A synthetic biology self-assembly vaccine production system and a method for producing a vaccine

Abstract

The present invention relates to a synthetic biology self-assembly vaccine production system and a method for producing a vaccine, wherein the system comprises a base sequence component capable of expressing an antigen, and the system is a sequence capable of enriching in host tissues and organs, expressing the antigen on the surface of a vesicle, and self-assembling into a vesicle complex, wherein the vesicle complex can induce an immune response. In the present invention, the base sequence component has the advantages of low production cost, stable physical and chemical properties, efficient antigen assembly and secretion process, fast response to pathogen mutation, 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 synthetic biology self-assembly vaccine production system, a vaccine, a carrier system and a method for producing a vaccine, which are used for the development of a new vaccine in the present application. Background Art

[0002] Vaccines refer to biological products made from various pathogenic microorganisms for use in vaccination. For the general public and families, vaccination is the most economical and effective way to prevent and control diseases, which can reduce the occurrence of diseases and reduce medical expenses.

[0003] At present, there are many types of vaccines, mainly including inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, and viral vector vaccines. However, for different diseases, these vaccines all have some shortcomings to a greater or lesser extent. For example, inactivated vaccines have the disadvantage of being unable to quickly respond to antigenic variations represented by RNA viruses. Once the key immune recognition sites of key viruses mutate, the antibodies produced by the inactivated vaccines will easily fail. The strains selected for live attenuated vaccines are alive, and in terms of safety, they are not easily accepted by the public. The sequences of nucleic acid vaccines are flexible and editable, but they are sometimes easily degraded in the body, and they also put forward higher requirements for transportation and storage conditions. The vaccine needs to be synthesized in vitro, and the cost is relatively high. Viral vector vaccines have high production costs, and viral vaccines represented by adenovirus vector vaccines may weaken their effects due to the pre-immunity phenomenon in the body. Moreover, the above vaccines generally have the problems of difficult development, long response 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 50-200nm. 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 there are 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 view of the defects of the prior art, the present invention provides a synthetic biology self-assembly vaccine production system and a method for producing a vaccine, aiming to overcome the deficiencies in 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 synthetic biology self-assembly vaccine production system, which includes a base sequence component capable of expressing antigens. The system is a sequence that can be enriched in tissues and organs in the host body and express antigens on the surface of vesicles and self-assemble into vesicle complexes, and the vesicle complexes can trigger an immune response.

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

[0010] Furthermore, the antigens include but are not limited to tuberculosis antigens, hepatitis A antigens, hepatitis B antigens, measles antigens, poliomyelitis antigens, diphtheria-tetanus-pertussis antigens, Japanese encephalitis antigens, pertussis antigens, diphtheria antigens, tetanus antigens, rabies antigens, influenza antigens, pneumonia antigens, varicella antigens, rotavirus antigens, hand, foot and mouth disease antigens, meningitis antigens, epidemic encephalitis B antigens, pneumonia antigens, new coronavirus antigens, HPV antigens, tumor antigens, mumps antigens, rubella antigens or antigens of mutants of the above diseases, etc. Correspondingly, the base sequence components also include base sequences that can express the aforementioned antigens, and these base sequences include sequences that express antigens and sequences that are more than 70% homologous to the sequences that express antigens, and some mutants can be included.

[0011] Furthermore, the system also includes a signal peptide base sequence fragment capable of expressing the antigen on the surface of the vesicle. Preferably, the base sequence component is located downstream of the signal peptide base sequence fragment, that is, located at the 3' end of the signal peptide.

[0012] 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.

[0013] Furthermore, the system also includes a transmembrane protein base sequence fragment that enables the antigen to anchor on the vesicle surface 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, located at the 5' end of the transmembrane protein.

[0014] 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.

[0015] Furthermore, the system also includes a promoter, which may be a CMV promoter; the promoter is disposed upstream of the base sequence component, and if a signal peptide base sequence fragment is contained, the promoter is located upstream of the signal peptide base sequence fragment.

