CN114213508B - Polypeptide, polypeptide complex nanoparticle, nucleic acid vaccine and application - Google Patents

Polypeptide, polypeptide complex nanoparticle, nucleic acid vaccine and application Download PDF

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CN114213508B
CN114213508B CN202111029790.2A CN202111029790A CN114213508B CN 114213508 B CN114213508 B CN 114213508B CN 202111029790 A CN202111029790 A CN 202111029790A CN 114213508 B CN114213508 B CN 114213508B
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polypeptide
nucleic acid
arg arg
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rna
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CN114213508A (en
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张龙贵
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Shenzhen Houcun Nano Pharmaceutical Co ltd
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    • A61K9/51—Nanocapsules; Nanoparticles
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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    • A61K9/513—Organic macromolecular compounds; Dendrimers
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    • C07—ORGANIC CHEMISTRY
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    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • A61K2039/53—DNA (RNA) vaccination
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

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Abstract

The invention provides a polypeptide, polypeptide complex nanoparticles thereof, a nucleic acid vaccine and application, and belongs to the field of drug delivery. The polypeptide has the functions of compressing and protecting nucleic acid from degradation, promoting nucleic acid to penetrate cell membranes and the like, has higher antibody effect, and can be used for in-vitro and in-vitro cell gene transfection and vaccine preparation.

Description

Polypeptide, polypeptide complex nanoparticle, nucleic acid vaccine and application
Technical Field
The invention relates to the field of drug delivery, in particular to a polypeptide, polypeptide complex nanoparticles thereof, a nucleic acid vaccine and application.
Background
Gene transfection is a technique that transfers or transports nucleic acids with biological functions into cells and maintains the nucleic acids in the cells for their biological functions. A gene vector refers to a means for introducing an exogenous therapeutic gene into a biological cell. Nucleic acid vaccines are new vaccines developed in recent years. Nucleic acid vaccines are a new approach to inducing specific immune responses by introducing nucleic acids encoding antigenic proteins into cells, and synthesizing the proteins by the expression system of the cells. Although most cells can spontaneously ingest nucleic acids, the efficiency is low and saturation occurs at low doses. In addition, since a large amount of RNase exists in nature, RNA is very unstable in vivo and in vitro and is easily degraded. Thus, there is a need for suitable formulations to protect nucleic acids from extracellular RNase-mediated degradation and to facilitate their entry into cells. In the development of prophylactic and therapeutic nucleic acid vaccines, the delivery of nucleic acids of specific sequences to Dendritic Cells (DCs) makes safe, efficient, and sufficient expression critical to the efficacy of the vaccine.
Cell penetrating peptides (CELL PENETRATING PEPTIDES, CPPs) are a class of polypeptides that are capable of penetrating directly into cells through the cell membrane in a receptor-independent manner, in a non-classical endocytotic manner, without causing damage to the cell membrane, typically no more than 30 amino acids in length and rich in basic amino acids, the amino acid sequence being generally positively charged, such as the human immunodeficiency virus transcription activator TAT type 1 (human immunodeficiency virus-1 transcription activator,HIV-1 TAT) (vitamins et al, J.biol. Chem.1997;272, 16010). The common properties of CPPs are: a net positive charge or charge neutrality, and both hydrophilic and hydrophobic (amphiphilic); the film penetrating delivery efficiency is high; low cytotoxicity; cell-free type restriction; different bioactive substances can be introduced into cells through chemical combination or gene fusion and the like, so that the bioactive substances have potential to become multifunctional targeted drug carriers. Specific transmembrane mechanisms of different CPPs have been studied to find that specific amino acid sequences bind to mRNA and interfere with and reduce cell membrane stability, thereby carrying biologically active substances to penetrate cell membranes, such as arginine-alanine-leucine-alanine Residue (RALA) sequences (Pardi et al, currOpin in immunol.2020, 65:14-20). Current research of CPPs in the field of mRNA delivery or vaccine development, compared to other modes of delivery, remains unreported.
The polypeptide prepared by connecting amide bonds (peptide bonds) has the advantages that the polypeptide composite nanoparticle has high transfection efficiency and low cytotoxicity, and can be degraded into amino acid in vivo, but no polypeptide composite nanoparticle delivery system for enabling mRNA gene drugs to be marketed exists at present.
The present invention addresses the shortcomings of existing delivery systems by synthesizing non-naturally occurring polypeptides, preparing polypeptide complex nanoparticles to provide improved gene vectors for mRNA delivery, and nanodelivery solutions suitable for animal vaccine or human mRNA drug development.
Disclosure of Invention
Brief description of the invention
In order to solve the problems described above, the present invention provides, in a first aspect, a polypeptide compound for nucleic acid drug delivery. In a second aspect, the present invention provides a polypeptide complex nanoparticle comprising the polypeptide compound. In a third aspect, the invention provides the use of polypeptide complex nanoparticles for in vivo and in vitro nucleic acid delivery. In a fourth aspect, the invention provides a nucleic acid vaccine comprising said polypeptide complex nanoparticle. In a fifth aspect, the present invention provides the use of the polypeptide complex nanoparticle in the preparation of a medicament or kit.
Detailed Description
In a first aspect, the present invention provides a polypeptide compound having the general structure:
(Xaa) x-Arg-Val-Gln-Pro-Thr-Glu-Ser-lle-Val-Arg-(Yaa)y (formula I),
Wherein: x is an integer of 1 to 25, and y is an integer of 0 to 9;
(Xaa) x can be a polypeptide segment consisting of any amino acid.
In some embodiments Xaa is selected from at least one of Arg (R), trp (W), cys (C), lys (K), leu (L), phe (F), pro (P), or His (H), x is the number of amino acids, and x is an integer from 1 to 20. In some embodiments, (Xaa) x is Arg. In some embodiments, (Xaa) x is (Xa 'a') n(Arg)1-10(Xa'a')n, wherein Xa 'a' is selected from at least one of Arg (R), trp (W), cys (C), lys (K), leu (L), phe (F), pro (P), or His (H), and n is an integer from 0 to 10. In certain embodiments, xaa consists of (Arg) 1-10, trp (W), and/or Cys (C). In other embodiments, xaa consists of (Arg) 1-10, trp (W), cys (C), his (H), and/or Pro (P) may be either before (Arg) 1-10, after (Arg) 1-10, or interspersed among one or several (Arg) 1-10.
The amino acid sequence of Arg (R), trp (W), cys (C), lys (K), leu (L), phe (F), pro (P) or His (H) is not limited in the general formula I.
(Yaa) y is a polypeptide fragment consisting of any amino acid; in some embodiments, yaa is selected from at least one of Arg (R), trp (W), phe (F), or Cys (C), y is the number of amino acids, and y is an integer from 0 to 10.
The x may be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.
The y may be 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
The n may be 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
In some embodiments, the amino acid sequence of the polypeptide compound is ：Seq.01、Seq.02、Seq.03、Seq.04、Seq.05、Seq.06、Seq.07、Seq.08、Seq.09、Seq.10、Seq.11、Seq.12、Seq.13、Seq.14、Seq.15、Seq.16、Seq.17、Seq.18、Seq.19、Seq.20、Seq.21、Seq.22、Seq.23、Seq.24、Seq.25、Seq.26、Seq.27、Seq.28、Seq.29、Seq.30、Seq.31、Seq.32、Seq.33、Seq.34、Seq.35、Seq.36、Seq.37、Seq.38、Seq.39、Seq.40、Seq.41、Seq.42、Seq.43、Seq.44、Seq.45、Seq.46、Seq.47、Seq.48、Seq.49、Seq.50、Seq.51、Seq.52 or seq.53. In some preferred embodiments, the amino acid sequence of the polypeptide compound is: seq.05, seq.12, seq.46, seq.47, seq.49 or seq.53.
In some embodiments, formula (I) is at least 50% similar to any of seq.01-seq.53 and it improves delivery of the nucleic acid molecule into the cell by at least 20%.
In some embodiments, formula (I) is at least 75% similar to any of seq.01-seq.53 and it improves delivery of the nucleic acid molecule into the cell by at least 50%.
In some embodiments, formula (I) is at least 90% similar to any of seq.01-seq.53 and it improves delivery of the nucleic acid molecule into the cell by at least 100%.
In some embodiments, formula (1) is at least 90% similar to either RRRRRWCRVQPTESIVR,RRRRRWFCRVQPTESIVR,FCRWCRRVQPTESIVRRCWRCF,FCRWCRRVQPTESIVCWRRRCF,HKRWCRRWCRVQPTESIVRC or WCRRRVQPTESIVRRRWC.
In some embodiments, the polypeptide of formula (I) comprises 10-35 amino acids, which is characterized in that it improves the delivery of the nucleic acid molecule into a cell by at least 10%, in some embodiments, the polypeptide of formula (I) comprises 10-35 amino acids, which is characterized in that it improves the delivery of the nucleic acid molecule into a cell by between about 50% to about 100%; in some embodiments, the polypeptide of formula (I) comprises 10-35 amino acids, which is characterized in that it provides between about 75% to about 500% improvement in delivery of the nucleic acid molecule into the cell.
The present invention provides novel non-naturally occurring polypeptides having the functions of compressing and protecting nucleic acids from degradation, promoting nucleic acid penetration through cell membranes, and the like, as well as polypeptide complex nanoparticles comprising the polypeptides, and methods of using the same for in vivo and in vitro cell gene transfection, and methods of using the same in vaccine formulations.
In a second aspect, the present invention provides a polypeptide complex nanoparticle.
A polypeptide complex nanoparticle comprising:
a) At least one polypeptide according to the first aspect of the invention, and
B) Nucleic acid.
In some embodiments, a polypeptide complex nanoparticle comprising:
a) A composition of at least one polypeptide according to the first aspect of the invention and at least one auxiliary material; and
B) Nucleic acid.
The nucleic acid may be chemically modified or non-chemically modified DNA, single-or double-stranded DNA, coding or non-coding DNA. In some embodiments, the nucleic acid is selected from the group consisting of a plasmid, an oligodeoxynucleotide, genomic DNA, a DNA primer, a DNA probe, an immunostimulatory DNA, an aptamer, or any combination thereof.
The nucleic acid may be chemically modified or non-chemically modified RNA, single-or double-stranded RNA, coding or non-coding RNA. In some embodiments, the nucleic acid is selected from messenger RNA (mRNA), oligoribonucleotide, viral RNA, replicon RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), immunostimulatory RNA (isRNA), microrna, small interfering RNA (siRNA), micronuclear RNA (snRNA), small hairpin RNA (shRNA) or riboswitch, RNA aptamer, RNA decoy, antisense RNA, ribozyme, or any combination thereof. In some preferred embodiments, the nucleic acid is a chemically modified messenger RNA (mRNA).
The nucleic acid sequence of the RNA may include all of the nucleic acid sequences listed in patent US9254311B2, as well as all of the sequences listed in the long sequence appendix of that patent. In some embodiments, the RNA sequences of the invention can be obtained by the nucleic acid synthesis methods set forth in patent US9254311B2 or CN106659803 a.
