Vaccines targeting the novel coronavirus

Abstract

The present invention relates to the field of biotechnology, and in particular to vaccines targeting novel coronaviruses. The present invention relates to a vaccine for the precise prevention and treatment of novel coronaviruses, which recombinantly fuses the B cell antigen epitopes of the S\M\N proteins of SARS-COV-2 with the chemokine CX3CL1. The experimental results show that this recombinant vaccine can efficiently induce specific humoral immunity and cellular immune responses in vivo, allowing the immune system to kill a large number of viruses, and can effectively prevent or treat novel coronavirus infection.

Description

Technical Field

[0001] The present invention relates to the field of vaccine research and development, and in particular to a vaccine targeting the novel coronavirus. Background Art

[0002] Coronavirus (Coronaviridae) is a type of single-stranded positive-strand RNA virus with a genome size of 27 to 32kb. Among all known RNA viruses (such as influenza virus, polio virus, HIV, etc.), the coronavirus genome is the largest. The virus is named because the viral envelope contains a spike-shaped envelope protein S, which resembles a pearl inlaid on a crown in the electron microscope structure. According to different phylogenetic developments, coronaviruses can be divided into four genera: α / β / γ / δ. Each genus has different strains. α / β coronaviruses infect mammals, while γ / δ coronaviruses infect birds.

[0003] The latest sequence research analysis shows that SARS-CoV-2 isolated from clinical patients and SARS coronavirus have 79.3% identity. The coronavirus genome structure is relatively conservative, containing 11 functional open reading frames, namely ORF1a, ORF1b, S, ORF3, ORF4a, ORF4b, ORF5, E, M, ORF8b and N. Among them, ORF1a and ORF1b encode two replicase PL2pro and 3CLpro, which are conserved in all coronaviruses. ORF3, 4a, 4b, 5 and 8b encode five accessory proteins of the virus, respectively. ORFs S, E, M and N encode four major structural proteins - Spike Protein (S), Envelope Protein (E), Membrane Protein (M) and Nucleocapsid Protein (N). Among them, E protein is a transmembrane protein that forms an ion channel on the surface of the virus and is related to the virulence of the virus. N protein is the main component of the viral genome RNA replication complex and binds to the C-terminal domain of M protein. M protein is a protein that makes up the shape of coronavirus particles and is involved in the integration of viral components into viral particles. S protein protrudes from the surface of viral particles and is a type I transmembrane glycoprotein that is associated with viral particle adsorption and membrane fusion. According to relevant research reports, S protein is the main immunogen that induces the body to produce neutralizing antibodies. S protein later became an important target for the development of MERS vaccine and genetic engineering drugs for Middle East Respiratory Syndrome virus. Based on previous work experience, S protein became the preferred antigen for SARS-COV-2. However, a large number of related virus defense studies have also revealed that antibodies against nucleocapsid protein can also protect the body from viral attacks, such as some hepatitis B vaccines against nucleocapsid protein.

[0004] In addition, after being taken up by antigen presenting cells, antigen proteins will be degraded by proteasomes or lysosomes into polypeptide sequences that can bind to MHCII or MHCI, which are called antigen epitopes. Compared with full-length proteins, directly selecting antigen epitopes can theoretically enhance the possibility of being presented. For example, in the development of tumor vaccines, there has been a process from using whole tumor cells to overexpressing proteins in a single tumor and predicting multiple antigen epitopes by bioinformatics methods. Initial B cells only exist in secondary lymphoid tissues such as lymph nodes, which are important places for immune responses. Only when exogenous antigens are taken up and processed by professional antigen presenting cells can they be effectively presented to the cell surface and activate initial B cells. Although dendritic cells (DCs) have been found to be powerful antigen presenting cells, different subpopulations of DCs have different abilities to induce antigen-specific immune responses. In recent years, a large number of studies have shown that in mice, CD8α+DC mainly presents antigens to Th1 CD4 T cells. Th1 CD4 T cells can secrete IFNr to promote B cells to secrete IgG2 antibodies, which are the main components of neutralizing antibodies, while other groups of DC cells present antigens to Th2 CD4 T cells, mainly secreting IL-10 to mediate the production of immunosuppressive IgG1. Therefore, in the process of antiviral antigen delivery, delivering antigens to secondary lymph nodes can, on the one hand, be taken up by CD8α+DC cells to promote the development of auxiliary Th1 CD4 T, and on the other hand, be directly phagocytosed by B cells to start immunity. The combination of the two can efficiently produce neutralizing antibodies against viruses. There is also a group of similar cells in the human body, namely CD141+DC (also known as BDCA3+DC). However, these cells also mainly exist in secondary lymphoid tissues and it is difficult for them to directly take up antigens from peripheral tissues. Even if a small amount of antigens in peripheral tissues enter the local draining lymph nodes through the draining lymphatic vessels, it is difficult for them to contact and be taken up by CD8α+DC cells in the paracortical area inside the lymph nodes due to the compactness of the lymph node anatomical structure. Therefore, there is currently a lack of ways to actively deliver viral protein immunogens to CD8α+DC cells to induce T and B cell immunity against antigens.