[0016] Furthermore, the system also includes a terminator, which is located downstream of the base sequence component; if the system contains the transmembrane protein base sequence fragment, the terminator is located downstream of the transmembrane protein base sequence fragment, and the terminator may be an HSV terminator.

[0017] 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.

[0018] Furthermore, 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.

[0019] Another invention of the present invention is to provide a method for producing a vaccine by biological 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 responses. Specifically, the base sequence components are first synthesized, and then integrated with other sequences according to the description of the present application.

[0020] Another aspect of the present invention is to provide a vaccine, which comprises the system described in any one of the above paragraphs.

[0021] Furthermore, the administration methods of the vaccine include but are not limited to oral administration, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.

[0022] The types of vaccines include but are not limited to BCG, hepatitis A vaccine, hepatitis B vaccine, measles vaccine, polio vaccine, diphtheria, pertussis, tetanus, rabies vaccine, influenza vaccine, pneumonia vaccine, varicella vaccine, rotavirus vaccine, hand, foot and mouth disease vaccine, meningitis vaccine, epidemic encephalitis B, pneumonia vaccine, tuberculosis vaccine, new coronavirus vaccine, HPV vaccine, tumor vaccine, mumps vaccine, and rubella.

[0023] 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 capable of loading or carrying the system. The vector includes but is not limited to plasmids and viruses.

[0024] The main beneficial effects of the present invention are:

[0025] In the vaccine production system of the present invention, corresponding base sequence components (such as gene components) are selected according to the needs of different diseases. 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.

[0026] 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.

[0027] 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, the immunity of related diseases is achieved, so its antigen assembly and secretion process is efficient, the immune response triggered is strong, and it is safe and protective, etc. It also avoids some toxic and side effects in the prior art.

[0028] The base sequence components in this system are highly editable, and can be edited to respond quickly to a certain disease or any mutation thereof, and can have a higher or faster response even to a mutation or sudden disease. Therefore, it can avoid the current problem that vaccines become ineffective due to mutations of certain diseases, and new vaccines need to be re-developed and studied over a long period of time.

[0029] The system and method of the present invention have good overall effects. It is a brand-new vaccine and vaccine synthesis method, which is a huge breakthrough in the biological and medical fields and has milestone significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the antigen expression of hpv-L1 detected in the serum described in the example of the present invention.

[0031] Figure 2 Schematic diagram of antibodies produced by HPV antigens detected in serum as described in an embodiment of the present invention.

[0032] Figure 3 This is a schematic diagram of the expression of HBsAg antigen detected in the serum described in the embodiment of the present invention.

[0033] Figure 4 This is a schematic diagram of the amount of antibodies produced by hepatitis B virus HBV antigen detected in the serum described in the embodiment of the present invention.

[0034] Figure 5 Schematic diagram of the expression of HPV16 E7 antigen detected in the serum described in the examples of the present invention.

[0035] Figure 6 This is a schematic diagram of the amount of antibodies produced by human papillomavirus HPV16 antigen detected in the serum described in the examples of the present invention.

[0036] Figure 7 Schematic diagram of the expression of H1N1 HA antigen detected in serum according to the embodiment of the present invention.

[0037] Figure 8 This is a schematic diagram of the amount of antibodies produced by influenza A virus H1N1 antigen detected in the serum described in the examples of the present invention.

[0038] Fig. 9 This is a diagram showing different RBD expression levels produced by different transmembrane protein base sequences in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] 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.

[0040] In the present invention, the upstream refers to the 5' end, and the downstream refers to the 3' end.

[0041] Example 1

[0042] One of the inventions of the present invention is to provide a synthetic biology self-assembly vaccine production system, the system includes a base sequence component capable of expressing an antigen, the base sequence component is an editable or synthesizable gene component. The system is a sequence that can be enriched in tissues and organs in the host body and express antigens on the surface of vesicles and self-assemble into vesicle complexes, and the vesicle complexes are vaccines that can induce immune response reactions.