The polypeptide complex nanoparticles are capable of encapsulating mRNA and allowing their efficient introduction into different cell lines in vitro, and are capable of efficient transfection in vivo. The polypeptide complex nanoparticle can carry mRNA encoding immunogenic peptide into cells, effectively release mRNA, express antigen and effectively realize the aim of immunotherapy or immunoprophylaxis.
The invention provides novel non-naturally occurring polypeptides having the functions of compressing and protecting nucleic acids from degradation, promoting nucleic acid penetration through cell membranes, and the like, as well as polypeptide complex nanoparticles comprising the polypeptides, and methods of using the same for in vivo and in vitro cell gene transfection, and methods of using the same in vaccine formulations.
In some embodiments of the invention, a polypeptide complex nanoparticle comprises:
a) At least one of the polypeptides of the invention,
B) Nucleic acid, and
C) Auxiliary materials.
The auxiliary material may include: lipid or PEG derivative.
The lipid may be a naturally occurring or synthetic phospholipid or a structural lipid.
The PEG derivative may be poloxamer, poloxamer derivatives, or PEG lipids.
The poloxamer may be selected from at least one of the following: Or/> The poloxamer derivative is synthesized by referring to patent CN 111285845B.
The poloxamer of the present invention may be selected from at least one of the following: poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403 and poloxamer 407.
The phospholipid may be selected from: 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sR-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sR-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (dpp), 1, 2-dioleoyl-s-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sR-glycero-3-phosphorylcholine (DUPC), 1-dioleoyl-2-dioleoyl-sR-glycero-3-phosphorylcholine (npro-phosphorylcholine (npc), 1, 2-dioleoyl-2-glycero-3-phosphorylcholine (npro-2-phosphoryl-2-phosphorylcholine (16), 1, 2-bis-docosahexaenoic acid-sn-glycerol-3-phosphorylcholine, 1, 2-bis-phytanic acid-sn-glycerol-3-phosphoethanolamine (ME 16.0 PE), 1, 2-bis-stearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-bis-linoleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-bis-arachidonic acid-sn-glycerol-3-phosphoethanolamine, 1, 2-bis-docosahexaenoic acid-sn-glycerol-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phosphate-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, or lecithin (PC) and mixtures thereof;
The structural lipid may be selected from: cholesterol (Chol), stigmasterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, and mixtures thereof;
The PEG lipid may be selected from any one of the PEG lipids described by patent No. CN111281981B, CN111315359A, CN111356444a, for example: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified cholesterol such as 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG), such as mPEG5000-C-CLS (PEG-CLS), such as mPEG2000-DSPE (PEG-DSPE).
The structural formula of the 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG) is shown as the following formula:
The structural formula of the mPEG5000-C-CLS (PEG-CLS) is shown as follows:
the structural formula of the mPEG2000-DSPE (PEG-DSPE) is shown as follows:
The mass ratio of the nucleic acid to the polypeptide may be less than or equal to about 1:1. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:1 to about 1:52. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:48. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:40. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:32. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:24. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:16. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:10. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:8. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:5. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2 to about 1:4. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1∶1、1∶2、1∶3、1∶2、1∶4、1∶5、1∶6、1∶7、1∶8、1∶9、1∶10、1∶11、1∶12、1∶13、1∶14、1∶15、1∶16、1∶17、1∶18、1∶19、1∶20、1∶21、1∶22、1∶23、1∶24、1∶25、1∶26、1∶27、1∶28、1∶29、1∶30、1∶31、1∶32、1∶33、1∶34、1∶35、1∶36、1∶37、1∶38、1∶39、1∶40、1∶41、1∶42、1∶43、1∶44、1∶45、1∶46、1∶47、1∶48、1∶49、1∶50、1∶51 or 1:52.
In some embodiments, the amino acid sequence of the polypeptide is seq.05 and the mass ratio of the nucleic acid to the polypeptide is less than or equal to 1:4. In some embodiments, the amino acid sequence of the polypeptide is seq.05 and the mass ratio of the nucleic acid to the polypeptide is from about 1:4 to about 1:52.
In some embodiments, the amino acid sequence of the polypeptide is seq.12 and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1:about 4. In some embodiments, the amino acid sequence of the polypeptide is seq.12 and the mass ratio of the nucleic acid to the polypeptide is from about 1:4 to about 1:52.
In some embodiments, the amino acid sequence of the polypeptide is Seq46 and the mass ratio of the nucleic acid to the polypeptide is less than or equal to 1:2. In some embodiments, the amino acid sequence of the polypeptide is seq.46 and the mass ratio of the nucleic acid to the polypeptide is from about 1:2 to about 1:52.
In some embodiments, the amino acid sequence of the polypeptide is seq.47 and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1:2. In some embodiments, the amino acid sequence of the polypeptide is seq.47 and the mass ratio of the nucleic acid to the polypeptide is from about 1:2 to about 1:52.
In some embodiments, the amino acid sequence of the polypeptide is seq.49 and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1:16. In some embodiments, the amino acid sequence of the polypeptide is seq.49 and the mass ratio of the nucleic acid to the polypeptide is from about 1:16 to about 1:52.
In some embodiments, the amino acid sequence of the polypeptide is seq.53 and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1:4. In some embodiments, the amino acid sequence of the polypeptide is seq.53 and the mass ratio of the nucleic acid to the polypeptide is from about 1:4 to about 1:52.
The mass ratio of the nucleic acid to the auxiliary material may be less than or equal to 1:2. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material may be less than or equal to about 1:50. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2 to about 1:800. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2 to about 1:500. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2 to 1:400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2 to about 1:50. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2 to about 1:33. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2 to 1:10. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2-1:6. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2, 1:3, 1:4, 1:5, or 1:6. in some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:33 to 1:400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:50 to 1:800.
In some embodiments, the auxiliary material is a PEG derivative and the mass ratio of the nucleic acid to the auxiliary material is 1:50-1:800. In some embodiments, the auxiliary material is a PEG derivative and the mass ratio of the nucleic acid to the auxiliary material is 1:100, 1:200, or 1:500. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is 1:2, the auxiliary material is a PEG derivative, and the mass ratio of the nucleic acid to the auxiliary material is 1:50-1:800.
In some embodiments, the auxiliary material is a PEG derivative, a phospholipid, and a structural lipid, and the mass ratio of the nucleic acid to the auxiliary material is about 1:2 to about 1:6. In some embodiments, the auxiliary material is a PEG derivative, a phospholipid, and a structural lipid, and the mass ratio of the nucleic acid to the auxiliary material is about 1:2, 1:3, 1:4, 1:5, or 1:6. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is 1:2-1:30, the auxiliary material is a PEG derivative, a phospholipid, and a structural lipid, and the mass ratio of the nucleic acid to the auxiliary material is 1:2-1:6.
In some embodiments, the auxiliary material is a PEG derivative and a phospholipid, and the mass ratio of the nucleic acid to the auxiliary material is about 1:33 to about 1:400 or about 1:33 to about 1:370. In some embodiments, the auxiliary material is a PEG derivative and a phospholipid, the mass ratio of the nucleic acid to the auxiliary material is about 1:33 to about 1:400 or about 1:33 to about 1:370, and the mass ratio of the PEG derivative to the phospholipid is 32:1 to 700:1. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2, the auxiliary material is a PEG derivative and a phospholipid, the mass ratio of the nucleic acid to the auxiliary material is 1:33-1:400 or 1:33-1:370, and the mass ratio of the PEG derivative and the phospholipid is 32:1-700:1.
In some embodiments, a polypeptide complex nanoparticle comprises: nucleic acid, polypeptide compound with amino acid sequence of seq.05 and auxiliary material, wherein the auxiliary material isAnd lecithin; the nucleic acid and the polypeptide compound with the amino acid sequence of seq.05 are shown in the specificationAnd lecithin at a mass ratio of about 1:2:322:1.
In some embodiments, a polypeptide complex nanoparticle comprises: nucleic acid, a polypeptide compound having the amino acid sequence seq.49 and an adjunct material which is 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and cholesterol; the mass ratio of the nucleic acid, the polypeptide compound with the amino acid sequence of seq.49, the 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, the 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and the cholesterol is about 10:300:8:16:31.
In some embodiments, a polypeptide complex nanoparticle comprises: nucleic acid, polypeptide compound with amino acid sequence of seq.53 and auxiliary materials, wherein the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxy polyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and cholesterol; the mass ratio of the nucleic acid, the polypeptide compound with the amino acid sequence of seq.53, the 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, the 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and the cholesterol is about 10:40:8:16:5.
In some embodiments of the invention, a polypeptide complex nanoparticle comprises at least one non-naturally occurring peptide and nucleic acid of the invention.
In some embodiments of the invention, a polypeptide complex nanoparticle comprises at least one non-naturally occurring peptide, nucleic acid, and at least one lipid or PEG derivative of the invention.
The nucleic acid may be chemically modified or non-chemically modified DNA, single-or double-stranded DNA, coding or non-coding DNA. In some embodiments, the nucleic acid is selected from the group consisting of a plasmid, an oligodeoxynucleotide, genomic DNA, a DNA primer, a DNA probe, an immunostimulatory DNA, an aptamer, or any combination thereof.
The nucleic acid may be chemically modified or non-chemically modified RNA, single-or double-stranded RNA, coding or non-coding RNA. In some embodiments, the nucleic acid is selected from messenger RNA (mRNA), oligoribonucleotide, viral RNA, replicon RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), immunostimulatory RNA (isRNA), microrna, small interfering RNA (siRNA), micronuclear RNA (snRNA), small hairpin RNA (shRNA) or riboswitch, RNA aptamer, RNA decoy, antisense RNA, ribozyme, or any combination thereof. In some preferred embodiments, the nucleic acid is a chemically modified messenger RNA (mRNA).
The nucleic acid sequence of the RNA may include all of the nucleic acid sequences listed in patent US9254311B2, as well as all of the sequences listed in the long sequence appendix of that patent. In some embodiments, the RNA sequences of the invention can be obtained by the nucleic acid synthesis methods set forth in patent US9254311B2 or CN106659803 a.
In some embodiments, the polypeptide complex nanoparticle may further comprise at least one pharmaceutically acceptable excipient.
The polypeptide complex nanoparticle of the present invention is stable in aqueous solution and may be contacted with human or animal tissue after formation, or may be stored for a period of time prior to contact with the cell or tissue. The polypeptide complex nanoparticle is stable and can be stored for a period of time of at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year. It is understood that the pot life may be between any of these time periods, for example between 31 minutes and 1 hour or between 1 hour and 24 hours.
In a third aspect, the invention provides the use of polypeptide complex nanoparticles for in vivo and in vitro nucleic acid delivery.
Use of a polypeptide complex nanoparticle according to the second aspect for in vivo and in vitro nucleic acid delivery.