[0005] CX3CL1 is an immune cell chemokine first discovered in the human genome in 1997 (Bazan, Bacon et al. 1997). The family classification of this chemokine is very unique, and it has certain sequence differences from the protein structure of other conventional CC family and CXC family chemokines. CX3CL1 was first discovered as a transmembrane protein similar to cell membrane glycoproteins containing a transmembrane region in the full-length sequence. This protein was later proven to have two different structural patterns in human tissue distribution. One is a full-length glycoprotein form distributed on the surface of the cell membrane, containing the chemotactic structure of its N-terminal head, the middle glycosyl structure and the cell membrane anchoring structure at the C-terminus. The other form is a secretory protein form that is cleaved and contains only the chemotactic structure and the glycosyl structure. It has been proven that the latter secretory protein form is the main form of CX3CL1 as a chemokine in tissue distribution. This form can drive the chemotactic ability of immune cells like other common chemokines of the CC family or CXC family. Many existing studies have shown that the chemotactic form of CX3CL1 has a very efficient ability to attract monocytes such as T cells and B cells, and does not respond to neutrophils. It is speculated that this may be related to its unique glycosyl stem structure, which enables it to exert a stronger chemokine ability.

[0006] Since SARS-COV-2 is a new virus for humans, the protective immune response process of the human body to SARS-COV-2 is still unclear, so it is impossible to determine which type of vaccine will achieve the best protection. An effective strategy is to develop multiple vaccine platforms and multiple immune methods in parallel in order to obtain safe and effective vaccines as soon as possible. Current research has not yet used CX3CL1 chemokines in combination with new coronavirus antigens for new coronavirus applications, and it is unknown whether it can significantly improve the performance of new coronavirus antigens. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a vaccine targeting the new coronavirus.

[0008] The present invention provides a recombinant antigen, which includes a CX3CL1 chemokine and a novel coronavirus B cell antigen. The novel coronavirus B cell antigen, referred to as antigen (or S / M / N antigen), includes an S antigen epitope, an N antigen epitope and an M antigen epitope;

[0009] The amino acid sequence of the S antigen epitope is shown in SEQ ID NO: 11;

[0010] The amino acid sequence of the M antigen epitope is shown in SEQ ID NO: 12;

[0011] The amino acid sequence of the N antigen epitope is shown in SEQ ID NO:13.

[0012] The S antigen epitope, N antigen epitope and M antigen epitope are independently connected by a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO:10.

[0013] Furthermore, in the present invention, in the recombinant antigen,

[0014] The novel coronavirus B cell antigen has an amino acid sequence as shown in SEQ ID NO: 5;

[0015] The CX3CL1 chemokine has a nucleic acid sequence as shown in SEQ ID NO:6.

[0016] The novel coronavirus B cell antigen described in the present invention is obtained through bioinformatics prediction of B cell epitopes. It is an internationally recognized epitope. Its distribution is not only present in the RBD region of the novel coronavirus, but also includes other positions of the S protein and the M and N proteins. Therefore, its extensibility and versatility are stronger than other novel coronavirus-specific antigens.

[0017] In the recombinant antigen of the present invention, the CX3CL1 chemokine and the novel coronavirus B cell antigen are connected by a linker, and the linker is (G5S)n, where n is 1 to 10. In some specific embodiments of the present invention, the linker is GGGGGSGGGGG (as shown in SEQ ID NO: 8).

[0018] Furthermore, the recombinant antigen of the present invention further comprises an IgE signal peptide and / or a Flag tag at the C-terminus. The amino acid sequence of the IgE signal peptide is MDWTWILFLVAAATRVHS, as shown in SEQ ID NO:7, and its addition to the N-terminus of the recombinant protein promotes the secretion of the fusion protein to the extracellular space; the amino acid sequence of the Flag tag is DYKDDDDK, as shown in SEQ ID NO:9, and this sequence is only a tag for identifying protein expression and does not affect the immune effect of the sequence.