[0043] The antigens include but are not limited to tuberculosis antigens, hepatitis A antigens, hepatitis B antigens, measles antigens, poliomyelitis antigens, diphtheria-tetanus-pertussis antigens, Japanese encephalitis antigens, pertussis antigens, diphtheria antigens, tetanus antigens, rabies antigens, influenza antigens, pneumonia antigens, varicella antigens, rotavirus antigens, hand, foot and mouth disease antigens, meningitis antigens, epidemic encephalitis B antigens, pneumonia antigens, new coronavirus antigens, HPV antigens, mumps antigens, tumor antigens, rubella antigens or antigens of mutants of the above diseases, etc. Correspondingly, the base sequence components also include base sequences that can express the aforementioned antigens, and these base sequences include sequences that express antigens and sequences that are more than 70% homologous to the sequences that express antigens, such as 70%, 75%, 80%, 85%, 90%, 95%, etc., and some mutants can be included.

[0044] The antigens that can be expressed in this example are listed as follows:

[0045] 1. Hepatitis B virus HBV:

[0046] Seq No.8: (HBsAg antigen sequence)

[0047] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGST TTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI*;

[0048] Seq No.9: (HBsAg expression sequence)

[0049] atggagaacatcacatcaggattcctaggacccctgctcgtgttacaggcggggtttttcttgttgacaagaatcctcacaataccgcagagtctagactcgtggtggacttctctcaattttctagggggatctcccgtgtgtcttggccaaaattcgcagtccccaacctccaatcactcaccaacctcctgtcctccaatttgtcctggttatcgctggatgtgtctgcggcgttttatcatattcctcttcatcctgctgctatgcctcatcttcttattggttcttctggattatcaaggtatgttgcccgtttgtcctctaattccaggatcaacaacaaccagtacgggaccatgcaaaacctgcacgactcctgctcaaggcaactctatgtttccctcatgttgctgtacaaaacctacggatggaaattgcacctgtattcccatcccatcgtcctgggctttcgcaaaatacctatgggagtgggcctcagtccgtttctcttggctcagtttactagtgccatttgttcagtggttcgtagggctttcccccactgtttggctttcagctatatggatgatgtggtattgggggccaagtctgtacagcatcgtgagtccctttataccgctgttaccaattttcttttgtctctgggtatacatttaa。

[0050] 2. Human papillomavirus HPV16:

[0051] Seq No.10: (HPV16 E7 antigen sequence)

[0052] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCK CDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP*;

[0053] Seq No.11: (HPV16 E7 expression sequence)

[0054] atgcatggagatacacctacattgcatgaatatatgttagatttgcaaccagagacaactgatctctactgttatgagcaattaaatgacagctcagaggaggaggatgaaatagatggtccagctggacaagcagaaccggacagagcccattacaatattgtaaccttttgttgcaagtgtgactctacgcttcggttgtgcgtacaaagcacacacgtagacattcgtactttggaagacctgttaatgggcacactaggaattgtgtgccccatctgttctcagaaaccataa。

[0055] 3. Influenza A virus H1N1

[0056] Seq No.12: (H1N1 HA antigen sequence)

[0057] MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTTKGVTAACSHAGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPSNSKDQQNIYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPRYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPFQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI*;

[0058] Seq No.13: (H1N1 HA antigen expression sequence)

[0059]

[0060] As a further preferred embodiment, the system further comprises a signal peptide base sequence fragment capable of expressing the antigen 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.

[0061] The signal peptide base sequence in this example is listed as follows:

[0062] Seq No.14: (TPA expression sequence)

[0063] atggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccagccaggaaa tccatgcccgattcagaagaggagccaga;

[0064] Seq No.15: (Sp signal peptide)

[0065] gatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccagccaggaaatcc atgcccgattcagaaga.

[0066] 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 has higher efficiency, better antigen concentration and antibody titer, and the antibody can be comparable to the current mature vaccine. Therefore, this sequence is preferred.

