CN114213508A

CN114213508A - Polypeptide and polypeptide compound nanoparticle thereof, nucleic acid vaccine and application

Info

Publication number: CN114213508A
Application number: CN202111029790.2A
Authority: CN
Inventors: 张龙贵
Original assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Current assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Priority date: 2020-09-03
Filing date: 2021-09-02
Publication date: 2022-03-22
Also published as: WO2022048619A1

Abstract

The invention provides a polypeptide and a polypeptide compound nanoparticle thereof, a nucleic acid vaccine and application, belonging to the field of drug delivery. The polypeptide has the functions of compressing and protecting nucleic acid from being degraded, promoting the nucleic acid to penetrate cell membranes and the like, has higher antibody effect, and can be used for in vivo and in vitro cell gene transfection and preparation of vaccine preparations.

Description

Polypeptide and polypeptide compound nanoparticle thereof, nucleic acid vaccine and application

Technical Field

The invention relates to the field of drug delivery, and in particular relates to a polypeptide and a polypeptide compound nanoparticle thereof, a nucleic acid vaccine and application.

Background

Gene transfection is a technique by which a nucleic acid having a biological function is transferred or transported into a cell and the nucleic acid is maintained in the cell for its biological function. A gene vector refers to a means for introducing a foreign therapeutic gene into a biological cell. Nucleic acid vaccines are new vaccines that have been developed in recent years. Nucleic acid vaccines, as a novel approach, introduce nucleic acids encoding antigenic proteins into cells, and synthesize the proteins by the expression system of the cells, thereby inducing specific immune responses. Although most cells can spontaneously take up nucleic acids, the efficiency is low and at low doses it saturates. In addition, because of the existence of a large amount of RNase in nature, RNA is very unstable in vitro and in vivo and is easily degraded. Thus, there is a need for suitable agents to protect nucleic acids from extracellular RNase mediated degradation and to facilitate their entry into cells. In the development process of preventive nucleic acid vaccines and therapeutic nucleic acid vaccines, it is important that nucleic acids with specific sequences are delivered to Dendritic Cells (DCs) so that the nucleic acids are expressed safely, efficiently and in sufficient quantities to produce the efficacy of the vaccines.

Cell Penetrating Peptides (CPPs) are a class of polypeptides that are capable of passing directly across a cell membrane into a cell in a receptor-independent, non-classical endocytic manner without causing damage to the cell membrane, are generally no longer than 30 amino acids in length and are rich in basic amino acids, the amino acid sequence being generally positively charged, such as human immunodeficiency virus type 1 transcriptional activator TAT (HIV-1 TAT) (videos et al, j.biol.chem.1997; 272, 16010). The common properties of CPPs are: a net positive charge or charge neutrality, while being hydrophilic and hydrophobic (amphiphilic); the transmembrane delivery efficiency is higher; low cytotoxicity; no limitation of cell type; different bioactive substances can be introduced into cells by means of chemical combination or gene fusion and the like, so that the bioactive substances have the potential to become multifunctional targeted drug carriers. The specific transmembrane mechanisms of different CPPs vary, and it has been found that specific amino acid sequences bind mRNA and interfere with and decrease cell membrane stability, thereby carrying bioactive substances that penetrate the cell membrane, such as the arginine-alanine-leucine-alanine Residue (RALA) sequence (Pardi et al, Curropin in Immunol. 2020, 65: 14-20). Compared with other delivery modes, the current researches on CPPs in the field of mRNA delivery or vaccine development are still lack of reports.

The polypeptide prepared into the polypeptide compound nanoparticles by amide bond (peptide bond) connection has the advantages that the polypeptide compound nanoparticles can be degraded into amino acids in vivo while ensuring high transfection efficiency and low cytotoxicity, but at present, no polypeptide compound nanoparticle delivery system for enabling mRNA gene drugs to be on the market exists.

Aiming at the defects of the existing delivery system, the invention synthesizes non-naturally occurring polypeptide, prepares polypeptide compound nanoparticles, provides an improved gene vector for mRNA delivery, and is suitable for a nano delivery solution for animal vaccines or human mRNA drug development.

Disclosure of Invention

Brief description of the invention

In order to solve the above problems, 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 an application of a polypeptide compound nanoparticle in vivo and in vitro nucleic acid delivery. In a fourth aspect, the invention provides a nucleic acid vaccine containing the polypeptide complex nanoparticles. In a fifth aspect, the invention provides an application of the polypeptide compound nanoparticle in preparation of a medicament or a 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(general formula I) is shown in the specification,

wherein: x is an integer of 1 to 25, and y is an integer of 0 to 9;

(Xaa)_xcan 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)_xIs Arg. In some embodiments, (Xaa)_xIs (Xa 'a')_n(Arg)_1-10(Xa’a’)_nWherein Xa 'a' is at least one selected from Arg (R), Trp (W), Cys (C), Lys (K), Leu (L), Phe (F), Pro (P) or His (H), and n is an integer of 0-10. In certain embodiments, Xaa consists of (Arg)_1-10Trp (W) and/or Cys (C). In other embodiments, Xaa consists of (Arg)_1-10Trp (W), Cys (C), His (H), and/or Pro (P), Trp (W), Cys (C), His (H), and/or Pro (P) may be at (Arg)_1-10The above may also be at (Arg)_1-10Behind, or interspersed with one or more (Arg)_1-10In the middle.

In the general formula I, the sequence of Arg (R), Trp (W), Cys (C), Lys (K), Leu (L), Phe (F), Pro (P) or His (H) is not limited.

(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 can 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.47, seq.48, seq.49, seq.48, seq.47, seq.48. 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, general formula (I) is at least 50% similar to any of seq.01-seq.53 and which improves delivery of the nucleic acid molecule into a cell by at least 20%.