[0019] In some specific embodiments, the composition of the recombinant antigen of the present invention may include any one of the following a) to e):

[0020] a) Antigen;

[0021] b), CX3CL1 chemokine and antigen;

[0022] c), IgE signal peptide, CX3CL1 chemokine and antigen;

[0023] d), CX3CL1 chemokine, antigen and Flag tag;

[0024] e), IgE signal peptide, CX3CL1 chemokine, antigen and Flag tag.

[0025] Furthermore, the recombinant antigen of the present invention has an amino acid sequence as shown in SEQ ID NO:3.

[0026] In the present invention, CX3CL1 is a chemokine that exercises the chemotactic ability of immune cells. After analyzing the protein structure and sequence of CX3CL1, the present invention reconstructs the CX3CL1 chemokine. The results show that among the reconstructed CX3CL1 chemokine variants, CX3CL1-b has a higher chemotactic activity and can significantly improve the cross-presentation effect of cell antigen molecules. Its amino acid sequence is shown in SEQ ID NO:6.

[0027] The recombinant antigen of the present invention and the fusion expression of CX3CL1-b chemokine improve the efficiency of S\M\N antigen epitopes entering secondary lymph nodes and the efficiency of phagocytosis, processing and presentation by immune cells and B cells, and improve the effect of inducing specific antibody response. The experimental results show that the antigen and recombinant antigen of the present invention have high protein expression, and after being injected into mice, they can effectively induce antibodies against S\M\N in the serum, indicating that the effect of fusion gene immunization is significant and effective; and the serum from its immune source has a strong ability to prevent and treat the new coronavirus, among which the serum from the CX3CL1-S / M / N recombinant antigen is significantly higher than the serum from the S / M / N antigen.

[0028] The present invention provides a nucleic acid encoding the recombinant antigen.

[0029] Furthermore, in the nucleic acid encoding the recombinant antigen,

[0030] The nucleic acid encoding CX3CL1 chemokine is shown in SEQ ID NO:4;

[0031] The nucleic acid encoding the new coronavirus B cell antigen is shown in SEQ ID NO:1.

[0032] In some specific embodiments of the present invention, the nucleic acid may be CX3CL1-antigen;

[0033] In other embodiments of the present invention, the nucleic acid of the present invention may be CX3CL1-linker-antigen;

[0034] In other embodiments of the present invention, the nucleic acid of the present invention may be IgE signal peptide-CX3CL1-linker-antigen;

[0035] In other embodiments of the present invention, the nucleic acid of the present invention may be a CX3CL1-linker-antigen-Flag tag;

[0036] In other embodiments of the present invention, the nucleic acid described in the present invention may be an IgE signal peptide-CX3CL1-linker-antigen-Flag tag.

[0037] In the present invention, the nucleic acid has a nucleic acid sequence as shown in SEQ ID NO:2.

[0038] The nucleic acid encoding the recombinant antigen of the present invention may be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. Nucleic acid may include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acid may be linear or circular in topology. Nucleic acid may be, for example, a part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid may be obtained directly from a natural source, or may be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0039] In the present invention, the DNA sequence encoding the recombinant antigen is optimized, and these optimizations include but are not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0040] The present invention also provides a transcription unit of the recombinant antigen, which refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc.

[0041] The present invention provides a recombinant vector, which comprises a vector backbone and the nucleic acid of the present invention.

[0042] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably linked coding gene in a specific host organism. The nucleic acid sequence or element necessary for the expression in a model animal or mammalian cell includes a promoter, a ribosome binding site and possible other sequences. Known eukaryotic cells utilize promoters, enhancers and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or only potential genomic insert. In a specific embodiment, the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various eukaryotic expression vectors. For example, its backbone vector can be a pVR series vector (see Chinese Patent ZL202110624820.8).

[0043] Furthermore, in some specific embodiments, the recombinant vector of the present invention may include any one of the following:

[0044] pVR-CX3CL1-antigen;

[0045] pVR-antigen.

[0046] The present invention provides a host transformed or transfected with the recombinant vector. The host cell is transformed or transfected with a vector constructed using recombinant DNA technology, so that the transformed host cell has the ability to replicate the protein-encoding vector or express the desired protein.

[0047] Furthermore, the host includes model animals or mammalian cells. The model animals include mice, rats, rabbits and other commonly used experimental animals in the biomedical field, and the present invention does not limit this. The mammalian cells include CHO cells, BHK cells, Sp2 / 0, HEK293, and HEK293T.