[0067] Seq1 is ATGTGCCTCTTCCCGGTTAAAGGCGCAAAGCTCATCCTGATCTTTCTGTTCCTAGGAGCCGTTCAGTCCAATGCATTGATAGTTAATTTGACAGATTCAAAGGGTACTTGCCTTTATGCTCGA.

[0068] As a further preferred embodiment, the system further comprises a transmembrane protein base sequence fragment that enables the antigen to be anchored on the vesicle surface and self-assembled 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.

[0069] 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%, 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 has higher efficiency, better antigen concentration and antibody titer, and the antibody can be comparable to the current mature vaccine. Therefore, this sequence is preferred.

[0070]

[0071] 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:

[0072] Seq No.16: (CD81 backbone sequence)

[0073] atgggggtggagggctgcaccaaatgcatcaaatacctgctcttcgtcttcaatttcgtcttctggctggctggaggcgtg atcctaggtgtagctctgtggttgcgtcatgatccacagaccaccagcctgctgtacctggaactg—AGATCTCTAGCCACC (the middle is the antigen sequence position) ACCGGTGGAGCTCGAATCAGATCT—;

[0074] Seq No.17: (CD9 backbone sequence)

[0075] atgccggtcaaaggaggtagcaagtgcatcaaatacctgctcttcggatttaacttcatcttctggctcgctggcat tgcagtgcttgctattggactatggctccgattcgactctcagaccaagagcatcttcgagcaagagaataac—AGATCTCTAGCCACC (the middle is the antigen sequence position) ACCGGTGGAGCTCGAATCAGATCT—;

[0076] The expression results of the above different transmembrane protein skeletons in exosomes are as follows Fig. 9 shown.

[0077] As a further preferred embodiment, the system further comprises a promoter, and the promoter is located upstream of the signal peptide base sequence fragment.

[0078] 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 has higher efficiency, better antigen concentration and antibody titer, and the antibody can be comparable to the current mature vaccine. Therefore, this sequence is preferred.

[0079] seq3:.

[0080] As a further preferred embodiment, the system further comprises a terminator, and the terminator is located downstream of the transmembrane protein base sequence fragment.

[0081] 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 signal fragments can be selected, it has been found through experiments that the use of seq4 or its homologous sequence in combination with other gene fragments has higher efficiency, better antigen concentration and antibody titer, and the antibody can be comparable to the current mature vaccine. Therefore, this sequence is preferred.

[0082] seq4: CGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTC.

[0083] 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.

[0084] As a further preferred embodiment, the tissues and organs include all cells, tissues and organs of the host, including but not limited to blood, muscle, liver, spleen, lung, stomach, intestine, gallbladder, pancreas, brain, heart, kidney, etc., the vesicle is an exosome, and the vesicle complex is an exosome complex.

[0085] As a further preferred embodiment, the system of the present invention is a vaccine, and the administration of the vaccine includes oral administration, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.

[0086] The types of vaccines include but are not limited to BCG vaccine, hepatitis A vaccine, hepatitis B vaccine, measles vaccine, polio vaccine, diphtheria, pertussis, tetanus, rabies vaccine, influenza vaccine, pneumonia vaccine, varicella vaccine, rotavirus vaccine, hand, foot and mouth disease vaccine, meningitis vaccine, epidemic encephalitis Japanese, pneumonia vaccine, tumor vaccine, tuberculosis vaccine, new coronavirus vaccine, HPV vaccine, mumps vaccine, and rubella.

[0087] Example 2

[0088] Based on Example 1, a method for producing a biological self-assembly vaccine comprises constructing a system as described in any of the above paragraphs, which is 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, and the vesicle complexes are 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.

[0089] 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.

[0090] Example 3

[0091] 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.

