A novel coronavirus vaccine production system and method based on synthetic biology self-assembly

Abstract

The present invention relates to a novel coronavirus vaccine production system and method based on synthetic biology self-assembly, the system comprising a base sequence component capable of expressing novel coronavirus antigens, the system being a sequence capable of being enriched in tissues and organs in the host body and expressing antigens on the surface of vesicles and self-assembling into vesicle complexes, the vesicle complexes being capable of inducing an immune response. In the present invention, the base sequence component has the advantages of low production cost, stable physicochemical properties, efficient antigen assembly and secretion process, fast response to pathogen mutations, strong immune response induced, high safety and protection, etc., providing a new type of vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a system and method for producing a new coronavirus vaccine based on synthetic biology self-assembly, which is used for the development of a new coronavirus vaccine. Background Art

[0002] COVID-19 is a severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a new coronavirus. Currently, there is still a lack of specific drugs for the new coronavirus. The development and production of a safe and effective new coronavirus vaccine has become the top priority for controlling the new coronavirus epidemic.

[0003] At present, the new crown vaccines under development and those that have entered clinical trials mainly include inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, and viral vector vaccines. Among them, mRNA vaccines belong to nucleic acid vaccines and are currently the most promising new crown vaccines. Their sequences are flexible and editable, and they can quickly respond to various mutations that may occur in the new coronavirus. However, due to the unstable chemical properties of mRNA itself and its easy degradation in the body, the delivery of mRNA vaccines requires the protection of carriers, and also places higher requirements on transportation and storage conditions. In addition, mRNA vaccines need to be synthesized in vitro and are relatively expensive. The strains used in live attenuated vaccines are alive, and in terms of safety, they are not easily accepted by the public. Other types of vaccines also generally have problems such as long research and development cycles and high costs.

[0004] Therefore, if a new type of vaccine can be developed, it may be a major change in this field, which will bring many new advantages and new hopes.

[0005] Exosomes are a type of double-layer membrane vesicles with a diameter of 50-200 nm. Almost all cells can secrete exosomes. Many studies have shown that exosomes play an important role in intercellular communication. The applicant and the inventor team have always been in a leading position in the study of exosomes, and have also achieved many research results. In recent related studies, it was found that editable gene components can be delivered in vivo, and specific polypeptides / proteins can be displayed on the surface of exosomes by self-assembly of biological tissues or organs, and can be transported to major tissues and organs throughout the body through the circulatory system. Based on the above information, the inventor team has developed a new type of vaccine, which uses a method of in vivo exosome self-assembly of related base sequences such as gene components, and uses exosomes to present antigens and induce immune responses to achieve in vivo immunity. This new type of vaccine and the method for preparing the vaccine are expected to become a new vaccine with excellent application prospects. Summary of the invention

[0006] In response to the defects of the prior art, the present invention provides a system and method for producing a new coronavirus vaccine based on synthetic biology self-assembly, aiming to overcome the shortcomings of the prior art.

[0007] The purpose of the present invention is to be achieved through the following technical solutions:

[0008] One inventive point of the present invention is to provide a new coronavirus vaccine production system based on synthetic biology self-assembly, wherein the system includes a base sequence component capable of expressing new coronavirus antigens. The system is a sequence that can be enriched in tissues and organs in the host body, express antigens on the surface of vesicles and self-assemble into vesicle complexes, and the vesicle complexes are vaccines that can trigger an immune response.

[0009] Furthermore, the base sequence component is an editable or synthesizable gene component.

[0010] Furthermore, the new coronavirus includes the virus itself and its mutants, and the new coronavirus antigen includes the existing new coronavirus antigen and the sequence after any one or several amino acid mutations in the existing antigen sequence.

[0011] Furthermore, the base sequence component is selected from at least one of the following sequences:

[0012] Q1: pcDNA6.2-RBD-HIS or a sequence with more than 70% homology to the pcDNA6.2-RBD-HIS sequence;

[0013] Q2: pcDNA6.2-HIS or a sequence with more than 70% homology to the pcDNA6.2-HIS sequence;

[0014] Q3: pcDNA6.2-RBD (WT) or a sequence that is more than 70% homologous to the pcDNA6.2-RBD (WT) sequence; the homologous sequences include but are not limited to pcDNA6.2-RBD (E484K), pcDNA6.2-RBD (N501Y), pcDNA6.2-S1 (D614G), etc. These sequences have been discovered, and many that have not been discovered can be included.