In some embodiments, general formula (I) is at least 75% similar to any of seq.01-seq.53 and which improves delivery of the nucleic acid molecule into a cell by at least 50%.

In some embodiments, general formula (I) is at least 90% similar to any of seq.01-seq.53 and which improves delivery of the nucleic acid molecule into a 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 in that it improves delivery of the nucleic acid molecule into the cell by at least 10%, in some embodiments, the polypeptide of formula (I) comprises 10-35 amino acids in that it improves delivery of the nucleic acid molecule into the cell by between about 50% to about 100%; in some embodiments, the polypeptide of formula (I) comprises 10-35 amino acids, which is characterized by an improvement in its delivery of the nucleic acid molecule into the cell of between about 75% to about 500%.

The present invention provides novel non-naturally occurring polypeptides having the functions of compressing and protecting nucleic acids from degradation, promoting penetration of nucleic acids through cell membranes, etc., as well as polypeptide complex nanoparticles comprising the polypeptides, methods for using the same for gene transfection of cells in vivo and in vitro, and methods for applying the same to vaccine preparations.

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) a nucleic acid.

In some embodiments, a polypeptide complex nanoparticle comprising:

a) a combination of at least one polypeptide according to the first aspect of the invention and at least one auxiliary material; and

b) a nucleic acid.

The nucleic acid may be chemically modified or chemically unmodified DNA, single-or double-stranded DNA, coding or non-coding DNA. In some embodiments, the nucleic acid is selected from 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 chemically unmodified RNA, single-or double-stranded RNA, coding or non-coding RNA. In some embodiments, the nucleic acid is selected from messenger RNA (mrna), oligoribonucleotides, viral RNA, replicon RNA, transfer RNA (trna), ribosomal RNA (rrna), immunostimulatory RNA (isrna), microrna, small interfering RNA (sirna), small nuclear RNA (snrna), small hairpin RNA (shrna) or riboswitches, RNA aptamers, RNA decoys, antisense RNA, ribozymes, or any combination thereof. In some preferred embodiments, the nucleic acid is 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 nucleic acid synthesis methods as set forth in patents US9254311B2 or CN 106659803A.

The polypeptide complex nanoparticles are capable of encapsulating mRNA and allowing its efficient introduction into different cell lines in vitro, and are capable of efficient transfection in vivo. The polypeptide compound nanoparticle can carry mRNA encoding immunogenic peptide to enter cells, effectively release the mRNA, express antigen and effectively achieve the aim of immunotherapy or immunoprophylaxis.

The novel non-naturally occurring polypeptide provided by the invention has the functions of compressing and protecting nucleic acid from degradation, promoting the nucleic acid to penetrate cell membranes and the like, polypeptide compound nanoparticles containing the polypeptide, a method for gene transfection of in vivo and in vitro cells by using the polypeptide, and a method for applying the polypeptide to vaccine preparations.

In some embodiments of the invention, a polypeptide complex nanoparticle comprises:

a) at least one polypeptide according to the invention,

b) nucleic acid, and

c) an auxiliary material.

The auxiliary material may include: a lipid or a PEG derivative.

The lipid may be a naturally occurring or synthetic phospholipid or a structural lipid.

The PEG derivative may be poloxamine, a poloxamine derivative, a poloxamer derivative or a PEG lipid.

The poloxamines of the present invention may be selected from at least one of the following poloxamines:

or

The poloxamine derivative is synthesized by referring to patent CN 111285845B.

The poloxamers 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-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-diundecenyl-sR-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sR-glycero-3-phosphocholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18: 0 diether PC), 1-oleoyl-2-cholestanyl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysopC), 1, 2-dioleyl-sn-glycero-3-phosphocholine, 1, 2-diacyl-sn-glycero-3-phosphocholine, 1, 2-didedodecaenoyl-sn-glycero-3-phosphocholine, 1, 2-diacyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dianeotetraenoic acid-sn-glycero-3-phosphoethanolamine, 1, 2-didodecanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), sphingomyelin, or lecithin (PC) and mixtures thereof;

the structural lipid may be selected from: cholesterol (Chol), coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof;

the PEG lipid may be selected from any one of the PEG lipids described by patent nos. 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-methoxy polyethylene glycol 2000(DMG-PEG) is shown as the following formula:

the structural formula of the mPEG5000-C-CLS (PEG-CLS) is shown as the following formula:

the structural formula of the mPEG2000-DSPE (PEG-DSPE) is shown as the following formula:

the mass ratio of the nucleic acid to the polypeptide can be less than or equal to about 1: 1. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from about 1: 1 to about 1: 52. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from about 1: 2 to about 1: 48. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from about 1: 2 to about 1: 40. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from about 1: 2 to about 1: 32. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from about 1: 2 to about 1: 24. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from about 1: 2 to about 1: 16. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from about 1: 2 to about 1: 10. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is from 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: 50, 1: 49, 1: 51, or 1: 51.

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 adjunct material can be less than or equal to about 1: 50. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is about 1: 2 to about 1: 800. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is about 1: 2 to about 1: 500. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is from 1: 2 to 1: 400. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is about 1: 2 to about 1: 50. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is about 1: 2 to about 1: 33. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is from 1: 2 to 1: 10. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is from 1: 2 to 1: 6. In some embodiments, the mass ratio of the nucleic acid to the adjunct 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 adjunct material is from 1: 33 to 1: 400. In some embodiments, the mass ratio of the nucleic acid to the adjunct material is from 1: 50 to 1: 800.

In some embodiments, the adjunct material is a PEG derivative and the mass ratio of the nucleic acid to the adjunct material is from 1: 50 to 1: 800. In some embodiments, the adjunct material is a PEG derivative and the mass ratio of the nucleic acid to the adjunct 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 adjunct material is a PEG derivative, and the mass ratio of the nucleic acid to the adjunct material is 1: 50 to 1: 800.