[0092] Example 4: In vitro experiments

[0093] The present invention synthesizes pcDNA6.2-HPV-L1, pcDNA6.2-HPV-16, pcDNA6.2-HBV-S, and pcDNA6.2-H1N1-NA gene components in vitro, respectively, sets the signal peptide of the present invention upstream of the above gene components, sets the lamp2b of the present invention downstream, connects the promoter upstream of the signal peptide, and connects the termination signal downstream of lamp2b, respectively, to form four groups of systems in the present application, corresponding to two HPV vaccines, HBV vaccine, and influenza A H1N1 vaccine. Transfected cells verify that the gene components can correctly assemble exosomes containing antigens;

[0094] ① Experimental process / method: 293T and huh-7 cell lines were transfected, and exosomes from the culture medium were extracted using a kit for Western Blot, using antibodies against the corresponding antigens as primary antibodies.

[0095] ELISA experiments were performed to detect the expression of corresponding antigens.

[0096] ② Experimental process / method: Through immunoprecipitation experiment, pull down experiment was performed using corresponding antibodies.

[0097] The results showed that the protein could be correctly expressed on the surface of exosomes.

[0098] This embodiment only uses HPV, hepatitis B, and influenza A as examples for illustration. In fact, other types of disease antigens or antigen genes may also be used, including but not limited to the diseases listed in this application.

[0099] Example 5: In vivo experiments

[0100] The system containing pcDNA6.2-HPV-L1, pcDNA6.2-HPV-16, pcDNA6.2-HBV-S, and pcDNA6.2-H1N1-NA gene components was injected into the muscles and tail veins in vivo, and the expression of antigens and antibodies was detected to verify that it can play the role of vaccine. The four plasmids here correspond to HPV vaccines (two types), HBV vaccines, and influenza A H1N1 vaccines.

[0101] Experimental process: A system containing the above gene components was synthesized in vitro, wherein the antigen genes used were genes that could express the L1 protein of human papillomavirus HPV, the HPV16 protein of human papillomavirus, the S protein of human hepatitis B virus, and the H1N1-NA protein antigen of influenza A virus. Gene components were extracted using the Tiangen endotoxin-free plasmid extraction kit: pcDNA6.2-HPV-L1, pcDNA6.2-HPV-16, pcDNA6.2-HBV-S, pcDNA6.2-H1N1-NA, and injected at 5 mg / kg, respectively, by tail vein and intramuscular injection, and intramuscular + lipo injection, and a commercial vaccine volume of 50-100 μl, physiological saline was used as a negative control (Ctrl), and the mice were bled at the eye sockets 6, 9, 12, and 24 hours after injection, and the expression of antigens was detected by ELISA. The blood was collected from the eye sockets on days 7, 14, and 28, and the corresponding antigen and antibody expression was detected by ELISA.

[0102] The specific antigen tests are as follows:

[0103] 1. The unmodified antibody corresponding to the antigen was coated at a concentration of 5 μg / ml, 100 μl / well, and incubated overnight at 4°C;

[0104] 2. Wash three times with pbsT (PBS with 0.05% Tween20), 300 μl per well, add 5% BSA / PBS, and block at 4°C for 12 h;

[0105] 3. Discard the solution and wash 3 times with PBST;

[0106] 4. Add 100 μl of serially diluted serum and corresponding antigen standard to each well and incubate at 37°C for 1 hour.

[0107] 4. Wash three times with PBST, and incubate with the corresponding biotin-modified primary antibody at 37°C for 1 hour. 5. Wash three times with PBST, and dilute with the corresponding secondary antibody modified with streptomycin, add 100 μl to each well, and add 100 μl TMB colorimetric solution after 1 hour at room temperature, and incubate at 37°C in the dark for 10-30 minutes.

[0108] 6. Add an equal volume of 1 M sulfuric acid and detect OD450 / 650. (OD = OD450-650).

[0109] The results showed that the expression of the above four antigens could be detected in serum exosomes after injection. Figure 1 This is a graph of antigen expression in serum given using hpv-L1 as an example. Figure 3 This is a graph of antigen expression in serum, taking HBsAg as an example. Figure 5 This is a graph of antigen expression in serum using HPV16 E7 as an example. Figure 7 This is a graph of antigen expression in serum using H1N1 HA as an example.