[0015] The above homologous sequences include sequences obtained by adding one or several bases, reducing one or several bases, or replacing any one or several bases on the basis of the original sequence; several includes 2 or more bases.

[0016] Furthermore, the system also includes a signal peptide base sequence fragment that enables the novel coronavirus antigen to be expressed on the surface of the vesicle. Preferably, the base sequence component is located downstream of the signal peptide base sequence fragment, that is, at the 3' end of the signal peptide.

[0017] Furthermore, the signal peptide base sequence fragment preferably includes a first sequence, which is a sequence containing seq1 or a sequence that is more than 70% homologous to the seq1 sequence; the homologous sequence includes a sequence obtained by adding one or several bases, reducing one or several bases, or replacing any one or several bases on the basis of seq1; several includes 2 and more.

[0018] Furthermore, the system also includes a transmembrane protein base sequence fragment that enables the new coronavirus antigen to anchor on the surface of the vesicle and self-assemble to form a vesicle complex. Preferably, the base sequence component is located upstream of the transmembrane protein base sequence fragment, that is, at the 5' end of the transmembrane protein.

[0019] Furthermore, the transmembrane protein base sequence fragment includes a second sequence, which is a sequence containing seq2 or a sequence that is more than 70% homologous to the seq2 sequence; the homologous sequence includes a sequence obtained by adding one or several bases, reducing one or several bases, or replacing any one or several bases on the basis of seq2; several includes 2 and more.

[0020] Furthermore, the system also includes a promoter, and the promoter is preferably a CMV promoter; the promoter is arranged upstream of the base sequence component, and if it contains a signal peptide base sequence fragment, the promoter is located upstream of the signal peptide base sequence fragment.

[0021] The system further comprises a termination signal, which is located downstream of the transmembrane protein base sequence fragment, and the termination signal is preferably an HSV termination signal.

[0022] Furthermore, any one or several bases in the system are independently modified bases or unmodified bases; wherein the modifications include thio modification, fluoro modification, amino modification, and methoxy modification.

[0023] Furthermore, the tissues and organs include all cells, tissues and organs of the host, the vesicles are exosomes, and the vesicle complexes are exosome complexes.

[0024] Furthermore, the system is delivered via a plasmid or virus system.

[0025] Another inventive point of the present invention is to provide a method for producing a virus vaccine based on synthetic biology self-assembly, the method comprising constructing a system as described in any of the above paragraphs, the system being a sequence that can be enriched in host cells, tissues and / or organs, express antigens on the surface of vesicles and self-assemble into vesicle complexes, the vesicle complexes being vaccines that can induce immune response reactions. Specifically, the synthetic base sequence components and other sequences are synthesized according to any of the arrangements described in this application.

[0026] Another aspect of the present invention is to provide a vaccine, which comprises the system described in any one of the above paragraphs.

[0027] Furthermore, the administration methods of the vaccine include but are not limited to oral administration, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.

[0028] The last invention of the present invention is to provide a vector system, which comprises the system described in any of the above paragraphs and a vector for loading or carrying the system. The vector includes but is not limited to plasmids and viruses.

[0029] The main beneficial effects of the present invention are:

[0030] In the vaccine production system of the present invention, the corresponding base sequence components (such as gene components) can be selected according to the needs of the virus itself or its different mutants, and only the corresponding sequences need to be synthesized. The production cost of the base sequence components is low, the cycle is short, the physical and chemical properties are stable, the preservation conditions are mild, the preservation is convenient, the preservation cost is low, and it is easy to store and transport.

[0031] The vaccine production system is capable of enriching in cells, tissues or organs in an organism, and through base sequence components, the antigens of the disease corresponding to the sequence components are displayed on the surface of vesicles (such as exosomes), and self-assembled to form vesicle complexes (such as exosome complexes), and the vesicles (such as exosomes) are used to deliver the antigens systemically and induce a specific immune response.

[0032] Since the system of the present invention can self-assemble into vesicle complexes, which are not artificially synthesized, and the vesicle complexes can be transported to the main tissues and organs of the whole body through the circulatory system, and then under the original action of the base sequence components, immunity to the new coronavirus is achieved, so its antigen assembly and secretion process is efficient, the immune response triggered is strong, and it is safe and protective, etc. And some toxic and side effects in the prior art are avoided.