The polypeptide complex nanoparticles are capable of encapsulating mRNA and allowing their efficient introduction into different cell lines in vitro, and are capable of efficient transfection in vivo. The polypeptide complex nanoparticle can carry mRNA encoding immunogenic peptide into cells, effectively release mRNA, express antigen and effectively realize the aim of immunotherapy or immunoprophylaxis.
The invention provides novel non-naturally occurring polypeptides having the functions of compressing and protecting nucleic acids from degradation, promoting nucleic acid penetration through cell membranes, and the like, as well as polypeptide complex nanoparticles comprising the polypeptides, and methods of using the same for in vivo and in vitro cell gene transfection, and methods of using the same in vaccine formulations.
In a fourth aspect, the invention provides a nucleic acid vaccine comprising a polypeptide complex nanoparticle according to the second aspect.
A nucleic acid vaccine comprising the polypeptide complex nanoparticle of the second aspect.
The polypeptide complex nanoparticle may include at least one RNA.
The nucleic acid vaccine can be used for treating or preventing diseases.
The RNA includes at least coding RNA.
The coding RNA may comprise RNA that may encode at least one coding region of at least one therapeutic protein, therapeutic polypeptide, immunogenic protein or immunogenic peptide. In some embodiments, the coding RNA is mRNA.
The present invention provides ribonucleic acid vaccine with RNA (such as messenger RNA (mRNA)) as core and polypeptide complex nano particle as delivery carrier, including bacteria, virus and other infectious pathogen vaccine and tumor vaccine, which can induce the specific immune system of organism to produce almost any target protein or its fragment. In some embodiments, the RNA is modified. The disclosed nucleic acid vaccines can be used to induce immune responses, including cellular and humoral immune responses, against infectious pathogens or cancers without the risk of potentially causing insertional mutagenesis. Depending on the incidence of infectious disease pathogens and cancer, nucleic acid vaccines with polypeptide complex nanoparticles as delivery vehicles can be used in a variety of disease types. The nucleic acid vaccine can be used for preventing and/or treating infectious pathogens or cancers of various stages or degrees of metastasis.
In some embodiments of the invention, a nucleic acid vaccine comprising the polypeptide complex nanoparticle of the second aspect; the polypeptide complex nanoparticle comprises at least one RNA; the RNA is messenger RNA (mRNA); the messenger RNA (mRNA) can safely direct the cellular machinery of the body to produce virtually any protein of interest, from natural proteins to antibodies and other entirely novel protein constructs that may be therapeutically active inside and outside the cell.
The nucleic acid vaccine may be used in a variety of contexts depending on the prevalence of infection or the extent or level of unmet medical need. The nucleic acid vaccines are useful for the treatment and/or prevention of HPV of various genotypes, strains and isolates. The nucleic acid vaccine is advantageous in that it produces a much greater antibody titer than commercially available antiviral therapeutic treatments, and also produces a response earlier. While not wishing to be bound by theory, it is believed that, like the mRNA polynucleotides, the RNA vaccine is more optimally designed to produce the appropriate protein configuration by translation as the RNA vaccine assigns the natural cellular mechanisms. Unlike traditional vaccines that are ex vivo and can trigger adverse cellular responses, the nucleic acid vaccine provides a template for the expression of protein antigens by the cellular system in a more natural manner.
In some embodiments of the invention, a nucleic acid vaccine comprising the polypeptide complex nanoparticle of the second aspect; the polypeptide complex nanoparticle comprises at least one RNA; the nucleotide sequence of the RNA is a nucleotide sequence encoding an antigen of any pathogen. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is mRNA and the nucleotide sequence of the mRNA encodes the S spike protein of the novel coronavirus SARS-CoV-2.
In some embodiments of the invention, a nucleic acid vaccine comprising the polypeptide complex nanoparticle of the second aspect; the polypeptide complex nanoparticle contains artificially synthesized pathogen antigen polypeptide. In some embodiments, the antigenic polypeptide is fused to other polypeptides that enhance transfection and delivery efficiency and/or enhance immune responses.
The dosage form of the nucleic acid vaccine can be injection, tablet, inhalation, suppository, eye drop or suspension, etc.
The nucleic acid vaccines of the present invention can be administered by any route that produces a therapeutically effective result. Such routes include, but are not limited to, intradermal, subcutaneous, intraperitoneal, oral, intramuscular, intranasal, intraocular, upper respiratory, intravenous, vaginal, rectal administration. In some embodiments, the mRNA vaccines of the present invention are administered using an injection.
In a fifth aspect, the present invention provides the use of a polypeptide complex nanoparticle according to the second aspect for the preparation of a medicament or kit.
Use of a polypeptide complex nanoparticle according to the second aspect for the preparation of a medicament or kit.
In some embodiments, the use of a polypeptide complex nanoparticle of the second aspect for the manufacture of a medicament for the prevention, treatment and/or amelioration of a disease selected from the group consisting of: cancer or neoplastic diseases, infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenic genetic diseases, or genetic diseases in general, diseases which have a genetic background and are typically caused by defined genetic defects and are inherited according to the mendelian rule, cardiovascular diseases, neuronal diseases, respiratory diseases, digestive diseases, skin diseases, musculoskeletal disorders, connective tissue disorders, tumors, immunodeficiency, endocrine, nutritional and metabolic diseases, eye diseases and ear diseases.
The infectious disease may include a viral infectious disease, a bacterial infectious disease, or a protozoal infectious disease.
Drawings
FIG. 1 shows a transmission electron microscope image of a polypeptide complex nanoparticle according to example III; in the figure, A represents recipe Rp.05, B represents recipe Rp.28, C represents recipe Rp.43, and the white scale is 200nm.
FIG. 2 shows the result of agarose gel electrophoresis of polypeptide complex nanoparticles in example four; in the figure, mRNA refers to mRNA positive control groups, and the mass ratio of 1, 2, 4, 8, 16, 32 and 64 refers to polypeptide to mRNA is 1:1, 2:1, 4:1, 8:1, 16:1, 32:1 and 64:1; the minimum mass ratios that each polypeptide guarantees complete compression of the mRNA are: seq.05 was 4, seq.12 was 4, seq.46 was 2, seq.47 was 2, seq.49 was 16, and seq.53 was 4.
FIG. 3 shows transfection of FLuc-mRNA polypeptide complex nanoparticles in DC2.4 cells in example five; the abscissa in the figure represents the polypeptide nanoparticle compositions of different prescriptions, and the ordinate represents the relative fluorescence intensities expressed after 24h transfection of the polypeptide nanoparticle composition containing the FLuc-mRNA at the same dose.
FIG. 4 shows the viability of DC2.4 cells after treatment according to the different prescriptions in example five; the abscissa represents the different polypeptide complex nanoparticle prescriptions, and the ordinate represents cell viability, the higher the cell activity, the less cytotoxicity.
FIG. 5 shows transfection of Luc-pDNA polypeptide complex nanoparticles in DC2.4 cells in example five; the abscissa represents the different prescriptions and the ordinate represents the relative fluorescence intensities expressed by DC2.4 cells 24h, 48h, 72h, after transfection of the same dose of Luc-pDNA.
FIG. 6 shows the expression of luciferase in mice by IVIS assay polypeptide complex nanoparticles in example six.
FIG. 7 shows serum IgG antibody levels of mice after nanoparticle immunization with the polypeptide complex of example seven; the abscissa represents the difference in OD values of the optical densities at two wavelengths at day 28 and day 49 after the first immunization of the different prescriptions, and the OD values are an index for determining the IgG antibody level in serum, reflecting the level of anti-S protein IgG in serum.
FIG. 8 shows serum IgG antibody titers of mice immunized with polypeptide complex nanoparticles of example seven; the abscissa represents the different dilutions of serum of different prescriptions after 49 days after the first immunization, and the ordinate represents the difference in OD (optical density) values at the two wavelengths. The 2x Baseline (double background) was used as the demarcation value to distinguish positive and negative results, and the maximum dilution with OD values above this value was titer.
Definition of terms
The terms used throughout the specification generally have their ordinary meanings in the art within the context of the present invention and in the specific context of each term used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing various embodiments of the invention and how to make and use aspects of the embodiments. It should be appreciated that the same concepts can be expressed in more than one way. Thus, alternative language and synonyms may be used for any one or more of the terms discussed herein, whether or not a term is detailed or discussed herein, and no particular meaning is given. Synonyms for certain terms may be provided. The recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or any illustrated terms.
In the context of the present invention, all numbers disclosed herein are approximations, whether or not the word "about" or "about" is used. The numerical value of each number may vary by less than 10% or reasonably as considered by those skilled in the art, such as 1%, 2%, 3%, 4% or 5%.
The term "polypeptide" means a polymer of amino acid residues (natural or unnatural) that are often joined together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. The polypeptide may be a single molecule or may be a multi-molecular complex, such as a dimer, trimer or tetramer. They may also comprise single or multi-chain polypeptides such as antibodies or insulin, and may be associated or linked. The most common disulfide bond is found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid.
The term "protein" is a polymer consisting essentially of any of the 20 amino acids. While "polypeptides" are generally used to refer to relatively larger polypeptides, and "peptides" are generally used to refer to small polypeptides, the use of these terms in the art overlaps and varies. The terms "peptide," "protein," and "polypeptide" are sometimes used interchangeably herein.
The term "hydrophilic" refers to being water-soluble under certain conditions, including readily water-soluble, and sparingly water-soluble.
The term "hydrophobic" means poorly soluble in water under certain conditions.
As used herein, the term "amino acid" generally refers to naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon to which hydrogen, carboxyl, amino, and R groups are bound), such as homoserine, norleucine, methionine sulfoxide, methionine, and methylsulfonium. Such analogs have modified R groups (e.g., norleucine or norvaline) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid but that function in a manner similar to a naturally occurring amino acid. The term "amino acid" may refer to an amino acid or derivative thereof (e.g., amino acid analog) as well as D and L forms thereof. Examples of such amino acids include glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine, N-acetylcysteine.
"Kit" refers to a transfection, DNA, RNAi or other bioactive agent (e.g., protein or anionic molecule) delivery or protein expression or gene knock-out (knockdown) kit comprising one or more agents of the invention or mixtures thereof. The kit may include one or more non-naturally occurring peptides described herein, or optionally in combination with one or more lipids or PEG derivatives. In some embodiments, the peptide and lipid reagents may be provided in a single formulation. In other embodiments, the complex material and peptide may be provided separately, along with instructions for the user to combine the reagents at the time of use. Such kits may comprise a carrying device that is partitioned to hold one or more container devices (e.g., vials, tubes, etc.) in a tightly constrained manner. Each of such container means comprises the component or mixture of components required for performing the transfection. Such kits may optionally include one or more components selected from any bioactive molecule, such as a nucleic acid (one or more expression vectors, DNA molecules, RNA molecules, or RNAi molecules in some embodiments), a cell, one or more compounds of the invention, a lipid compound, a transfection enhancer, a bioactive substance, and the like.