In some embodiments, the adjunct material is a PEG derivative, a phospholipid, and a structural lipid, and the mass ratio of the nucleic acid to the adjunct material is about 1: 2 to about 1: 6. In some embodiments, the adjunct material is a PEG derivative, a phospholipid and a structural lipid, and the mass ratio of the nucleic acid to the adjunct 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 to 1: 30, the adjunct material is a PEG derivative, a phospholipid and a structural lipid, and the mass ratio of the nucleic acid to the adjunct material is 1: 2 to 1: 6.

In some embodiments, the adjunct material is a PEG derivative and a phospholipid, and the mass ratio of the nucleic acid to the adjunct material is from about 1: 33 to about 1: 400 or from about 1: 33 to about 1: 370. In some embodiments, the adjunct material is a PEG derivative and a phospholipid, the mass ratio of the nucleic acid to the adjunct material is from about 1: 33 to about 1: 400 or from about 1: 33 to about 1: 370, and the mass ratio of the PEG derivative to the phospholipid is from 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 to 1: 400 or 1: 33 to 1: 370, and the mass ratio of the PEG derivative to the phospholipid is 32: 1 to 700: 1.

In some embodiments, a polypeptide complex nanoparticle, comprising: nucleic acid, a polypeptide compound with an amino acid sequence of seq.05 and an auxiliary material

And lecithin; the nucleic acid, the polypeptide compound with the amino acid sequence of seq.05,

And lecithin in a mass ratio of about 1: 2: 322: 1.

In some embodiments, a polypeptide complex nanoparticle, comprising: nucleic acid, a polypeptide compound with the amino acid sequence of seq.49 and auxiliary materials, wherein the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphocholine and cholesterol; the mass ratio of the nucleic acid, the polypeptide compound with the amino acid sequence of seq.49, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and cholesterol is about 10: 300: 8: 16: 31.

In some embodiments, a polypeptide complex nanoparticle, comprising: nucleic acid, a polypeptide compound with the amino acid sequence of seq.53 and auxiliary materials, wherein the auxiliary materials are 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.53, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and 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 a 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 described herein.

In some embodiments, the polypeptide complex nanoparticle may further comprise at least one pharmaceutically acceptable excipient.

The polypeptide complex nanoparticles of the invention are stable in aqueous solution and can be contacted with human or animal cellular tissue after formation, or can be stored for a period of time prior to contact with the cells or tissue. The polypeptide complex nanoparticles are 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 will be appreciated that the storage period 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 an application of a polypeptide compound nanoparticle in vivo and in vitro nucleic acid delivery.

Use of a nanoparticle of a polypeptide complex of the second aspect for nucleic acid delivery in vivo or in vitro.

In a fourth aspect, the present invention provides a nucleic acid vaccine comprising the polypeptide complex nanoparticle of the second aspect.

A nucleic acid vaccine comprising a nanoparticle of the polypeptide complex 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 include RNA that may encode at least one coding region for at least one therapeutic protein, therapeutic polypeptide, immunogenic protein, or immunogenic peptide. In some embodiments, the coding RNA is mRNA.

The invention provides ribonucleic acid vaccines which can safely induce a specific immune system naturally existing in an organism to generate almost any target protein or fragment thereof, take RNA (such as messenger RNA (mRNA)) as a core and take polypeptide compound nanoparticles as a delivery carrier, and the ribonucleic acid vaccines comprise infectious pathogen vaccines such as bacteria and viruses and tumor vaccines. In some embodiments, the RNA is modified. The nucleic acid vaccines disclosed herein are useful for inducing immune responses, including cellular and humoral immune responses, against infectious pathogens or cancer without the risk of potentially leading to insertional mutagenesis. Nucleic acid vaccines with polypeptide complex nanoparticles as delivery vectors can be used in a variety of disease types depending on the incidence of infectious pathogens and cancer. The nucleic acid vaccines are useful for the prevention and/or treatment of infectious pathogens or cancers of various metastatic stages or degrees.

In some embodiments of the invention, a nucleic acid vaccine comprising a 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 almost any protein of interest, from natural proteins to antibodies and other entirely novel protein constructs that can have therapeutic activity both inside and outside the cell.

The nucleic acid vaccines can be used in a variety of contexts depending on the prevalence of infection or the degree or level of unmet medical need. The nucleic acid vaccine may be used for the treatment and/or prevention of HPV of various genotypes, strains and isolates. The nucleic acid vaccine is advantageous in that it produces much greater antibody titers and responds much earlier than commercially available treatments for antiviral therapy. While not wishing to be bound by theory, it is believed that like mRNA polynucleotides, RNA vaccines are more optimally designed to generate the appropriate protein configuration upon translation when the RNA vaccine assigns native cellular machinery. Unlike traditional vaccines that are made ex vivo and can trigger adverse cellular responses, the nucleic acid vaccines provide a template for expression of protein antigens by cellular systems in a more natural manner.

In some embodiments of the invention, a nucleic acid vaccine comprising a 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, the nucleotide sequence of which encodes the S spike protein of the novel coronavirus SARS-CoV-2.

In some embodiments of the invention, a nucleic acid vaccine comprising a polypeptide complex nanoparticle of the second aspect; the polypeptide compound nanoparticles contain artificially synthesized pathogen antigen polypeptides. In some embodiments, the antigenic polypeptides are 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 preparation, suppository, eye drop or suspension, etc.

The nucleic acid vaccines of the present invention may 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 injections.

In a fifth aspect, the invention provides a use of the polypeptide complex nanoparticle of the second aspect in the preparation of a medicament or a kit.

