CN113234744B - MRNA (messenger ribonucleic acid) for encoding secreted angiotensin converting enzyme 2 and application thereof - Google Patents
MRNA (messenger ribonucleic acid) for encoding secreted angiotensin converting enzyme 2 and application thereof Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
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Abstract
The invention relates to mRNA encoding secreted angiotensin converting enzyme 2 and application thereof. The mRNA includes a coding region of 70% or more of the signal peptide of angiotensin converting enzyme 2 and a coding region of 70% or more of the peptidase domain. The mRNA encoding the secreted angiotensin converting enzyme 2 can express the secreted ACE2 by utilizing a translation system of a host cell, the secreted ACE2 can be secreted outside cells 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 combining 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 beta coronavirus named by world health organization, the virus has envelope, the particles are round or oval, the main structural proteins comprise Spike protein (Spike, S), messenger protein (Envelop, E), membrane protein (Membrane, M) and nucleocapsid protein (Nucleocapsid, N), and the SARS-CoV-2 is found to enter host cells through binding with angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE 2) receptor for virus infection and replication.
The human ACE2 gene encodes an ACE2 protein receptor of 805 amino acids in full length. The receptor consists of an extracellular domain (amino acids 1-740), a single transmembrane helix (amino acids 741-763) and a cytoplasmic domain (amino acids 764-805), wherein the extracellular domain comprises a nitrogen-terminal signal peptide (amino acids 1-17), a peptidase domain (amino acids 18-615), a dimer domain (amino acids 616-726), and a linker region (amino acids 727-740).
Conventional vaccines are mostly in the form of inactivated viruses, which often have a number of disadvantages in their use, including: the preparation process is complex, the virus is required to be produced by a cell culture method, and certain potential safety hazards exist; the quality control and process amplification requirements are high; the effective antigen amount generated by the virus is low, and in order to ensure the effect, the virus titer is required to be improved, so that the production cost is increased, and therefore, the provision of an improved vaccine for the new coronaries pneumonia has important significance.
MRNA-based protein replacement therapy shows good application prospects in infectious diseases, mRNA vaccines usually use nanotechnology to deliver mRNA sequences encoding novel coronavirus (SARS-CoV-2) spike protein (S protein) or spike protein partial sequences (such as S1 and RBD), then express SARS-CoV-2 antigen through a host translation system, induce generation of corresponding antibodies, and finally achieve the aim of immune protection.
CN111218458a discloses a method for preparing mRNA and vaccine for encoding SARS-CoV-2 virus antigen and vaccine, the mRNA for encoding SARS-CoV-2 virus antigen at least contains at least one protein of S protein and N protein for encoding SARS-CoV-2 virus and/or coding region of fragment of at least one protein, and the mRNA can be delivered into body to make organism produce immune reaction, however for patient already infected with SARS-CoV-2, it is difficult to effectively treat, and still the virus is destroyed mainly by body' S own immune system.
In summary, the provision of a strategy that can effectively neutralize and treat SARS-CoV-2 infection is of great significance in the area of treatment of SARS-CoV-2.
Disclosure of Invention
In view of the shortcomings and actual needs of the prior art, the present invention provides an mRNA encoding secreted angiotensin converting enzyme 2, which can express secreted ACE2 in cells using a translation system, and the secreted ACE2 can be secreted extracellularly and serve as a decoy receptor, having the ability to competitively inhibit the activity of SARS-CoV-2, i.e. to bind to the S protein receptor binding domain of SARS-CoV-2, thereby competitively inhibiting entry of SARS-CoV-2 into cells.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect. The present invention provides an mRNA encoding secreted angiotensin converting enzyme 2, which comprises a coding region of 70% or more of the signal peptide of angiotensin converting enzyme 2 and a coding region of 70% or more of the peptidase domain.
The mRNA encoding the secreted angiotensin converting enzyme 2 has a coding region of an extracellular domain of ACE2, can express the secreted ACE2 by utilizing a translation system of a host cell, and can secrete the secreted ACE2 to the outside and be combined with an S protein receptor binding domain of SARS-CoV-2 as a decoy receptor, thereby competitively inhibiting the SARS-CoV-2 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 dimer domain and/or a coding region for a linker region of angiotensin converting enzyme 2.
Preferably, the mRNA further comprises any one or a combination of at least two of the mutation sites Q18R, T Y/W, K31F, N33 8238 34S, E35Q, W69R, Q76R, L79T, N330Y, H L or a386L, wherein typical but non-limiting combinations include a combination of T27Y, H79Y, H330Y, H L and a386L, a combination of Q18Y, H31Y, H33Y, H34Y, H35Y, H69Y, H R and H345L, a combination of T27Y, H79Y, H345L and a386L, a combination of T27Y, H79Y, H69Y, H330Y, H345L and a386L, or a combination of Q18Y, H31Y, H33Y, H35 69Y, H76R and H345L.