The media, methods, kits and compositions of the invention are suitable for monolayer or suspension culture, transfection and incubation of cells, and for expression of proteins in monolayer or suspension cultured cells. In some embodiments, the media, methods, kits and compositions of the invention are used for suspension culture, transfection and incubation of cells, and for expression of protein products in suspension cultured cells.
Immune response: the immune response may typically be a specific response of the adaptive immune system against a specific antigen (so-called specific or adaptive immune response) or a non-specific response of the innate immune system (so-called non-specific or innate immune response). (Fotin-Mleczek Mariola et al, CN 108064176A)
Vaccine: vaccines are typically understood to be prophylactic or therapeutic substances that provide at least one antigen or antigenic function. The antigen or antigen function may stimulate the adaptive immune system of the body to provide an adaptive immune response. (Fotin-Mleczek Mariola et al, CN 108064176A)
MRNA providing antigen: the mRNA providing the antigen may typically be an mRNA having at least one open reading frame that can be translated by the cell or organism provided with the mRNA. The translated product is a peptide or protein that can be used as an antigen (preferably as an immunogen). The product may also be a fusion protein consisting of more than one immunogen, for example a fusion protein consisting of two or more epitopes, peptides or proteins, wherein the epitopes, peptides or proteins may be linked by a linker sequence. (Fotin-MleczekMariola et al, CN 108064176A)
Nucleic acid: the term "nucleic acid" refers to any DNA or RNA molecule and is used synonymously with "polynucleotide". Wherever reference is made herein to a nucleic acid or nucleic acid sequence encoding a particular protein and/or peptide, the nucleic acid or nucleic acid sequence, respectively, preferably also includes regulatory and/or other sequences that allow for its expression and/or stability in a suitable host (e.g., human), i.e., transcription and/or translation of the nucleic acid sequence encoding the particular protein or peptide. (Fotin-Mleczek Mariola et al, CN 108064176A)
Peptide: peptides are polymers of amino acid monomers. Typically, monomers are linked by peptide bonds. The term "peptide" does not limit the length of the polymer chain of amino acids. In some embodiments of the invention, the peptide may, for example, contain less than 50 monomer units. Longer peptides, which may also be referred to as polypeptides, typically have 50 to 600 monomer units, more specifically 50 to 300 monomer units. (Fotin-Mleczek Mariola et al, CN 108064176A)
Pharmaceutically effective amount of: in the context of the present invention, a pharmaceutically effective amount is typically understood to be an amount sufficient to induce an immune response or trigger a desired therapeutic effect. (Fotin-Mleczek Mariola et al, CN 108064176A)
Chemical synthesis of RNA: chemical synthesis of relatively short fragments of oligonucleotides having defined chemical structures provides a quick and inexpensive method of obtaining custom oligonucleotides of any desired sequence. Although enzymes synthesize DNA and RNA only in the 5 'to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often done in the opposite (i.e., 3 'to 5') direction. Currently, this process is carried out in solid phase synthesis using the phosphoramidite method and phosphoramidite building blocks derived from protected nucleotides (A, C, G and U) or chemically modified nucleotides. (Fotin-Mleczek Mariola et al, CN 108064176A)
To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide strand on the solid phase in the order required for the product sequence in a fully automated process. After chain assembly is completed, the product is released from the solid phase into solution, deprotected, and collected. The presence of side reactions sets practical limits (up to about 200 nucleotide residues) on the length of the synthesized oligonucleotide, as the number of errors increases with the length of the synthesized oligonucleotide. The product is typically isolated by HPLC to obtain the desired oligonucleotide in high purity. (Fotin-MleczekMariola et al, CN 108064176A)
RNA in vitro transcription: the term "RNA in vitro transcription (RNA in vitro transcription)" or "(in vitro transcription (in vitro transcription)" relates to a process in which RNA is synthesized (in vitro) in a cell-free system, DNA, in particular plasmid DNA, is used as template for the production of RNA transcripts, RNA can be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, preferably a linearized plasmid DNA template according to the invention, the promoter for controlling in vivo transcription can be any promoter for any DNA-dependent RNA polymerase, specific examples of DNA-dependent RNA polymerase are T7, T3 and SP6RNA polymerase, DNA templates for in vitro RNA transcription can be obtained by cloning nucleic acids, in particular cDNA, corresponding to the respective RNA to be transcribed in vitro, and introducing them into an appropriate vector for in vitro transcription (for example into plasmid DNA), in a preferred embodiment of the invention the DNA template is linearized with an appropriate restriction endonuclease, which is subsequently transcribed in vitro, cDNA can be obtained by reverse transcription or chemical synthesis of mRNA, furthermore, DNA templates for in vitro RNA synthesis can also be obtained by gene synthesis (Fotin, 3234 et al
Methods for in vitro transcription are known in the art (see, e.g., geall et al (2013) Semin. Immunol.25 (2): 152-159; brunelle et al (2013) Methods enzymes. 530: 101-14).
RNA, mRNA: RNA is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are typically Adenosine Monophosphate (AMP), uridine Monophosphate (UMP), guanosine Monophosphate (GMP) and Cytidine Monophosphate (CMP) monomers or analogues thereof, which are linked to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (i.e., ribose) of a first monomer and the phosphate moiety of a second, adjacent monomer. The specific order of monomers, i.e., the order of bases attached to the sugar/phosphate backbone, is referred to as the RNA sequence. In general, RNA can be obtained by transcription of a DNA sequence (e.g., in a cell). In eukaryotic cells, transcription is typically performed in the nucleus or mitochondria. In vivo, transcription of DNA usually produces so-called pre-mature RNAs (also called pre-mRNA, pre-mRNA or heterologous nuclear RNAs), which must be processed into so-called messenger RNAs (commonly abbreviated as mRNA). For example, in eukaryotic organisms, the processing of pre-mature RNA includes a variety of different post-transcriptional modifications, such as splicing, 5' -capping, polyadenylation, derivation from the nucleus or mitochondria, and the like. The sum of these processes is also known as RNA maturation. Mature messenger RNAs typically provide nucleotide sequences that can be translated into the amino acid sequence of a particular peptide or protein. Typically, the mature mRNA comprises a 5' -cap, optionally a 5' UTR, an open reading frame, optionally a 3' UTR, and a poly (A) tail (Fotin-Mleczek Mariola et al, CN 108064176A).
In addition to messenger RNAs, there are several non-coding types of RNAs that can be involved in the regulation of transcription and/or translation as well as in immune stimulation. Within the present invention, the term "RNA" further includes any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers, riboswitches, immunostimulatory/immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), micronuclear RNA (snRNA), micronucleolar RNA (snoRNA), microrna (miRNA) and Piwi-interacting RNA (piRNA) (Fotin-Mleczek Mariola et al, CN108064176 a).
The term "chemically modified" refers to modification of A, G, U or C ribonucleotides. Generally, in this context, these terms are not intended to refer to modifications of ribonucleotides in the cap portion of the naturally occurring 5' terminal mRNA. The modification may be a variety of different modifications. In some embodiments, wherein the nucleic acid is an mRNA, the coding region, flanking region, and/or terminal region may comprise one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide introduced into the cell may exhibit reduced degradation in the cell as compared to the unmodified polynucleotide.
The term "amino acid" as used herein refers to a molecule having a side chain, an amino group, and an acid group (e.g., -a carboxyl group of CO2H or a sulfo group of-SO 3 H), wherein the amino acid is attached to the parent molecular group via the side chain, the amino group, or the acid group (e.g., the side chain). In some embodiments, the amino acid is attached to the parent molecular group through a carbonyl group, wherein the side chain or amino group is attached to the carbonyl group. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkylaryl, alkylheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl groups. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine. The amino acid group may be optionally substituted with 1,2,3 or in the case of amino acid groups of 2 or more carbons with 4 substituents independently selected from: (1) C1-6 alkoxy; (2) C1-6 alkylsulfinyl; (3) Amino, as defined herein (e.g., unsubstituted amino.
Delivery (Delivery): as used herein, "delivery" refers to the act or manner of delivering a compound, substance, entity, moiety, load (cargo), or payload.
Delivery vehicle (DELIVERY AGENT): as used herein, "delivery vehicle" refers to any substance that at least partially facilitates in vivo delivery of a polynucleotide to a target cell.
Expression (Expresslon): as used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) Generating an RNA template from the DNA sequence (e.g., by transcription); (2) Processing of the RNA transcript (e.g., by cleavage, editing, 5 'cap formation, and/or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.
Formulation (Formulation): as used herein, "formulation" includes at least one polynucleotide and a delivery agent.
In vitro (ln vitro): the term "in vitro" as used herein refers to events that occur in an artificial environment, such as in a test tube or reactor, in a cell culture, in a petri dish (PETRI DISH), etc., rather than within an organism (e.g., an animal, plant, or microorganism).
In vivo (ln vivo): the term "in vivo" as used herein refers to an event that occurs within an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). Modified (Modified): as used herein, "modified" refers to an altered state or structure of a molecule of the invention. The molecules may be modified in a variety of ways, including chemical, structural and functional modifications. In one embodiment, the mRNA molecules of the invention are modified, for example, by the introduction of non-natural nucleosides and/or nucleotides as they relate to natural ribonucleotides A, U, G and C. Atypical nucleotides such as cap structures are not considered "modified" although they differ from the chemical structure of A, C, G, U ribonucleotides.
Naturally occurring (Naturally occurring): as used herein, "naturally occurring" means that it exists in nature and does not require manual assistance.
Pharmaceutically acceptable (Pharmaceutically acceptable): the phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
Prevention (PREVENTING): the term "preventing" as used herein refers to partially or completely delaying the onset of an infection, disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delay progression of an infection, a particular disease, disorder, and/or condition; and/or reduce the risk of developing pathologies associated with infections, diseases, disorders and/or conditions.
Target protein (Protein ofinterest): as used herein, the term "protein of interest" or "protein of interest" includes the proteins provided herein and fragments, mutants, variants and alterations thereof.
Treatment (treatment): the term "treatment" as used herein refers to the partial or complete alleviation, amelioration, alleviation of a particular infection, disease, disorder, and/or condition; partially or completely delaying the onset of a particular infection, disease, disorder, and/or condition; inhibiting, partially or completely, the progression of a particular infection, disease, disorder, and/or condition; partially or completely reducing the severity of a particular infection, disease, disorder, and/or condition; and/or reduce the incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example, "treating" cancer may refer to inhibiting the survival, growth, and/or spread of a tumor. For the purpose of reducing the risk of developing a pathology associated with a disease, disorder and/or condition, the treatment may be administered to a subject that does not exhibit signs of the disease, disorder and/or condition and/or to a subject that exhibits only early signs of the disease, disorder and/or condition.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
In order to better understand the technical solution of the present invention, some non-limiting examples are further disclosed below to further describe the present invention in detail.
The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.
