus S protein RBD neutralizing antibodies on different experimental group samples according to some embodiments of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the terms "comprises," "comprising," and the like are intended to cover the presence of an element or article that appears before the term and that is listed after the term and its equivalents, without excluding other elements or articles.

Aiming at the problems existing 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 an antigenic peptide or protein of at least one virus, in whole or in part; dividing the coding region of said full or partial antigen 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 or a non-immunogenic glycine or serine linker coding sequence such that 5-15 amino acid residues overlap; and then connecting a plurality of partially overlapped nucleic acids in a tandem mode to form nucleic acid vaccine fragments.

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 encoding sequence or a non-immunogenic glycine or serine linker encoding sequence such that the 6-14 amino acid residue encoding sequences overlap; and then connecting a plurality of partially overlapped nucleic acids in a tandem mode to form nucleic acid vaccine fragments.

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 encoding sequence or a non-immunogenic glycine or serine linker encoding sequence such that 7-9 amino acid residue encoding sequences overlap; and then connecting a plurality of partially overlapped nucleic acids in a tandem mode to form nucleic acid vaccine fragments.

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 subtilisin-like protein precursor converting enzyme. It is the main processing enzyme in the secretory pathway, located at the network site on the reverse side of the golgi apparatus. The substrate comprises blood coagulation factors, serum proteins, growth factor receptors and the like, and the shortest cleavage recognition site is: arg-X-X-Arg.

The preparation method of the virus vaccine further comprises the following steps: operably linking the linking elements sequentially or randomly from the 5 'end to the 3' end to form a nucleic acid sequence; the nucleic acid sequence is subcloned into a conventional plasmid sequence by a molecular biological method, and the nucleic acid vaccine is prepared under a clean environment.

In some embodiments of the invention, the linking element comprises an untranslated fragment at the 5 'end, a Kozak sequence, a start codon sequence, the nucleic acid vaccine fragment, a stop codon sequence, a Poly (a) signal sequence, and an untranslated fragment at the 3' end, 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 being operably linked sequentially or randomly in a5 'to 3' direction 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 under a clean environment.

Specifically, between the above-mentioned start codon sequence and the nucleic acid vaccine fragment, a signal peptide of MHC class I molecule and a transport region coding sequence may be further added.

In some embodiments of the 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, wherein in step S3, 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 in a5 'to 3' direction to form the nucleic acid sequence; subcloning the nucleic acid sequence into a conventional plasmid sequence through a molecular biological method, and transcribing the nucleic acid sequence into an mRNA sequence in vitro under a clean environment to prepare the mRNA vaccine.

Specifically, between the above-mentioned start codon sequence and the nucleic acid vaccine fragment, a signal peptide of MHC class I molecule and a transport region coding sequence may be further added.

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

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

In some embodiments of the invention, a plurality of partially overlapping nucleic acid sequences designed based on the nucleic acid sequence encoding the novel coronavirus spike protein S1 subunit are ligated in tandem to form a nucleic acid vaccine fragment sequence, 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, namely a metabolic gene activator protein (catabolite gene activatorprotein, CAP) analog.

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

In some specific 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.

Fragments of various nucleic acid sequences described in embodiments of the invention are contiguous or non-contiguous nucleotide fragments of the corresponding full-length nucleic acid sequences. Variants or derivatives of the various types of nucleic acid sequences are formed by deletion, insertion, addition or substitution of at least one nucleotide.

In some embodiments of the invention, the partially overlapping nucleic acid sequences are derived from artificially modified nucleic acids to enhance the stability of the nucleic acid sequences. The coding region of the nucleic acid sequence has a codon adaptation index of 0.5 to 1 to suit human codon usage. The codons so modified optimize antigen peptide or protein expression and reduce the possibility of mutation by recombination with endogenous viruses.

The antigenic peptide or protein according to the embodiments of the present invention refers to a substance that is recognized by the immune system of a mammal and is 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 peptide or protein is a fragment, variant or derivative of a peptide or protein comprising at least one epitope.

In particular, the epitope is also referred to as an epitope, and some embodiments of the invention have T cell epitopes or protein portions with 8-11 amino acid residues, some of which contain amino acid residues of the same or similar nature, and others of which contain any amino acid residue, or longer or shorter, but still bind efficiently 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 in the form of a complex of peptide fragments and MHC class I molecules.

The T cell epitope or protein portion of some embodiments of the invention has 10-25 amino acid residues, or 13-18 amino acid residues, wherein some sites contain amino acid residues of the same or similar nature, while other sites contain any amino acid residues, or longer or shorter, but still allow for efficient 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, which fragments are normally recognized by T cells in the form of a complex of peptide fragments and MHC class II molecules.

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

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 the spike protein.