[0033] The base sequence components in this system are highly editable, and can be quickly edited to respond to the new coronavirus or any of its mutations. Therefore, it can avoid the current shortcomings of vaccines being ineffective due to mutations, and new vaccines needing to be re-developed and studied over a long period of time.

[0034] The system and method in the present invention have good overall effects. It is a new type of new crown vaccine and a vaccine synthesis method. It is a huge breakthrough in the fields of biology and medicine and has milestone significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the plasmid constructed according to the embodiment of the present invention.

[0036] Figure 2 This is a diagram showing different RBD expression levels produced by different transmembrane protein base sequences in an embodiment of the present invention.

[0037] Figure 3 This is the ELISA test result that shows that the gene assembly can correctly express the RBD antigen in the embodiment of the present invention.

[0038] Figure 4 This is the ELISA test result of the embodiment of the present invention, which shows that the lamp2b skeleton can present the RBD antigen on the exosomes.

[0039] Figure 5 In the examples of the present invention, in vivo experiments demonstrate that the gene component can induce RBD to present the detection results of exosomes.

[0040] Figure 6 In the present embodiment, in vivo experiments verified that the gene assembly can induce an immune response to produce antibodies.

[0041] Figure 7 In the examples of the present invention, mice were killed 10 days after the second round of immunization, and the response results of RBD-specific T cells after stimulation with the peptide library were summarized. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the specific embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In the present invention, the upstream refers to the 5' end, and the downstream refers to the 3' end.

[0044] Example 1

[0045] A novel coronavirus vaccine production system based on synthetic biology self-assembly, the system includes gene components capable of expressing novel coronavirus antigens, the gene components are editable or synthesizable because they are base sequences. The system containing these gene components can enrich in tissues and organs in the host body and express antigens on the surface of vesicles and self-assemble into vesicle complex sequences, and the vesicle complex can induce an immune response, then the system or the vesicle complex can be called a vaccine.

[0046] The above-mentioned novel coronavirus includes the virus itself and its mutants. Correspondingly, the novel coronavirus antigen includes the currently existing novel coronavirus antigen and any one or several amino acid mutation sequences in the existing antigen sequence, where several refers to 2 or more.

[0047] The novel coronavirus antigen sequences and mutant sequences that can be expressed in this example are listed as follows:

[0048] 1. Novel Coronavirus N501Y:

[0049] Seq No.5: (nucleic acid sequence encoding the novel coronavirus N501Y antigen)

[0050] ;

[0051] Seq.

[0052] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPGVTYGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF。

[0053] 2、coronavirus DETAILS:

[0054] Seq.

[0055] ;

[0056] Seq.

[0057] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT GCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF.

[0058] 3. Novel Coronavirus OMICRON:

[0059] Seq No.9: (Nucleic acid sequence encoding novel coronavirus OMICRON antigen)

[0060] agagtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttgatgaagtttttaacgccaccaaatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattttgcaccatttttcacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaatattgctgattataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaagcttgattctaaggttagtggtaattataattacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtaacaaaccttgtaatggtgttgcaggttttaattgttactttcctttacgatcatatggtttccgacccacttatggtgttggtcaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttc;

[0061] Seq No.10: (Amino acid sequence of the OMICRON antigen of the novel coronavirus)

[0062] RVQPTESIVRFPNITNLCPFDEVFNATKFASVYAWNRKRISNCVADYSVLYNFAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF。

[0063] As a further preferred embodiment, the gene component is selected from at least one of the following sequences: Q1: pcDNA6.2-RBD-HIS or a sequence more than 70% homologous to the pcDNA6.2-RBD-HIS sequence;

[0064] Q2: pcDNA6.2-HIS or a sequence with more than 70% homology to the pcDNA6.2-HIS sequence;

[0065] Q3: pcDNA6.2-RBD (WT) or a sequence with more than 70% homology to the pcDNA6.2-RBD (WT) sequence; the homologous sequences include but are not limited to pcDNA6.2-RBD (E484K), pcDNA6.2-RBD (N501Y), pcDNA6.2-S1 (D614G), etc. These sequences have been discovered, and others that have not been discovered or may appear in the future are also within this range.

[0066] For example, the gene component can be selected from one or more of Q1, Q2 or Q3. If there is more than one, it can be selected from Q1 and Q2, Q2 and Q3, Q1 and Q3, or Q1, Q2 and Q3, and so on.

