CN113234744A - mRNA (messenger ribonucleic acid) for coding secretory angiotensin converting enzyme 2 and application thereof - Google Patents
mRNA (messenger ribonucleic acid) for coding secretory angiotensin converting enzyme 2 and application thereof Download PDFInfo
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- CN113234744A CN113234744A CN202110552971.7A CN202110552971A CN113234744A CN 113234744 A CN113234744 A CN 113234744A CN 202110552971 A CN202110552971 A CN 202110552971A CN 113234744 A CN113234744 A CN 113234744A
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Abstract
The invention relates to mRNA for coding secreted angiotensin converting enzyme 2 and application thereof. The mRNA includes a coding region of 70% or more of a signal peptide of angiotensin converting enzyme 2 and a coding region of 70% or more of a peptidase domain. The mRNA coding the secretory angiotensin converting enzyme 2 can express the secretory ACE2 by utilizing a translation system of a host cell, the secretory ACE2 can be secreted to the extracellular space and can be used as a decoy receptor to be combined with an S protein receptor binding domain of SARS-CoV-2, so that the SARS-CoV-2 is competitively inhibited from being combined with an endogenous ACE2 receptor, and a potential intervention strategy is provided for treating SARS-CoV-2.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to mRNA for coding secreted angiotensin converting enzyme 2 and application thereof.
Background
The novel coronavirus SARS-CoV-2 is a novel coronavirus of beta genus named by the world health organization, the virus has envelope, the particle is circular or elliptical, the main structural proteins comprise Spike protein (Spike, S), messenger protein (envelope, E), Membrane protein (Membrane, M) and Nucleocapsid protein (N), and it has been found that SARS-CoV-2 enters host cells by binding with Angiotensin converting enzyme 2 (ACE 2) receptor to infect and replicate the virus.
The human ACE2 gene encodes an ACE2 protein receptor with the full length of 805 amino acids. The receptor consists of an extracellular domain (amino acids 1-740), a single-pass transmembrane helix (amino acids 741-.
Most of the conventional vaccines are in the form of inactivated viruses, and generally have many disadvantages in their use, including: the preparation process is complex, a cell culture method is needed for producing the virus, and certain potential safety hazards exist; the requirements on quality control and process amplification are high; the effective antigen amount generated by the virus is low, and the virus titer is required to be improved to ensure the effect, so that the production cost is increased, and therefore, the improvement of the vaccine for the new coronary pneumonia is of great significance.
The protein replacement therapy based on mRNA shows good application prospect in infectious diseases, mRNA vaccines generally use nanotechnology to deliver mRNA sequences coding the spike protein (S protein) of a novel coronavirus (SARS-CoV-2) or a part sequence (such as S1 and RBD) of the spike protein, then SARS-CoV-2 antigen is expressed through a translation system of a host, the generation of corresponding antibodies is induced, and finally the aim of immune protection is achieved.
CN111218458A discloses a mRNA encoding SARS-CoV-2 virus antigen and vaccine and preparation method thereof, wherein the mRNA encoding SARS-CoV-2 virus antigen at least contains coding region encoding at least one protein of S protein and N protein of SARS-CoV-2 virus, and/or at least one fragment of protein, and the mRNA is delivered into the body to enable the body to generate immune response, however, for the patient already infected with SARS-CoV-2, the treatment is difficult to be effective, and the virus is still killed mainly by the body' S own immune system.
In conclusion, the strategy for effectively neutralizing and treating SARS-CoV-2 infection is provided, and has important significance for the field of SARS-CoV-2 treatment.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides mRNA for coding secreted angiotensin converting enzyme 2, which can express secreted ACE2 in cells by using a translation system, and secreted ACE2 can be secreted to the outside of cells and used as a decoy receptor, and has the activity of competitively inhibiting SARS-CoV-2, namely can be combined with an S protein receptor binding domain of SARS-CoV-2, so as to competitively inhibit SARS-CoV-2 from entering cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
a first aspect. The present invention provides an mRNA encoding a secreted angiotensin converting enzyme 2, the mRNA including a coding region of 70% or more of a signal peptide of angiotensin converting enzyme 2 and a coding region of 70% or more of a peptidase domain.
The mRNA for coding the secretory angiotensin converting enzyme 2 has a coding region of an ACE2 extracellular domain, can express the secretory ACE2 by utilizing a translation system of a host cell, and can secrete the secretory ACE2 to the outside of the cell and be used as a decoy receptor to be combined with an S protein receptor binding domain of SARS-CoV-2, so that the SARS-CoV-2 is competitively inhibited from entering the cell.
Preferably, the coding region of the signal peptide comprises the nucleic acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.
SEQ ID NO:1：
augucuaguucuagcuggcugcugcugagccugguggccgucaccgccgcc。
SEQ ID NO:2：
augagcagcagcagcuggcugcugcugagccugguggccgugaccgccgcu。
SEQ ID NO:3：
augaguucuagcagcuggcugcugcugagccucguggccgugaccgccgcc。
Preferably, the coding region of the peptidase domain comprises the nucleic acid sequence shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.
SEQ ID NO:4：
cagagcaccaucgaggagcaggccaagaccuuccuggauaaguucaaccacgaggccgaagaucuguucuaccagucaagccuugcuagcuggaacuacaacaccaacaucacagaggaaaauguccagaacaugaacaacgccggagacaaguggagcgcuuuccugaaggaacagagcacccuggcccagauguacccccuccaagagauccagaaccugacagugaagcugcagcugcaggcccugcaacagaauggcagcagcgugcuaagcgaggacaagagcaagcggcugaacaccauccuuaauaccaugagcacgaucuacagcacaggcaaggucugcaacccugacaauccccaggagugccuccugcucgagccaggccugaacgagaucauggccaauagccuggacuacaacgagagacugugggccugggaaagcuggagaagcgaggugggaaaacaguugagaccucuguacgaagaguacguggugcugaagaaugaaauggcuagagccaaccacuacgaagauuacggcgacuacuggcgcggcgacuacgaggugaacggcguggacggcuacgacuacagcagaggccagcugaucgaggaugucgagcauacauucgaggaaauuaagccucuguaugagcaccugcacgccuaugugagggccaagcugaugaacgccuauccuagcuacaucagccccaucggaugucugccugcccaccugcugggcgacauguggggcagauucuggaccaaccuguacucucugaccgugccauucggccagaagcccaacaucgacgugacagacgccaugguggaucaagccugggaugcccagcggaucuucaaagaggccgagaaauucuucguguccgugggacugccuaacaugacccagggcuucugggagaacaguaugcugaccgacccuggaaauguucaaaaagcugugugccacccuaccgccugggaccugggcaagggcgacuucagaauccugaugugcaccaaggugaccauggacgacuuucugacagcucaccacgagaugggccacauccaguacgacauggccuacgcugcccagcccuuccugcugagaaacggggcuaaugagggcuuccacgaggccgugggcgagaucaugucgcugagcgccgcuacaccuaagcaucugaaauccaucggccugcugucuccagacuuucaggaggauaacgaaaccgagaucaacuuccugcugaagcaggcucugacaaucgugggcacccugcccuucaccuacaugcuggaaaaauggagauggaugguguucaagggcgaaaucccuaaggaccaguggaugaaaaaguggugggagaugaaacgggaaaucgugggcgugguggaaccugugccccacgacgagacauauugcgacccugccucucuguuucacguguccaacgacuacagcuuuauucgguacuacacccggacacuguaccaguuccaguuccaagaagcccugugucaggcugccaagcacgaggggccucugcacaagugcgauaucagcaacagcacagaagccggccagaaacuguuuaauaugcugagacuggguaagucugaacccuggacccuggcccuggaaaacguggugggcgcuaagaacaugaacgugagaccucugcugaacuacuucgagccucuguuuaccuggcugaaggaccaaaacaagaacagcuucgugggaugguccaccgacuggagcccuuacgccgac。
SEQ ID NO:5：
cagagcaccaucgaggagcaagccaaguacuuccuggacaaguucaaccacgaggccgaggaccuguucuaucagagcagccuggcaagcuggaacuacaacaccaacaucaccgaggagaacgugcagaacaugaacaacgccggcgacaaguggagcgccuuccugaaggagcagagcaccaccgcucagauguacccccugcaagagauucagaaccugaccgugaagcugcagcugcaagcccugcagcagaacggcagcagcgugcugagcgaggacaagagcaagagacugaacaccauccugaacaccaugagcaccaucuacagcaccggcaaggugugcaaccccgacaacccccaagagugccugcugcuggagcccggccugaacgagaucauggccaauagccuggacuacaacgagagacugugggccugggagagcuggagaagcgaggugggcaagcagcugagaccccuguacgaagaguacguggugcugaagaacgagauggcuagagccaaccacuacgaggacuacggcgacuacuggagaggcgacuacgaggugaacggcguggacggcuacgacuacagcagagggcagcugaucgaggacguggagcacaccuucgaggagaucaagccccuguacgagcaccugcacgccuacgugagagccaagcugaugaacgccuacccuagcuacaucagccccaucggcugccugcccgcccaccugcugggcgacauguggggcagauucuggaccaaccuguacagccugaccgugcccuucgggcagaagcccaacaucgacgugaccgacgccaugguggaccaagccugggacgcucagagaaucuucaaggaggccgagaaguucuucgugagcgugggccugcccaacaugacccaaggcuucugggaguacagcaugcugaccgaccccggcaacgugcagaaggccgugugccugcccaccgccugggaccugggcaagggcgacuucagaauccugaugugcaccaaggugaccauggacgacuuccugaccgcccaccacgagaugggccacauucaguacgacauggccuaccuggcucagcccuuccugcugagaaacggcgccaacgagggcuuccacgaggccgugggcgagaucaugagccugagcgccgccacccccaagcaccugaagagcaucggccugcugagccccgacuuccaagaggacaacgagaccgagaucaacuuccugcugaagcaagcccugaccaucgugggcacccugcccuucaccuacaugcuggagaaguggagauggaugguguucaagggcgagauccccaaggaucaguggaugaagaaguggugggagaugaagagagagaucgugggcgugguggagcccgugccccacgacgagaccuacugcgaccccgcaagccuguuccacgugagcaacgacuauagcuuuaucagauacuacacaagaacccuguaucaguuucaguuccaagaggcccugugccaagccgccaagcacgagggcccccugcacaagugcgauauuagcaacagcaccgaggccgggcagaagcuguucaacaugcugagacugggcaagagcgagcccuggacccuggcccuggagaacguggugggcgccaagaacaugaacgugagaccccugcugaacuacuucgagccccuguucaccuggcugaaggaucagaacaagaacagcuucgugggcuggagcaccgacuggagccccuacgccgau。