[0048] The present invention provides a method for preparing the recombinant antigen, which comprises culturing the host of the present invention to obtain a culture containing the recombinant antigen.

[0049] The present invention provides the use of at least one of the following I) to V) in the preparation of a vaccine for preventing and treating a novel coronavirus:

[0050] I), the recombinant antigen of the present invention;

[0051] II), the nucleic acid of the present invention;

[0052] III), the recombinant vector of the present invention;

[0053] IV), the host of the present invention;

[0054] V), a culture containing a recombinant antigen obtained by the preparation method of the present invention.

[0055] The present invention provides a novel coronavirus vaccine, the raw materials of which include any one or more of the following i) to v):

[0056] i), the recombinant antigen of the present invention;

[0057] ii), the nucleic acid of the present invention;

[0058] iii), the recombinant vector of the present invention;

[0059] iv), the host of the present invention;

[0060] v), a culture containing a recombinant antigen obtained by the preparation method of the present invention.

[0061] The vaccine described in the present invention may be a DNA vaccine, an RNA vaccine or a protein vaccine, and the present invention is not limited to this.

[0062] Furthermore, the vaccine of the present invention further includes adjuvants or buffers; the adjuvants are used to maintain the activity of the vaccine or help the vaccine to work. The adjuvants include but are not limited to carriers, surfactants and reducing agents, etc.; the carriers include lentivirus auxiliary vectors, adenovirus auxiliary vectors and other vectors that assist in assembly or integration; the surfactants include but are not limited to triton; the reducing agents include but are not limited to DTT or β-mercaptoethanol, etc. The buffers of the present invention include PBS buffer, KCl buffer, NaCl buffer, Tris buffer, etc.

[0063] Furthermore, the vaccine described in the present invention also includes the transcription mRNA or translation initiation product of the nucleic acid, etc., which is not limited by the present invention.

[0064] Furthermore, the vaccine also includes lipid nanoparticles containing the nucleic acid or mRNA. The preparation process of the lipid nanoparticles includes encapsulating the nucleic acid or mRNA using a transfection reagent in an animal body to form a vaccine in the form of lipid nanoparticles containing the nucleic acid or mRNA for the prevention and treatment of the new coronavirus.

[0065] The present invention provides a method for preventing and treating a novel coronavirus, which comprises administering the vaccine of the present invention. The administration method may include injection.

[0066] The present invention relates to a nucleic acid vaccine for the precise prevention and treatment of novel coronavirus. The vaccine uses a functionally active variant of chemokine CX3CL1 after sequence optimization as an adjuvant, and uses a B cell epitope of a unique S\M\N protein of a novel coronavirus as a targeting antigen. The results show that the novel vaccine can induce efficient specific humoral and cellular immune responses in vivo, and ultimately enables the immune system to kill a large number of viruses, which can effectively prevent or treat novel coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The comparison of the ability of CX3CL1 and its various functionally active variants to attract various professional antigen-presenting cells is shown;

[0068] Figure 2 The plasmid maps of pVR-CX3CL1-S / M / N and pVR-S / M / N are shown;

[0069] Figure 3 The expression of the target gene in plasmids pVR-CX3CL1-S / M / N and pVR-S / M / N is shown;

[0070] Figure 4 The results show the neutralizing antibodies produced by mice after they were immunized with pVR-CX3CL1-S / M / N and pVR-S / M / N plasmids;

[0071] Figure 5 The figure shows the effect of serum from mice immunized with different fusion genes in neutralizing the new coronavirus, where the vertical axis indicates the highest dilution multiple at which various sera can completely neutralize viral infection. DETAILED DESCRIPTION

[0072] The present invention provides a vaccine targeting the novel coronavirus, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0073] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0074] Antigenic epitope, also known as "antigenic determinant cluster" or "antigenic determinant site". The size of the antigenic epitope is suitable for the antigen binding site of the corresponding antibody. The specificity of an antigenic epitope is determined by all the residues that make it up, but some of the residues play a greater role than other residues when binding to the antibody. These residues are called immunodominant groups.