[0110] The specific antibody tests are as follows:

[0111] Serum samples were inactivated at 56°C for 30 min before use.

[0112] 1. Dilute the four antigens HPV-L1, HPV16, HBV-S, and H1N1-NA to 5 μg / ml with coating solution (0.75 g sodium carbonate, 1.46 g sodium bicarbonate, 500 ml ddW, pH 9.6), 100 μl per well, and let stand overnight at 4 degrees;

[0113] 2. Add 300 μl of PBST (0.05% tween in PBS) to each well, let stand for 30 seconds, discard the solution and add new PBST, repeat washing 3 times; pat dry on absorbent paper;

[0114] 3.5% (2%) BSA (PBS), blocked at 4°C for 12 h (2 h at room temperature);

[0115] 4. Discard the solution and wash twice with PBST;

[0116] 5. Dilute the serum 100 times with PBS and then dilute it in 2-fold gradients, 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);

[0117] 6. Wash three times with PBST, dilute goat anti-mouse IgG at 1:5000 in PBS, add 100 μl to each well, and incubate at 37 degrees for 30 minutes;

[0118] 7. Wash 5 times with PBST, add 100 μl TMB reaction solution and incubate at 37 degrees for 10-30 minutes, add 100 μl (50 μl) 2M sulfuric acid to terminate the reaction and measure at 450nm wavelength. Calculate the titer with OD>2 (1.4) times of negative serum as the termination point.

[0119] The results showed that antibodies produced by the above antigens could be detected in the serum after injection. Figure 2 The amount of antibodies induced by HPV-L1 antigen. Figure 4 The amount of antibodies induced by HBV antigens. Figure 6 The amount of antibodies induced by HPV16 antigen. Figure 8 The amount of antibodies induced by H1N1 antigen.

[0120] 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.

[0121] In this embodiment, the sequences of HPV-L1, HBV-S, and H1N1-NA are Seq No. 5, Seq No. 6, and Seq No. 7, respectively.

[0122] This embodiment only uses HPV, hepatitis B, and influenza A as examples for illustration. In fact, other types of disease antigens or antigen genes may also be used, including but not limited to the diseases listed in this application.

[0123] In the present invention, some related structures are given clear sequences. However, those skilled in the art should understand that if small changes are made on the basis of the above sequences, they are all within the scope of protection of the present invention. That is, all sequences in this application include adding, lacking or replacing one or more bases or amino acids on the basis of them, and multiple here refers to 2 or more than 2.

[0124] It should also be noted that the antigens in this application are not limited, and theoretically any antigen can be used. The ones listed are only some antigen types that the inventors have more or less studied for the system of this application.

[0125] 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 synthetic biology self-assembly vaccine production system, characterized in that: The system is capable of expressing base sequence components of antigens, which are sequences that can be enriched in host tissues and organs, express antigens on the surface of vesicles, and self-assemble into vesicle complexes, which can trigger an immune response. The system is a system containing a signal peptide base sequence fragment capable of expressing the antigen on the surface of the vesicle, The signal peptide base sequence fragment is a first sequence, the first sequence is a sequence of seq1, seq1 is ATGTGCCTCTCTCCGGTTAAAGGCGCAAAGCTCATCCTGATC TTTCTGTTCCTAGGAGCCGTTCAGTCCAATGCATTGATAGTTA ATTTGACAGATTCAAAGGGTACTTGCCTTTATGCTCGA; The system is a system containing transmembrane protein base sequence fragments that can anchor antigens on the surface of vesicles and self-assemble to form vesicle complexes. The transmembrane protein base sequence fragment is a second sequence, and the second sequence is a sequence of seq2, and seq2 isACCTGTCTGCTGGCTACCATGGGGCTGCAGCTGAACATCACTGAGGAGAAGGTGCCTTTCATTTTTAACATCAACCCTGCCACAACCAACTTCACCGGCAGCTGTCAACCTCAAAGTGCTCAACTTAGGCTGAACAACAGCCAAATTAAGTATCTTGACTTTATCTTTGCTGTGAAAAATGAAAAACGGTTCTATCTGAAGGAAGTGAATGTCTACATGTATTTGGCTAATGGCTCAGCTTTCAACATTTCCAACAAGAACCTTAGCTTCTGGGATGCCCCTCTGGGAAGTTCTTATATGTGCAACAAAGAGCAGGTGCTTTCTGTGTCTAGAGCGTTTCAGATCAACACCTTTAACCTAAAGGTGCAACCTTTTAATGTGACAAAAGGACAGTATTCTACAGCCCAGGAGTGTTCGCTGGATGATGACACCATTCTAATACCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACCTAATTGGCAGAAGAAAGACCTATGCTGGATATCAGACTCTGTAACACTAA; The system is a system containing a promoter, The sequence of the promoter is the third sequence, and the third sequence is the sequence of seq3, The system is a system containing a terminator, The sequence of the terminator is the fourth sequence, and the fourth sequence is the sequence of seq4; The vector selected in the system is pcDNA6.