SEQ ID NO:6：
agaucuaccaucgaggaacaggccaagaccuuccuggacuucuucgacagccaggcugaggaucuguucuaccagagcagccuggccagcuggaacuacaacaccaauaucacagaagagaacgugcagaauaugaacaacgccggcgacaagagaagcgccuuccucaaagagcgguccacccuggcucagauguacccucugcaggagauucagaaucugacagugaagcugcagcuccaggcccugcagcagaacggcagcagcgugcugagcgaggacaagagcaagagacuaaacaccauccugaauaccaugagcacaaucuacagcacaggcaagguguguaauccugacaacccccaagaaugccugcugcuggaaccaggccugaaugagaucauggccaauagcuuggauuauaacgaaagacugugggccugggaaagcuggagaagcgaggugggaaaacaacucagaccccuuuaugaagaguacguugugcugaaaaacgagauggccagagccaaccacuacgaagauuacggcgacuacuggcggggagacuacgaggugaacggcguggacggcuacgacuacagcagagggcagcugaucgaagaugucgaacacaccuuugaggaaauuaagccucuguaugagcaccugcacgccuaugugcgggccaaacugaugaaugccuacccuagcuacaucaguccuaucggcugccugccagcccaucugcugggcgacauguggggcagauucuggaccaaccuguacucccugacagugccuuucggccaaaaaccuaacaucgacgugacugaugccauggucgaccaagcuugggaugcccaaagaaucuucaaggaagcugaaaaguucuucguguccgugggccugccaaacaugacacagggauucugggagaacagcaugcugacagaccccggcaacguccagaaggccguuugucugcccaccgccugggaccugggcaagggcgauuucagaauccugaugugcaccaaggugacaauggacgacuuucugaccgcccaccacgagaugggccacauccaguacgauauggcauaugccgcccagccuuuccugcuuagaaacggcgcuaacgagggcuuccacgaggccgugggagaaaucaugagccugagcgccgcuacaccuaagcaccugaagagcaucggacugcugucuccugauuuucaggaggauaacgagacagagaucaauuuccugcugaaacaggcccugaccaucgugggcacccugccuuuuaccuacaugcuggaaaaguggcgcuggaugguguucaagggcgaaaucccuaaggaccaguggaugaaaaaguggugggagaugaagcgggaaaucgucggagugguggaaccugugccccacgacgagacguacugcgacccugcuucucuguuucauguguccaacgacuacagcuucaucagauacuacaccagaacccuguaccaguuccaauuucaggaggcccugugccaggccgccaagcacgagggcccucugcacaagugcgacaucagcaacuccaccgaggcaggccagaagcuguucaacaugcugagacugggcaaaucugagccuuggacacuggcccuggaaaacguggugggcgccaagaacaugaacgugcggccacugcugaacuacuucgagccccuguucaccuggcugaaggaccagaacaagaacuccuucgugggcugguccaccgauuggagccccuacgcugau。
Preferably, the mRNA comprises the nucleic acid sequence shown as SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
SEQ ID NO:7：
augucuaguucuagcuggcugcugcugagccugguggccgucaccgccgcccagagcaccaucgaggagcaggccaagaccuuccuggauaaguucaaccacgaggccgaagaucuguucuaccagucaagccuugcuagcuggaacuacaacaccaacaucacagaggaaaauguccagaacaugaacaacgccggagacaaguggagcgcuuuccugaaggaacagagcacccuggcccagauguacccccuccaagagauccagaaccugacagugaagcugcagcugcaggcccugcaacagaauggcagcagcgugcuaagcgaggacaagagcaagcggcugaacaccauccuuaauaccaugagcacgaucuacagcacaggcaaggucugcaacccugacaauccccaggagugccuccugcucgagccaggccugaacgagaucauggccaauagccuggacuacaacgagagacugugggccugggaaagcuggagaagcgaggugggaaaacaguugagaccucuguacgaagaguacguggugcugaagaaugaaauggcuagagccaaccacuacgaagauuacggcgacuacuggcgcggcgacuacgaggugaacggcguggacggcuacgacuacagcagaggccagcugaucgaggaugucgagcauacauucgaggaaauuaagccucuguaugagcaccugcacgccuaugugagggccaagcugaugaacgccuauccuagcuacaucagccccaucggaugucugccugcccaccugcugggcgacauguggggcagauucuggaccaaccuguacucucugaccgugccauucggccagaagcccaacaucgacgugacagacgccaugguggaucaagccugggaugcccagcggaucuucaaagaggccgagaaauucuucguguccgugggacugccuaacaugacccagggcuucugggagaacaguaugcugaccgacccuggaaauguucaaaaagcugugugccacccuaccgccugggaccugggcaagggcgacuucagaauccugaugugcaccaaggugaccauggacgacuuucugacagcucaccacgagaugggccacauccaguacgacauggccuacgcugcccagcccuuccugcugagaaacggggcuaaugagggcuuccacgaggccgugggcgagaucaugucgcugagcgccgcuacaccuaagcaucugaaauccaucggccugcugucuccagacuuucaggaggauaacgaaaccgagaucaacuuccugcugaagcaggcucugacaaucgugggcacccugcccuucaccuacaugcuggaaaaauggagauggaugguguucaagggcgaaaucccuaaggaccaguggaugaaaaaguggugggagaugaaacgggaaaucgugggcgugguggaaccugugccccacgacgagacauauugcgacccugccucucuguuucacguguccaacgacuacagcuuuauucgguacuacacccggacacuguaccaguuccaguuccaagaagcccugugucaggcugccaagcacgaggggccucugcacaagugcgauaucagcaacagcacagaagccggccagaaacuguuuaauaugcugagacuggguaagucugaacccuggacccuggcccuggaaaacguggugggcgcuaagaacaugaacgugagaccucugcugaacuacuucgagccucuguuuaccuggcugaaggaccaaaacaagaacagcuucgugggaugguccaccgacuggagcccuuacgccgac。
SEQ ID NO:8：
augagcagcagcagcuggcugcugcugagccugguggccgugaccgccgcucagagcaccaucgaggagcaagccaaguacuuccuggacaaguucaaccacgaggccgaggaccuguucuaucagagcagccuggcaagcuggaacuacaacaccaacaucaccgaggagaacgugcagaacaugaacaacgccggcgacaaguggagcgccuuccugaaggagcagagcaccaccgcucagauguacccccugcaagagauucagaaccugaccgugaagcugcagcugcaagcccugcagcagaacggcagcagcgugcugagcgaggacaagagcaagagacugaacaccauccugaacaccaugagcaccaucuacagcaccggcaaggugugcaaccccgacaacccccaagagugccugcugcuggagcccggccugaacgagaucauggccaauagccuggacuacaacgagagacugugggccugggagagcuggagaagcgaggugggcaagcagcugagaccccuguacgaagaguacguggugcugaagaacgagauggcuagagccaaccacuacgaggacuacggcgacuacuggagaggcgacuacgaggugaacggcguggacggcuacgacuacagcagagggcagcugaucgaggacguggagcacaccuucgaggagaucaagccccuguacgagcaccugcacgccuacgugagagccaagcugaugaacgccuacccuagcuacaucagccccaucggcugccugcccgcccaccugcugggcgacauguggggcagauucuggaccaaccuguacagccugaccgugcccuucgggcagaagcccaacaucgacgugaccgacgccaugguggaccaagccugggacgcucagagaaucuucaaggaggccgagaaguucuucgugagcgugggccugcccaacaugacccaaggcuucugggaguacagcaugcugaccgaccccggcaacgugcagaaggccgugugccugcccaccgccugggaccugggcaagggcgacuucagaauccugaugugcaccaaggugaccauggacgacuuccugaccgcccaccacgagaugggccacauucaguacgacauggccuaccuggcucagcccuuccugcugagaaacggcgccaacgagggcuuccacgaggccgugggcgagaucaugagccugagcgccgccacccccaagcaccugaagagcaucggccugcugagccccgacuuccaagaggacaacgagaccgagaucaacuuccugcugaagcaagcccugaccaucgugggcacccugcccuucaccuacaugcuggagaaguggagauggaugguguucaagggcgagauccccaaggaucaguggaugaagaaguggugggagaugaagagagagaucgugggcgugguggagcccgugccccacgacgagaccuacugcgaccccgcaagccuguuccacgugagcaacgacuauagcuuuaucagauacuacacaagaacccuguaucaguuucaguuccaagaggcccugugccaagccgccaagcacgagggcccccugcacaagugcgauauuagcaacagcaccgaggccgggcagaagcuguucaacaugcugagacugggcaagagcgagcccuggacccuggcccuggagaacguggugggcgccaagaacaugaacgugagaccccugcugaacuacuucgagccccuguucaccuggcugaaggaucagaacaagaacagcuucgugggcuggagcaccgacuggagccccuacgccgau。
SEQ ID NO:9：
augaguucuagcagcuggcugcugcugagccucguggccgugaccgccgccagaucuaccaucgaggaacaggccaagaccuuccuggacuucuucgacagccaggcugaggaucuguucuaccagagcagccuggccagcuggaacuacaacaccaauaucacagaagagaacgugcagaauaugaacaacgccggcgacaagagaagcgccuuccucaaagagcgguccacccuggcucagauguacccucugcaggagauucagaaucugacagugaagcugcagcuccaggcccugcagcagaacggcagcagcgugcugagcgaggacaagagcaagagacuaaacaccauccugaauaccaugagcacaaucuacagcacaggcaagguguguaauccugacaacccccaagaaugccugcugcuggaaccaggccugaaugagaucauggccaauagcuuggauuauaacgaaagacugugggccugggaaagcuggagaagcgaggugggaaaacaacucagaccccuuuaugaagaguacguugugcugaaaaacgagauggccagagccaaccacuacgaagauuacggcgacuacuggcggggagacuacgaggugaacggcguggacggcuacgacuacagcagagggcagcugaucgaagaugucgaacacaccuuugaggaaauuaagccucuguaugagcaccugcacgccuaugugcgggccaaacugaugaaugccuacccuagcuacaucaguccuaucggcugccugccagcccaucugcugggcgacauguggggcagauucuggaccaaccuguacucccugacagugccuuucggccaaaaaccuaacaucgacgugacugaugccauggucgaccaagcuugggaugcccaaagaaucuucaaggaagcugaaaaguucuucguguccgugggccugccaaacaugacacagggauucugggagaacagcaugcugacagaccccggcaacguccagaaggccguuugucugcccaccgccugggaccugggcaagggcgauuucagaauccugaugugcaccaaggugacaauggacgacuuucugaccgcccaccacgagaugggccacauccaguacgauauggcauaugccgcccagccuuuccugcuuagaaacggcgcuaacgagggcuuccacgaggccgugggagaaaucaugagccugagcgccgcuacaccuaagcaccugaagagcaucggacugcugucuccugauuuucaggaggauaacgagacagagaucaauuuccugcugaaacaggcccugaccaucgugggcacccugccuuuuaccuacaugcuggaaaaguggcgcuggaugguguucaagggcgaaaucccuaaggaccaguggaugaaaaaguggugggagaugaagcgggaaaucgucggagugguggaaccugugccccacgacgagacguacugcgacccugcuucucuguuucauguguccaacgacuacagcuucaucagauacuacaccagaacccuguaccaguuccaauuucaggaggcccugugccaggccgccaagcacgagggcccucugcacaagugcgacaucagcaacuccaccgaggcaggccagaagcuguucaacaugcugagacugggcaaaucugagccuuggacacuggcccuggaaaacguggugggcgccaagaacaugaacgugcggccacugcugaacuacuucgagccccuguucaccuggcugaaggaccagaacaagaacuccuucgugggcugguccaccgauuggagccccuacgcugau。
Preferably, the mRNA further comprises a coding region for the dimeric domain of angiotensin converting enzyme 2 and/or a coding region for the linking region.
Preferably, the mRNA further comprises any one of or a combination of at least two of the mutation sites Q18R, T27Y/W, K31F, N33D, H34S, E35Q, W69R, Q76R, L79T, N330Y, H345L or a386L, wherein typical but non-limiting combinations include a combination of T27Y, L79Y, N330Y, H345Y and a 386Y, Q18Y, K31Y, N33Y, H34Y, E35Y, W69Y, a combination of Q76Y and H345Y, T27Y, L79Y, a combination of H345Y and a Y, T27Y, L79Y, W69Y, N330Y, H Y and a Y or a combination of Q18, K345, N386Y, N386Y and a Y.
In the invention, mutation sites are introduced into the mRNA, so that the affinity of the expressed secretory ACE2 can be further enhanced, and the SARS-CoV-2S protein can be more efficiently combined.
Preferably, the mRNA further comprises any one of a cap structure, an untranslated region, or a polyadenylation sequence, or a combination of at least two.
Preferably, the nucleotide sequence of the mRNA coding region is codon optimized.
Preferably, the nucleotides of the mRNA coding region contain unmodified nucleotides and/or modified nucleotides.
Preferably, the modified nucleotide comprises 5-moUTP, 5mCTP or ψ UTP.
Preferably, the mRNA comprises the nucleic acid sequence shown as SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12.