[0075] The present invention provides antigenic epitopes of the spike protein, envelope protein, and nucleocapsid protein of the new coronavirus SARS-COV-2. In the present invention, the S protein, spike protein, SARS-COV-2 S protein, and SARS-COV-2 spike protein have the same meaning and can be used interchangeably herein. The M protein, envelope protein, SARS-COV-2 M protein, or envelope protein have the same meaning and can be used interchangeably herein. The N protein, nucleocapsid protein, SARS-COV-2 N protein, or SARS-COV-2 nucleocapsid protein have the same meaning and can be used interchangeably herein. In the present invention, the antigenic epitope of the spike protein of SARS-COV-2 is also referred to as the antigenic epitope of the S protein of SARS-COV-2 or the antigenic epitope of the S protein or the S epitope; the antigenic epitope of the envelope protein of SARS-COV-2 is also referred to as the antigenic epitope of the M protein of SARS-COV-2 or the antigenic epitope of the M protein or the M epitope; the antigenic epitope of the nucleocapsid protein of SARS-COV-2 is also referred to as the antigenic epitope of the N protein of SARS-COV-2 or the antigenic epitope of the N protein or the N epitope.

[0076] In the present invention, the connecting peptide or linker refers to a connector that connects two protein fragments in a fusion protein. In some embodiments, the connection between antigenic epitopes is achieved through the tripeptide AAY, and the antigenic epitope and the chemokine protein are connected through (GGGGS)n.

[0077] In the present invention, the recombinant antigen refers to the expression product obtained after gene recombination through DNA recombination technology.

[0078] In some embodiments, the recombinant antigen provided by the present invention includes an antigenic epitope of the S protein of SARS-COV-2, an antigenic epitope of the N protein of SARS-COV-2, and an antigenic epitope of the M protein of SARS-COV-2, which are denoted as S\M\N. In the present invention, the connection order of the S epitope, the M epitope, and the N epitope in the fusion protein is not limited. In the embodiment of the present invention, from the N-terminus to the C-terminus, the fusion protein includes the S epitope, the M epitope, and the N epitope in sequence, wherein the S epitope and the M epitope are connected by a connecting peptide AAY, and the M epitope and the S epitope are connected by a connecting peptide AAY.

[0079] In other embodiments, the recombinant antigen provided by the present invention includes the antigenic epitope of the S protein of CX3CL1 chemokine protein and SARS-COV-2, the antigenic epitope of the N protein of SARS-COV-2, and the antigenic epitope of the M protein of SARS-COV-2; it is recorded as CX3CL1-S\M\N, and the connection order of the CX3CL1 protein, S epitope, N epitope, and M epitope is not limited. In some embodiments of the present invention, CX3CL1 and S\M\N epitope fragments are connected with linker, and the epitope fragments are connected with a connecting peptide AAY. In some specific embodiments, in the sequence of CX3CL1-S\N\M, from N-terminus to C-terminus, it is CX3CL1 protein-linker-S epitope-AAY-M epitope-AAY-N epitope, and its amino acid sequence is shown in SEQ 3, and the nucleic acid sequence encoding the fusion protein is shown in SEQ 2.

[0080]

[0081]

[0082] The amino acid sequence of the CX3CL1-S / M / N fusion protein is: QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLEGGGGGSGGGGGVDAVDCALDPLSETKCTLKSFTVEKGIYQTSNAAYVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVIAAYGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSAAYFSQILPDPSKPSKRSFIEAAYFGAGAALQIPFAMQMAYRFNGIAAYMADSNGTITVEELKKLLEQWNLVIAAYPLLESELVIGAVILRGHLRIAAYRPQGLPNNTASWFTALTQHGKAAYNNNAATVLQLPQGTTLPKGFAAYNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAADM (as shown in SEQ ID NO:3).

[0083] The CX3CL1 nucleotide sequence is: cagcaccacggagttaccaagtgtaacattacctgcagtaagatgacatccaagattcccgttgccctgttgattcactatcagcaaaaccaggcctcttgtggaaaaagggctattattctggagactcgacagcacaggctcttttgcgccgacccaaaggagcagtgggtcaaagacgccatgcagcacctggaccgccaggctgccgccctcaccagaaatggggggacattcgagaagcagattggggaagtcaaaccccgcaccacccccgcggccggcggcatggacgaatccgttgtcctcgag (as shown in SEQ ID NO:4).

[0084] The amino acid sequence of the S / M / N antigen is: VDAVDCALDPLSETKCTLKSFTVEKGIYQTSNAAYVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVIAAYGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSAAYFSQILPDPSKPS KRSFIEAAYFGAGAALQIPFAMQMAYRFNGIAAYMADSNGTITVEELKKLLEQWNLVIAAYPLLESELVIGAVILRGHLRIAAYRPQGLPNNTASWFTALTQHGKAAYNNNAATVLQLPQGTTLPKGFAAYNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAADM (as SEQ ID NO:5).