[0067] The above homologous sequences include sequences obtained by adding one or more bases, reducing one or more bases, or replacing any one or more bases on the basis of the original sequence; several includes 2 and more. Therefore, the preparation of the new coronavirus and its mutant vaccines are currently within the scope of protection of this application.

[0068] As a further preferred embodiment, the vaccine production system also includes a signal peptide base sequence fragment that enables the new coronavirus antigen to be expressed on the surface of the vesicle. Preferably, the above-mentioned gene component is located downstream of the signal peptide base sequence fragment, that is, at the 3' end of the signal peptide.

[0069] The signal peptide base sequence in this example is listed as follows:

[0070] Seq No.11: (TPA expression sequence)

[0071] atggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccagccaggaaatccatgcccgattcagaagaggagccaga;

[0072] Seq No.12: (Sp signal peptide)

[0073] gatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccagccaggaaatccatgcccgattcagaaga.

[0074] As a further preferred embodiment, the signal peptide base sequence fragment preferably includes a first sequence, which is a sequence containing seq1 or a sequence that is more than 70% homologous to the seq1 sequence, such as 70%, 75%, 80%, 85%, 90%, 95%, etc.; the homologous sequence includes a sequence obtained by adding one or more bases, reducing one or more bases, or replacing any one or more bases on the basis of seq1; several includes 2 and more. Although other types of signal peptides can be selected, it has been found through experiments that the use of seq1 or its homologous sequence in combination with other gene fragments of the present application has higher efficiency, better antigen concentration and antibody titer, and the antibody can be comparable to mature vaccines in the prior art. Therefore, this sequence is preferred.

[0075] Seq1 is ATGTGCCTTCCTCCGGTTAAAGGCGCAAAGCTCATCCTGATC TTTCTGTTCCTAGGAGCCGTTCAGTCCAATGCATTGATAGTTAATTTGACAGATTCAAAGGGTACTTGCCTTTATGCTCGA.

[0076] As a further preferred embodiment, the vaccine production system also includes a transmembrane protein base sequence fragment that enables the new coronavirus antigen to anchor on the surface of the vesicle and self-assemble to form a vesicle complex. Preferably, the gene component is located upstream of the transmembrane protein base sequence fragment, that is, at the 5' end of the transmembrane protein.

[0077] As a further preferred embodiment, the transmembrane protein base sequence fragment includes a second sequence, which is a sequence containing seq2 or a sequence that is more than 70% homologous to the seq2 sequence, such as 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, etc.; the homologous sequence includes a sequence obtained by adding one or more bases, reducing one or more bases, or replacing any one or more bases on the basis of seq2; several includes 2 and more. Although other types of transmembrane proteins can be selected, it has been found through experiments that the use of seq2 or its homologous sequence in combination with other gene fragments of the present application has higher efficiency, better antigen concentration and antibody titer, and the antibody can be comparable to mature vaccines in the prior art. Therefore, this sequence is preferred.

[0078] The transmembrane protein is lamp2b transmembrane protein. The gene assembly can correctly express the RBD antigen and the lamp2b skeleton can present the RBD antigen on the exosomes. Figure 3-4 shown.

[0079]

[0080] In this embodiment, the transmembrane protein base sequence that enables the antigen to be anchored on the surface of the vesicle and self-assembled to form a vesicle complex can also be two skeletons, CD9 and CD81. These two skeletons do not require signal peptides and can directly present the antigen to the exosomes. The specific sequences are as follows:

[0081] Seq No.13: (CD81 backbone sequence)

[0082] atgggggtggagggctgcaccaaatgcatcaaatacctgctcttcgtcttcaatttcgtcttctggctggctggaggcgtgatcctaggtgtagctctgtggttgcgtcatgatccacagaccaccagcctgctgtacctggaactg— AGATCTCTAGCCACC (the middle is the antigen sequence position) ACCGGTGGAGCTCGAATCAGATCT —;

[0083] Seq No.14: (CD9 backbone sequence)

[0084] atgccggtcaaaggaggtagcaagtgcatcaaatacctgctcttcggatttaacttcatcttctggctcgctggcattgcagtgcttgctattggactatggctccgattcgactctcagaccaagagcatcttcgagcaagagaataac— AGATCTCTAGCCACC (the middle is the antigen sequence position) ACCGGTGGAGCTCGAATCAGATCT— ;

[0085] The expression results of the above different transmembrane protein skeletons in exosomes are as follows Figure 2 shown.