SEQ ID NO:10：
augucuaguucuagcuggcugcugcugagccugguggccgucaccgccgcccagagcaccaucgaggagcaggccaagaccuuccuggauaaguucaaccacgaggccgaagaucuguucuaccagucaagccuugcuagcuggaacuacaacaccaacaucacagaggaaaauguccagaacaugaacaacgccggagacaaguggagcgcuuuccugaaggaacagagcacccuggcccagauguacccccuccaagagauccagaaccugacagugaagcugcagcugcaggcccugcaacagaauggcagcagcgugcuaagcgaggacaagagcaagcggcugaacaccauccuuaauaccaugagcacgaucuacagcacaggcaaggucugcaacccugacaauccccaggagugccuccugcucgagccaggccugaacgagaucauggccaauagccuggacuacaacgagagacugugggccugggaaagcuggagaagcgaggugggaaaacaguugagaccucuguacgaagaguacguggugcugaagaaugaaauggcuagagccaaccacuacgaagauuacggcgacuacuggcgcggcgacuacgaggugaacggcguggacggcuacgacuacagcagaggccagcugaucgaggaugucgagcauacauucgaggaaauuaagccucuguaugagcaccugcacgccuaugugagggccaagcugaugaacgccuauccuagcuacaucagccccaucggaugucugccugcccaccugcugggcgacauguggggcagauucuggaccaaccuguacucucugaccgugccauucggccagaagcccaacaucgacgugacagacgccaugguggaucaagccugggaugcccagcggaucuucaaagaggccgagaaauucuucguguccgugggacugccuaacaugacccagggcuucugggagaacaguaugcugaccgacccuggaaauguucaaaaagcugugugccacccuaccgccugggaccugggcaagggcgacuucagaauccugaugugcaccaaggugaccauggacgacuuucugacagcucaccacgagaugggccacauccaguacgacauggccuacgcugcccagcccuuccugcugagaaacggggcuaaugagggcuuccacgaggccgugggcgagaucaugucgcugagcgccgcuacaccuaagcaucugaaauccaucggccugcugucuccagacuuucaggaggauaacgaaaccgagaucaacuuccugcugaagcaggcucugacaaucgugggcacccugcccuucaccuacaugcuggaaaaauggagauggaugguguucaagggcgaaaucccuaaggaccaguggaugaaaaaguggugggagaugaaacgggaaaucgugggcgugguggaaccugugccccacgacgagacauauugcgacccugccucucuguuucacguguccaacgacuacagcuuuauucgguacuacacccggacacuguaccaguuccaguuccaagaagcccugugucaggcugccaagcacgaggggccucugcacaagugcgauaucagcaacagcacagaagccggccagaaacuguuuaauaugcugagacuggguaagucugaacccuggacccuggcccuggaaaacguggugggcgcuaagaacaugaacgugagaccucugcugaacuacuucgagccucuguuuaccuggcugaaggaccaaaacaagaacagcuucgugggaugguccaccgacuggagcccuuacgccgaccagagcaucaaggugcggaucucucuuaagagcgcccugggcgauaaggccuacgaguggaacgacaacgagauguaccuguuuagaagcuccguugccuaugccaugagacaguauuuccucaaggucaagaaucagaugauccuguucggagaagaggaugugcgaguggcaaaccugaagccucggauuagcuucaacuucuucgugacagcccccaagaacgugucugauaucauccccagaaccgaaguggaaaaggccaucagaauguccagaagcagaaucaacgacgccuuccggcugaacgauaauagccuggaauuccugggcauccagccaacucugggccccccuaaccagccuccuguguccuga。
SEQ ID NO:11：
augagcagcagcagcuggcugcugcugagccugguggccgugaccgccgcucagagcaccaucgaggagcaagccaaguacuuccuggacaaguucaaccacgaggccgaggaccuguucuaucagagcagccuggcaagcuggaacuacaacaccaacaucaccgaggagaacgugcagaacaugaacaacgccggcgacaaguggagcgccuuccugaaggagcagagcaccaccgcucagauguacccccugcaagagauucagaaccugaccgugaagcugcagcugcaagcccugcagcagaacggcagcagcgugcugagcgaggacaagagcaagagacugaacaccauccugaacaccaugagcaccaucuacagcaccggcaaggugugcaaccccgacaacccccaagagugccugcugcuggagcccggccugaacgagaucauggccaauagccuggacuacaacgagagacugugggccugggagagcuggagaagcgaggugggcaagcagcugagaccccuguacgaagaguacguggugcugaagaacgagauggcuagagccaaccacuacgaggacuacggcgacuacuggagaggcgacuacgaggugaacggcguggacggcuacgacuacagcagagggcagcugaucgaggacguggagcacaccuucgaggagaucaagccccuguacgagcaccugcacgccuacgugagagccaagcugaugaacgccuacccuagcuacaucagccccaucggcugccugcccgcccaccugcugggcgacauguggggcagauucuggaccaaccuguacagccugaccgugcccuucgggcagaagcccaacaucgacgugaccgacgccaugguggaccaagccugggacgcucagagaaucuucaaggaggccgagaaguucuucgugagcgugggccugcccaacaugacccaaggcuucugggaguacagcaugcugaccgaccccggcaacgugcagaaggccgugugccugcccaccgccugggaccugggcaagggcgacuucagaauccugaugugcaccaaggugaccauggacgacuuccugaccgcccaccacgagaugggccacauucaguacgacauggccuaccuggcucagcccuuccugcugagaaacggcgccaacgagggcuuccacgaggccgugggcgagaucaugagccugagcgccgccacccccaagcaccugaagagcaucggccugcugagccccgacuuccaagaggacaacgagaccgagaucaacuuccugcugaagcaagcccugaccaucgugggcacccugcccuucaccuacaugcuggagaaguggagauggaugguguucaagggcgagauccccaaggaucaguggaugaagaaguggugggagaugaagagagagaucgugggcgugguggagcccgugccccacgacgagaccuacugcgaccccgcaagccuguuccacgugagcaacgacuauagcuuuaucagauacuacacaagaacccuguaucaguuucaguuccaagaggcccugugccaagccgccaagcacgagggcccccugcacaagugcgauauuagcaacagcaccgaggccgggcagaagcuguucaacaugcugagacugggcaagagcgagcccuggacccuggcccuggagaacguggugggcgccaagaacaugaacgugagaccccugcugaacuacuucgagccccuguucaccuggcugaaggaucagaacaagaacagcuucgugggcuggagcaccgacuggagccccuacgccgaucagagcaucaaggugagaaucagccugaagagcgcccugggcgacaaggccuacgaguggaacgacaacgagauguaccuguucagaagcagcguggccuacgccaugagacaguacuuccugaaggugaagaaucagaugauccuguucggcgaggaggacgugagaguggccaaccugaagccuagaaucagcuucaacuucuucgugaccgcccccaagaacgugagcgacaucaucccuagaaccgagguggagaaggccaucagaaugagcagaagcagaaucaacgacgccuucagacugaacgacaacucacuggaguuccugggcauucagcccacccugggcuga。
SEQ ID NO:12：
augaguucuagcagcuggcugcugcugagccucguggccgugaccgccgccagaucuaccaucgaggaacaggccaagaccuuccuggacuucuucgacagccaggcugaggaucuguucuaccagagcagccuggccagcuggaacuacaacaccaauaucacagaagagaacgugcagaauaugaacaacgccggcgacaagagaagcgccuuccucaaagagcgguccacccuggcucagauguacccucugcaggagauucagaaucugacagugaagcugcagcuccaggcccugcagcagaacggcagcagcgugcugagcgaggacaagagcaagagacuaaacaccauccugaauaccaugagcacaaucuacagcacaggcaagguguguaauccugacaacccccaagaaugccugcugcuggaaccaggccugaaugagaucauggccaauagcuuggauuauaacgaaagacugugggccugggaaagcuggagaagcgaggugggaaaacaacucagaccccuuuaugaagaguacguugugcugaaaaacgagauggccagagccaaccacuacgaagauuacggcgacuacuggcggggagacuacgaggugaacggcguggacggcuacgacuacagcagagggcagcugaucgaagaugucgaacacaccuuugaggaaauuaagccucuguaugagcaccugcacgccuaugugcgggccaaacugaugaaugccuacccuagcuacaucaguccuaucggcugccugccagcccaucugcugggcgacauguggggcagauucuggaccaaccuguacucccugacagugccuuucggccaaaaaccuaacaucgacgugacugaugccauggucgaccaagcuugggaugcccaaagaaucuucaaggaagcugaaaaguucuucguguccgugggccugccaaacaugacacagggauucugggagaacagcaugcugacagaccccggcaacguccagaaggccguuugucugcccaccgccugggaccugggcaagggcgauuucagaauccugaugugcaccaaggugacaauggacgacuuucugaccgcccaccacgagaugggccacauccaguacgauauggcauaugccgcccagccuuuccugcuuagaaacggcgcuaacgagggcuuccacgaggccgugggagaaaucaugagccugagcgccgcuacaccuaagcaccugaagagcaucggacugcugucuccugauuuucaggaggauaacgagacagagaucaauuuccugcugaaacaggcccugaccaucgugggcacccugccuuuuaccuacaugcuggaaaaguggcgcuggaugguguucaagggcgaaaucccuaaggaccaguggaugaaaaaguggugggagaugaagcgggaaaucgucggagugguggaaccugugccccacgacgagacguacugcgacccugcuucucuguuucauguguccaacgacuacagcuucaucagauacuacaccagaacccuguaccaguuccaauuucaggaggcccugugccaggccgccaagcacgagggcccucugcacaagugcgacaucagcaacuccaccgaggcaggccagaagcuguucaacaugcugagacugggcaaaucugagccuuggacacuggcccuggaaaacguggugggcgccaagaacaugaacgugcggccacugcugaacuacuucgagccccuguucaccuggcugaaggaccagaacaagaacuccuucgugggcugguccaccgauuggagccccuacgcugaucagagcaucaaggugcggauuucucugaagagcgcccugggcgacaaggccuacgaguggaaugacaacgagauguaccuguuccggucuucuguggcuuacgccaugagacaguacuuccugaaagugaagaaccagaugauccuguuuggcgaggaggacgugcggguggccaaccugaagccccggaucagcuuuaacuucuucgugaccgccccuaagaaugucucugauaucauccccaggaccgagguggaaaaagccauccggaugagcagaagccgcaucaacgacgcuuuucggcugaacgacaauucacuggaauuccugggaauccagccuacacugggcuga。
In a second aspect, the present invention provides a secretory expression system, mRNA encoding secretory angiotensin converting enzyme 2, and a delivery vector carrying the mRNA encoding secretory angiotensin converting enzyme 2.
Preferably, the mRNA encoding secreted angiotensin converting enzyme 2 comprises the mRNA encoding secreted angiotensin converting enzyme 2 according to the first aspect and/or the wild-type mRNA encoding secreted angiotensin converting enzyme 2.
Preferably, the wild-type mRNA encoding secreted angiotensin converting enzyme 2 comprises more than 70% of the coding region of the signal peptide and more than 70% of the coding region of the peptidase domain of wild-type angiotensin converting enzyme 2.
Preferably, the coding region for the signal peptide and the coding region for the peptidase domain of the wild-type angiotensin converting enzyme 2 are derived from any one of Homo sapiens (human), Pan troglytes (chimpanzee), Ponga abelii (Sumatran organistan), Macaca multta (Rhesus monkey), Chlorocebus sabaeus (green monkey), Sus scrofa (pig), Bos Taurus (cat), Ovis aries (sheet), Equus calbauus (horse), Oryctolagus cuniculus (rabbit), Canis lupulis family (dog), Felis cat (dog), Gallus Gallus (chicken), Mus mulus (horse mouse), or Danibrio (horse).
Preferably, the coding region of the signal peptide of the wild-type angiotensin-converting enzyme 2 comprises the sequence shown in SEQ ID NO 13.
SEQ ID NO:13：
augucaagcucuuccuggcuccuucucagccuuguugcuguaacugcugcu。
Preferably, the coding region of the peptidase domain of wild-type angiotensin converting enzyme 2 comprises the sequence shown in SEQ ID NO. 14.
SEQ ID NO:14：
caguccaccauugaggaacaggccaagacauuuuuggacaaguuuaaccacgaagccgaagaccuguucuaucaaaguucacuugcuucuuggaauuauaacaccaauauuacugaagagaauguccaaaacaugaauaaugcuggggacaaauggucugccuuuuuaaaggaacaguccacacuugcccaaauguauccacuacaagaaauucagaaucucacagucaagcuucagcugcaggcucuucagcaaaaugggucuucagugcucucagaagacaagagcaaacgguugaacacaauucuaaauacaaugagcaccaucuacaguacuggaaaaguuuguaacccagauaauccacaagaaugcuuauuacuugaaccagguuugaaugaaauaauggcaaacaguuuagacuacaaugagaggcucugggcuugggaaagcuggagaucugaggucggcaagcagcugaggccauuauaugaagaguauguggucuugaaaaaugagauggcaagagcaaaucauuaugaggacuauggggauuauuggagaggagacuaugaaguaaaugggguagauggcuaugacuacagccgcggccaguugauugaagauguggaacauaccuuugaagagauuaaaccauuauaugaacaucuucaugccuaugugagggcaaaguugaugaaugccuauccuuccuauaucaguccaauuggaugccucccugcucauuugcuuggugauaugugggguagauuuuggacaaaucuguacucuuugacaguucccuuuggacagaaaccaaacauagauguuacugaugcaaugguggaccaggccugggaugcacagagaauauucaaggaggccgagaaguucuuuguaucuguuggucuuccuaauaugacucaaggauucugggaaaauuccaugcuaacggacccaggaaauguucagaaagcagucugccaucccacagcuugggaccuggggaagggcgacuucaggauccuuaugugcacaaaggugacaauggacgacuuccugacagcucaucaugagauggggcauauccaguaugauauggcauaugcugcacaaccuuuucugcuaagaaauggagcuaaugaaggauuccaugaagcuguuggggaaaucaugucacuuucugcagccacaccuaagcauuuaaaauccauuggucuucugucacccgauuuucaagaagacaaugaaacagaaauaaacuuccugcucaaacaagcacucacgauuguugggacucugccauuuacuuacauguuagagaaguggagguggauggucuuuaaaggggaaauucccaaagaccaguggaugaaaaaguggugggagaugaagcgagagauaguuggggugguggaaccugugccccaugaugaaacauacugugaccccgcaucucuguuccauguuucuaaugauuacucauucauucgauauuacacaaggacccuuuaccaauuccaguuucaagaagcacuuugucaagcagcuaaacaugaaggcccucugcacaaaugugacaucucaaacucuacagaagcuggacagaaacuguucaauaugcugaggcuuggaaaaucagaacccuggacccuagcauuggaaaauguuguaggagcaaagaacaugaauguaaggccacugcucaacuacuuugagcccuuauuuaccuggcugaaagaccagaacaagaauucuuuugugggauggaguaccgacuggaguccauaugcagac。
Preferably, the wild-type mRNA encoding secreted angiotensin converting enzyme 2 comprises the nucleic acid sequence shown in SEQ ID NO. 15.