[0085] The amino acid sequence of CX3CL1 is: QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLE (as shown in SEQ ID NO: 6).

[0086] The amino acid sequence of IgE is: MDWTWILFLVAAATRVHS (as shown in SEQ ID NO: 7).

[0087] The linker amino acid sequence is: GGGGGSGGGGG (as shown in SEQ ID NO: 8).

[0088] The amino acid sequence of the Flag tag is: DYKDDDDK (as shown in SEQ ID NO: 9).

[0089] Connecting peptide: AAY (as shown in SEQ ID NO: 10).

[0090] The amino acid sequence of the S antigen epitope is: VDAVDCALDPLSETKCTLKSFTVEKGIYQTSNAAYVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVIAAYGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSAAYFSQILPDPSKPSKRSFIEAAYFGAGAALQIPFAMQMAYRFNGI (shown in SEQ ID NO: 11).

[0091] The amino acid sequence of the M antigen epitope is: MADSNGTITVEELKKLLEQWNLVIAAYPLLESELVIGAVILRGHLRI (shown in SEQ ID NO: 12).

[0092] The amino acid sequence of the N antigen epitope is: RPQGLPNNTASWFTALTQHGKAAYNNNAATVLQLPQGTTLPKGFAAYNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAADM (shown in SEQ ID NO: 13).

[0093] In the embodiments of the present invention, the amino acid sequences of the involved fragments and the encoded nucleic acid fragments are shown in Table 1:

[0094]

[0095] The reagents and consumables used in the present invention are all common commercial products and can be purchased in the market.

[0096] The present invention will be further described below in conjunction with embodiments:

[0097] Example 1 Structural analysis and screening of CX3CL1 chemokine protein

[0098] The protein structure and sequence analysis of CX3CL1 chemokine was performed. The analysis revealed that the molecule has three structural domains, namely: the chemokine domain at the N-terminus, the glycosyl domain in the middle, and the transmembrane domain at the C-terminus. In order to find out the most efficient chemokine sequence for various immune cells, multiple sequences of the CX3CL1 protein itself were constructed and the chemotactic ability of the protein molecule was tested at the same time. Since the C-terminal domain is a transmembrane domain, the antigen protein molecule cannot be secreted out of the cell, so the C-terminal transmembrane region was completely deleted in different variant tests, and the amino acid sequence of CX3CL1 protein from 25 to 338 was defined as the full-length CX3CL1 molecule. Based on this, combined with protein structural analysis, the following molecular variants were designed (amino acid numbers are shown in brackets): CX3CL1-a (25-100); CX3CL1-b (25-128); CX3CL1-c (25-185); CX3CL1-d (25-265); CX3CL1-e (25-309); CX3CL1-f (25-338, full length) and CX3CL1-g (37-388). Sequences of all the above molecular variants were synthesized and constructed into the Escherichia coli pET28a vector for protein expression and purification, and finally crude pure samples of all 7 protein molecules were obtained for subsequent experiments.

[0099] Monocytes and T cell subsets were isolated from the mouse bone marrow and peripheral blood. Among them, bone marrow monocytes were induced to differentiate into macrophages and DC cells by adding M-CSF, GM-CSF and IL4, respectively, and then chemotaxis experiments were performed. The above-mentioned isolated or induced differentiated cells were placed in the upper chamber of the chemotaxis chamber (carbonate membrane Transwell chamber: 5μm; Costar, Cat: 3422), and the number of cells added was 1× 106 / 100μl / well based on the previous work of the laboratory. At the same time, a spontaneous migration control group and a CX3CL1 cytokine full functional variant group were set up, and the number of cells added was the same. Based on the previous work of the laboratory, 100ng / ml is the optimal chemotaxis efficiency dose. After 4 hours, the cells in the chemotaxis lower chamber were collected, and the chemotaxis ability of CX3CL1 functional variants on various immune cells was analyzed by flow cytometry. The results showed that different molecular variants of CX3CL1 could effectively recruit various immune cells from the upper chamber to the lower chamber (P < 0.001), and the chemotactic ability of different protein molecules was different. The functionally active variant with the strongest chemotactic ability was not the chemokine domain protein understood by traditional sequence analysis, but the molecular variant CX3CL1-b ( Figure 1 ), all of the following CX3CL1 functional variants or CX3CL1 genes and proteins represent the molecular variant CX3CL1-b, whose amino acid sequence is shown in SEQ ID NO:6, and whose nucleic acid sequence is shown in SEQ ID NO:4.