In the invention, mutation sites are introduced into the mRNA, so that the affinity of expressed secreted ACE2 can be further enhanced, and the mRNA can be combined with S protein of SARS-CoV-2 more efficiently.
Preferably, the mRNA further includes any one or a combination of at least two of a cap structure, an untranslated region, or a polyadenylation sequence.
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 nucleotides comprise 5-moUTP, 5mCTP or ψutp.
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, an mRNA encoding a secreted angiotensin converting enzyme 2 and a delivery vehicle carrying the mRNA encoding the secreted angiotensin converting enzyme 2.
Preferably, the mRNA encoding the secreted angiotensin converting enzyme 2 comprises the mRNA encoding the secreted angiotensin converting enzyme 2 according to the first aspect and/or the mRNA encoding the secreted angiotensin converting enzyme 2 wild-type.
Preferably, the wild-type mRNA encoding secreted angiotensin converting enzyme 2 comprises more than 70% of the coding region of the signal peptide of wild-type angiotensin converting enzyme 2 and more than 70% of the coding region of the peptidase domain.
Preferably, the coding region of the signal peptide and the coding region of the peptidase domain of wild-type angiotensin converting enzyme 2 are derived from either Homo sapiens(human)、Pan troglodytes(chimpanzee)、Pongo abelii(Sumatran orangutan)、Macaca mulatta(Rhesus monkey)、Chlorocebus sabaeus(green monkey)、Sus scrofa(pig)、Bos Taurus(cattle)、Ovis aries(sheep)、Equus caballus(horse)、Oryctolagus cuniculus(rabbit)、Canis lupus familiaris(dog)、Felis catus(domestic cat)、Gallus gallus(chicken)、Mus musculus(house mouse) or Danio rerio (zebrafish).
Preferably, the coding region of the signal peptide of 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 encoding secreted ACE2, as shown in figure 1, the lipid-like nanoparticle loaded with mRNA capable of encoding secreted ACE2 can enter cells, after entering cells, the mRNA capable of encoding secreted ACE2 can express the secreted ACE2 in the cells by using a translation system, and the secreted ACE2 can be secreted out 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 is structurally similar to, but not classifiable in, a lipid molecule.
Preferably, the molar ratio of lipid-like molecules to 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 ratio of nitrogen to phosphorus of the lipid-like molecule and 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 molecule comprises any one or a 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, with the structural formulae shown below.
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-phosphorylethanolamine, sodium salt of 1, 2-dioleoyl-rac- (1-glycero), 1, 2-palmitoyl-phosphatidylglycerol, 1-palmitoyl-2-oleoyl lecithin, 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), distearoyl-2-oleoyl phosphatidylethanolamine, 1-stearoyl-2-oleoyl phosphatidylethanolamine, 1-oleoyl-stearoyl-2-phosphatidylethanolamine, sphingomyelin-2-phosphatidylethanolamine, or a combination of at least one of sphingomyelin, or two of sphingomyelin-2-phosphatidyl-2, preferably, a combination of sphingomyelin-of at least one of the two of the foregoing.
In a third aspect, the present invention provides a method for producing the secretory expression system of the second aspect, the method comprising:
mixing the delivery vector with the mRNA encoding the secreted angiotensin converting enzyme 2, and incubating to obtain the secreted expression vector.
Preferably, the delivery vehicle comprises a mixture of lipid-like molecules and lipid molecules.
Preferably, the molar ratio of lipid-like molecules to 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 ratio of nitrogen to phosphorus of the lipid-like molecule and 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 (which may be, for example, 4, 5, 6 or 7).
Preferably, the buffer comprises Phosphate (PBS) buffer.
Preferably, the incubation time is 10 to 20 minutes, including but not limited to 11, 12, 13, 14, 15, 16, 18, or 19 minutes.
In a fourth aspect, the present invention provides a recombinant cell comprising an mRNA encoding secreted angiotensin converting enzyme 2 according to the first aspect or a secretory expression system according to the second aspect.
In a fifth aspect, the present invention provides a pharmaceutical composition comprising an mRNA encoding secreted angiotensin converting enzyme 2 according to the first aspect, a secretory expression system according to the second aspect or a 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 an mRNA encoding secreted angiotensin converting enzyme 2 according to the first aspect, a secretory expression system according to the second aspect, a recombinant cell according to the fourth aspect or a pharmaceutical composition according to the fifth aspect for the preparation of a medicament for the treatment of SARS-CoV-2.