SEQ ID NO:15：
augucaagcucuuccuggcuccuucucagccuuguugcuguaacugcugcucaguccaccauugaggaacaggccaagacauuuuuggacaaguuuaaccacgaagccgaagaccuguucuaucaaaguucacuugcuucuuggaauuauaacaccaauauuacugaagagaauguccaaaacaugaauaaugcuggggacaaauggucugccuuuuuaaaggaacaguccacacuugcccaaauguauccacuacaagaaauucagaaucucacagucaagcuucagcugcaggcucuucagcaaaaugggucuucagugcucucagaagacaagagcaaacgguugaacacaauucuaaauacaaugagcaccaucuacaguacuggaaaaguuuguaacccagauaauccacaagaaugcuuauuacuugaaccagguuugaaugaaauaauggcaaacaguuuagacuacaaugagaggcucugggcuugggaaagcuggagaucugaggucggcaagcagcugaggccauuauaugaagaguauguggucuugaaaaaugagauggcaagagcaaaucauuaugaggacuauggggauuauuggagaggagacuaugaaguaaaugggguagauggcuaugacuacagccgcggccaguugauugaagauguggaacauaccuuugaagagauuaaaccauuauaugaacaucuucaugccuaugugagggcaaaguugaugaaugccuauccuuccuauaucaguccaauuggaugccucccugcucauuugcuuggugauaugugggguagauuuuggacaaaucuguacucuuugacaguucccuuuggacagaaaccaaacauagauguuacugaugcaaugguggaccaggccugggaugcacagagaauauucaaggaggccgagaaguucuuuguaucuguuggucuuccuaauaugacucaaggauucugggaaaauuccaugcuaacggacccaggaaauguucagaaagcagucugccaucccacagcuugggaccuggggaagggcgacuucaggauccuuaugugcacaaaggugacaauggacgacuuccugacagcucaucaugagauggggcauauccaguaugauauggcauaugcugcacaaccuuuucugcuaagaaauggagcuaaugaaggauuccaugaagcuguuggggaaaucaugucacuuucugcagccacaccuaagcauuuaaaauccauuggucuucugucacccgauuuucaagaagacaaugaaacagaaauaaacuuccugcucaaacaagcacucacgauuguugggacucugccauuuacuuacauguuagagaaguggagguggauggucuuuaaaggggaaauucccaaagaccaguggaugaaaaaguggugggagaugaagcgagagauaguuggggugguggaaccugugccccaugaugaaacauacugugaccccgcaucucuguuccauguuucuaaugauuacucauucauucgauauuacacaaggacccuuuaccaauuccaguuucaagaagcacuuugucaagcagcuaaacaugaaggcccucugcacaaaugugacaucucaaacucuacagaagcuggacagaaacuguucaauaugcugaggcuuggaaaaucagaacccuggacccuagcauuggaaaauguuguaggagcaaagaacaugaauguaaggccacugcucaacuacuuugagcccuuauuuaccuggcugaaagaccagaacaagaauucuuuugugggauggaguaccgacuggaguccauaugcagaccaaagcaucaaagugaggauaagccuaaaaucagcucuuggagauaaagcauaugaauggaacgacaaugaaauguaccuguuccgaucaucuguugcauaugcuaugaggcaguacuuuuuaaaaguaaaaaaucagaugauucuuuuuggggaggaggaugugcgaguggcuaauuugaaaccaagaaucuccuuuaauuucuuugucacugcaccuaaaaaugugucugauaucauuccuagaacugaaguugaaaaggccaucaggaugucccggagccguaucaaugaugcuuuccgucugaaugacaacagccuagaguuucuggggauacagccaacacuuggaccuccuaaccagcccccuguuuccuga。
Preferably, the delivery vehicle comprises lipid-like nanoparticles.
The invention utilizes a delivery carrier such as a lipid-like nanoparticle to load mRNA capable of coding secretory ACE2, as shown in figure 1, the lipid-like nanoparticle loaded with the mRNA coding secretory ACE2 can enter cells, after entering the cells, the mRNA coding secretory ACE2 can express secretory ACE2 in the cells by utilizing a translation system, and the secretory ACE2 can be secreted to the outside of the cells and combined with a SARS-CoV-2 receptor binding domain, thereby achieving the purpose of preventing SARS-CoV-2 from infecting the cells.
Preferably, the delivery vehicle comprises a lipid-like molecule and a lipid molecule, wherein a lipid-like molecule refers to a compound that has a structure similar to a lipid molecule, but cannot be classified as a lipid molecule.
Preferably, the molar ratio of the lipid-like molecules to the lipid molecules is (0.1-10): 1, including but not limited to 0.2:1, 0.3:1, 0.4:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 8.5:1 or 9: 1.
Preferably, the nitrogen-to-phosphorus ratio of the lipoid molecule to the mRNA is (1-20): 1, including but not limited to 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 17:1, 18:1, or 19: 1.
Preferably, the lipid-like molecules comprise any one or combination of at least two of tB-UC18, tB-C18, tB-C16, tB-C14, qtB-UC18, qtB-C18, qtB-C16, qtB-C14, pB-UC18, pB-C18, pB-C16, pB-C14, qpB-UC18, qpB-C18, qpB-C16 and qpB-C14, preferably tB-UC18, and the structural formulas are respectively shown as follows.
tB-UC18
tB-C18
tB-C16
tB-C14
qtB-UC18
qtB-C18
qtB-C16
qtB-C14
pB-UC18
pB-C18
pB-C16
pB-C14
qpB-UC18
qpB-C18
qpB-C16
qpB-C14
Preferably, the lipid molecule comprises 1, 2-dioleyl-sn-glycero-3-phosphorylethanolamine (DOPE), cholesterol, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine, 1,2- (myristoyl-sn-glycero-3-phosphorylethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoryl-rac- (1-glycero) sodium salt, or a pharmaceutically acceptable salt thereof, 1, 2-palmitoyl phosphatidylglycerol, 1-palmitoyl-2-oleoyl lecithin, 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine, 1-stearoyl-2-oleoyl phosphatidylcholine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), sphingomyelin, ceramide, cephalin, cerebroside, diacylglycerol, and sphingomyelin, preferably 1-palmitoyl-2-oleoyl phosphatidylethanolamine.
In a third aspect, the present invention provides a method for preparing the secretory expression system of the second aspect, the method comprising:
and mixing the delivery vector and the mRNA for coding the secretory angiotensin converting enzyme 2, and incubating to obtain the secretory expression vector.
Preferably, the delivery vehicle comprises a mixture of lipid-like molecules and lipid molecules.
Preferably, the molar ratio of the lipid-like molecules to the lipid molecules is (0.1-10): 1, including but not limited to 0.2:1, 0.3:1, 0.4:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 8.5:1 or 9: 1.
Preferably, the lipid-like molecules and lipid molecules are dissolved in an organic solvent.
Preferably, the organic solvent comprises ethanol.
Preferably, the nitrogen-to-phosphorus ratio of the lipoid molecule to the mRNA is (1-20): 1, including but not limited to 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 17:1, 18:1, or 19: 1.
Preferably, the mRNA is dissolved in a buffer having a pH of 3 to 8 (e.g., 4, 5, 6 or 7).
Preferably, the buffer comprises a Phosphate (PBS) buffer.
Preferably, the incubation time is 10-20 min, including but not limited to 11min, 12min, 13min, 14min, 15min, 16min, 18min or 19 min.
In a fourth aspect, the present invention provides a recombinant cell comprising the mRNA encoding secreted angiotensin-converting enzyme 2 according to the first aspect or the secretory expression system according to the second aspect.
In a fifth aspect, the present invention provides a pharmaceutical composition comprising the mRNA encoding secreted angiotensin-converting enzyme 2 according to the first aspect, the secretory expression system according to the second aspect or the recombinant cell according to the fourth aspect.
Preferably, the pharmaceutical composition further comprises an adjuvant.
Preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
In a sixth aspect, the present invention provides the use of the mRNA encoding secreted angiotensin-converting enzyme 2 according to the first aspect, the secretory expression system according to the second aspect, the recombinant cell according to the fourth aspect, or the pharmaceutical composition according to the fifth aspect, in the preparation of a medicament for treating SARS-CoV-2.
Compared with the prior art, the invention has the following beneficial effects:
(1) the mRNA for coding the secretory angiotensin converting enzyme 2 can utilize a translation system of a host cell to express the secretory ACE2, and the secretory ACE2 can be secreted to the extracellular space and can be used as a decoy receptor to be combined with an S protein receptor binding domain of SARS-CoV-2, so that the combination of SARS-CoV-2 and an endogenous ACE2 receptor is competitively inhibited, and a potential intervention strategy is provided for treating SARS-CoV-2;
(2) the invention introduces mutation sites into mRNA encoding secretory angiotensin converting enzyme 2, which can further improve the affinity of secretory ACE2, thereby combining with SARS-CoV-2 more efficiently;
(3) the secretion expression system constructed by the invention can load mRNA for coding secretory angiotensin converting enzyme 2 into cells, so that the cells can express and secrete ACE2, thereby competitively inhibiting the combination of SARS-CoV-2 and endogenous ACE2 receptor.
Drawings
FIG. 1 is a schematic diagram of the delivery, secretory expression and neutralization of SARS-CoV-2 virus of mRNA encoding secreted ACE 2;
FIG. 2 is a graph of ACE2 enzyme activity of cell supernatants 24 hours after cells were treated with mRNA delivery vectors encoding secreted angiotensin converting enzyme 2, with error bars marked on the curves, with the undisplayed curves having smaller errors and the length of the error bars being smaller than the marked pattern on the curves;
FIG. 3 is a graph of the inhibitory effect of the ACE2 specific inhibitor MLN-4760 on ACE2 enzyme activity in cell supernatants 24 hours after treatment of cells with mRNA delivery vectors encoding secreted angiotensin converting enzyme 2;
FIG. 4 is a graph of the effect of cell supernatants at 48 hours after treatment of cells with mRNA delivery vectors encoding secreted angiotensin converting enzyme 2 on competitive inhibition of SARS-CoV-2 binding to the ACE2 receptor.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1
This example prepares mRNA encoding secreted angiotensin-converting enzyme 2, which has the nucleic acid sequence shown in SEQ ID NO: 11.
The preparation method of the mRNA comprises the following steps:
(1) carrying out codon optimization according to the combination of the amino acid sequences of a signal peptide, a peptidase domain, a dimer domain and a connecting region of the extracellular domain of the ACE2 receptor and a specific mutation site to obtain an mRNA coding sequence for coding a secretory ACE2 decoy receptor;
(2) synthesizing a DNA sequence containing a T7 RNA polymerase promoter and an untranslated region;
(3) taking 1 microgram of DNA in the step (2) as a transcription template of mRNA in a 20 mu L system, adding 2 mu L of RNA T7 RNA polymerase to transcribe the mRNA in vitro, incubating at 37 ℃ for 60min, and incubating at 37 ℃ for 15min by using 2 mu L of RNA enzyme 1;
(4) purifying the reaction product obtained in the step (3) by using a NEB RNA purification kit to obtain mRNA, measuring the concentration of the mRNA, subpackaging and storing at-80 ℃.