Use of a nanoparticle of a polypeptide complex of the second aspect in the preparation of a medicament or kit.

In some embodiments, a polypeptide complex nanoparticle of the second aspect for use in the manufacture of a medicament for preventing, treating and/or ameliorating a disease selected from the group consisting of: cancer or tumor 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 a defined genetic defect and are inherited according to Mendelian's Law, 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 micrograph of a nanoparticle of a polypeptide complex according to example III; in the figure, A represents the prescription Rp.05, B represents the prescription Rp.28, C represents the prescription Rp.43, and the white scale is 200 nm.

FIG. 2 shows the result of agarose gel electrophoresis of the nanoparticles of the polypeptide complex of example IV; in the figure, mRNA refers to the positive control group of mRNA, and 1, 2, 4, 8, 16, 32, 64 refer to the mass ratio of polypeptide to mRNA is 1: 1, 2: 1, 4: 1, 8: 1, 16: 1, 32: 1, 64: 1; the lowest mass ratio for each polypeptide that ensures that the mRNA can be completely compressed is: 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-loaded polypeptide complex nanoparticles in DC2.4 cells in example five; the abscissa in the figure represents polypeptide nanoparticle compositions of different formulations, and the ordinate represents the relative fluorescence intensity expressed 24h after transfection of the polypeptide nanoparticle composition containing the same dose of FLuc-mRNA.

FIG. 4 shows the survival rate of DC2.4 cells after treatment with different prescriptions in the fifth example; the abscissa represents different polypeptide compound nanoparticle formulations, and the ordinate represents cell viability, with higher cell activity showing less cytotoxicity.

FIG. 5 illustrates transfection of Luc-pDNA polypeptide complex-loaded nanoparticles in DC2.4 cells in example five; the abscissa represents the different prescriptions and the ordinate is the relative fluorescence intensity expressed by DC2.4 cells 24h, 48h, 72h after transfection with the same dose of Luc-pDNA.

FIG. 6 shows the expression of luciferase in mice by IVIS detection polypeptide complex nanoparticles in example six.

FIG. 7 shows serum IgG antibody levels of mice after nanoparticle immunization with the polypeptide complexes of example seven; the abscissa represents the difference between the OD values at two wavelengths of the optical density on the 28 th and 49 th days after the first immunization for different prescriptions, and the OD value is an index for judging the IgG antibody level in serum and reflects the anti-S protein IgG level in serum.

FIG. 8 shows the serum IgG antibody titer of mice immunized with the nanoparticles of the polypeptide complex of example VII; the abscissa represents the different dilution of the serum for different prescriptions after 49 days after the first immunization, and the ordinate represents the difference in OD (optical density) values at the two wavelengths. 2x Baseline (twice background) was used as a cut-off to distinguish between positive and negative results, and the maximum dilution at which the OD was higher than this was the titer.

Definition of terms

The terms used throughout this specification generally have their ordinary meanings in the art within the context of the present invention and in the specific circumstances where each term is 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 such embodiments. It should be appreciated that the same concept 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, regardless of whether a term is detailed or discussed herein, nor is any particular meaning assigned. Synonyms for certain terms may be provided. Reciting one or more synonyms does not preclude 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 exemplified terms.

In the context of the present invention, all numbers disclosed herein are approximate values, regardless of whether the word "about" or "approximately" is used. There may be differences below 10% in the value of each number or reasonably considered by those skilled in the art, such as differences of 1%, 2%, 3%, 4% or 5%.

The term "polypeptide"Meaning polymers of amino acid residues (natural or non-natural) that are many times linked 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 multimolecular 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 bonds are 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 "polypeptide" is generally used to refer to relatively larger polypeptides, and "peptide" is 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"It is meant to be soluble in water under specified conditions, including readily soluble in water, sparingly soluble in water.

The term "hydrophobic"It means that it is hardly soluble in water under specific 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 mimetics that function in a manner similar to the 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, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid (i.e., the alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, methionine, and methyl sulfonium. 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 functions in a manner similar to a naturally occurring amino acid. The term "amino acid" may refer to amino acids or derivatives thereof (e.g., amino acid analogs) as well as the 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-acetyl cysteine.

By "kit" is meant a transfection, DNA, RNAi or other bioactive (e.g., protein or anionic molecule) delivery or protein expression or gene knock-down 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 agents may be provided in a single formulation. In other embodiments, the composite material and peptide may be provided separately, along with instructions directing the user to combine the agents at the time of use. Such kits may comprise a carrier device partitioned to hold one or more container devices (e.g., vials, tubes, etc.) in a tightly constrained manner. Each of such container devices contains 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 (in some embodiments one or more expression vectors, DNA molecules, RNA molecules, or RNAi molecules), a cell, one or more compounds of the invention, a lipid compound, a transfection-enhancing agent, 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 reaction of the adaptive immune system against a specific antigen (so-called specific or adaptive immune response) or a non-specific reaction of the innate immune system (so-called non-specific or innate immune response). (Fotin-Mleczek Mariola et al, CN108064176A)

Vaccine: a vaccine is typically understood as a prophylactic or therapeutic substance that provides at least one antigen or antigenic function. The antigen or antigenic function may stimulate the adaptive immune system of the body to provide an adaptive immune response. (Fotin-Mleczek Mariola et al, CN108064176A)

mRNA providing antigen: the mRNA providing the antigen may typically be an mRNA having at least one open reading frame which can be translated by the cell or organism to which the mRNA is provided. The product of this translation 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, e.g. 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, CN108064176A)

Nucleic acid (A): 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 comprises regulatory and/or other sequences which allow its expression and/or stability in a suitable host (e.g. human), i.e. the transcription and/or translation of the nucleic acid sequence encoding the particular protein or peptide. (Fotin-Mleczek Mariola et al, CN108064176A)