Compared with the prior art, the invention has the following beneficial effects:
(1) The mRNA for encoding the secreted angiotensin converting enzyme 2 can express the secreted ACE 2by utilizing a translation system of a host cell, the secreted ACE2 can be secreted outside cells 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 combining with an endogenous ACE2 receptor, and a potential intervention strategy is provided for treating SARS-CoV-2;
(2) The introduction of mutation sites in mRNA encoding secreted angiotensin converting enzyme 2 can further improve the affinity of secreted ACE2, so that the mRNA encoding secreted ACE2 can be combined with SARS-CoV-2 more efficiently;
(3) The secretion expression system constructed by the invention can load mRNA encoding the secretion type angiotensin converting enzyme 2 into cells, so that the cells can express and secrete ACE2, thereby competitively inhibiting SARS-CoV-2 from being combined with endogenous ACE2 receptor.
Drawings
FIG. 1 is a schematic representation of delivery, secretory expression and neutralization of SARS-CoV-2 virus by mRNA encoding secreted ACE 2;
FIG. 2 is a graph of ACE2 enzyme activity of cell supernatants from cells treated with mRNA delivery vehicle encoding secreted angiotensin converting enzyme 2 for 24 hours, with error bars marked on the curve, the curve not shown being a graph with less error and error bar length less than the mark on the curve;
FIG. 3 is a graph showing the inhibition of ACE2 enzyme activity of ACE2 specific inhibitor MLN-4760 on cell supernatants after 24 hours of treatment of cells with mRNA delivery vehicle encoding secreted angiotensin converting enzyme 2;
FIG. 4 is a graph showing the effect of competitive inhibition of SARS-CoV-2 binding to ACE2 receptor by cell supernatants after 48 hours of treatment of cells with mRNA delivery vehicle encoding secreted angiotensin converting enzyme 2.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
This example prepares mRNA encoding secreted angiotensin converting enzyme 2, the sequence of which is the nucleic acid sequence shown in SEQ ID NO. 11.
The preparation method of the mRNA comprises the following steps:
(1) Codon optimization is carried out according to the amino acid sequences of the signal peptide, the peptidase domain, the dimer domain and the connecting region of the extracellular domain of the ACE2 receptor and specific mutation sites, so that an mRNA coding sequence for encoding the secreted ACE2 decoy receptor is obtained;
(2) Synthesizing a DNA sequence containing a T7 RNA polymerase promoter and an untranslated region;
(3) Taking 1 microgram of the DNA in the step (2) as a transcription template of mRNA in a 20 microgram system, adding 2 mu LNEB T RNA polymerase to transcribe the mRNA in vitro, incubating for 60 minutes at 37 ℃, and incubating for 15 minutes at 37 ℃ by using 2 mu LDNA enzyme 1;
(4) Purifying the reaction product obtained in the step (3) by using a NEB RNA purification kit to obtain mRNA, determining the concentration of the mRNA, and subpackaging and storing at-80 ℃.
Example 2
This example prepares 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 the preparation of a 96-well single-well dose as an example, 240ng of the mRNA prepared in example 1 was dissolved in 10.8. Mu.L of PBS (pH=7.4);
(2) Dissolving a lipid-like molecule tB-UC18 and a lipid molecule 1, 2-dioleyl-sn-glycerol-3-phosphorylethanolamine (DOPE) in ethanol according to a molar ratio of 1:1;
(3) Taking 1.2 mu L of ethanol mixed solution containing the lipid-like molecules and the lipid molecules with corresponding mass according to the ratio of the total phosphorus mole number of the mRNA to the total nitrogen mole number of the lipid-like molecules as a reference, adding the ethanol mixed solution into the mRNA solution prepared in the step (1), quickly blowing and uniformly mixing, and incubating at 25 ℃ for 15min to obtain the lipid-like nanoparticle loaded with the secreted ACE2 mRNA, namely the secretory expression system.
Example 3
This example detects secreted ACE2 enzyme activity.
The specific process comprises the following steps:
(1) Taking 10 mu L of the secretion expression system prepared in the example 2, adding the secretion expression system into a 96-well plate paved with 293T cells, placing the 96-well plate into a cell culture box, and respectively setting an experimental group added with PBS, nanoparticles loaded with mRNA encoding beta-galactosidase and no substances (blank) as a control;
(2) After 24h, 10 μl of cell supernatant was taken and the enzyme activity of ACE2 was detected according to the method for detecting angiotensin converting enzyme 2 activity fluorescence detection kit (P0319S-100T) of bi yun, the results are shown in fig. 2;
(3) The enzyme activity of ACE2 was further determined by the same method by adding different concentrations (0 μm, 0.01 μm, 0.1 μm, 1 μm, 10 μm) of the ACE 2-specific inhibitor MLN-4760 to the cell supernatant, and then measuring the enzyme activity of ACE2 in a dose-dependent inhibition, and the results are shown in fig. 3.