Example 2
This example prepared a secretory expression system comprising the mRNA encoding secreted angiotensin-converting enzyme 2 prepared in example 1 and a delivery vector.
The preparation method of the secretory expression line comprises the following steps:
(1) taking a 96-well single-well dose as an example, 240ng of the mRNA prepared in example 1 was dissolved in 10.8 μ L PBS (pH 7.4);
(2) dissolving lipid-like molecule tB-UC18 and lipid molecule 1, 2-dioleyl-sn-glycerol-3-phosphorylethanolamine (DOPE) in ethanol at a molar ratio of 1: 1;
(3) taking 1.2 mu L of ethanol mixed solution containing lipid sample molecules and lipid molecules with corresponding mass according to the standard that the ratio of the total mole number of phosphorus to the total mole number of nitrogen of the mRNA molecules is 1:1.5, adding the ethanol mixed solution into the mRNA solution prepared in the step (1), quickly blowing, uniformly mixing, and incubating for 15min at 25 ℃ to obtain the lipid-like nanoparticles loaded with secretory ACE2 mRNA, namely the secretory expression system.
Example 3
This example measures secreted ACE2 enzyme activity.
The specific process comprises the following steps:
(1) adding 10 mu L of the secretion expression system prepared in the embodiment 2 into a 96-well plate paved with 293T cells, putting the 96-well plate into a cell culture box, and respectively setting an experimental group which is added with PBS, added with nanoparticles loaded with encoding beta-galactosidase mRNA and not added with any substance (blank) as a control;
(2) after 24h, taking 10 μ L of cell supernatant, and detecting the enzyme activity of ACE2 according to the detection method of an angiotensin converting enzyme 2 activity fluorescence detection kit (P0319S-100T) in Byunnan, wherein the result is shown in figure 2;
(3) the enzyme activity of ACE2 was further determined as dose-dependent inhibition by adding different concentrations (0. mu.M, 0.01. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M) of the ACE2 specific inhibitor MLN-4760 to the cell supernatants and then testing the enzyme activity of ACE2 in the same manner, the results are shown in FIG. 3.
As can be seen from FIG. 2, the supernatant of the cells cultured with the secretion expression vector of the present invention has high ACE2 enzyme activity, which indicates that the secretion expression system constructed by the present invention can successfully enter the host cell, and the loaded mRNA can express the secreted ACE2 by using the translation system of the host cell.
As can be seen from fig. 3, as the concentration of the added ACE 2-specific inhibitor MLN-4760 increased, the ACE2 enzyme activity of the supernatant of the cells cultured with the addition of the secretion expression vector of the present invention gradually decreased, further indicating that the cell supernatant has ACE2 enzyme activity, i.e., is capable of expressing secreted ACE2 in the cells.
Example 4
This example examines the ability of secreted ACE2 to neutralize SARS-CoV-2.
The specific process comprises the following steps:
(1) adding 200 mu L of the secretion expression system prepared in the embodiment 2 into a 293T cell paved 6-well plate, and putting the 6-well plate into a cell culture box;
(2) after 48h of culture, the cell supernatant was concentrated 30-fold, 100. mu.L of the concentrate was used to detect the neutralizing activity of secreted ACE2 against SARS-CoV-2 according to the detection method of the SARS-CoV-2 Virus Neutralization assay Kit (SARS-CoV-2 Virus Neutralization Test (sVNT) Kit, L00847-A) of Kinry, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the inhibition rate of the supernatant of the cells cultured by adding the secretion expression vector of the invention on SARS-CoV-2 is close to the positive control (SARS-CoV-2 neutralizing antibody) result in the kit and is much higher than the negative control result in the kit, which indicates that the secretion expression system constructed by the invention can effectively express the secretory ACE2 in the cells, and the secretory ACE2 can be secreted to the outside of the cells and effectively neutralize SARS-CoV-2.
In conclusion, the secretory expression system constructed by the invention can load mRNA coding secretory ACE2 to enter cells, after entering the cells, the mRNA coding secretory ACE2 can express secretory ACE2, and secretory ACE2 can be secreted to the outside of the cells, and can effectively neutralize SARS-CoV-2, thereby providing a brand new idea for treating SARS-CoV-2.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Shenzhen citizen hospital
<120> mRNA encoding secreted angiotensin converting enzyme 2 and use thereof
<130> 20210520
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 51
<212> RNA
<213> Artificial sequence
<400> 1
augucuaguu cuagcuggcu gcugcugagc cugguggccg ucaccgccgc c 51
<210> 2
<211> 51
<212> RNA
<213> Artificial sequence
<400> 2
augagcagca gcagcuggcu gcugcugagc cugguggccg ugaccgccgc u 51
<210> 3
<211> 51
<212> RNA
<213> Artificial sequence
<400> 3
augaguucua gcagcuggcu gcugcugagc cucguggccg ugaccgccgc c 51
<210> 4
<211> 1794
<212> RNA
<213> Artificial sequence
<400> 4
cagagcacca ucgaggagca ggccaagacc uuccuggaua aguucaacca cgaggccgaa 60
gaucuguucu accagucaag ccuugcuagc uggaacuaca acaccaacau cacagaggaa 120
aauguccaga acaugaacaa cgccggagac aaguggagcg cuuuccugaa ggaacagagc 180
acccuggccc agauguaccc ccuccaagag auccagaacc ugacagugaa gcugcagcug 240
caggcccugc aacagaaugg cagcagcgug cuaagcgagg acaagagcaa gcggcugaac 300
accauccuua auaccaugag cacgaucuac agcacaggca aggucugcaa cccugacaau 360
ccccaggagu gccuccugcu cgagccaggc cugaacgaga ucauggccaa uagccuggac 420
uacaacgaga gacugugggc cugggaaagc uggagaagcg aggugggaaa acaguugaga 480
ccucuguacg aagaguacgu ggugcugaag aaugaaaugg cuagagccaa ccacuacgaa 540
gauuacggcg acuacuggcg cggcgacuac gaggugaacg gcguggacgg cuacgacuac 600
agcagaggcc agcugaucga ggaugucgag cauacauucg aggaaauuaa gccucuguau 660
gagcaccugc acgccuaugu gagggccaag cugaugaacg ccuauccuag cuacaucagc 720
cccaucggau gucugccugc ccaccugcug ggcgacaugu ggggcagauu cuggaccaac 780
cuguacucuc ugaccgugcc auucggccag aagcccaaca ucgacgugac agacgccaug 840
guggaucaag ccugggaugc ccagcggauc uucaaagagg ccgagaaauu cuucgugucc 900
gugggacugc cuaacaugac ccagggcuuc ugggagaaca guaugcugac cgacccugga 960
aauguucaaa aagcugugug ccacccuacc gccugggacc ugggcaaggg cgacuucaga 1020
auccugaugu gcaccaaggu gaccauggac gacuuucuga cagcucacca cgagaugggc 1080
cacauccagu acgacauggc cuacgcugcc cagcccuucc ugcugagaaa cggggcuaau 1140
gagggcuucc acgaggccgu gggcgagauc augucgcuga gcgccgcuac accuaagcau 1200
cugaaaucca ucggccugcu gucuccagac uuucaggagg auaacgaaac cgagaucaac 1260
uuccugcuga agcaggcucu gacaaucgug ggcacccugc ccuucaccua caugcuggaa 1320
aaauggagau ggaugguguu caagggcgaa aucccuaagg accaguggau gaaaaagugg 1380
ugggagauga aacgggaaau cgugggcgug guggaaccug ugccccacga cgagacauau 1440
ugcgacccug ccucucuguu ucacgugucc aacgacuaca gcuuuauucg guacuacacc 1500
cggacacugu accaguucca guuccaagaa gcccuguguc aggcugccaa gcacgagggg 1560
ccucugcaca agugcgauau cagcaacagc acagaagccg gccagaaacu guuuaauaug 1620
cugagacugg guaagucuga acccuggacc cuggcccugg aaaacguggu gggcgcuaag 1680
aacaugaacg ugagaccucu gcugaacuac uucgagccuc uguuuaccug gcugaaggac 1740
caaaacaaga acagcuucgu gggauggucc accgacugga gcccuuacgc cgac 1794
<210> 5
<211> 1794
<212> RNA
<213> Artificial sequence
<400> 5
cagagcacca ucgaggagca agccaaguac uuccuggaca aguucaacca cgaggccgag 60
gaccuguucu aucagagcag ccuggcaagc uggaacuaca acaccaacau caccgaggag 120
aacgugcaga acaugaacaa cgccggcgac aaguggagcg ccuuccugaa ggagcagagc 180
accaccgcuc agauguaccc ccugcaagag auucagaacc ugaccgugaa gcugcagcug 240
caagcccugc agcagaacgg cagcagcgug cugagcgagg acaagagcaa gagacugaac 300
accauccuga acaccaugag caccaucuac agcaccggca aggugugcaa ccccgacaac 360
ccccaagagu gccugcugcu ggagcccggc cugaacgaga ucauggccaa uagccuggac 420
uacaacgaga gacugugggc cugggagagc uggagaagcg aggugggcaa gcagcugaga 480
ccccuguacg aagaguacgu ggugcugaag aacgagaugg cuagagccaa ccacuacgag 540
gacuacggcg acuacuggag aggcgacuac gaggugaacg gcguggacgg cuacgacuac 600
agcagagggc agcugaucga ggacguggag cacaccuucg aggagaucaa gccccuguac 660
gagcaccugc acgccuacgu gagagccaag cugaugaacg ccuacccuag cuacaucagc 720
cccaucggcu gccugcccgc ccaccugcug ggcgacaugu ggggcagauu cuggaccaac 780
cuguacagcc ugaccgugcc cuucgggcag aagcccaaca ucgacgugac cgacgccaug 840
guggaccaag ccugggacgc ucagagaauc uucaaggagg ccgagaaguu cuucgugagc 900
gugggccugc ccaacaugac ccaaggcuuc ugggaguaca gcaugcugac cgaccccggc 960
aacgugcaga aggccgugug ccugcccacc gccugggacc ugggcaaggg cgacuucaga 1020
auccugaugu gcaccaaggu gaccauggac gacuuccuga ccgcccacca cgagaugggc 1080
cacauucagu acgacauggc cuaccuggcu cagcccuucc ugcugagaaa cggcgccaac 1140
gagggcuucc acgaggccgu gggcgagauc augagccuga gcgccgccac ccccaagcac 1200
cugaagagca ucggccugcu gagccccgac uuccaagagg acaacgagac cgagaucaac 1260
uuccugcuga agcaagcccu gaccaucgug ggcacccugc ccuucaccua caugcuggag 1320
aaguggagau ggaugguguu caagggcgag auccccaagg aucaguggau gaagaagugg 1380
ugggagauga agagagagau cgugggcgug guggagcccg ugccccacga cgagaccuac 1440