The amino acid sequence of the polypeptide synthesized by the embodiment of the invention:
Table 1: the polypeptides of the general formula
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Table 1 illustrates various peptide sequences that can be used to practice the invention, but it will be understood by those of ordinary skill in the art that the list of peptide sequences in table 1 is provided by way of example only and is not intended to limit the scope of the invention to only those sequences explicitly written. Rather, it will be readily apparent to such persons that, based on the teachings set forth above with respect to the polypeptides of the general formula, there may be a large number of peptides potentially suitable for use in practicing the invention set forth herein. Furthermore, it is well within the knowledge of the skilled artisan to determine whether a given peptide sequence falls within the scope of the present invention using standard techniques in the art without undue experimentation. Furthermore, it is to be understood that various variants of the peptide sequences presented in Table 1 are also within the scope of the present invention, provided that such variants meet the structural and functional features set forth above. The peptide sequence variants appearing in table 1 or any other candidate peptide variants not explicitly recited in table 1 but meeting the structural and functional requirements set forth above may include deletions, insertions, use of naturally occurring or non-protein amino acid substitutions.
Embodiment one: method for preparing the polypeptide of the present invention
The non-naturally occurring peptides of the invention are produced by any previously known peptide synthesis method known to those of ordinary skill in the art, including but not limited to recombinant methods or peptide synthesis chemistry, such as solid phase peptide synthesis. Solid phase synthesis methods (MARRIFIELD, journal of the american society of chemistry (j.am. Chem. Soc.), 85, 2149-2154, 1963) can be labeled as just one example of such peptide synthesis methods. Currently, peptides can be produced simply and in a relatively short period of time using an automated universal peptide synthesizer based on those principles. Alternatively, peptides may be produced using well known recombinant protein production techniques, which are widely known to the skilled artisan.
The simple synthesis method and specific process of the polypeptide of the invention are described as follows (taking sequence seq.05 as an example):
(1) Resin treatment
① Swelling of the resin: fmoc-Arg (pbf) -2-Chlorotrityl Resin (molar substitution 0.317 mmol/g) was selected as starting resin, added to 50ml reaction column, soaked in DCM (dichloromethane), and pumped down to complete the swelling of the resin.
② Deprotection: adding 20% (g/100 ml) of piperidine in DMF (N, N-dimethylformamide), stirring for 30min by introducing N 2 (nitrogen), and filtering off the solvent; and washing the resin by using DMF (N, N-dimethylformamide) for 6 times, and pumping out to finish deprotection of the resin.
(2) Amino acid coupling reaction
The reaction monitoring and detecting method comprises the following steps: the progress of the reaction was monitored by ninhydrin method.
The raw materials and the reagents used are as follows:
Name of the name Feed amount (mmol)
Protecting amino acids 2
O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU) 2
N, N-Diisopropylethylamine (DIEA) 2
The specific operation process is as follows:
Weighing corresponding amounts of TBTU and protected amino acid in a beaker, and adding DMF for dissolution; then, the reaction solution was added to the resin deprotected in step (1), DIEA was further added thereto, and the mixture was blown for about 90 minutes through N 2, and the reaction was detected by ninhydrin reaction. After the reaction was completed, the solvent was removed, and the resin was washed 3 times with DMF. Then adding DMF solution containing 20% (g/100 ml) piperidine into the resin, introducing N 2, continuously blowing for 30 minutes, then removing the solvent, and washing the resin with DMF for 6 times to finish the coupling of the amino acid.
The above reaction procedure was repeated until all the condensation reactions of the protected amino acids were completed. After the coupling to the last protected amino acid is completed, the polypeptide is contracted, DMF, DCM and methanol are washed for 3 times in sequence, and the weight is weighed after pumping.
The condensation method comprises the following steps: tbtu+diea, condensing agent: TBTU:0.64g, DIEA:0.7ml.
Table 2: abbreviations for amino acids and corresponding protected amino acids
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(3) TFA cleavage (cleavage of polypeptide from resin and removal of amino acid side chain protecting groups)
The resin finally obtained by the amino acid coupling reaction (2) was added to a previously prepared cleavage solution (containing 86% TFA, 5% EDT (ethylene dithiol), 5% phenylsulfide, 3% phenol and 2% pure water) and stirred for 150 minutes. Then separating the resin from the lysate, and adding diethyl ether into the separated lysate to fully separate out the polypeptide; filtering, washing with diethyl ether for 6 times to obtain crude peptide, and purifying to obtain polypeptide shown in sequence seq.05. And so forth to synthesize other polypeptides provided herein.
(4) Simple purification step
Dissolving the crude peptide obtained in the step (3) by using a mixed solution of acetonitrile and water in a volume ratio of 3mi of 1:1, clarifying, filtering, and detecting the purity and molecular weight of the polypeptide by adopting liquid chromatography-mass spectrometry (HPLC-MS). The results are shown in Table 1, and the purity of the polypeptides is above 98.00%.
Embodiment two: preparation of polypeptide complex nanoparticles
The polypeptide compound nanoparticle can be prepared by referring to patent CN111249476A, CN111281981B, CN111281982A, CN111285845B, CN111588637A or a nanoparticle preparation method with application number 202110713076.9.
The preparation method comprises the following steps: dissolving polypeptide with ultra-pure water without ribozyme to obtain 1mg/ml solution, mixing polypeptide and mRNA according to the mass ratio shown in Table 3, stirring for 15min, and standing to obtain polypeptide compound nanoparticle, wherein the specific prescription is shown in Table 3:
TABLE 3 polypeptide nanoparticle formulation and particle size, potential and encapsulation efficiency thereof
The preparation method comprises the following steps: taking out the PEG derivative auxiliary material from a refrigerator at the temperature of minus 20 ℃ to be balanced to 25 ℃ according to the prescription design in the table 4, weighing at the temperature of 25 ℃, adding the ultra-pure water without the ribozyme, dissolving in a 15mL centrifuge tube without the ribozyme, enabling the concentration to be 100mg/mL, and fully oscillating for 5min by a vortex instrument to obtain a stock solution A; and taking out the lipid auxiliary material from the refrigerator at the temperature of minus 20 ℃ to be balanced to 25 ℃, weighing at the temperature of 25 ℃, dissolving the lipid auxiliary material into a 1.5mL centrifuge tube without the nuclease at the temperature of 25 ℃ by using ethanol (chromatographic grade), and fully oscillating for 5min by using a vortex instrument to obtain a stock solution B.
Adding the corresponding polypeptide in the table 4 into the non-ribozyme ultrapure water to dissolve into a solution of 5mg/ml, mixing with mRNA for 10min according to the mass ratio shown in the table 4 in sequence to obtain a stock solution C, mixing the stock solution A with the stock solution B for 1min according to the mass ratio shown in the table 4, adding the stock solution C, and fully oscillating for 1min by using a vortex instrument to obtain polypeptide compound nano particles, wherein the specific prescription is shown in the table 4:
TABLE 4 polypeptide nanoparticle formulation and particle size, potential and encapsulation efficiency thereof
And the preparation method comprises the following steps: weighing 5.0mg of DMG-PEG, adding 1mL of chromatographic pure ethanol for dissolution, weighing 10.3mg of DSPC (distearoyl phosphatidylcholine) and adding 1mL of chromatographic pure ethanol for dissolution; 19.4mg of cholesterol was weighed and dissolved in 2mL of chromatographically pure ethanol.
Dissolving polypeptide with non-ribozyme ultrapure water to obtain 1mg/mL solution, respectively mixing polypeptide and mRNA uniformly according to the corresponding mass ratio in table 5, adding the solution into a citrate buffer solution with a value of 20mLpH of 5.4 after 5min, continuously stirring at 1500rpm, dropwise adding ethanol solution of DMG-PEG and DSPC into the citrate buffer solution, dropwise adding a certain volume (100 uL, 200uL, 250uL or 500 uL) of ethanol solution of cholesterol with a concentration of 19.4mg/mL, continuously stirring for 30min, and removing ethanol by reduced pressure rotary evaporation at 40 ℃ to obtain polypeptide compound nanoparticles, wherein the specific prescription is shown in table 5:
TABLE 5 polypeptide nanoparticle formulation and particle size, potential and encapsulation efficiency thereof
The preparation method is as follows: 10mg of the corresponding auxiliary material (DMG-PEG, PEG-CLS, or PEG-DSPE) in Table 6 was weighed, and dissolved in nuclease-free ultrapure water to a concentration of 1mg/mL to obtain stock solution A. The polypeptide was dissolved in non-ribozyme ultrapure water to obtain a 1mg/ml solution, and stock solution B was obtained. Mixing stock solution A and stock solution B according to the corresponding mass ratio in Table 6 for 1min, mixing the mixed solution with mRNA for 10min, 15min, 30min or 60min respectively, preferably selecting for 10min, and sufficiently oscillating for 20min by a vortex instrument to obtain polypeptide compound nanoparticles, wherein the specific prescription is shown in Table 6:
TABLE 6 polypeptide nanoparticle formulation and particle size, potential and encapsulation efficiency thereof
Mixing the prepared polypeptide compound nanoparticle aqueous solution with a freeze-drying agent, and freeze-drying by a freeze dryer (CHRIST ALPHA LD plus, germany) to prepare the freeze-drying agent, wherein the freeze-drying agent can be trehalose or sucrose, and storing in a refrigerator at 4 ℃ for standby.
Embodiment III: characterization of polypeptide Complex nanoparticles according to the present invention
Morphology of nanoparticles: preparing polypeptide compound nanoparticles by using EGFP-mRNA as model mRNA according to the preparation method I, the preparation method II, the preparation method III, the preparation method IV and the corresponding prescriptions in the second embodiment, and testing the nanoparticle morphology of the representative polypeptide compound nanoparticle aqueous solution by using a transmission electron microscope (model FEI Talos F200X). Immersing copper bars without any dyeing into freshly prepared polypeptide compound nano particle aqueous solution, naturally drying at 25 ℃ to obtain a sample, and testing to obtain the polypeptide compound nano particle, wherein the result shows that the polypeptide compound nano particles have better dispersibility, are in regular or irregular spherical structures and have the particle size range of 60-120 nm, as shown in figure 1. The results are shown in FIG. 1.
Particle size and potential: polypeptide complex nanoparticles were prepared as described in example two using EGFP-mRNA as model mRNA, and the dynamic light scattering particle size (size), surface Potential (Zeta Potential) and Polydispersity (PDI) of the polypeptide complex nanoparticles were tested using a Markov nanoparticle sizer (Malvern Zetasizer Nano ZSE) at 25 ℃. The results are shown in tables 3 to 6, and the results show that the polypeptide compound nanoparticle has good dispersibility and surface charge of the nanoparticle ranging from-15 mV to 5mV, and the particle size of the polypeptide compound nanoparticle ranges from 56nm to 273 nm.