[0086] As a further preferred embodiment, the system further comprises a promoter, which is preferably a CMV promoter; the promoter is arranged upstream of the base sequence component, and if it contains a signal peptide base sequence fragment, the promoter is located upstream of the signal peptide base sequence fragment.

[0087] The sequence of the promoter includes a third sequence, which is a sequence containing seq3 or a sequence that is more than 70% homologous to the seq3 sequence, such as 70%, 75%, 80%, 85%, 90%, 95%, etc.; the homologous sequence includes a sequence obtained by adding one or more bases, reducing one or more bases, or replacing any one or more bases on the basis of seq3; several includes 2 and more. Although other types of promoters can be selected, it has been found through experiments that the use of seq3 or its homologous sequence in combination with other gene fragments of the present application has higher efficiency, better antigen concentration and antibody titer, and the antibody can be comparable to mature vaccines in the prior art. Therefore, this sequence is preferred.

[0088] seq3:.

[0089] As a further preferred embodiment, the system further comprises a termination signal, wherein the termination signal is located downstream of the transmembrane protein base sequence fragment, and the termination signal is preferably an HSV termination signal.

[0090] The sequence of the termination signal includes a fourth sequence, which is a sequence containing seq4 or a sequence that is more than 70% homologous to the seq4 sequence; such as 70%, 75%, 80%, 85%, 90%, 95%, etc. The homologous sequence includes a sequence obtained by adding one or more bases, reducing one or more bases, or replacing any one or more bases on the basis of seq4; several includes 2 and more. Although other types of termination signals can be selected, it has been found through experiments that the use of seq4 or its homologous sequence in combination with other gene fragments of the present application is more efficient, has better antigen concentration and antibody titer, and the antibody can be comparable to mature vaccines in the prior art. Therefore, this sequence is preferred.

[0091] seq4: CGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTC.

[0092] As a further preferred embodiment, any one or several bases in the system are independently modified bases or unmodified bases; wherein the modification includes thio modification, fluoro modification, amino modification, and methoxy modification. The stability of the base sequence can be increased by appropriate modification.

[0093] In this embodiment, the tissues and organs include all cells, tissues and organs of the host, including but not limited to blood, muscles, liver, spleen, lungs, stomach, intestines, gallbladder, pancreas, brain, heart, kidneys, etc., the vesicles are exosomes, and the vesicle complexes are exosome complexes.

[0094] The administration methods of the above system or the above vaccine include but are not limited to oral administration, inhalation, subcutaneous injection, intramuscular injection, intravenous injection, etc.

[0095] Example 2

[0096] On the basis of Example 1, a method for producing a virus vaccine based on synthetic biology self-assembly, the method comprising constructing a system as described in any of the above paragraphs, the system being a sequence that can be enriched in host cells, tissues and / or organs, express antigens on the surface of vesicles and self-assemble into vesicle complexes, the vesicle complexes being vaccines that can induce an immune response. Specifically, the synthetic base sequence components and other sequences are synthesized according to any of the arrangements described in this application.

[0097] The method in this embodiment is simple and rapid, and can synthesize a large number of different sequences in a short period of time. Therefore, the vaccine production system of the present invention can produce rapidly and efficiently.

[0098] Example 3

[0099] Based on Example 1 or 2, a vector system is provided, which comprises the vaccine production system described in Example 1 or 2 and a vector for loading or carrying the system. The vector may be a plasmid or a virus, or other types of vectors.

[0100] Example 4: In vitro experiments

[0101] The present invention synthesizes pcDNA6.2-RBD-HIS and pcDNA6.2-HIS gene components in vitro, wherein the upstream of the gene component is a signal peptide and the downstream is lamp2b, and a promoter is connected to the upstream of the signal peptide and a termination signal is connected to the downstream of lamp2b to form the system in the present application, and constructs plasmids containing the above two systems respectively; transfects cells and injects mice to verify that the gene components can correctly assemble exosomes containing antigens.

[0102] ① Experimental process / method: 293T and huh-7 cell lines were transfected, and exosomes were extracted from the culture medium using a kit for Western Blot. SARS-CoV / SARS-CoV-2 Spike Antibody, Rabbit PAb, AntigenAffinity Purified (40150-T62-COV2), and His-Tag Monoclonal Antibody were used as primary antibodies, respectively.