Specifically, the spike protein is in a long rod shape and is protruded on the surface of coronavirus particles, and is used as a protective antigen of viruses to be responsible for interacting with specific protein receptors of hosts to mediate fusion of the viruses and protein membranes, so that genomes of the viruses enter cells.

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

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

In some embodiments of the invention, the antigenic peptide or protein of the virus further comprises hemagglutinin (HA protein), neuraminidase (NA protein), envelope proteins, membrane proteins, nucleocapsid proteins, and other viral structural proteins, or any one or more of fragments, variants, or derivatives of these viral structural proteins. The beneficial effects are that the effective assembly and the cell outlet of the virus play an important role in recognizing host cell receptors, 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 tract infection virus, digestive tract infection virus, hepatitis virus, encephalitis 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 invention, the pharmaceutical composition comprises a nucleic acid vaccine obtained by the method of preparing a viral vaccine for use in antagonizing viral infections.

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

The immune system refers to a system that protects an organism from infection. The innate immune system provides immediate but non-specific responses if pathogens break through the physical barrier of an organism into the organism. If the pathogen escapes this innate response, the vertebrate has a second layer of protection, the adaptive immune system.

The adaptive immune system is composed of highly specialized, systemic cells and processes that can eliminate or prevent pathogenic growth. Adaptive immune responses provide the vertebrate immune system with the ability to recognize and memorize a particular pathogen, i.e., to generate an immune response, and initiate a stronger challenge each time a pathogen is encountered. The system is very adaptable due to somatic mutation 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 gene recombination results in irreversible changes in the DNA of each cell, all the offspring of that 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 generally be a specific response of the adaptive immune system to a specific antigen or a non-specific response of the innate immune system.

Adaptive immune response is generally understood to be antigen-specific. Antigen specificity allows for the generation of responses to specific antigens, pathogens, or pathogen-infected cells. The ability of these tailored responses is maintained in vivo by "memory cells". If the pathogen infects the human body more than once, these specific memory cells are used to rapidly eliminate it.

Cellular immunity or cellular immune responses are typically associated with activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and release of various cytokines in an antigenic response. In general, cellular immunity is not related to antibodies, but rather to activation of cells of the immune system. Cellular immune responses are characterized by the ability to induce apoptosis in 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 to enable 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 concomitant ancillary processes. The humoral immune response is typically characterized by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell production.

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

In some embodiments of the present invention, the pharmaceutical composition further comprises an adjuvant to induce a long-term and efficient specific immune response in the body, thereby improving the protective ability of the body, reducing the dosage of immune substances, and reducing the production cost of the vaccine.

Specifically, the adjuvant includes any 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, ISCOMATRIX, juvllmmux, lipoVac, MF59, monophosphoryl lipid a, meng Dani de IMS1312, meng Dani de ISA206, meng Dani de ISA50V, meng Dani de ISA-51, ok-432, om-174, om-197-MP-EC, ONTAK, PLGA microparticles, requimod, SRL172, viral micelles and other virus-like particles, YF-17d, vegf trap, R848, β -glucan, pam3Cys, ajqua QS21 excitons, vadizen or AsA404 (dmea). Specifically, the immunomodulatory oligonucleotide is Amplivax.

The pharmaceutical composition of the embodiments of the present invention can be stored and transported without a cold chain and can be produced quickly and extendably.

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

FIG. 1 is a schematic representation of the structure of partially overlapping nucleic acid sequences encoding novel coronavirus spike protein S1 subunit fragments, including receptor binding domains, during the course of several of the examples described herein. Although the S1 subunit has good immunogenicity, it contains more than 650 amino acid residues and may cause serious side reactions after entering the human body. Referring to FIG. 1, the pJM908 circular DNA plasmid has an S1 subunit NTD-encoding nucleic acid fragment, which contains 11 partially overlapping nucleic acid sequences, each of which encodes 36 amino acids, forming a partially overlapping 36 amino acid residue encoding nucleic acid sequence 13, and downstream of which, with the adjacent partially overlapping region, repeatedly encodes 7 amino acids, forming a 7 amino acid residue overlapping encoding nucleic acid sequence 14. The 1 st sequence of the 5 'end contains a start codon 11 and an MHC class I molecule signal peptide coding nucleic acid sequence 12, each sequence end contains a furin cleavage site coding nucleic acid sequence 15, the 3' end sequence contains an MHC class I molecule transfer 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 the complete nucleic acid vaccine fragment is formed by gene synthesis and subcloning. The pJM909 circular DNA plasmid has an S1 subunit RBD coding nucleic acid fragment and a small part of S2 subunit coding nucleic acid fragment, contains 13 partial overlapping nucleic acid sequences, has the other structures identical to those of pJM908, and is formed by gene synthesis and subcloning. Two candidate DNA vaccines were prepared in a clean environment. After the prepared candidate DNA vaccine is injected and introduced into animals and human bodies, a plurality of partial overlapped nucleic acid sequences and furin cleavage site coding nucleic acid fragments at the downstream of the partial overlapped nucleic acid sequences can be identified and translated through in vivo cell treatment, so that the nucleic acid vaccine fragments are treated by furin to further form a plurality of small fragment antigen peptides, and the small fragment antigen peptides are directly combined with tissue compatibility complex MHCI or II molecules, thereby effectively avoiding serious side effects possibly caused by S1 subunit large fragment or full-length S protein vaccine, and maintaining the immunogenicity of the antigen peptides or proteins, and further effectively antagonizing antiviral infection. Meanwhile, the small fragment antigen peptide component can be correspondingly and rapidly adjusted according to the mutation of the novel coronavirus S protein, so that the nucleic acid vaccine can be also effective to the novel 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 the step S2, each of the tens of partially overlapping nucleic acid sequences is composed of 108 nucleotides to encode 36 amino acid residues, each partially overlapping nucleic acid sequence is separated by a furin cleavage site encoding sequence, the 7 amino acid residue encoding sequences are overlapped, and then connected in series to form a nucleic acid vaccine fragment.