Encapsulation efficiency: polypeptide complex nanoparticles were prepared according to the preparation method described in example two using FLuc-mRNA as model mRNA, and the encapsulation efficiency of each prescription on mRNA was determined using the Quant-iTRiboGreen RNA detection kit (ThermoFische company), and the brief processing method of the invention was as follows: centrifuging each prescription at 4 ℃ and 20000rpm for 2 hours by using a low-temperature high-speed centrifuge, collecting supernatant and quantifying the volume thereof by using a pipette, and recording as V1; measuring the concentration of mRNA in the supernatant with a Quant-iTRiboGreen RNA detection kit, designated as C1; dissolving the centrifuged precipitate in 25ul of chromatographic pure DMSO, continuously adding 0.9% physiological saline injection, mixing uniformly, standing at 25deg.C for 2h, recording total volume V2, measuring mRNA concentration with Quant-iTRiboGreen RNA detection kit, and recording as C2; the calculation formula of the entrapment rate of each prescription is as follows: the encapsulation efficiency=100% - (v1c1)/(v1c1+v2c2) ×100%, and the results are shown in tables 3 to 6, the formulation has a good encapsulation effect on mRNA, and the encapsulation efficiency is 98.0% or more.
Embodiment four: agarose gel electrophoresis to detect the compression capacity of polypeptide to mRNA
Agarose gel with a mass-to-volume ratio of 1% (agarose 0.4g:1 xTAE buffer solution 40 ml) was prepared, and was melted twice by microwaves, and 4 mu l SyBR SAFE DNA GEL STAIN dye (Lot No.1771519, invitrogen, USA) was added to agarose at a ratio of 1:10000, and poured into the corresponding gel tank (15-well stuck tank) after being sufficiently uniform, and cooled for 20min for use.
The preparation method of the mRNA positive control group comprises the following steps: with EGFP-mRNA as model mRNA, 1. Mu.l of mRNA solution (i.e., 100 ng) at a concentration of 100 ng/. Mu.l was added, then 9. Mu.l of nuclease-free ultrapure water was added to make up the system volume to 10. Mu.l, and finally 2. Mu.l of 6X Loading Buffer was added to mix well.
The sample group configuration method comprises the following steps: through different mass ratios of polypeptide to mRNA, 1 μl of mRNA solution (i.e. 100 ng) with a concentration of 100ng/ul was added to the polypeptide solution (1 μg/μl) and mixed well, then nuclease-free ultrapure water was added to make up the system volume to 10 μl, and after 10min of mixing, 2 μl of 6 x Loading Buffer was added to each sample and mixed well. After mixing the samples, 12 μl of the system was applied to each well, and run for 25min using an 80V voltage electrophoresis apparatus and observed with a gel imager, and the experimental results are shown in fig. 2.
Conclusion: the minimum mass ratios (polypeptide: mRNA) that each polypeptide guarantees complete compression of the mRNA are: seq.05 was 4, seq.12 was 4, seq.46 was 2, seq.47 was 2, seq.49 was 16, and seq.53 was 4.
Fifth embodiment: in vitro cell transfection experiment and cytotoxicity investigation of polypeptide complex nanoparticles
Cell transfection of mRNA (with FLuc-mRNA as model mRNA): the logarithmic growth phase DC2.4 cell suspension was packed into 96-well plates at a density of 4X 10 4 cells per well, and placed in a 5% CO 2 incubator at 37℃for stationary culture. After 24h, FLuc-mRNA at a concentration of 1. Mu.g/. Mu.l was diluted to 0.1. Mu.g/. Mu.l with nuclease-free ultrapure water, and polypeptide complex nanoparticles were prepared by the preparation method of the different formulations described in example two, respectively, and then diluted to 88. Mu.l of a polypeptide nanoparticle composition mixture containing 10 ng/. Mu. lFLuc-mRNA with nuclease-free ultrapure water, and after 10min of standing, each sample was added to a 96-well plate containing 180. Mu. lopti-MEM medium at a volume of 20. Mu.l per well, respectively, and 4 wells were repeated for each sample. After 4h of administration, the medium aspirated into the 96-well plate was replaced with complete medium. The incubation was continued for 24h, complete medium was aspirated and rinsed once with PBS, 100. Mu.l of D-Luciferin working solution (working concentration 250. Mu.g/ml) was added to each 96-well plate, incubation was continued for 5min at 37℃in an incubator, and the fluorescence expression intensity of FLuc-mRNA was tested by imaging with an Omega-FLuostar microplate reader. The results are shown in FIG. 3.
Cytotoxicity experiment: the DC2.4 cell suspension in logarithmic growth phase was packed into 96-well plates at a density of 4X 10 4 cells per well, and placed in a 5% CO 2 incubator at 37℃for stationary culture. After 24h, FLuc-mRNA at a concentration of 1. Mu.g/. Mu.l was diluted to 0.1. Mu.g/. Mu.l with nuclease-free ultrapure water, and polypeptide complex nanoparticles were prepared by the preparation method according to the different prescriptions described in example two, respectively, and then diluted to 88. Mu.l of a polypeptide nanoparticle composition mixture containing 10 ng/. Mu. lFLuc-mRNA with nuclease-free ultrapure water, respectively, and after standing for 10min, 20. Mu.l of each well was added to 96-well plates containing 180. Mu.l of opti-MEM medium, respectively, and 4 wells were repeated for each sample. After 4h of administration, the medium aspirated into the 96-well plate was replaced with complete medium. The culture was continued for 48h, complete medium was aspirated and rinsed three times with PBS, cell-free CCK-8 medium wells served as negative controls, cell-free CCK-8 medium wells served as blank controls, 90. Mu.l of serum-free medium and 10. Mu. lCCK-8 solution were added to each well and incubation was continued for 2h in the incubator. The absorbance at 450nm was measured with an Ome ga-FLuostar microplate reader. Cell viability calculation formula:
Cell viability% = [ a (dosed) -a (blank) ]/[ a (non-dosed) -a (blank) ] × 100%;
a (dosing): absorbance of DC2.4 cells, prescription solution and CCK-8 solution was added per well;
a (blank): absorbance of CCK-8 solution alone per well;
a (no drug addition): absorbance of DC2.4 cells and CCK-8 solution was added to each well;
* Cell viability: cell proliferation activity or cytotoxicity activity.
The results are shown in FIG. 4.
Conclusion: the result shows that the survival rate of cells is above 90%, which indicates that the polypeptide compound nanoparticle prescription has no obvious cytotoxicity, has good biocompatibility and can be used for subsequent in vivo experiments of animals. Cell transfected DNA (model DN a with Luc-pDNA): the DC2.4 cell suspension in logarithmic growth phase was packed into 96-well plates at a density of 4X 10 4 cells per well, and placed in a 5% CO 2 incubator at 37℃for stationary culture. After 24 hours, luc-pDNA at a concentration of 1. Mu.g/. Mu.l was diluted to 0.1. Mu.g/. Mu.l with nuclease-free ultrapure water. Polypeptide compound nanoparticles are prepared by the preparation methods of different prescriptions as described in the second example, and then diluted with nuclease-free ultrapure water to 88 μl of a polypeptide nanoparticle composition mixture containing 15 ng/. Mu.l of Luc-pDNA, and after standing for 30min, the mixture is added into 96-well plates containing 180 μm lopti-MEM medium per well in a volume of 20 μl per well, and 4 wells are repeated for each sample. After 4h of administration, the medium aspirated into the 96-well plate was replaced with complete medium. Continuously culturing for 24h, sucking out the complete culture medium, adding 100 μl of D-Luciferin solution with working concentration of 250 μg/ml into each 96-well plate, continuously culturing in an incubator at 37 ℃ for 5min, imaging with an Omega-FLuostar microplate reader, testing the fluorescence expression intensity of Luc-pDNA, repeating the test every 24h, sucking out the culture medium containing D-Luciferin after each test, adding fresh complete culture medium, continuously culturing for 24h, adding D-Luciferin, and repeating the test for three days. The results are shown in FIG. 5, with the abscissa representing the different prescriptions and the ordinate representing the relative fluorescence intensities expressed by the same dose of Luc-pDNA after 24h, 48h, 72h transfection. The results are shown in FIG. 5.
Conclusion: as shown in FIG. 5, the polypeptide complex nanoparticles entrapped with Luc-pDNA show better expression levels at the cellular level, the expression level is highest on the next day, and the expression level decreases from the third day, wherein Rp.01, rp.27 and Rp.34 are superior to other prescriptions.
Example six: transfection of polypeptide complex nanoparticles in mice by fluorescence imaging detection of small animals
Three female BALB/c mice per group were prescribed polypeptide complex nanoparticles containing FLuc-mRNA by the method of the present invention using FLuc-mRNA as model mRNA. The experimental group used an insulin needle to inject 50 μl of polypeptide complex nanoparticle formulation containing 5 μ gFLuc-mRNA into each mouse. Wherein the administration mode of the prescription Rp.01, rp.07, rp.11, pp.12, rp.13, rp.17, rp.20, rp.22 and Rp.24 groups is subcutaneous injection, and the injection site is subcutaneous on the back of the mice; the modes of administration of the prescription Rp.33, rp.36, rp.37, rp.42 groups are intraperitoneal injections; the other prescription group was given by intramuscular injection, and the injection site was the thigh muscle of the mice. The blank group was denoted NC, and insulin needle was injected intramuscularly with 50. Mu.l PBS buffer. After 6 hours of administration, a proper amount of substrate D-Luciferin is taken, diluted by PBS to prepare a solution with the concentration of 25mg/ml, the solution is kept away from light for later use, 125 mu l of substrate is injected into the abdominal cavity of each mouse, the mice are placed in a small animal anesthesia box, a ventilation valve is opened, and isoflurane anesthetized mice are released. The mice were subjected to whole-body in vivo imaging bioluminescence image detection using a small animal in vivo imaging system (PERKINELMER, IVIS lumine SERIES III) 5min after substrate injection. Wherein the prescription groups Rp.33, rp.36, rp.37, rp.42 take abdominal bioluminescence images of the mice and the remaining prescription groups take dorsal bioluminescence images of the mice. Results as shown in fig. 6, one for each group of representative mice, and the experimental group of polypeptide complex nanoparticle formulations showed luciferase expression in whole body in vivo imaging, with greater fluorescence intensity and greater luciferase expression.
Conclusion: as shown in FIG. 6, the polypeptide complex nanoparticles of each experimental group with the FLuc-mRNA are better expressed by luciferase in mice, and are effectively expressed by intraperitoneal injection, subcutaneous injection and intramuscular injection. In the experimental group prescription, rp.27, rp.33, rp.41 of luciferase was superior to other prescriptions.
Embodiment seven: humoral immunity effect evaluation of polypeptide complex nanoparticle in mouse
The novel crown S-mRNA is used as model mRNA, and provided by Shanghai megadimension technology development Co., ltd (Hongene Biotech Corporation), and the nucleotide sequence of the novel crown S-mRNA (cap 1 structure, N1-me-pseudo U modified) is shown in the S-mRNA in the sequence table.
Specific information for S-mRNA stock solution is:
product name: COVID-19Spike Protein,Full Length-mRNA;
description of the product: a length of 4088 nucleotides;
modifications (modification): fully substituted with N1-Me-pseudo UTP; (all replaced with N1-Me-pseudo UTP);
concentration: 1.0mg/ml;
Storage environment: 1mM sodium citrate, pH 6.4;
Storage requirements are: -40 ℃ or less.