[0103] The results showed that the exosomes contained his expression and RBD expression, indicating that the system of the present invention can express the antigen corresponding to the above gene fragment and express it on the exosomes.

[0104] ② Experimental process / methods: 293T and huh-7 cell lines were transfected with mock, HIS (negative control), HIS-RBD (positive control), and RBD (negative control), and the culture medium and exosomes extracted from the culture medium were collected, respectively, and ELISA experiments were performed to detect the expression of RBD.

[0105] The results showed that transfection of the plasmid allowed cells to secrete exosomes containing RBD on their surface.

[0106] ③ Experimental process / method: Through immunoprecipitation experiment, pull down experiment was performed using His-Tag monoclonal antibody and 40150-T62-COV2.

[0107] The results showed that the protein was correctly expressed on the surface of exosomes.

[0108] Example 5: In vivo experiments

[0109] The systems containing pcDNA6.2-RBD (WT), pcDNA6.2-RBD (E484K), pcDNA6.2-RBD (N501Y), and pcDNA6.2-S1 (D614G) gene components were injected intramuscularly and intravenously in vivo, and the expression of antigens and antibodies was detected to verify that they could play the role of vaccine.

[0110] Experimental process: pcDNA6.2-RBD (WT), pcDNA6.2-RBD (E484K), pcDNA6.2-RBD (N501Y), pcDNA6.2-S1 (D614G) gene components were synthesized in vitro. For each gene component, the upstream was a signal peptide and the downstream was lamp2b. A promoter was connected to the upstream of the signal peptide and a termination signal was connected to the downstream of lamp2b to form the system in this application. Plasmids containing the above four systems were constructed respectively.

[0111] The above gene components were extracted in large quantities using the Tiangen Endotoxin-Free Plasmid Extraction Kit: pcDNA6.2-RBD (WT), pcDNA6.2-RBD (E484K), pcDNA6.2-RBD (N501Y), and pcDNA6.2-S1 (D614G). They were injected at 5 mg / kg via tail vein and intramuscular injection, as well as intramuscular + lipo injection, with a volume of 50-100 μl. Orbital blood was collected from mice at 6, 9, 12, and 24 hours after injection, and the expression of antigens was detected by ELISA. Orbital blood was collected on days 7, 14, and 28, and antigen and antibody expression was detected by ELISA.

[0112] The specific antigen tests are as follows:

[0113] 1. ACE2 (ACE-HM401) was coated at a concentration of 5 μg / ml, 100 μl / well, and incubated overnight at 4°C;

[0114] 2. Wash three times with PBST (0.05% Tween20 in PBS), 300 μl per well, add 5% BSA / PBS, and block at 4 degrees Celsius for 12 hours;

[0115] 3. Discard the solution and wash twice with PBST;

[0116] 4. Add 100 μl of serially diluted serum and RBD standard (SARS-CoV-2 RBD-HisRecombinant Protein 40592-V08B) to each well and incubate at room temperature for 1 hour.

[0117] 4. Wash three times with PBS, and use SARS-CoV-2 (2019-nCoV) Spike S1 Antibody (SinoBiological) in PBST at 1:5000 for 1 hour at room temperature;

[0118] 5. Wash three times with PBST, dilute Goat Anti-Rabbit IgG-Fc Secondary Antibody (HRP) at a ratio of 0.2μg / ml, add 100μl to each well, incubate at room temperature for 1h, add 100μl TMB colorimetric solution, and incubate at 37 degrees in the dark for 10-30min;

[0119] 6. Add an equal volume of 1M sulfuric acid and detect OD450 / 650. (OD = OD450-650)

[0120] The results showed that: Figure 5 As shown, the expression of antigens corresponding to the above genes can be detected in serum exosomes after injection.

[0121] The specific antibody tests are as follows:

[0122] Serum samples were inactivated at 56°C for 30 min before use.