[0100] Example 2 Antigen design and construction and preparation of mammalian expression plasmid

[0101] Antigen design scheme for fusion gene or protein vaccine and construction and preparation of mammalian expression plasmid

[0102] Construction of pVR-CX3CL1-S / M / N plasmid: The S / M / N specific BCR antigen sequence uses the amino acid sequence shown in SEQ ID NO: 5, and the human CX3CL1 protein uses the amino acid sequence shown in SEQ ID NO: 6. An IgE signal peptide (SEQ ID NO: 7) with an amino acid sequence of MDWTWILFLVAAATRVHS is connected to the N-terminus of the fusion protein CX3CL1-S / M / N; a Flag tag consisting of 8 amino acids of DYKDDDDK (as shown in SEQ ID NO: 9) is connected to the C-terminus of the fusion protein CX3CL1-S / M / N.

[0103] The final fusion protein includes, from N-terminus to C-terminus, the following: IgE signal peptide, human CX3CL1 protein sequence, linker sequence (GGGGGSGGGGG), S / M / N sequence and Flag tag sequence.

[0104] The amino acid sequence of the fusion protein was codon-optimized for mammalian cell expression, and the fusion gene sequence was determined to be SEQ ID NO: 2. The fusion gene sequence was synthesized and then constructed into the corresponding multiple cloning site region of the pVR plasmid vector so that the fusion protein could be expressed in the correct codon translation order. The final constructed plasmid was named pVR-CX3CL1-S / M / N plasmid. Figure 2 As shown in A.

[0105] Construction of pVR-S / M / N plasmid: an IgE signal peptide with an amino acid sequence of MDWTWILFLVAAATRVHS was connected before the S / M / N specific BCR antigen sequence, followed by a Flag tag consisting of 8 amino acids DYKDDDDK.

[0106] The final fusion protein includes, from N-terminus to C-terminus, the IgE signal peptide, the S / M / N-specific BCR protein sequence, and the Flag tag sequence. Figure 2 As shown in B.

[0107] The amino acid sequence of the fusion protein was codon-optimized for mammalian cell expression preference, and the fusion gene sequence was determined to be SEQ ID NO: 1. The fusion gene sequence was synthesized, and then the entire sequence was constructed into the corresponding multiple cloning site region of the pVR plasmid vector, so that the fusion protein could be expressed in the correct codon translation order. The final constructed plasmid was named pVR-S / M / N plasmid.

[0108] Example 3 In vitro cell transfection experiment of constructed plasmid

[0109] 24 hours before transfection, 2.5×10 5 HEK293T cells were added, and the transfection experiment was started when the cell density grew to 60%~70%. During transfection, the cell culture medium and serum-free Opti-MEM medium were preheated in a 37℃ water bath in advance. During transfection, 5 micrograms of empty vector (Vector), pVR-CX3CL1-S / M / N expression vector, pVR-S / M / N expression vector and 20μL PEI transfection reagent were added to 200 μL serum-free Opti-MEM successively, mixed evenly, and allowed to stand at room temperature for 20 minutes. The cells to be transfected were replaced with fresh culture medium, gently added with the above transfection system, and gently shaken. The cells were returned to the cell culture incubator and cultured for 6 hours and the medium was changed. After 48 hours of transfection, the cells were collected and the expression effect of the fusion gene plasmid in HEK293T cells was detected by Western Blot.

[0110] Collect the cells and add 60 μL of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor. Resuspend the cells thoroughly and lyse the cells at 4°C for 30 minutes. Centrifuge the lysate at 12000 rpm and 4°C for 10 minutes, collect the supernatant into a new 1.5 mL EP tube, and discard the precipitate. Add 5× SDS-PAGE protein loading buffer according to the actual volume of the sample, mix well, heat the sample in an air bath at 100°C for 10 minutes, and immediately perform Western blot and detect using Flag tag antibody (Sigma, F3165). The results are as follows: Figure 3 (Western blot was used to detect the expression of fusion protein with a Flag tag at the C-terminus) The empty vector (Vector) showed no protein expression, and the size and position of the protein expressed by pVR-CX3CL1-S / M / N were significantly higher than those of pVR-S / M / N, indicating that both the experimental group plasmid pVR-CX3CL1-S / M / N and the control group plasmid pVR-S / M / N can be successfully and normally expressed in mammalian cells.