Peptide: peptides are polymers of amino acid monomers. Typically, the 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, a peptide may, for example, contain less than 50 monomer units. Longer peptides, which may also be referred to as polypeptides, typically have from 50 to 600 monomer units, more particularly from 50 to 300 monomer units. (Fotin-Mleczek Mariola et al, CN108064176A)

A pharmaceutically effective amount: 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 to trigger a desired therapeutic effect. (Fotin-Mleczek Mariola et al, CN108064176A)

Chemical synthesis of RNA: the chemical synthesis of relatively short fragments of oligonucleotides having defined chemical structures provides a fast and inexpensive method of obtaining custom oligonucleotides of any desired sequence. Although enzymes only synthesize DNA and RNA in the 5 'to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it most often proceeds in the opposite (i.e., 3 'to 5') direction. Currently, this process is performed as 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, CN108064176A)

To obtain the desired oligonucleotide, building blocks are sequentially coupled to the growing oligonucleotide chain on the solid phase in the order required for the product sequence in a fully automated process. After completion of the chain assembly, the product is released from the solid phase into solution, deprotected, and collected. The presence of side reactions sets a practical limit on the length of the synthetic oligonucleotide (up to about 200 nucleotide residues) because the number of errors increases with the length of the synthetic oligonucleotide. The product is usually separated by HPLC to obtain the desired oligonucleotide in high purity. (Fotin-MleczekMariola et al, CN108064176A)

RNA in vitro transcription: the term "RNA in vitro transcription" or "in vitro transcription" relates to a process in which RNA is synthesized (in vitro) in a cell-free system.A DNA, in particular a plasmid DNA, is used as a template for the production of RNA transcripts.RNA is obtainable by DNA-dependent in vitro transcription of a suitable DNA template, which according to the invention is preferably a linearized plasmid DNA template.A promoter for controlling in vivo transcription can be any promoter for any DNA-dependent RNA polymerase.specific examples of DNA-dependent RNA polymerases are T7, T3 and SP6RNA polymerase.A DNA template for in vitro RNA transcription can be obtained by cloning a nucleic acid, in particular a cDNA, corresponding to the corresponding RNA to be transcribed in vitro and introducing it into a suitable vector for in vitro transcription (e.g.into a plasmid DNA). in a preferred embodiment of the invention, the DNA template is linearized with a suitable restriction enzyme and subsequently transcribed in vitro. cDNA can be obtained by reverse transcription of mRNA or by chemical synthesis. In addition, a DNA template for in vitro RNA synthesis can also be obtained by gene synthesis. (Fotin-Mleczek Mariola et al, CN108064176A)

Methods for in vitro transcription are known in the art (see, e.g., Geall et al (2013) Semin. Immunol.25 (2): 152-.

RNA, mRNA: RNA is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer composed of nucleotide monomers. These nucleotides are typically Adenosine Monophosphate (AMP), Uridine Monophosphate (UMP), Guanosine Monophosphate (GMP) and Cytidine Monophosphate (CMP) monomers or analogs thereof, which are linked to each other along a so-called backbone. The backbone is formed by the phosphodiester bond 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. Typically, RNA is obtained by transcription of a DNA sequence (e.g., within a cell). In eukaryotic cells, transcription typically occurs in the nucleus or mitochondria. In vivo, transcription of DNA typically produces so-called mature pre-RNA (also known as pre-mRNA, pre-mRNA or heterologous nuclear RNA), which must be processed into so-called messenger RNA (often abbreviated mRNA). For example, the processing of pre-mature RNA in eukaryotic organisms 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 called maturation of RNA. Mature messenger RNA typically provides a nucleotide sequence that can be translated into an amino acid sequence of a particular peptide or protein. Typically, the mature mRNA comprises a 5 ' -cap, an optional 5 ' UTR, an open reading frame, an optional 3 ' UTR, and a poly (A) tail (Fotin-Mleczek Mariola et al, CN 108064176A).

In addition to messenger RNA, there are several non-coding types of RNA that may 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 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), small nuclear RNA (snrna), small nucleolar RNA (snorna), micro RNA (mirna), and Piwi-interacting RNA (pirna) (pit et al, CN 108064176A).

The term "chemically modified" refers to modifications to A, G, U or the C ribonucleotide. Generally, in this context, these terms are not intended to refer to modifications of ribonucleotides in the cap portion of mRNA at the naturally occurring 5' end. The modification may be a variety of different modifications. In some embodiments, where the nucleic acid is an mRNA, the coding region, flanking regions, and/or terminal regions may comprise one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide introduced into a cell may exhibit reduced degradation in the cell compared to an unmodified polynucleotide.

The term "amino acid" as used herein refers to a compound having a side chain, an amino group, and an acid group (e.g., -carboxyl group of-CO 2H or-SO)₃H, a sulfo group) wherein the amino acid is attached to the parent molecular moiety through a side chain, an amino group, or an acidic group (e.g., a side chain). In some embodiments, the amino acid is attached to the parent molecular group through a carbonyl group to which a side chain or amino group is attached. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl groups. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxy norvaline, 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 2 or more carbon amino acid groups, 4 substituents independently selected from: (1) c1-6 alkoxy; (2) c1-6 alkylsulfinyl; (3) an amino group, as defined herein (e.g., an unsubstituted amino group.

Delivery (Delivery): as used herein, "delivery" refers to the act or manner of delivering a compound, substance, entity, moiety, cargo (cargo) or payload.

Delivery vehicle (Delivery Agent): as used herein, "delivery vehicle" refers to any substance that at least partially facilitates the 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 RNA transcripts (e.g., by cleavage, editing, 5 'cap formation, and/or 3' end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modifications of the polypeptide or protein.