The experimental process comprises the following steps:
Step 1: mice were immunized for the first time: day 0, 5-6 week female BALB/c mice were divided into 9 groups (5 animals per group) and each were intramuscular injected with 75. Mu.l PBS (blank), 5. Mu.g of a combination of naked S-mRNA and 5. Mu. g S protein (positive control) and 5. Mu.g of polypeptide complex nanoparticle formulations Rp.21, rp.25, rp.27, rp.41, rp.01, RP.08, 75. Mu.l, each.
Step 2: first serum collection: on day 28, mice were bled from the outer canthus. After serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at the temperature of 5000 Xg at 4 ℃, taking supernatant, centrifuging for 5 minutes at the temperature of 4 ℃ at the temperature of 10000 Xg at the speed of 10000 Xg, taking supernatant, adding the supernatant into eight rows of PCR tubes, sub-packaging, and freezing at-20 ℃ for standby.
Step 3: secondary immunization of mice: on day 28, mice were given intramuscular injections of 75 μl PBS (blank control), 5 μg of a combination of naked S-mRNA and 5 μ g S protein (positive control) and 5 μ g S-mRNA-entrapped polypeptide complex nanoparticle formulations Rp.21, rp.25, rp.27, rp.41, rp.01, RP.08, 75 μl, respectively, after outer canthus blood collection. The process of the first immunization was repeated.
Step 4: secondary serum collection: 21 days after the second immunization, the mice were bled from the outer canthus. After serum is solidified for 1h at 4 ℃, the serum is centrifugated for 5 minutes at 4 ℃ at a rotation speed of 5000 Xg (5000 times of gravity acceleration), the supernatant is taken out, and then centrifugated for 5 minutes at 4 ℃ at a rotation speed of 10000 Xg, the supernatant is taken out and added into eight rows of PCR tubes for sub-packaging, and the supernatant is frozen for standby at-20 ℃.
Step 5: ELISA detection of serum IgG content: s protein was diluted in PBS, ELISA plates were coated with 100. Mu.l of the dilution (containing 1. Mu. g S protein) per well, and coated at 4℃for 6h. After removing the liquid from the plate and adding 200. Mu.l of PBST wash plate 3 times per well, 200. Mu.1 PBS blocking solution containing 5% BSA was added to each well and blocked for 2h at 25 ℃. After removing the blocking solution and washing the plate 1 time with 200. Mu.l of PBST per well, 100. Mu.l of serum diluted 200-fold with PBS was added and incubated for 2h at 25 ℃. After discarding serum and washing the plates 3 times with 200. Mu.l of PBST per well, 100. Mu.l of antibody (antibody diluted 1:1000 with PBS) was added per well and incubated for 1h at 25 ℃. After discarding the antibody and washing the plate 3 times with 200. Mu.l of PBST per well, 50. Mu.l of TMB color development solution per well was added to react in the dark, and after the positive control well became blue or reacted for 10 minutes, 50. Mu.l of 2M sulfuric acid was added to terminate the reaction, the optical densities at wavelengths of 450nm and 630nm were detected by a microplate reader, and the difference in OD values was calculated to reflect the level of anti-S protein IgG in serum. The results are shown in FIG. 7.
The small knot: the result shows that the OD value corresponding to the prescription Rp14 is higher than that of the control group after two immunizations, and the prescription nanoparticle has stronger serum conversion efficiency and humoral immunity activation function.
Step 6: ELISA detection of serum IgG titres: s protein was diluted in PBS, ELISA plates were coated with 100. Mu.l of the dilution (1. Mu.g S protein) per well, and coated at 4℃for 6h. After removing the liquid from the plate and adding 200. Mu.l of PBST wash plate 1 time per well, 200. Mu.l of PBS blocking solution containing 5% BSA was added to each well and blocked for 2 hours at 25 ℃. After removing the blocking solution and washing the plate 3 times with 200. Mu.l of PBST per well, serum diluted 50, 250, 1250, 6250, 31250, 156250, 781250, 3906250 times with PBS was added and incubated for 2h at 25℃in a shaker. After discarding serum and washing the plates 3 times with 200. Mu.l of PBST per well, 100. Mu.l of antibody (antibody diluted 1:1000 with PBS) was added per well and incubated for 1h at 25 ℃. After discarding the antibody and washing the plate 3 times with 200. Mu.l of PBST per well, 50. Mu.l of TMB color development liquid per well was added to react in the dark, and after the positive control well became blue or reacted for 10 minutes, 50. Mu.l of 2M sulfuric acid was added to terminate the reaction, and the optical densities at 450nm and 630nm were detected by an ELISA reader. The results are shown in FIG. 8.
The small knot: the invention takes 2 times of the average OD value of the PBS group as a base line, and the OD value of the Rp.08 group is still higher than the base line by 2 times when the PBS group is diluted to 3906250 times, which indicates that the prescription Rp.08 has stronger serum conversion efficiency and humoral immunity activation function.
Example eight: gene transfection kit
The gene transfection kit is a multipurpose transfection reagent, can be composed of any prescription of the invention, and can provide efficient transfection in various adherent and suspension cell lines. Suitable for all common cell lines and many cell lines which are difficult to transfect, and can be used for a culture medium containing or not containing serum. The kit uses the 96-well cell culture plate to transfect mammalian cells. The method comprises the following specific steps:
1. One day before transfection, 1X 10 4 to 10X 10 4 cells were seeded in 96-well cell culture plates with 200. Mu.l of medium per well to achieve a cell growth density of over 80% at the time of transfection.
2. For each transfected sample, the following complexes were prepared:
a. Diluting 200ng DNA or RNA to 15 μl with sterile nuclease-free water, and gently mixing;
b. The transfection reagents were gently mixed in the prescribed proportions prior to use, and then diluted to 15 μl with sterile nuclease-free water in appropriate amounts;
c. The diluted DNA or RNA was gently mixed with the diluted transfection reagent in the prescribed ratio (total volume=30 μl), incubated at 25 ℃ for 10 to 30 minutes to obtain nucleic acid-polypeptide complex nanoparticles.
3. After rinsing the 96-well plate with PBS, 170. Mu.l of opti-MEM medium and 30. Mu.l of nucleic acid-polypeptide complex nanoparticles were added to each well, the final volume of medium being 200. Mu.l.
4. Placing the cells in a carbon dioxide incubator to incubate, replacing the original culture medium with the complete culture medium after 4 hours, continuing to incubate for 12 to 72 hours in the carbon dioxide incubator, and finally detecting the expression quantity of the nucleic acid.
All references, including patent references, disclosed herein are incorporated by reference.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Shenzhen thick memory nano pharmaceutical Co., ltd
<120> Polypeptide, polypeptide complex nanoparticle, nucleic acid vaccine and application thereof
<130> PCT002
<141> 2021-09-02
<160> 54
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> (Synthesis)
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Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10
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<211> 14
<212> PRT
<213> (Synthesis)
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Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10
<210> 3
<211> 15
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10 15
<210> 4
<211> 16
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10 15
<210> 5
<211> 17
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val
1 5 10 15
Arg
<210> 6
<211> 18
<212> PRT
<213> (Synthesis)
<400> 6
Arg Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg
<210> 7
<211> 19
<212> PRT
<213> (Synthesis)
<400> 7
Arg Arg Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser
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Ile Val Arg
<210> 8
<211> 20
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg
20
<210> 9
<211> 22
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro
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Thr Glu Ser Ile Val Arg
20
<210> 10
<211> 27
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Trp
1 5 10 15
Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
20 25
<210> 11
<211> 32
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
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Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
20 25 30
<210> 12
<211> 18
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Trp Phe Cys Arg Val Gln Pro Thr Glu Ser Ile
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Val Arg
<210> 13
<211> 18
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg His Phe Cys Arg Val Gln Pro Thr Glu Ser Ile
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Val Arg
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<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Trp Cys Phe Arg Val Gln Pro Thr Glu Ser Ile
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Val Arg
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<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Cys Pro Trp Arg Val Gln Pro Thr Glu Ser Ile
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Val Arg
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<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
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<213> (Synthesis)
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Arg Arg Arg Arg Arg Cys Tyr Pro Trp Cys Arg Val Gln Pro Thr Glu
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Ser Ile Val Arg
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<213> (Synthesis)
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Arg Arg Arg Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg
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<213> (Synthesis)
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Arg Arg Arg Arg Arg Cys Pro Arg Val Gln Pro Thr Glu Ser Ile Val
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Arg
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<213> (Synthesis)
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Arg Arg Arg Arg Arg Trp Pro Cys Arg Val Gln Pro Thr Glu Ser Ile
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Val Arg
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<211> 18
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Pro Phe Cys Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg
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<211> 18
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Arg Arg Arg Trp Cys Val Gln Pro Thr Glu Ser Ile Val
1 5 10 15
Arg Cys
<210> 23
<211> 17
<212> PRT
<213> (Synthesis)
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Trp Cys Arg Arg Arg Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val
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Arg
<210> 24
<211> 20
<212> PRT
<213> (Synthesis)
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Arg Arg Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg
20
<210> 25
<211> 20
<212> PRT
<213> (Synthesis)
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His Lys Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg
20
<210> 26
<211> 20
<212> PRT
<213> (Synthesis)
<400> 26
Trp Cys Arg Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg
20
<210> 27
<211> 20
<212> PRT
<213> (Synthesis)
<400> 27
Trp Cys Lys Leu Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg
20
<210> 28
<211> 22
<212> PRT
<213> (Synthesis)
<400> 28
Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10 15
Cys Trp Arg Arg Arg Arg
20
<210> 29
<211> 22
<212> PRT
<213> (Synthesis)
<400> 29
Trp Cys Arg Arg Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10 15
Arg Arg Arg Arg Cys Trp
20
<210> 30
<211> 26
<212> PRT
<213> (Synthesis)
<400> 30
Arg Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg Cys Trp Arg Arg Cys Trp Arg Arg
20 25
<210> 31
<211> 28
<212> PRT
<213> (Synthesis)
<400> 31
Phe Cys Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu Ser
1 5 10 15
Ile Val Arg Arg Arg Arg Cys Trp Arg Arg Cys Phe
20 25
<210> 32
<211> 28
<212> PRT
<213> (Synthesis)
<400> 32
Phe Cys Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu Ser
1 5 10 15
Ile Val Arg Cys Trp Arg Arg Arg Arg Arg Cys Phe
20 25
<210> 33
<211> 21
<212> PRT
<213> (Synthesis)
<400> 33
Arg Arg Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg Cys
20
<210> 34
<211> 21
<212> PRT
<213> (Synthesis)
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His Lys Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg Cys
20
<210> 35
<211> 21
<212> PRT
<213> (Synthesis)
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Trp Cys Arg Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg Cys
20
<210> 36
<211> 21
<212> PRT
<213> (Synthesis)
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Trp Cys Lys Leu Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu
1 5 10 15
Ser Ile Val Arg Cys
20
<210> 37
<211> 24
<212> PRT
<213> (Synthesis)
<400> 37
Cys Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val
1 5 10 15
Arg Cys Trp Arg Arg Arg Arg Cys
20
<210> 38
<211> 22
<212> PRT
<213> (Synthesis)
<400> 38
Trp Cys Arg Arg Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10 15
Arg Arg Arg Arg Trp Cys
20
<210> 39
<211> 18
<212> PRT
<213> (Synthesis)
<400> 39
Arg Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg
<210> 40
<211> 18
<212> PRT
<213> (Synthesis)
<400> 40
His Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg
<210> 41
<211> 17
<212> PRT
<213> (Synthesis)
<400> 41
Trp Cys Arg Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val
1 5 10 15
Arg
<210> 42
<211> 18
<212> PRT
<213> (Synthesis)
<400> 42
Trp Cys Lys Leu Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg
<210> 43
<211> 18
<212> PRT
<213> (Synthesis)
<400> 43
Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg Cys Trp Arg
1 5 10 15
Arg Arg
<210> 44
<211> 17
<212> PRT
<213> (Synthesis)
<400> 44
Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg Arg Arg Cys
1 5 10 15
Trp
<210> 45
<211> 22
<212> PRT
<213> (Synthesis)
<400> 45
Arg Trp Cys Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10 15
Cys Trp Arg Cys Trp Arg
20
<210> 46
<211> 22
<212> PRT
<213> (Synthesis)
<400> 46
Phe Cys Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg
1 5 10 15
Arg Cys Trp Arg Cys Phe
20
<210> 47
<211> 22
<212> PRT
<213> (Synthesis)
<400> 47
Phe Cys Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val Cys
1 5 10 15
Trp Arg Arg Arg Cys Phe
20
<210> 48
<211> 19
<212> PRT
<213> (Synthesis)
<400> 48
Arg Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg Cys
<210> 49
<211> 20
<212> PRT
<213> (Synthesis)
<400> 49
His Lys Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser
1 5 10 15
Ile Val Arg Cys
20
<210> 50
<211> 18
<212> PRT
<213> (Synthesis)
<400> 50
Trp Cys Arg Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val
1 5 10 15
Arg Cys
<210> 51
<211> 18
<212> PRT
<213> (Synthesis)
<400> 51
Trp Cys Lys Leu Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile
1 5 10 15
Val Arg
<210> 52
<211> 20
<212> PRT
<213> (Synthesis)
<400> 52
Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg Cys
1 5 10 15
Trp Arg Arg Cys
20
<210> 53
<211> 18
<212> PRT
<213> (Synthesis)
<400> 53
Trp Cys Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg Arg Arg
1 5 10 15
Trp Cys
<210> 54
<211> 4088
<212> RNA
<213> (Synthesis)
<400> 54
gagaauaaac uaguauucuu cuggucccca cagacucaga gagaacccgc caccauguuc 60
guguuccugg ugcugcugcc ucuggugucc agccagugug ugaaccugac caccagaaca 120
cagcugccuc cagccuacac caacagcuuu accagaggcg uguacuaccc cgacaaggug 180
uucagaucca gcgugcugca cucuacccag gaccuguucc ugccuuucuu cagcaacgug 240
accugguucc acgccaucca cguguccggc accaauggca ccaagagauu cgacaacccc 300
gugcugcccu ucaacgacgg gguguacuuu gccagcaccg agaaguccaa caucaucaga 360
ggcuggaucu ucggcaccac acuggacagc aagacccaga gccugcugau cgugaacaac 420
gccaccaacg uggucaucaa agugugcgag uuccaguucu gcaacgaccc cuuccugggc 480
gucuacuacc acaagaacaa caagagcugg auggaaagcg aguuccgggu guacagcagc 540
gccaacaacu gcaccuucga guacgugucc cagccuuucc ugauggaccu ggaaggcaag 600
cagggcaacu ucaagaaccu gcgcgaguuc guguuuaaga acaucgacgg cuacuucaag 660
aucuacagca agcacacccc uaucaaccuc gugcgggauc ugccucaggg cuucucugcu 720
cuggaacccc ugguggaucu gcccaucggc aucaacauca cccgguuuca gacacugcug 780
gcccugcaca gaagcuaccu gacaccuggc gauagcagca gcggauggac agcuggugcc 840
gccgcuuacu augugggcua ccugcagccu agaaccuucc ugcugaagua caacgagaac 900
ggcaccauca ccgacgccgu ggauugugcu cuggacccuc ugagcgagac aaagugcacc 960
cugaaguccu ucaccgugga aaagggcauc uaccagacca gcaacuuccg ggugcagccc 1020
accgaaucca ucgugcgguu ccccaauauc accaaucugu gccccuucgg cgagguguuc 1080
aaugccacca gauucgccuc uguguacgcc uggaaccgga agcggaucag caauugcgug 1140
gccgacuacu ccgugcugua caacuccgcc agcuucagca ccuucaagug cuacggcgug 1200
uccccuacca agcugaacga ccugugcuuc acaaacgugu acgccgacag cuucgugauc 1260
cggggagaug aagugcggca gauugccccu ggacagacag gcaagaucgc cgacuacaac 1320
uacaagcugc ccgacgacuu caccggcugu gugauugccu ggaacagcaa caaccuggac 1380
uccaaagucg gcggcaacua caauuaccug uaccggcugu uccggaaguc caaucugaag 1440
cccuucgagc gggacaucuc caccgagauc uaucaggccg gcagcacccc uuguaacggc 1500
guggaaggcu ucaacugcua cuucccacug caguccuacg gcuuucagcc cacaaauggc 1560
gugggcuauc agcccuacag agugguggug cugagcuucg aacugcugca ugccccugcc 1620
acagugugcg gcccuaagaa aagcaccaau cucgugaaga acaaaugcgu gaacuucaac 1680
uucaacggcc ugaccggcac cggcgugcug acagagagca acaagaaguu ccugccauuc 1740
cagcaguuug gccgggauau cgccgauacc acagacgccg uuagagaucc ccagacacug 1800
gaaauccugg acaucacccc uugcagcuuc ggcggagugu cugugaucac cccuggcacc 1860
aacaccagca aucagguggc agugcuguac caggacguga acuguaccga agugcccgug 1920
gccauucacg ccgaucagcu gacaccuaca uggcgggugu acuccaccgg cagcaaugug 1980
uuucagacca gagccggcug ucugaucgga gccgagcacg ugaacaauag cuacgagugc 2040
gacaucccca ucggcgcugg aaucugcgcc agcuaccaga cacagacaaa cagcccucgg 2100
agagccagaa gcguggccag ccagagcauc auugccuaca caaugucucu gggcgccgag 2160
aacagcgugg ccuacuccaa caacucuauc gcuaucccca ccaacuucac caucagcgug 2220
accacagaga uccugccugu guccaugacc aagaccagcg uggacugcac cauguacauc 2280
ugcggcgauu ccaccgagug cuccaaccug cugcugcagu acggcagcuu cugcacccag 2340
cugaauagag cccugacagg gaucgccgug gaacaggaca agaacaccca agagguguuc 2400
gcccaaguga agcagaucua caagaccccu ccuaucaagg acuucggcgg cuucaauuuc 2460
agccagauuc ugcccgaucc uagcaagccc agcaagcgga gcuucaucga ggaccugcug 2520
uucaacaaag ugacacuggc cgacgccggc uucaucaagc aguauggcga uugucugggc 2580
gacauugccg ccagggaucu gauuugcgcc cagaaguuua acggacugac agugcugccu 2640
ccucugcuga ccgaugagau gaucgcccag uacacaucug cccugcuggc cggcacaauc 2700
acaagcggcu ggacauuugg agcaggcgcc gcucugcaga uccccuuugc uaugcagaug 2760
gccuaccggu ucaacggcau cggagugacc cagaaugugc uguacgagaa ccagaagcug 2820
aucgccaacc aguucaacag cgccaucggc aagauccagg acagccugag cagcacagca 2880
agcgcccugg gaaagcugca ggacgugguc aaccagaaug cccaggcacu gaacacccug 2940
gucaagcagc uguccuccaa cuucggcgcc aucagcucug ugcugaacga uauccugagc 3000
agacuggacc cuccugaggc cgaggugcag aucgacagac ugaucacagg cagacugcag 3060
agccuccaga cauacgugac ccagcagcug aucagagccg ccgagauuag agccucugcc 3120
aaucuggccg ccaccaagau gucugagugu gugcugggcc agagcaagag aguggacuuu 3180
ugcggcaagg gcuaccaccu gaugagcuuc ccucagucug ccccucacgg cgugguguuu 3240
cugcacguga cauaugugcc cgcucaagag aagaauuuca ccaccgcucc agccaucugc 3300
cacgacggca aagcccacuu uccuagagaa ggcguguucg uguccaacgg cacccauugg 3360
uucgugacac agcggaacuu cuacgagccc cagaucauca ccaccgacaa caccuucgug 3420
ucuggcaacu gcgacgucgu gaucggcauu gugaacaaua ccguguacga cccucugcag 3480
cccgagcugg acagcuucaa agaggaacug gacaaguacu uuaagaacca cacaagcccc 3540
gacguggacc ugggcgauau cagcggaauc aaugccagcg ucgugaacau ccagaaagag 3600
aucgaccggc ugaacgaggu ggccaagaau cugaacgaga gccugaucga ccugcaagaa 3660
cuggggaagu acgagcagua caucaagugg cccugguaca ucuggcuggg cuuuaucgcc 3720
ggacugauug ccaucgugau ggucacaauc augcuguguu gcaugaccag cugcuguagc 3780
ugccugaagg gcuguuguag cuguggcagc ugcugcaagu ucgacgagga cgauucugag 3840
cccgugcuga agggcgugaa acugcacuac acaugagcug gagccucggu ggccaugcuu 3900
cuugccccuu gggccucccc ccagccccuc cuccccuucc ugcacccgua cccccguggu 3960
cuuugaauaa agucugaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080
aaaaaaaa 4088

Claims (5)

1. A polypeptide compound having the amino acid sequence: seq ID NO: 05.
2. A polypeptide complex nanoparticle comprising:
a) The polypeptide compound of claim 1; and
B) The nucleic acid is a nucleic acid,
The nucleic acid is a nucleic acid drug.
3. The polypeptide complex nanoparticle of claim 2, comprising: the nucleic acid, polypeptide compound with the amino acid sequence of Seq ID NO: 05 and auxiliary materials, wherein the auxiliary materials are Tetronic 90R4 and lecithin; the mass ratio of the nucleic acid to the polypeptide compound with the amino acid sequence of Seq ID NO: 05 to the Tetronic cube 90R4 to the lecithin is 1:2:322:1.
4. A nucleic acid vaccine comprising the polypeptide complex nanoparticle of any one of claims 2-3.
5. Use of a polypeptide complex nanoparticle according to any one of claims 2 or 3 in the manufacture of a medicament or kit.
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