ugcgaccccg caagccuguu ccacgugagc aacgacuaua gcuuuaucag auacuacaca 1500
agaacccugu aucaguuuca guuccaagag gcccugugcc aagccgccaa gcacgagggc 1560
ccccugcaca agugcgauau uagcaacagc accgaggccg ggcagaagcu guucaacaug 1620
cugagacugg gcaagagcga gcccuggacc cuggcccugg agaacguggu gggcgccaag 1680
aacaugaacg ugagaccccu gcugaacuac uucgagcccc uguucaccug gcugaaggau 1740
cagaacaaga acagcuucgu gggcuggagc accgacugga gccccuacgc cgau 1794
<210> 6
<211> 1794
<212> RNA
<213> Artificial sequence
<400> 6
agaucuacca ucgaggaaca ggccaagacc uuccuggacu ucuucgacag ccaggcugag 60
gaucuguucu accagagcag ccuggccagc uggaacuaca acaccaauau cacagaagag 120
aacgugcaga auaugaacaa cgccggcgac aagagaagcg ccuuccucaa agagcggucc 180
acccuggcuc agauguaccc ucugcaggag auucagaauc ugacagugaa gcugcagcuc 240
caggcccugc agcagaacgg cagcagcgug cugagcgagg acaagagcaa gagacuaaac 300
accauccuga auaccaugag cacaaucuac agcacaggca agguguguaa uccugacaac 360
ccccaagaau gccugcugcu ggaaccaggc cugaaugaga ucauggccaa uagcuuggau 420
uauaacgaaa gacugugggc cugggaaagc uggagaagcg aggugggaaa acaacucaga 480
ccccuuuaug aagaguacgu ugugcugaaa aacgagaugg ccagagccaa ccacuacgaa 540
gauuacggcg acuacuggcg gggagacuac gaggugaacg gcguggacgg cuacgacuac 600
agcagagggc agcugaucga agaugucgaa cacaccuuug aggaaauuaa gccucuguau 660
gagcaccugc acgccuaugu gcgggccaaa cugaugaaug ccuacccuag cuacaucagu 720
ccuaucggcu gccugccagc ccaucugcug ggcgacaugu ggggcagauu cuggaccaac 780
cuguacuccc ugacagugcc uuucggccaa aaaccuaaca ucgacgugac ugaugccaug 840
gucgaccaag cuugggaugc ccaaagaauc uucaaggaag cugaaaaguu cuucgugucc 900
gugggccugc caaacaugac acagggauuc ugggagaaca gcaugcugac agaccccggc 960
aacguccaga aggccguuug ucugcccacc gccugggacc ugggcaaggg cgauuucaga 1020
auccugaugu gcaccaaggu gacaauggac gacuuucuga ccgcccacca cgagaugggc 1080
cacauccagu acgauauggc auaugccgcc cagccuuucc ugcuuagaaa cggcgcuaac 1140
gagggcuucc acgaggccgu gggagaaauc augagccuga gcgccgcuac accuaagcac 1200
cugaagagca ucggacugcu gucuccugau uuucaggagg auaacgagac agagaucaau 1260
uuccugcuga aacaggcccu gaccaucgug ggcacccugc cuuuuaccua caugcuggaa 1320
aaguggcgcu ggaugguguu caagggcgaa aucccuaagg accaguggau gaaaaagugg 1380
ugggagauga agcgggaaau cgucggagug guggaaccug ugccccacga cgagacguac 1440
ugcgacccug cuucucuguu ucaugugucc aacgacuaca gcuucaucag auacuacacc 1500
agaacccugu accaguucca auuucaggag gcccugugcc aggccgccaa gcacgagggc 1560
ccucugcaca agugcgacau cagcaacucc accgaggcag gccagaagcu guucaacaug 1620
cugagacugg gcaaaucuga gccuuggaca cuggcccugg aaaacguggu gggcgccaag 1680
aacaugaacg ugcggccacu gcugaacuac uucgagcccc uguucaccug gcugaaggac 1740
cagaacaaga acuccuucgu gggcuggucc accgauugga gccccuacgc ugau 1794
<210> 7
<211> 1845
<212> RNA
<213> Artificial sequence
<400> 7
augucuaguu cuagcuggcu gcugcugagc cugguggccg ucaccgccgc ccagagcacc 60
aucgaggagc aggccaagac cuuccuggau aaguucaacc acgaggccga agaucuguuc 120
uaccagucaa gccuugcuag cuggaacuac aacaccaaca ucacagagga aaauguccag 180
aacaugaaca acgccggaga caaguggagc gcuuuccuga aggaacagag cacccuggcc 240
cagauguacc cccuccaaga gauccagaac cugacaguga agcugcagcu gcaggcccug 300
caacagaaug gcagcagcgu gcuaagcgag gacaagagca agcggcugaa caccauccuu 360
aauaccauga gcacgaucua cagcacaggc aaggucugca acccugacaa uccccaggag 420
ugccuccugc ucgagccagg ccugaacgag aucauggcca auagccugga cuacaacgag 480
agacuguggg ccugggaaag cuggagaagc gaggugggaa aacaguugag accucuguac 540
gaagaguacg uggugcugaa gaaugaaaug gcuagagcca accacuacga agauuacggc 600
gacuacuggc gcggcgacua cgaggugaac ggcguggacg gcuacgacua cagcagaggc 660
cagcugaucg aggaugucga gcauacauuc gaggaaauua agccucugua ugagcaccug 720
cacgccuaug ugagggccaa gcugaugaac gccuauccua gcuacaucag ccccaucgga 780
ugucugccug cccaccugcu gggcgacaug uggggcagau ucuggaccaa ccuguacucu 840
cugaccgugc cauucggcca gaagcccaac aucgacguga cagacgccau gguggaucaa 900
gccugggaug cccagcggau cuucaaagag gccgagaaau ucuucguguc cgugggacug 960
ccuaacauga cccagggcuu cugggagaac aguaugcuga ccgacccugg aaauguucaa 1020
aaagcugugu gccacccuac cgccugggac cugggcaagg gcgacuucag aauccugaug 1080
ugcaccaagg ugaccaugga cgacuuucug acagcucacc acgagauggg ccacauccag 1140
uacgacaugg ccuacgcugc ccagcccuuc cugcugagaa acggggcuaa ugagggcuuc 1200
cacgaggccg ugggcgagau caugucgcug agcgccgcua caccuaagca ucugaaaucc 1260
aucggccugc ugucuccaga cuuucaggag gauaacgaaa ccgagaucaa cuuccugcug 1320
aagcaggcuc ugacaaucgu gggcacccug cccuucaccu acaugcugga aaaauggaga 1380
uggauggugu ucaagggcga aaucccuaag gaccagugga ugaaaaagug gugggagaug 1440
aaacgggaaa ucgugggcgu gguggaaccu gugccccacg acgagacaua uugcgacccu 1500
gccucucugu uucacguguc caacgacuac agcuuuauuc gguacuacac ccggacacug 1560
uaccaguucc aguuccaaga agcccugugu caggcugcca agcacgaggg gccucugcac 1620
aagugcgaua ucagcaacag cacagaagcc ggccagaaac uguuuaauau gcugagacug 1680
gguaagucug aacccuggac ccuggcccug gaaaacgugg ugggcgcuaa gaacaugaac 1740
gugagaccuc ugcugaacua cuucgagccu cuguuuaccu ggcugaagga ccaaaacaag 1800
aacagcuucg ugggaugguc caccgacugg agcccuuacg ccgac 1845
<210> 8
<211> 1845
<212> RNA
<213> Artificial sequence
<400> 8
augagcagca gcagcuggcu gcugcugagc cugguggccg ugaccgccgc ucagagcacc 60
aucgaggagc aagccaagua cuuccuggac aaguucaacc acgaggccga ggaccuguuc 120
uaucagagca gccuggcaag cuggaacuac aacaccaaca ucaccgagga gaacgugcag 180
aacaugaaca acgccggcga caaguggagc gccuuccuga aggagcagag caccaccgcu 240
cagauguacc cccugcaaga gauucagaac cugaccguga agcugcagcu gcaagcccug 300
cagcagaacg gcagcagcgu gcugagcgag gacaagagca agagacugaa caccauccug 360
aacaccauga gcaccaucua cagcaccggc aaggugugca accccgacaa cccccaagag 420
ugccugcugc uggagcccgg ccugaacgag aucauggcca auagccugga cuacaacgag 480
agacuguggg ccugggagag cuggagaagc gaggugggca agcagcugag accccuguac 540
gaagaguacg uggugcugaa gaacgagaug gcuagagcca accacuacga ggacuacggc 600
gacuacugga gaggcgacua cgaggugaac ggcguggacg gcuacgacua cagcagaggg 660
cagcugaucg aggacgugga gcacaccuuc gaggagauca agccccugua cgagcaccug 720
cacgccuacg ugagagccaa gcugaugaac gccuacccua gcuacaucag ccccaucggc 780
ugccugcccg cccaccugcu gggcgacaug uggggcagau ucuggaccaa ccuguacagc 840
cugaccgugc ccuucgggca gaagcccaac aucgacguga ccgacgccau gguggaccaa 900
gccugggacg cucagagaau cuucaaggag gccgagaagu ucuucgugag cgugggccug 960
cccaacauga cccaaggcuu cugggaguac agcaugcuga ccgaccccgg caacgugcag 1020
aaggccgugu gccugcccac cgccugggac cugggcaagg gcgacuucag aauccugaug 1080
ugcaccaagg ugaccaugga cgacuuccug accgcccacc acgagauggg ccacauucag 1140
uacgacaugg ccuaccuggc ucagcccuuc cugcugagaa acggcgccaa cgagggcuuc 1200
cacgaggccg ugggcgagau caugagccug agcgccgcca cccccaagca ccugaagagc 1260
aucggccugc ugagccccga cuuccaagag gacaacgaga ccgagaucaa cuuccugcug 1320
aagcaagccc ugaccaucgu gggcacccug cccuucaccu acaugcugga gaaguggaga 1380
uggauggugu ucaagggcga gauccccaag gaucagugga ugaagaagug gugggagaug 1440
aagagagaga ucgugggcgu gguggagccc gugccccacg acgagaccua cugcgacccc 1500
gcaagccugu uccacgugag caacgacuau agcuuuauca gauacuacac aagaacccug 1560
uaucaguuuc aguuccaaga ggcccugugc caagccgcca agcacgaggg cccccugcac 1620
aagugcgaua uuagcaacag caccgaggcc gggcagaagc uguucaacau gcugagacug 1680
ggcaagagcg agcccuggac ccuggcccug gagaacgugg ugggcgccaa gaacaugaac 1740
gugagacccc ugcugaacua cuucgagccc cuguucaccu ggcugaagga ucagaacaag 1800
aacagcuucg ugggcuggag caccgacugg agccccuacg ccgau 1845
<210> 9
<211> 1845
<212> RNA
<213> Artificial sequence
<400> 9
augaguucua gcagcuggcu gcugcugagc cucguggccg ugaccgccgc cagaucuacc 60
aucgaggaac aggccaagac cuuccuggac uucuucgaca gccaggcuga ggaucuguuc 120
uaccagagca gccuggccag cuggaacuac aacaccaaua ucacagaaga gaacgugcag 180
aauaugaaca acgccggcga caagagaagc gccuuccuca aagagcgguc cacccuggcu 240
cagauguacc cucugcagga gauucagaau cugacaguga agcugcagcu ccaggcccug 300
cagcagaacg gcagcagcgu gcugagcgag gacaagagca agagacuaaa caccauccug 360
aauaccauga gcacaaucua cagcacaggc aaggugugua auccugacaa cccccaagaa 420
ugccugcugc uggaaccagg ccugaaugag aucauggcca auagcuugga uuauaacgaa 480
agacuguggg ccugggaaag cuggagaagc gaggugggaa aacaacucag accccuuuau 540
gaagaguacg uugugcugaa aaacgagaug gccagagcca accacuacga agauuacggc 600
gacuacuggc ggggagacua cgaggugaac ggcguggacg gcuacgacua cagcagaggg 660
cagcugaucg aagaugucga acacaccuuu gaggaaauua agccucugua ugagcaccug 720
cacgccuaug ugcgggccaa acugaugaau gccuacccua gcuacaucag uccuaucggc 780
ugccugccag cccaucugcu gggcgacaug uggggcagau ucuggaccaa ccuguacucc 840
cugacagugc cuuucggcca aaaaccuaac aucgacguga cugaugccau ggucgaccaa 900
gcuugggaug cccaaagaau cuucaaggaa gcugaaaagu ucuucguguc cgugggccug 960
ccaaacauga cacagggauu cugggagaac agcaugcuga cagaccccgg caacguccag 1020
aaggccguuu gucugcccac cgccugggac cugggcaagg gcgauuucag aauccugaug 1080
ugcaccaagg ugacaaugga cgacuuucug accgcccacc acgagauggg ccacauccag 1140
uacgauaugg cauaugccgc ccagccuuuc cugcuuagaa acggcgcuaa cgagggcuuc 1200
cacgaggccg ugggagaaau caugagccug agcgccgcua caccuaagca ccugaagagc 1260
aucggacugc ugucuccuga uuuucaggag gauaacgaga cagagaucaa uuuccugcug 1320
aaacaggccc ugaccaucgu gggcacccug ccuuuuaccu acaugcugga aaaguggcgc 1380
uggauggugu ucaagggcga aaucccuaag gaccagugga ugaaaaagug gugggagaug 1440
aagcgggaaa ucgucggagu gguggaaccu gugccccacg acgagacgua cugcgacccu 1500
gcuucucugu uucauguguc caacgacuac agcuucauca gauacuacac cagaacccug 1560
uaccaguucc aauuucagga ggcccugugc caggccgcca agcacgaggg cccucugcac 1620
aagugcgaca ucagcaacuc caccgaggca ggccagaagc uguucaacau gcugagacug 1680