Formulation (Formulation): as used herein, a "formulation" includes at least one polynucleotide and a delivery agent.

In vitro (ln vitro): the term "in vitro" as used herein refers to an event that occurs in an artificial environment, e.g., in a test tube or reactor, in 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 occurring within an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). Modified (Modified): "modified" as used herein refers to an altered state or structure of a molecule of the invention. Molecules can 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 in chemical structure from A, C, G, U ribonucleotides.

Naturally occurring (Naturally occiring): as used herein, "naturally occurring" means occurring in nature and without the need for human assistance.

Pharmaceutically acceptable (Pharmaceutically acceptable): the phrase "pharmaceutically acceptable" refers herein 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 delaying, partially or completely, the onset of an infection, disease, disorder, and/or condition; partially or completely delaying the onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; delay, partially or completely, the onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; delay in the progression of infection, a particular disease, disorder, and/or condition, either partially or completely; and/or reducing the risk of developing a pathology associated with an infection, disease, disorder, and/or condition.

Protein of interest (Protein of interest): as used herein, the term "protein of interest" or "desired protein" includes the proteins provided herein and fragments, mutants, variants and alterations thereof.

Treatment (treting): the term "treating" as used herein refers to partially or completely alleviating, ameliorating, alleviating a particular infection, disease, disorder and/or condition; delay, partially or completely, the onset of a particular infection, disease, disorder, and/or condition; partially or completely inhibiting 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 reducing 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, treatment may be administered to a subject who does not exhibit signs of the disease, disorder, and/or condition and/or a subject who exhibits only early signs of the disease, disorder, and/or condition.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

In order to make the technical solutions of the present invention better understood by those skilled in the art, some non-limiting examples are further disclosed below to further explain the present invention in detail.

The reagents used in the present invention are either 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 is as follows:

table 1: the polypeptide of the general formula

Table 1 sets forth various peptide sequences useful in practicing the present 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 present invention to only those sequences explicitly written. Rather, such persons will readily appreciate that, based on the teachings set forth above with respect to polypeptides of the general formula, there may be a large number of peptides that are potentially useful in practicing the invention set forth herein. Moreover, determining whether a given peptide sequence falls within the scope of the present invention using standard techniques in the art without undue experimentation is well within the knowledge of the skilled artisan. 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, as long as such variants meet the structural and functional characteristics set forth above. Variants of the peptide sequences presented in table 1 or any other candidate peptide not specifically recited in table 1 but satisfying the structural and functional requirements set forth above may include deletions, insertions, use of naturally occurring or non-protein amino acid substitutions.

The first embodiment is as follows: method for producing 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, J.Am.chem.Soc.), 85, 2149-. Currently, peptides can be produced simply and in a relatively short period of time using automated universal peptide synthesizers based on those principles. In addition, peptides can be produced using well known recombinant protein production techniques, which are widely known to the skilled artisan.

The simple synthesis method and the specific process of the polypeptide of the invention are described as follows (taking sequence seq.05 as an example):

(1) resin treatment

Resin swelling: Fmoc-Arg (pbf) -2-Chlorotrityl Resin (molar substitution coefficient of 0.317mmol/g) is selected as a starting Resin, added into a 50ml reaction column, soaked by adding DCM (dichloromethane), and drained to complete the swelling of the Resin.

Deprotection: adding 20% (g/100ml) piperidine in DMF (N, N-dimethylformamide), and introducing N₂Stirring for 30 minutes (under nitrogen) and draining the solvent; the resin was washed with DMF (N, N-dimethylformamide) 6 times and then dried by suction to complete the deprotection of the resin.

(2) Amino acid coupling reaction

The reaction monitoring and detecting method comprises the following steps: the reaction progress was monitored by the indetrione method.

The raw materials and reagents used were as follows:

name (R)	Batch size (mmol)
		Protected amino acids	2
O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU)	2
		N, N-Diisopropylethylamine (DIEA)	2

The specific operation process is as follows:

weighing corresponding amount of TBTU and protected amino acid in a beaker, and adding DMF for dissolving; adding the reaction solution into the resin subjected to deprotection in the step (1), adding DIEA, and introducing N₂After blowing for about 90 minutes, the reaction was examined by ninhydrin reaction. After the reaction was complete, the solvent was removed and the resin was washed 3 times with DMF. Then, add 20% (g/100ml) piperidine in DMF to the resin and let in N₂Blowing is continued for 30 minutes, then the solvent is removed, and the resin is washed 6 times with DMF, namely the coupling of the amino acid is completed.

The above reaction procedure is repeated until the condensation reaction of all protected amino acids is completed. And (3) after the coupling is finished to the last protected amino acid, shrinking the polypeptide, washing the polypeptide for 3 times by DMF (dimethyl formamide), DCM (DCM) and methanol in sequence, draining, and weighing the polypeptide.

The condensation method comprises the following steps: TBTU + DIEA, condensing agent: TBTU: 0.64g, DIEA: 0.7 ml.

Table 2: abbreviations for amino acids and corresponding protected amino acids

(3) TFA cleavage (cleavage of the polypeptide from the resin and removal of amino acid side chain protecting groups)

The resin finally obtained in the step of (2) amino acid coupling reaction was added to a previously prepared lysate (containing 86% TFA, 5% EDT (ethylene dithiol), 5% thioanisole, 3% phenol and 2% pure water), and the mixture was stirred for 150 min. Then separating the resin from the lysate, and adding ether into the separated lysate to fully separate out the polypeptide; filtering, fully washing with ether for 6 times to obtain crude peptide, and purifying to obtain the polypeptide shown in sequence seq.05. And so on to synthesize other polypeptides provided by the invention.