[0123] 1. RBD (currently obtained 1.5mg / ml in PBS) was diluted to 5μg / ml with coating solution (0.75g sodium carbonate, 1.46g sodium bicarbonate, 500ml ddW, pH 9.6), 100μl per well (equivalent to 500ng RBD / well), and incubated overnight at 4 degrees; 2. PBST (0.05% tween in PBS) 300μl per well, incubated for 30s, discarded and added with new PBST, and washed 3 times; patted dry on absorbent paper;

[0124] 3. 5% (2%) BSA (PBS), block at 4°C for 12 h (2 h at room temperature);

[0125] 4. Discard the solution and wash twice with PBST;

[0126] 5. Dilute the serum 100 times with PBS and then dilute it in a 2-fold gradient, for a total of 7 gradients (covering 10,000 titers), add 100 μl to each well, dilution steps (do not count duplicate wells, use a separate plate to dilute first and then add to the ELISA plate). The starting well is 2 μl of original serum + 198 μl of PBS (3 μl + 247 μl), pipetting and mixing, the next well is aspirated 90 μl + 90 μl of PBS (120 + 120), pipetting and mixing, and each gradient is diluted in the same way. After the dilution is completed, aspirate 50 μl (100 μl) into the ELISA plate. Incubate in a 37-degree incubator for 30 minutes (37 degrees, 60 minutes)

[0127] Wash 3 times with PBST, dilute goat anti-mouse IgG in PBS at 1:5000, add 100μl to each well, and incubate at 37 degrees for 30 minutes; wash 5 times with PBST, add 100μl TMB reaction solution and incubate at 37 degrees for 10-30 minutes, add 100μl (50μl), 2M sulfuric acid to terminate the reaction, measure the wavelength at 450nm, and calculate the titer with OD>2 (1.4) times of negative serum as the termination point. The results showed that: Figure 6 As shown, antibodies elicited by the above antigens can be detected in the serum after injection.

[0128] At the same time, the inventors also verified the effect of peptide library stimulation on RBD-specific T cell response. Specifically, the same experimental mice were used as above, and they were killed 10 days after the second round of immunization. Blood was collected from the eye sockets. After stimulation with the peptide library, RBD-specific T cells (CD4 + 、CD8 + ) reaction results, such as Figure 7 shown.

[0129] From the above results, it can be seen that the injection of gene components causes mice to obtain high titers of specific antibodies and neutralizing antibodies, and the antibody titers are comparable to those of commercial vaccines.

[0130] The plasmid structure inserted into the system of the present invention used in Examples 4 and 5 is as follows Figure 1 Of course, the system of the present application can be inserted into other types of plasmids or viruses, etc., and can achieve better results.

[0131] In the present invention, the inventors also selected sequences after mutation of certain sites of signal peptides and sequences after mutation of certain sites of transmembrane proteins (such as lamp2b) and conducted experiments respectively. The results showed that the mutated sequences can also interact with antigen genes to achieve the production of antibodies in vivo.

[0132] The pcDNA6.2-RBD-HIS, pcDNA6.2-HIS, pcDNA6.2-RBD (WT) and the like in this application are all prior art, and are not listed in this application because the sequences are too long. The pcDNA6.2-RBD (E484K), pcDNA6.2-RBD (N501Y), and pcDNA6.2-S1 (D614G) are all sequences obtained after mutation of the specific sites shown in brackets based on pcDNA6.2-RBD (WT).

[0133] The present invention provides clear sequences for related structures. However, those skilled in the art should understand that minor changes based on the above sequences do not affect the protection of the sequences described in the present invention and are within the scope of protection of the present invention. That is, all sequences in this application include the addition, deletion or replacement of one or more bases or amino acids on the basis thereof, and the multiple here refers to 2 or more than 2.

[0134] Finally, it should be noted that the above-described embodiments are only used to illustrate the technical solution of the utility model, rather than to limit it. Although the utility model has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the utility model.