ggcaaaucug agccuuggac acuggcccug gaaaacgugg ugggcgccaa gaacaugaac 1740
gugcggccac ugcugaacua cuucgagccc cuguucaccu ggcugaagga ccagaacaag 1800
aacuccuucg ugggcugguc caccgauugg agccccuacg cugau 1845
<210> 10
<211> 2223
<212> RNA
<213> Artificial sequence
<400> 10
augucuaguu cuagcuggcu gcugcugagc cugguggccg ucaccgccgc ccagagcacc 60
aucgaggagc aggccaagac cuuccuggau aaguucaacc acgaggccga agaucuguuc 120
uaccagucaa gccuugcuag cuggaacuac aacaccaaca ucacagagga aaauguccag 180
aacaugaaca acgccggaga caaguggagc gcuuuccuga aggaacagag cacccuggcc 240
cagauguacc cccuccaaga gauccagaac cugacaguga agcugcagcu gcaggcccug 300
caacagaaug gcagcagcgu gcuaagcgag gacaagagca agcggcugaa caccauccuu 360
aauaccauga gcacgaucua cagcacaggc aaggucugca acccugacaa uccccaggag 420
ugccuccugc ucgagccagg ccugaacgag aucauggcca auagccugga cuacaacgag 480
agacuguggg ccugggaaag cuggagaagc gaggugggaa aacaguugag accucuguac 540
gaagaguacg uggugcugaa gaaugaaaug gcuagagcca accacuacga agauuacggc 600
gacuacuggc gcggcgacua cgaggugaac ggcguggacg gcuacgacua cagcagaggc 660
cagcugaucg aggaugucga gcauacauuc gaggaaauua agccucugua ugagcaccug 720
cacgccuaug ugagggccaa gcugaugaac gccuauccua gcuacaucag ccccaucgga 780
ugucugccug cccaccugcu gggcgacaug uggggcagau ucuggaccaa ccuguacucu 840
cugaccgugc cauucggcca gaagcccaac aucgacguga cagacgccau gguggaucaa 900
gccugggaug cccagcggau cuucaaagag gccgagaaau ucuucguguc cgugggacug 960
ccuaacauga cccagggcuu cugggagaac aguaugcuga ccgacccugg aaauguucaa 1020
aaagcugugu gccacccuac cgccugggac cugggcaagg gcgacuucag aauccugaug 1080
ugcaccaagg ugaccaugga cgacuuucug acagcucacc acgagauggg ccacauccag 1140
uacgacaugg ccuacgcugc ccagcccuuc cugcugagaa acggggcuaa ugagggcuuc 1200
cacgaggccg ugggcgagau caugucgcug agcgccgcua caccuaagca ucugaaaucc 1260
aucggccugc ugucuccaga cuuucaggag gauaacgaaa ccgagaucaa cuuccugcug 1320
aagcaggcuc ugacaaucgu gggcacccug cccuucaccu acaugcugga aaaauggaga 1380
uggauggugu ucaagggcga aaucccuaag gaccagugga ugaaaaagug gugggagaug 1440
aaacgggaaa ucgugggcgu gguggaaccu gugccccacg acgagacaua uugcgacccu 1500
gccucucugu uucacguguc caacgacuac agcuuuauuc gguacuacac ccggacacug 1560
uaccaguucc aguuccaaga agcccugugu caggcugcca agcacgaggg gccucugcac 1620
aagugcgaua ucagcaacag cacagaagcc ggccagaaac uguuuaauau gcugagacug 1680
gguaagucug aacccuggac ccuggcccug gaaaacgugg ugggcgcuaa gaacaugaac 1740
gugagaccuc ugcugaacua cuucgagccu cuguuuaccu ggcugaagga ccaaaacaag 1800
aacagcuucg ugggaugguc caccgacugg agcccuuacg ccgaccagag caucaaggug 1860
cggaucucuc uuaagagcgc ccugggcgau aaggccuacg aguggaacga caacgagaug 1920
uaccuguuua gaagcuccgu ugccuaugcc augagacagu auuuccucaa ggucaagaau 1980
cagaugaucc uguucggaga agaggaugug cgaguggcaa accugaagcc ucggauuagc 2040
uucaacuucu ucgugacagc ccccaagaac gugucugaua ucauccccag aaccgaagug 2100
gaaaaggcca ucagaauguc cagaagcaga aucaacgacg ccuuccggcu gaacgauaau 2160
agccuggaau uccugggcau ccagccaacu cugggccccc cuaaccagcc uccugugucc 2220
uga 2223
<210> 11
<211> 2199
<212> RNA
<213> Artificial sequence
<400> 11
augagcagca gcagcuggcu gcugcugagc cugguggccg ugaccgccgc ucagagcacc 60
aucgaggagc aagccaagua cuuccuggac aaguucaacc acgaggccga ggaccuguuc 120
uaucagagca gccuggcaag cuggaacuac aacaccaaca ucaccgagga gaacgugcag 180
aacaugaaca acgccggcga caaguggagc gccuuccuga aggagcagag caccaccgcu 240
cagauguacc cccugcaaga gauucagaac cugaccguga agcugcagcu gcaagcccug 300
cagcagaacg gcagcagcgu gcugagcgag gacaagagca agagacugaa caccauccug 360
aacaccauga gcaccaucua cagcaccggc aaggugugca accccgacaa cccccaagag 420
ugccugcugc uggagcccgg ccugaacgag aucauggcca auagccugga cuacaacgag 480
agacuguggg ccugggagag cuggagaagc gaggugggca agcagcugag accccuguac 540
gaagaguacg uggugcugaa gaacgagaug gcuagagcca accacuacga ggacuacggc 600
gacuacugga gaggcgacua cgaggugaac ggcguggacg gcuacgacua cagcagaggg 660
cagcugaucg aggacgugga gcacaccuuc gaggagauca agccccugua cgagcaccug 720
cacgccuacg ugagagccaa gcugaugaac gccuacccua gcuacaucag ccccaucggc 780
ugccugcccg cccaccugcu gggcgacaug uggggcagau ucuggaccaa ccuguacagc 840
cugaccgugc ccuucgggca gaagcccaac aucgacguga ccgacgccau gguggaccaa 900
gccugggacg cucagagaau cuucaaggag gccgagaagu ucuucgugag cgugggccug 960
cccaacauga cccaaggcuu cugggaguac agcaugcuga ccgaccccgg caacgugcag 1020
aaggccgugu gccugcccac cgccugggac cugggcaagg gcgacuucag aauccugaug 1080
ugcaccaagg ugaccaugga cgacuuccug accgcccacc acgagauggg ccacauucag 1140
uacgacaugg ccuaccuggc ucagcccuuc cugcugagaa acggcgccaa cgagggcuuc 1200
cacgaggccg ugggcgagau caugagccug agcgccgcca cccccaagca ccugaagagc 1260
aucggccugc ugagccccga cuuccaagag gacaacgaga ccgagaucaa cuuccugcug 1320
aagcaagccc ugaccaucgu gggcacccug cccuucaccu acaugcugga gaaguggaga 1380
uggauggugu ucaagggcga gauccccaag gaucagugga ugaagaagug gugggagaug 1440
aagagagaga ucgugggcgu gguggagccc gugccccacg acgagaccua cugcgacccc 1500
gcaagccugu uccacgugag caacgacuau agcuuuauca gauacuacac aagaacccug 1560
uaucaguuuc aguuccaaga ggcccugugc caagccgcca agcacgaggg cccccugcac 1620
aagugcgaua uuagcaacag caccgaggcc gggcagaagc uguucaacau gcugagacug 1680
ggcaagagcg agcccuggac ccuggcccug gagaacgugg ugggcgccaa gaacaugaac 1740
gugagacccc ugcugaacua cuucgagccc cuguucaccu ggcugaagga ucagaacaag 1800
aacagcuucg ugggcuggag caccgacugg agccccuacg ccgaucagag caucaaggug 1860
agaaucagcc ugaagagcgc ccugggcgac aaggccuacg aguggaacga caacgagaug 1920
uaccuguuca gaagcagcgu ggccuacgcc augagacagu acuuccugaa ggugaagaau 1980
cagaugaucc uguucggcga ggaggacgug agaguggcca accugaagcc uagaaucagc 2040
uucaacuucu ucgugaccgc ccccaagaac gugagcgaca ucaucccuag aaccgaggug 2100
gagaaggcca ucagaaugag cagaagcaga aucaacgacg ccuucagacu gaacgacaac 2160
ucacuggagu uccugggcau ucagcccacc cugggcuga 2199
<210> 12
<211> 2199
<212> RNA
<213> Artificial sequence
<400> 12
augaguucua gcagcuggcu gcugcugagc cucguggccg ugaccgccgc cagaucuacc 60
aucgaggaac aggccaagac cuuccuggac uucuucgaca gccaggcuga ggaucuguuc 120
uaccagagca gccuggccag cuggaacuac aacaccaaua ucacagaaga gaacgugcag 180
aauaugaaca acgccggcga caagagaagc gccuuccuca aagagcgguc cacccuggcu 240
cagauguacc cucugcagga gauucagaau cugacaguga agcugcagcu ccaggcccug 300
cagcagaacg gcagcagcgu gcugagcgag gacaagagca agagacuaaa caccauccug 360
aauaccauga gcacaaucua cagcacaggc aaggugugua auccugacaa cccccaagaa 420
ugccugcugc uggaaccagg ccugaaugag aucauggcca auagcuugga uuauaacgaa 480
agacuguggg ccugggaaag cuggagaagc gaggugggaa aacaacucag accccuuuau 540
gaagaguacg uugugcugaa aaacgagaug gccagagcca accacuacga agauuacggc 600
gacuacuggc ggggagacua cgaggugaac ggcguggacg gcuacgacua cagcagaggg 660
cagcugaucg aagaugucga acacaccuuu gaggaaauua agccucugua ugagcaccug 720
cacgccuaug ugcgggccaa acugaugaau gccuacccua gcuacaucag uccuaucggc 780
ugccugccag cccaucugcu gggcgacaug uggggcagau ucuggaccaa ccuguacucc 840
cugacagugc cuuucggcca aaaaccuaac aucgacguga cugaugccau ggucgaccaa 900
gcuugggaug cccaaagaau cuucaaggaa gcugaaaagu ucuucguguc cgugggccug 960
ccaaacauga cacagggauu cugggagaac agcaugcuga cagaccccgg caacguccag 1020
aaggccguuu gucugcccac cgccugggac cugggcaagg gcgauuucag aauccugaug 1080
ugcaccaagg ugacaaugga cgacuuucug accgcccacc acgagauggg ccacauccag 1140
uacgauaugg cauaugccgc ccagccuuuc cugcuuagaa acggcgcuaa cgagggcuuc 1200
cacgaggccg ugggagaaau caugagccug agcgccgcua caccuaagca ccugaagagc 1260
aucggacugc ugucuccuga uuuucaggag gauaacgaga cagagaucaa uuuccugcug 1320
aaacaggccc ugaccaucgu gggcacccug ccuuuuaccu acaugcugga aaaguggcgc 1380
uggauggugu ucaagggcga aaucccuaag gaccagugga ugaaaaagug gugggagaug 1440
aagcgggaaa ucgucggagu gguggaaccu gugccccacg acgagacgua cugcgacccu 1500
gcuucucugu uucauguguc caacgacuac agcuucauca gauacuacac cagaacccug 1560
uaccaguucc aauuucagga ggcccugugc caggccgcca agcacgaggg cccucugcac 1620
aagugcgaca ucagcaacuc caccgaggca ggccagaagc uguucaacau gcugagacug 1680
ggcaaaucug agccuuggac acuggcccug gaaaacgugg ugggcgccaa gaacaugaac 1740
gugcggccac ugcugaacua cuucgagccc cuguucaccu ggcugaagga ccagaacaag 1800
aacuccuucg ugggcugguc caccgauugg agccccuacg cugaucagag caucaaggug 1860
cggauuucuc ugaagagcgc ccugggcgac aaggccuacg aguggaauga caacgagaug 1920
uaccuguucc ggucuucugu ggcuuacgcc augagacagu acuuccugaa agugaagaac 1980
cagaugaucc uguuuggcga ggaggacgug cggguggcca accugaagcc ccggaucagc 2040
uuuaacuucu ucgugaccgc cccuaagaau gucucugaua ucauccccag gaccgaggug 2100
gaaaaagcca uccggaugag cagaagccgc aucaacgacg cuuuucggcu gaacgacaau 2160
ucacuggaau uccugggaau ccagccuaca cugggcuga 2199
<210> 13
<211> 51
<212> RNA
<213> Artificial sequence
<400> 13
augucaagcu cuuccuggcu ccuucucagc cuuguugcug uaacugcugc u 51
<210> 14
<211> 1794
<212> RNA
<213> Artificial sequence
<400> 14
caguccacca uugaggaaca ggccaagaca uuuuuggaca aguuuaacca cgaagccgaa 60
gaccuguucu aucaaaguuc acuugcuucu uggaauuaua acaccaauau uacugaagag 120
aauguccaaa acaugaauaa ugcuggggac aaauggucug ccuuuuuaaa ggaacagucc 180
acacuugccc aaauguaucc acuacaagaa auucagaauc ucacagucaa gcuucagcug 240
caggcucuuc agcaaaaugg gucuucagug cucucagaag acaagagcaa acgguugaac 300
acaauucuaa auacaaugag caccaucuac aguacuggaa aaguuuguaa cccagauaau 360
ccacaagaau gcuuauuacu ugaaccaggu uugaaugaaa uaauggcaaa caguuuagac 420
uacaaugaga ggcucugggc uugggaaagc uggagaucug aggucggcaa gcagcugagg 480
ccauuauaug aagaguaugu ggucuugaaa aaugagaugg caagagcaaa ucauuaugag 540
gacuaugggg auuauuggag aggagacuau gaaguaaaug ggguagaugg cuaugacuac 600