(4) Simple step of purification

And (3) dissolving the crude peptide obtained in the step (3) by using a mixed solution of acetonitrile and water with the volume ratio of 3mi being 1: 1, clarifying and filtering, and detecting the purity and the 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 polypeptide is above 98.00%.

Example two: preparation of polypeptide composite nanoparticles

The polypeptide compound nanoparticle can be prepared by referring to the preparation methods of patent CN111249476A, CN111281981B, CN111281982A, CN111285845B, CN111588637A or the nanoparticle with application number 202110713076.9.

The preparation method comprises the following steps: dissolving polypeptide with ultrapure water without ribozyme to obtain 1mg/ml solution, mixing polypeptide and mRNA according to the mass ratio in table 3, stirring for 15min, standing to obtain polypeptide compound nanoparticles, wherein the specific formula is shown in table 3:

TABLE 3 polypeptide nanoparticle formula and its particle size, potential, encapsulation efficiency

The second preparation method comprises the following steps: according to the prescription design in table 4, taking out the PEG derivative auxiliary material from a refrigerator at the temperature of-20 ℃, balancing to 25 ℃, weighing at the temperature of 25 ℃, adding ultrapure water without nuclease, dissolving in a 15mL centrifuge tube without nuclease to ensure that the concentration is 100mg/mL, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; the lipid-assisted material was then removed from the-20 ℃ freezer and equilibrated to 25 ℃, weighed at 25 ℃, dissolved in a nuclease-free 1.5mL centrifuge tube with ethanol (chromatographic grade) at 25 ℃ to a concentration of 10mg/mL, and shaken thoroughly for 5min with a vortexer to obtain stock solution B.

Adding ribozyme-free ultrapure water into the corresponding polypeptide in the table 4 to dissolve the polypeptide into a solution of 5mg/ml, mixing the solution with mRNA for 10min in sequence according to the mass ratio shown in the table 4 to obtain a stock solution C, mixing the stock solution A with the stock solution B according to the mass ratio shown in the table 4 for 1min, adding the stock solution C, and fully oscillating the mixture for 1min by using a vortex instrument to obtain the polypeptide compound nanoparticles, wherein the specific formula is shown in the table 4:

TABLE 4 polypeptide nanoparticle formula and its particle size, potential, encapsulation efficiency

The preparation method comprises the following steps: weighing 5.0mg DMG-PEG, adding 1mL of chromatographic pure ethanol for dissolving, weighing 10.3mg DSPC (distearoyl phosphatidylcholine), adding 1mL of chromatographic pure ethanol for dissolving; 19.4mg of cholesterol was weighed out and dissolved in 2mL of chromatographically pure ethanol.

Dissolving polypeptide with ribozyme-free ultrapure water to obtain a solution of 1mg/mL, respectively and sequentially mixing the polypeptide and mRNA according to corresponding mass ratios in table 5 uniformly, adding the mixture into a citrate buffer solution with a pH value of 20mL of 5.4 after 5min, continuously stirring at 1500rpm, dropwise adding ethanol solutions of DMG-PEG and DSPC into the citrate buffer solution, dropwise adding ethanol solutions of cholesterol with a certain volume (100uL, 200uL, 250uL or 500uL) of 19.4mg/mL, continuously stirring for 30min, and carrying out reduced pressure rotary evaporation at 40 ℃ to remove ethanol, thus obtaining polypeptide compound nanoparticles, wherein the specific formula is shown in table 5:

TABLE 5 polypeptide nanoparticle formula and its particle size, potential, encapsulation efficiency

The preparation method comprises the following steps: 10mg of the auxiliary material (DMG-PEG, PEG-CLS, or PEG-DSPE) corresponding to Table 6 was weighed, and dissolved in nuclease-free ultrapure water to a concentration of 1mg/mL, to obtain stock solution A. Dissolving the polypeptide with ribozyme-free ultrapure water to obtain a solution with the concentration of 1mg/ml, thus obtaining a stock solution B. Mixing the stock solution A and the stock solution B according to the corresponding mass ratio in the table 6 for 1min, respectively mixing the mixed solution and the mRNA for 10min, 15min, 30min or 60min, preferably 10min, and fully oscillating for 20min by using a vortex instrument to obtain the polypeptide compound nanoparticles, wherein the specific formula is shown in the table 6:

TABLE 6 polypeptide nanoparticle formula and its particle size, potential, encapsulation efficiency

Mixing the prepared polypeptide compound nanoparticle aqueous solution with a freeze-drying agent, and freeze-drying with a freeze-dryer (Christ Alpha LD plus, Germany) to obtain the freeze-drying agent, wherein the freeze-drying agent can be trehalose or sucrose, and is stored in a refrigerator at 4 deg.C for later use.

Example three: characterization of the polypeptide Complex nanoparticles of the invention

Morphology of nanoparticles: EGFP-mRNA is used as model mRNA, polypeptide compound nanoparticles are prepared according to the preparation method I, the preparation method II, the preparation method III, the preparation method IV and a corresponding prescription, and the shape of the nanoparticles of the representative polypeptide compound nanoparticle aqueous solution is tested by using a transmission electron microscope (model FEI Talos F200X). Immersing a copper grid without any dyeing into a freshly prepared polypeptide compound nanoparticle aqueous solution, naturally drying at 25 ℃ to obtain a sample, and testing to obtain the polypeptide compound nanoparticle, wherein the result shows that the polypeptide compound nanoparticle has better dispersibility, is in a regular or irregular spherical structure and has a 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 at 25 ℃ using a Malvern Zetasizer Nano ZSE. The results are shown in tables 3 to 6, and the results show that the polypeptide complex nanoparticles have the particle size ranging from 56nm to 273nm, have better dispersibility and have the surface charge ranging from-15 mV to 5 mV.