The plasmids used in the step S4 are pJM908 and pJM909, the plasmid maps of which are shown in FIGS. 2-3, respectively, and the sequences of which are shown in SEQ ID NOS 1-2, respectively.

In the embodiment 1 of the invention, the specific steps of the in vitro transfection 293T cell experiment of the DNA vaccine plasmid comprise: the frozen 293T cells were recovered, and the supernatant was centrifuged and inoculated into a petri dish and placed in an incubator for culturing. 293T cells were observed to grow to about 80% and electrotransport was performed. Collecting cells, diluting cell pellet with electrotransfer solution R according to count to obtain cell density of 1×10 ⁶ -5×10 ⁶ . 5ug of pJM908 and pJM909 plasmids were added to each well of cells, respectively, and the final transfection volume was 120ul. Electrotransformation was performed using a Neon electrotransformation apparatus (Invitrogen). Injecting the shocked cells into a new culture dish, adding a serum-free culture medium for culturing for 4 hours, changing the culture medium into a complete culture medium, changing the liquid respectively for 24 hours and 48 hours, and collecting cell supernatant for later use.

FIG. 4 shows graphs of the results of Western blot experiments of cell supernatants collected after transfection of 293T cells with pJM908 and pJM909 DNA vaccine plasmids. Wherein the primary antibody is mouse anti-new crown S protein RBD monoclonal antibody from Xinbo biosciences Co-Ltd; the secondary antibody is goat anti-mouse-HRP; the JP-396 polypeptide covering the RBD region served as a positive control. The results of this experiment show that S protein fragments, particularly small fragment antigenic peptides of the pJM909 plasmid, are expressed in transfected cells.

In the embodiment 2 of the invention, the specific steps of the injection immunization experiment of the pestis vaccine comprise:

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/dose, group 2 was injected with pJM909 vaccine alone at 100. Mu.g/dose, group 3 was injected with pJM908 and pJM909 vaccine of the same content in a mixed manner, at 60. Mu.g/dose, and group 4 was injected with pJM908 and pJM909 vaccine of the same content in a mixed manner, at 200. Mu.g/dose; group 5 was injected with empty pVax1 plasmid at 100 μg/dose.

Specifically, muscle cells were electroporated immediately after injection using a tarsha electrotransfer device (from Shanghai tarsha health technologies Co., ltd.).

Specifically, it was observed that BALB/c mice were repeatedly subjected to intramuscular injection of pJM909 and the mixed plasmid for 9 weeks (1 time every 2 weeks for 5 times total), and the body had no significant toxic reaction or significant irritation to the administration site.

All experimental mice survived to the planned dissection time, and after observation and dissection, compared with the control group mice, no macroscopic lesions were seen, no changes were observed in organ weights and histopathology, and the vaccine was found to be safe and reliable. The changes refer to inflammatory, mineralization and degenerative phenomena to a degree above a mild extent.

In example 2 of the present invention, ELISA titer levels of the novel coronavirus S protein RBD antibodies in serum of each group of experimental mice were detected 5-7 days after completion of the intramuscular injection, the number of detected animals in each group was 10, and the samples to be detected in the remaining groups except the blank group of group 5 were 1: a dilution of 100, a specific titer level, is characterized by absorbance at a wavelength of 450 nm. Specific detection methods are routine technical means for those skilled in the art.