2.

2. The synthetic biology self-assembly vaccine production system according to claim 1, characterized in that: The base sequence component is an editable or synthesizable gene component.

3. The synthetic biology self-assembly vaccine production system according to claim 1, characterized in that: The antigen is selected from one or more of tuberculosis antigen, hepatitis A antigen, hepatitis B antigen, measles antigen, poliomyelitis antigen, diphtheria-tetanus-pertussis antigen, Japanese encephalitis antigen, pertussis antigen, diphtheria antigen, tetanus antigen, rabies antigen, influenza antigen, varicella antigen, rotavirus antigen, hand, foot and mouth disease antigen, meningitis antigen, epidemic encephalitis B antigen, pneumonia antigen, new coronavirus antigen, HPV antigen, mumps antigen, rubella antigen and tumor antigen, and correspondingly, the base sequence component is selected as a base sequence capable of expressing the aforementioned antigens.

4. The synthetic biology self-assembly vaccine production system according to claim 1, characterized in that: The signal peptide base sequence fragment is located upstream of the base sequence component.

5. The synthetic biology self-assembly vaccine production system according to claim 1, characterized in that: The transmembrane protein base sequence fragment is located downstream of the base sequence component.

6. The synthetic biology self-assembly vaccine production system according to claim 1, characterized in that: The system is a system containing a promoter; the promoter is arranged upstream of the base sequence component; if the system contains a signal peptide base sequence fragment, the promoter is located upstream of the signal peptide base sequence fragment.

7. The synthetic biology self-assembly vaccine production system according to claim 1, characterized in that: The system is a system containing a terminator, and the terminator is located downstream of the base sequence component; if the system contains the transmembrane protein base sequence fragment, the terminator is located downstream of the transmembrane protein base sequence fragment.

8. The synthetic biology self-assembly vaccine production system according to claim 1, characterized in that: The vesicle is an exosome, and the vesicle complex is an exosome complex.

9. A method for producing vaccines by synthetic biology self-assembly, characterized in that: The method is to construct a system as described in any one of claims 1-8, which is 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, and the vesicle complexes can trigger an immune response.

10. A vaccine, characterized in that: The vaccine is a vaccine comprising the system according to any one of claims 1 to 8; The vaccine is administered by one or more of oral, inhalation, subcutaneous, intramuscular and intravenous injections; The type of vaccine is selected from one or more of BCG, hepatitis A vaccine, hepatitis B vaccine, measles vaccine, polio vaccine, diphtheria, pertussis, tetanus, rabies vaccine, influenza vaccine, pneumonia vaccine, varicella vaccine, rotavirus vaccine, hand, foot and mouth disease vaccine, meningitis vaccine, epidemic encephalitis B, tuberculosis vaccine, new coronavirus vaccine, tumor vaccine, HPV vaccine, mumps vaccine and rubella vaccine.

Images