As can be seen from fig. 2, the supernatant of the cell cultured by adding the secretory expression vector of the present invention has high ACE2 enzyme activity, which indicates that the secretory expression system constructed by the present invention can successfully enter host cells, and the loaded mRNA can express the secretory ACE2 by using the translation system of the host cells.
As can be seen from fig. 3, as the concentration of the added ACE2 specific inhibitor MLN-4760 increases, the ACE2 enzyme activity of the supernatant of the cells cultured with the secretion expression vector of the present invention gradually decreases, further indicating that the supernatant of the cells has ACE2 enzyme activity, i.e., can express secreted ACE2 in the cells.
Example 4
This example measures the ability of secreted ACE2 to neutralize SARS-CoV-2.
The specific process comprises the following steps:
(1) 200. Mu.L of the secretory expression system prepared in example 2 was added to a 6-well plate in which 293T cells were spread, and the 6-well plate was placed in a cell incubator;
(2) After 48h of culture, the cell supernatant was concentrated 30-fold, 100. Mu.L of the concentrate was used to detect the neutralization activity of secreted ACE2 on SARS-CoV-2 according to the method for detecting SARS-CoV-2 replacement virus neutralization detection Kit (SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) Kit, L00847-A), and the result is shown in FIG. 4.
As can be seen from FIG. 4, the inhibition rate of the supernatant of the cells cultured by adding the secretory expression vector of the present invention to SARS-CoV-2 is close to that of the positive control (SARS-CoV-2 neutralizing antibody) in the kit, which is far higher than that of the negative control of the kit, indicating that the secretory expression system constructed by the present invention can effectively express the secretory ACE2 in the cells, the secretory ACE2 can be secreted outside the cells, and the SARS-CoV-2 can be effectively neutralized.
In summary, the secretion expression system constructed by the invention can load mRNA encoding the secreted ACE2 into cells, after entering the cells, the mRNA encoding the secreted ACE2 can express the secreted ACE2, the secreted ACE2 can be secreted outside the cells, and SARS-CoV-2 can be effectively neutralized, thus providing a brand new idea for treating SARS-CoV-2.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
SEQUENCE LISTING
<110> Shenzhen City people Hospital
<120> An 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 (7)
1. A secretory expression system comprising mRNA encoding secreted angiotensin converting enzyme 2 and a delivery vector carrying the mRNA encoding secreted angiotensin converting enzyme 2;
the nucleic acid sequence of the mRNA for encoding the secreted angiotensin converting enzyme 2 is shown as SEQ ID NO. 11;
The delivery vehicle consists of a lipid-like molecule and a lipid molecule;
the molar ratio of the lipid-like molecules to the lipid molecules is 1:1;
the ratio of nitrogen to phosphorus of the lipid-like molecule to mRNA is 1.5:1;
The lipid-like molecule is tB-UC18;
the lipid molecule is 1, 2-dioleyl-sn-glycerol-3-phosphorylethanolamine;
the secretory expression system is a lipid-like nanoparticle loaded with secreted ACE2 mRNA.
2. A method of making a secretory expression system according to claim 1, comprising:
Mixing the delivery carrier with the mRNA encoding the secreted angiotensin converting enzyme 2, and incubating to obtain the secreted expression system.
3. A recombinant cell comprising the secretory expression system of claim 1.
4. A pharmaceutical composition comprising the secretory expression system of claim 1 or the recombinant cell of claim 3.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition further comprises an adjuvant.
6. The pharmaceutical composition of claim 4, further comprising a pharmaceutically acceptable carrier.
7. Use of the secretory expression system of claim 1, the recombinant cell of claim 3 or the pharmaceutical composition of any one of claims 4-6 in the manufacture of a medicament for treating 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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Patent 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 |
Non-Patent Citations (2)
| Title |
|---|
| Homo sapiens angiotensin converting enzyme 2 (ACE2), transcript variant 1, mRNA，GENBANK ACCESSION NO. NM_001371415;Mehdipour AR and Hummer G.;《GENBANK》;20210516;第3页TEATURES * |
| The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2;Procko E.;《bioRxiv [Preprint]》;第1-2页的第28-46行，第12页的第237-258行，第15页的第319-334行，表1，图2 * |
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