Specifically, FIG. 5 is a graph showing ELISA titer levels of the novel coronavirus S protein RBD antibodies in serum after inoculation of each group of experimental mice. Referring to fig. 5, the titer level of group 1 is significantly higher than that of group 5, and the titer levels of groups 2 to 4 are comparable to that of group 5, but since the samples to be tested of the remaining groups except the blank group of group 5 were subjected to 1:100, it was determined that the vaccine of the present application was effective in inducing the production of novel coronavirus S protein RBD antibodies in mice.

Example 3 of the invention a male rhesus monkey 3-6 years old and 3.5-8 kg body weight was intramuscular injected with reference to the mouse vaccine injection immunization experiment described above, forming the following experimental group:

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

group 8 and group 9: 2 rhesus monkeys were injected with pJM909 vaccine alone, with an injection amount of 2 mg/rhesus monkey;

group 10 and group 11: 2 rhesus monkeys were mixed with the same content of pJM908 vaccine and pJM909 vaccine, and the injection amounts were 4 mg/monkey.

Specifically, muscle cells were electroporated immediately after injection using a tarsha electrotransfer device (from Shanghai tarsha health technologies Co., ltd.).

Specifically, it was observed that the rhesus monkey was repeatedly injected with pJM909 and the mixed plasmid intramuscularly for 8 weeks (administered 1 time every 4 weeks for 4 times in total), and the body had no obvious toxic reaction or obvious stimulation to the administration site.

Serum samples from each animal of the above experimental group were subjected to 1:100 as a test sample, serum samples corresponding to non-injected animals were subjected to 1:100 as a control sample, and detecting the neutralizing antibody of the novel coronavirus S protein RBD. The specific process is as follows:

the test product and the control product are respectively mixed with HRP-RBD compound 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 anti-HRP-RBD.

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

Specifically, a PBS dialysate with a pH of 7.4 at 0.15 mol/L was used as the washing liquid for washing.

The substrate TMB solution was added sequentially as a developing solution to the capture plate, and the reaction was quenched by addition of a stop solution 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 investigate virus neutralizing antibody titer, i.e. OD value, in the test samples. The absorbance of the sample is inversely proportional to the titer.

The percent competitive inhibition was calculated by the following formula:

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

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

Referring to fig. 6, the antibody levels in the serum samples of groups 11 and example 2, groups 1 and 3, based on the average percent competitive inhibition of groups 6 and 7, were significantly higher than the baseline, demonstrating that the vaccine of the present application was effective in inducing not only mouse and rhesus novel coronavirus S protein RBD antibodies.

While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. 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 described herein is capable of other embodiments and of being practiced or of being carried out in various ways.

Sequence listing

<110> Shanghai Mongolian Biotechnology Co., ltd

Preparation method of <120> virus vaccine and pharmaceutical composition

<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 (6)

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 said full-length or partial antigen 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 such that the 5-15 amino acid residue coding sequences overlap, and ligating a plurality of said partially overlapping nucleic acid sequences in tandem to form a nucleic acid vaccine fragment;

s3: operably linking the linking elements sequentially or randomly from the 5 'end to the 3' end to form a nucleic acid sequence;

s4: the nucleic acid sequence is subjected to gene synthesis and subcloning to conventional plasmids through a molecular biological method, wherein the conventional plasmids are pJM908 and pJM909, the sequence of the pJM908 is shown as SEQ ID NO. 1, the sequence of the pJM909 is shown as SEQ ID NO. 2, and the nucleic acid vaccine is prepared in a clean environment.

2. The method of claim 1, wherein the antigenic peptides or proteins of the virus are the S1 and S2 subunits of spike proteins.

3. The method for preparing a viral vaccine according to claim 2, wherein the antigenic peptide or protein of the virus is derived from a novel coronavirus.

4. A pharmaceutical composition comprising a nucleic acid vaccine obtained by the method of preparing a viral vaccine according to claim 1 for use in antagonizing viral infections.

5. The pharmaceutical composition of claim 4, further comprising a substance formed by complexation of the nucleic acid vaccine with one or more liposomes.

6. The pharmaceutical composition of claim 4, further comprising an adjuvant that is any one or more of poly-ICLC, TLR,1018ISS, aluminum salt, immunomodulatory oligonucleotide, AS15, BCG, CP-870, CP-893, cpG7909, cyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, imuFact IMP321, ISPatch, ISS, ISCOMATRIX, juvlmmune, lipoVac, MF59, monophosphoryl lipid a, meng Dani dema 1312, meng Dani dema 206, meng Dani dema 50V, meng Dani dema-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, requimod, SRL172, viral microsomes, virus-like particles, YF-17D, VEGF, R848, β -glucan, pam3Cys, nyfra QS21 piercer, vadimezan, and AsA 404.

Images