[0111] Example 4 Detection of antibodies against S\M\N proteins in mouse serum after immunization with fusion protein particles

[0112] C57B6 (purchased from Tonglihua) week-old male mice were divided into three groups, one was injected with the experimental group plasmid pVR-CX3CL1-S / M / N and the other was injected with the control group plasmid pVR-S / M / N and PBS control, with 10 mice in each group. The right side of the mice near the inguinal lymph nodes were depilated with a depilatory cream. Afterwards, the plasmid was injected into the depilated area with an electroporator, 50 μg per mouse, once a week, for a total of four injections. Blood was collected from the inner canthus before the first injection, after the second injection, and one week after the fourth injection. The serum was obtained by centrifugation at 12000 rpm for 20 minutes and frozen at -80°C. After the last blood collection, ELISA was used to detect the antibodies against S\M\N proteins in the serum of each group of mice. The results are as follows Figure 4 As shown in the figure, immunization with XCL1-S\M\N plasmid can effectively induce antibodies against S\M\N protein in serum, indicating that the effect of fusion gene immunization is significant and effective.

[0113] Example 5 Detection of Neutralization Ability Against Live COVID-19 Virus in Mouse Serum after Fusion Protein Immunization

[0114] Antibody neutralization experiment: Take 20μL of mouse immune serum and mix it with the same volume of new coronavirus liquid at 37℃ for 1 hour, then add it to Vero cells cultured in 12-well plates. After 2 hours of infection, remove the infected cell culture medium, replace it with fresh cell culture medium and culture it at 37℃. After 3 days, the serum neutralization ability was determined by cytopathic effect CPE. Through experiments, we found that serum from pVR-CX3CL1-S / M / N immune sources can greatly reduce the efficiency of viral infection. After antibody neutralization, the ability of the virus to infect cells was significantly reduced, indicating that the specific antibodies in the serum can block the entry of new coronavirus particles mediated by S protein into cells. Figure 5 shown.

[0115] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant antigen, characterized in that It is formed by connecting CX3CL1 chemokine and novel coronavirus B cell antigen through a linker, and the recombinant antigen is CX3CL1 protein-linker-S epitope-AAY-M epitope-AAY-N epitope from N-terminus to C-terminus; The amino acid sequence of the novel coronavirus B cell antigen is shown in SEQ ID NO:

5. The amino acid sequence of the CX3CL1 chemokine is shown in SEQ ID NO:

6. The amino acid sequence of the S epitope is shown in SEQ ID NO: 11; The amino acid sequence of the M epitope is shown in SEQ ID NO: 12; The amino acid sequence of the N epitope is shown in SEQ ID NO:

13.

2. The recombinant antigen according to claim 1, characterized in that The linker is (G5S)n, where n is 1~10.

3. The recombinant antigen according to claim 1, characterized in that The recombinant antigen also includes an IgE signal peptide and / or a Flag tag.

4. The recombinant antigen according to claim 1 or 2, characterized in that The amino acid sequence of the recombinant antigen is shown in SEQ ID NO:

3.

5. A nucleic acid encoding the recombinant antigen according to any one of claims 1 to 4.

6. The nucleic acid according to claim 5, characterized in that The nucleic acid encoding CX3CL1 chemokine is shown in SEQ ID NO:4; The nucleic acid encoding the new coronavirus B cell antigen is shown in SEQ ID NO:

1.

7. The nucleic acid according to claim 5 or 6, characterized in that Its nucleic acid sequence is shown in SEQ ID NO:

2.

8. A recombinant vector comprising a vector backbone and the nucleic acid according to any one of claims 5 to 7.

9. The recombinant vector according to claim 8, characterized in that The vector backbone includes pVR.

10. Mammalian cells transformed or transfected with the recombinant vector according to claim 8 or 9.

11. The method for preparing a recombinant antigen according to any one of claims 1 to 4, characterized in that: Cultivate the mammalian cell according to claim 10 to obtain a culture containing the recombinant antigen.

12. Use of at least one of the following I) to V) in the preparation of a vaccine for preventing and treating novel coronavirus infection: I), the recombinant antigen according to any one of claims 1 to 4; II), the nucleic acid according to any one of claims 5 to 7; III), the recombinant vector according to claim 8 or 9; IV), the mammalian cell according to claim 10; V), a culture containing a recombinant antigen obtained by the preparation method according to claim 11.

13. A novel coronavirus vaccine, the raw materials of which include any one or more of the following i) to v): i), the recombinant antigen according to any one of claims 1 to 4; ii), the nucleic acid according to any one of claims 5 to 7; iii), the recombinant vector according to claim 8 or 9; iv), the mammalian cell according to claim 10; v), a culture containing a recombinant antigen obtained by the preparation method according to claim 11.

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