Claims

1. A new coronavirus vaccine production system based on synthetic biology, Its characteristics are: The system includes a base sequence component that can express antigens of the new coronavirus. The system is a sequence that can enrich in host tissues and organs and express antigens on the surface of vesicles and self-assemble them into vesicle complexes, which can trigger an immune response, The base sequence component is selected from at least one of the following sequences: Q1: pcDNA6.2-RBD-HIS; Q2: pcDNA6.2-RBD (WT), pcDNA6.2-RBD (E484K), pcDNA6.2-RBD (N501Y); The new coronavirus vaccine production system also includes a transmembrane protein base sequence fragment that can anchor the new coronavirus antigen on the vesicle surface and self-assemble to form a vesicle complex, and the signal peptide base sequence fragment is seq1: ATGTGCCTCTCTCTCCCGGTTAAAGGCGCAAAGCTCATCCTGATCTTTCTGTTCCTAGGAGCCGTTCAGTCCAATGCATTGATAGTTAATTTGACAGATTCAAAGGGTACTTGCCTTTATGCTCGA; the new coronavirus vaccine production system also includes a signal peptide base sequence fragment that can express the new coronavirus antigen on the vesicle surface; The sequence of the transmembrane protein base sequence fragment is the sequence of seq2: TCCGGAGGTGCAGAATGGGAGATGAATTTCACAATAACATATGAAACTACAAACCAAACCAATAAACTAAAACTATAAACCATTGCAGTACCTGACAAGGCGACACACGATGGAAGCAGTTGTGGGGATG ACCGGAATAGTGCCAAAATAATGATACAATTTGGATTCGCTGTCTCTTGGGCTGTGAATTTTACCAAGGAAGCATCTCATTATTCAATTCATGACATCGTGCTTTCCTACTACAACTAGTGATAGCACAGTATTTCCTGGTGCTGT AGCTAAAGGAGTTCATACTGTTAAAAATCCTGAGAATTTCAAAGTTCCATTGGATGTCATCTTTAAGTGCAATAGTGTTTTAACTTACAACCTGACTCCTGTCGTTCAGAAATATTGGGTATTCACCTGCAAGCTTTTGTCCAA AATGGTACAGTGAGTAAAAATGAACAAGTGTGTGAAGAAGACCAAACTCCCACCACTGTGGCACCCATCATTCACACCACTGCCCCGTCGACTACAACTACACTCACTCCAACTTCAACACCCACTCCAACTCCAACTCCAACTCC AACCGTTGGAAACTACAGCATTAGAAATGGCAATACTACCTGTCTGGCTACCATGGGCTGCAGCTGAACATCACTGAGGAGAAGGTGCCTTTCATTTTTAACATCAACCCCTGCCACAACCAACTTCACCGGCAGCTGTCAA CCTCAAAAGTGCTCAACTTAGGCTGAACAACAACAGCCAAATTAAGTATCTTGACTTTATCTTGCTGTGAAAAATGAAAAACGGTTCTATCTGAAGGAAGTGAATGTCTACATGTATTTGGCTCAGCTTCAACATTTCCA ACAAGAACCTTAGCTTCTGGGATGCCCCCTCTGGGAAGTTCTTATATGTGCAACAAAGAGCAGGTGCTTTCTGTGTCTAGAGCGTTTCAGATCAACCTTTAACCTTTAACCTAAAGGTGCAACCTTTTAATGTGACAAAAGGACAGTATTC TACAGCCCAGGAGTGTTCGCTGGATGATGACACCATTCTAATACCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACCTAATTGGCAGAAGAAAGACCTATGCTGGATATCAGACTCTGTAA; The system also includes a promoter, which is located upstream of the signal peptide base sequence fragment within the base sequence component, and the sequence of the promoter is seq3: GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT GGCTGACCGCCCAACGACCCCCCCGCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTT GACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGTAGGCGTGTACGGTGGGAGGTCTATAAGCAGAGCT; The system also includes a termination signal, which is located downstream of the nucleic protein base sequence fragment within the base sequence component, and the sequence of the termination signal is the sequence of seq4: CGGCAATAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTC.

2. The new coronavirus vaccine production system based on synthetic biology self-assembled according to claim 1, It is characterized in that Any one or several bases in the new coronavirus vaccine production system are independently modified bases or unmodified bases; among which, the modification includes thio modification, fluoro modification, ammonia modification, and methoxy modification.

3. The new coronavirus vaccine production system based on synthetic biology self-assembled according to claim 1, It is characterized in that The vesicle is an exosome, and the vesicle complex is an exosome complex.

4. A method for producing viral vaccines based on synthetic biology, Its characteristics are: The method comprises constructing a system of any one of claims 1-3, the system being a sequence that is able to enrich in host cells, tissues, and / or organs and express antigens on the surface of a vesicle and self-assemble into a vesicle complex, the vesicle complex being a vaccine capable of eliciting an immune response.

5. A vaccine, Its characteristics are: It includes the new coronavirus vaccine production system as claimed in any one of claims 1-4; The modes of administration of the vaccine include but are not limited to oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.

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