agccgcggcc aguugauuga agauguggaa cauaccuuug aagagauuaa accauuauau 660
gaacaucuuc augccuaugu gagggcaaag uugaugaaug ccuauccuuc cuauaucagu 720
ccaauuggau gccucccugc ucauuugcuu ggugauaugu gggguagauu uuggacaaau 780
cuguacucuu ugacaguucc cuuuggacag aaaccaaaca uagauguuac ugaugcaaug 840
guggaccagg ccugggaugc acagagaaua uucaaggagg ccgagaaguu cuuuguaucu 900
guuggucuuc cuaauaugac ucaaggauuc ugggaaaauu ccaugcuaac ggacccagga 960
aauguucaga aagcagucug ccaucccaca gcuugggacc uggggaaggg cgacuucagg 1020
auccuuaugu gcacaaaggu gacaauggac gacuuccuga cagcucauca ugagaugggg 1080
cauauccagu augauauggc auaugcugca caaccuuuuc ugcuaagaaa uggagcuaau 1140
gaaggauucc augaagcugu uggggaaauc augucacuuu cugcagccac accuaagcau 1200
uuaaaaucca uuggucuucu gucacccgau uuucaagaag acaaugaaac agaaauaaac 1260
uuccugcuca aacaagcacu cacgauuguu gggacucugc cauuuacuua cauguuagag 1320
aaguggaggu ggauggucuu uaaaggggaa auucccaaag accaguggau gaaaaagugg 1380
ugggagauga agcgagagau aguuggggug guggaaccug ugccccauga ugaaacauac 1440
ugugaccccg caucucuguu ccauguuucu aaugauuacu cauucauucg auauuacaca 1500
aggacccuuu accaauucca guuucaagaa gcacuuuguc aagcagcuaa acaugaaggc 1560
ccucugcaca aaugugacau cucaaacucu acagaagcug gacagaaacu guucaauaug 1620
cugaggcuug gaaaaucaga acccuggacc cuagcauugg aaaauguugu aggagcaaag 1680
aacaugaaug uaaggccacu gcucaacuac uuugagcccu uauuuaccug gcugaaagac 1740
cagaacaaga auucuuuugu gggauggagu accgacugga guccauaugc agac 1794
<210> 15
<211> 2223
<212> RNA
<213> Artificial sequence
<400> 15
augucaagcu cuuccuggcu ccuucucagc cuuguugcug uaacugcugc ucaguccacc 60
auugaggaac aggccaagac auuuuuggac aaguuuaacc acgaagccga agaccuguuc 120
uaucaaaguu cacuugcuuc uuggaauuau aacaccaaua uuacugaaga gaauguccaa 180
aacaugaaua augcugggga caaauggucu gccuuuuuaa aggaacaguc cacacuugcc 240
caaauguauc cacuacaaga aauucagaau cucacaguca agcuucagcu gcaggcucuu 300
cagcaaaaug ggucuucagu gcucucagaa gacaagagca aacgguugaa cacaauucua 360
aauacaauga gcaccaucua caguacugga aaaguuugua acccagauaa uccacaagaa 420
ugcuuauuac uugaaccagg uuugaaugaa auaauggcaa acaguuuaga cuacaaugag 480
aggcucuggg cuugggaaag cuggagaucu gaggucggca agcagcugag gccauuauau 540
gaagaguaug uggucuugaa aaaugagaug gcaagagcaa aucauuauga ggacuauggg 600
gauuauugga gaggagacua ugaaguaaau gggguagaug gcuaugacua cagccgcggc 660
caguugauug aagaugugga acauaccuuu gaagagauua aaccauuaua ugaacaucuu 720
caugccuaug ugagggcaaa guugaugaau gccuauccuu ccuauaucag uccaauugga 780
ugccucccug cucauuugcu uggugauaug ugggguagau uuuggacaaa ucuguacucu 840
uugacaguuc ccuuuggaca gaaaccaaac auagauguua cugaugcaau gguggaccag 900
gccugggaug cacagagaau auucaaggag gccgagaagu ucuuuguauc uguuggucuu 960
ccuaauauga cucaaggauu cugggaaaau uccaugcuaa cggacccagg aaauguucag 1020
aaagcagucu gccaucccac agcuugggac cuggggaagg gcgacuucag gauccuuaug 1080
ugcacaaagg ugacaaugga cgacuuccug acagcucauc augagauggg gcauauccag 1140
uaugauaugg cauaugcugc acaaccuuuu cugcuaagaa auggagcuaa ugaaggauuc 1200
caugaagcug uuggggaaau caugucacuu ucugcagcca caccuaagca uuuaaaaucc 1260
auuggucuuc ugucacccga uuuucaagaa gacaaugaaa cagaaauaaa cuuccugcuc 1320
aaacaagcac ucacgauugu ugggacucug ccauuuacuu acauguuaga gaaguggagg 1380
uggauggucu uuaaagggga aauucccaaa gaccagugga ugaaaaagug gugggagaug 1440
aagcgagaga uaguuggggu gguggaaccu gugccccaug augaaacaua cugugacccc 1500
gcaucucugu uccauguuuc uaaugauuac ucauucauuc gauauuacac aaggacccuu 1560
uaccaauucc aguuucaaga agcacuuugu caagcagcua aacaugaagg cccucugcac 1620
aaaugugaca ucucaaacuc uacagaagcu ggacagaaac uguucaauau gcugaggcuu 1680
ggaaaaucag aacccuggac ccuagcauug gaaaauguug uaggagcaaa gaacaugaau 1740
guaaggccac ugcucaacua cuuugagccc uuauuuaccu ggcugaaaga ccagaacaag 1800
aauucuuuug ugggauggag uaccgacugg aguccauaug cagaccaaag caucaaagug 1860
aggauaagcc uaaaaucagc ucuuggagau aaagcauaug aauggaacga caaugaaaug 1920
uaccuguucc gaucaucugu ugcauaugcu augaggcagu acuuuuuaaa aguaaaaaau 1980
cagaugauuc uuuuugggga ggaggaugug cgaguggcua auuugaaacc aagaaucucc 2040
uuuaauuucu uugucacugc accuaaaaau gugucugaua ucauuccuag aacugaaguu 2100
gaaaaggcca ucaggauguc ccggagccgu aucaaugaug cuuuccgucu gaaugacaac 2160
agccuagagu uucuggggau acagccaaca cuuggaccuc cuaaccagcc cccuguuucc 2220
uga 2223
Claims (10)
1. An mRNA encoding a secreted angiotensin converting enzyme 2, wherein the mRNA comprises a coding region of 70% or more of a signal peptide of angiotensin converting enzyme 2 and a coding region of 70% or more of a peptidase domain.
2. The mRNA of claim 1, wherein the coding region for the signal peptide comprises the nucleic acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3.
3. The mRNA of claim 1 or 2, wherein the coding region for the peptidase domain comprises the nucleic acid sequence of SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 6.
4. The mRNA of any one of claims 1 to 3, which comprises the nucleic acid sequence set forth in SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9;
preferably, the mRNA further comprises a coding region for the dimer domain and/or a coding region for the junction region of angiotensin-converting enzyme 2;
preferably, the mRNA further comprises any one of or a combination of at least two of mutation sites Q18R, T27Y/W, K31F, N33D, H34S, E35Q, W69R, Q76R, L79T, N330Y, H345L or a 386L.
5. The mRNA of any one of claims 1 to 4, which comprises the nucleic acid sequence of SEQ ID NO 10, 11 or 12.
6. A secretory expression system comprising mRNA encoding secretory angiotensin converting enzyme 2 and a delivery vector carrying the mRNA encoding secretory angiotensin converting enzyme 2;
preferably, the mRNA encoding secreted angiotensin converting enzyme 2 comprises the mRNA encoding secreted angiotensin converting enzyme 2 according to any of claims 1 to 5 and/or the wild-type mRNA encoding secreted angiotensin converting enzyme 2;
preferably, the delivery vehicle comprises lipid-like nanoparticles;
preferably, the delivery vehicle comprises a lipid-like molecule and a lipid molecule;
preferably, the molar ratio of the lipid-like molecules to the lipid molecules is (0.1-10): 1;
preferably, the nitrogen-phosphorus ratio of the lipid-like molecules to the mRNA is (1-20): 1;
preferably, the lipid-like molecules comprise any one or a combination of at least two of tB-UC18, tB-C18, tB-C16, tB-C14, pB-UC18, pB-C18, pB-C16, pB-C14, qtB-UC18, qtB-C18, qtB-C16, qtB-C14, qpB-UC18, qpB-C18, qpB-C16 and qpB-C14;
preferably, the lipid molecule comprises 1, 2-dioleyl-sn-glycero-3-phosphorylethanolamine, cholesterol, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine, 1,2- (myristoyl-sn-glycero-3-phosphorylethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoryl-rac- (1-glycero) sodium salt, or a pharmaceutically acceptable salt thereof, Any one or a combination of at least two of 1, 2-palmitoyl phosphatidylglycerol, 1-palmitoyl-2-oleoyl lecithin, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, 1-stearoyl-2-oleoyl phosphatidylcholine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, sphingomyelin, ceramide, cephalin, cerebroside, diacylglycerol, and sphingomyelin.
7. A method for preparing the secretory expression system of claim 6, comprising:
and mixing the delivery vector and the mRNA for coding the secretory angiotensin converting enzyme 2, and incubating to obtain the secretory expression vector.
8. A recombinant cell comprising the mRNA encoding secreted angiotensin converting enzyme 2 according to any of claims 1 to 5 or the secretory expression system according to claim 6.
9. A pharmaceutical composition comprising the mRNA encoding secreted angiotensin converting enzyme 2 according to any of claims 1 to 5, the secretory expression system according to claim 6 or the recombinant cell according to claim 8;
preferably, the pharmaceutical composition further comprises an adjuvant;
preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
10. Use of the mRNA encoding secreted angiotensin-converting enzyme 2 according to any of claims 1 to 5, the secretory expression system according to claim 6, the recombinant cell according to claim 8 or the pharmaceutical composition according to claim 9 for the manufacture of a medicament for the treatment of SARS-CoV-2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112138152A (en) * | 2020-09-21 | 2020-12-29 | 中吉智药(天津)生物技术有限公司 | AAV vector-based coronavirus infection universal gene therapy medicine and preparation method thereof |
| CN112522205A (en) * | 2020-12-15 | 2021-03-19 | 广东省农业科学院动物卫生研究所 | Cell line for over-expressing angiotensin converting enzyme 2 and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112138152A (en) * | 2020-09-21 | 2020-12-29 | 中吉智药(天津)生物技术有限公司 | AAV vector-based coronavirus infection universal gene therapy medicine and preparation method thereof |
| CN112522205A (en) * | 2020-12-15 | 2021-03-19 | 广东省农业科学院动物卫生研究所 | Cell line for over-expressing angiotensin converting enzyme 2 and preparation method and application thereof |
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| MEHDIPOUR AR AND HUMMER G.: "Homo sapiens angiotensin converting enzyme 2 (ACE2), transcript variant 1, mRNA，GENBANK ACCESSION NO. NM_001371415", 《GENBANK》 * |
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