Encapsulation efficiency: taking FLuc-mRNA as model mRNA, preparing polypeptide compound nanoparticles according to the preparation method described in the embodiment II, and determining the mRNA encapsulation rate of each prescription by using a Quant-iTRiboGreen RNA detection kit (ThermoFische company), wherein the specific method refers to the kit specification, and the brief processing method of the invention comprises the following steps: separating each prescription at 4 deg.C and 20000rpm by using low temperature high speed centrifugeAfter 2h, the supernatant was collected and its volume was quantified by pipette, as V1; measuring the concentration of mRNA in the supernatant by using a Quant-iTRiboGreen RNA detection kit, and marking as C1; dissolving the centrifuged precipitate in 25ul of chromatographic pure DMSO (dimethyl sulfoxide), continuously adding 0.9% physiological saline injection, uniformly mixing, standing at 25 ℃ for 2 hours, recording the total volume V2, and determining the concentration of mRNA (messenger ribonucleic acid) by using a Quant-iTRiboGreen RNA detection kit, wherein the concentration is marked as C2; the packet loading rate calculation formula of each prescription is as follows: the encapsulation efficiency is 100% - (V1C1)/(V1C1+ V2C2) × 100%, and the results are shown in tables 3 to 6, and the formulations all have a good encapsulation effect on mRNA, and the encapsulation efficiency is 98.0% or more.

Example four: agarose gel electrophoresis for detecting mRNA compression capacity of polypeptide

Agarose Gel with the mass volume ratio of 1% (0.4 g agarose: 1 XTAE buffer solution 40ml) is prepared, the agarose Gel is fully melted by microwave twice, 4 mul SyBR Safe DNA Gel Stain (Lot No.1771519, Invitrogen, USA) is added into the agarose according to the proportion of 1: 10000, and the agarose is poured into a corresponding glue groove (15-hole clamp groove) after being fully and uniformly mixed, and is cooled for 20min for standby.

The mRNA positive control group configuration method comprises the following steps: EGFP-mRNA was used as model mRNA, 1. mu.l of mRNA solution (100 ng) at 100 ng/. mu.l was added, 9. mu.l of nuclease-free ultrapure water was added to make up the volume of the system to 10. mu.l, and 2. mu.l of 6 × Loading Buffer was added and mixed.

The sample group configuration method comprises the following steps: through different mass ratios of polypeptide and mRNA, 1 mul of mRNA solution (namely 100ng) with the concentration of 100ng/ul is added into the polypeptide solution (1 mug/mul) for uniform mixing, then nuclease-free ultrapure water is added to make up the volume of the system to 10 mul, and after 10min of mixing, 2 mul of 6 Loading Buffer is added into each sample for uniform mixing. After mixing, the sample was loaded, 12. mu.l of the system was added to each well, and the gel was run for 25min using a 80V voltage electrophoresis apparatus and observed with a gel imager, and the experimental results are shown in FIG. 2.

And (4) conclusion: the minimum mass ratio (polypeptide: mRNA) for each polypeptide to ensure complete compression of the mRNA is: 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.

Example five: in-vitro cell transfection experiment and cytotoxicity investigation of polypeptide compound nanoparticles

Cell-transfected mRNA (with FLuc-mRNA as model mRNA): logarithmic growth phase DC2.4 cell suspension at 4X 10⁴The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO₂And (5) standing and culturing in an incubator. After 24h, the Fluc-mRNA with the concentration of 1 mug/mul is diluted to 0.1 mug/mul by nuclease-free ultrapure water, the Fluc-mRNA is taken to prepare polypeptide compound nanoparticles according to the preparation methods of different prescriptions described in example two, the polypeptide compound nanoparticles are respectively diluted to 88 mul by nuclease-free ultrapure water, 10 ng/mul of polypeptide nanoparticle composition mixed liquor containing the Fluc-mRNA is obtained, after standing for 10min, the polypeptide compound nanoparticles are respectively added into 96-well plates containing 180 mu lopti-MEM culture medium in the volume of 20 mul per well, and 4 wells are repeated for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. The culture was continued for 24h, the 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, the culture was continued in an incubator at 37 ℃ for 5min, and the Fluc-mRNA fluorescence expression intensity was measured by imaging with an Omega-Fluostar microplate reader. The results are shown in FIG. 3.

Cytotoxicity experiments: DC2.4 cell suspension in logarithmic growth phase at 4X 10⁴The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO₂And (5) standing and culturing in an incubator. After 24h, the Fluc-mRNA with the concentration of 1 mug/mul is diluted to 0.1 mug/mul by nuclease-free ultrapure water, the Fluc-mRNA is taken to prepare polypeptide compound nanoparticles according to the preparation methods of different prescriptions described in the second embodiment respectively, the polypeptide compound nanoparticles are diluted to 88 mul by nuclease-free ultrapure water respectively, 10 ng/mul of polypeptide nanoparticle composition mixed liquor containing the Fluc-mRNA is obtained, after standing for 10min, the polypeptide compound nanoparticles are added to 96-well plates containing 180 mul of opti-MEM culture medium in the volume of 20 mul per well respectively, and 4 wells are repeated for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. The culture was continued for 48h, the complete medium was aspirated and rinsed three times with PBS, wells without the prescription were used as negative controls and wells with CCK-8 medium without cells were used as blank controls, and 90. mu.l serum-free medium and 10. mu.l CCK-8 solution were added to each well and the incubation was continued in the incubator for 2 h. Absorbance at 450nm was measured with an Ome ga-Fluostar microplate readerAnd (4) luminosity. Cell viability calculation formula:

cell viability = [ a (dosed) -a (blank) ]/[ a (not dosed) -a (blank) ] × 100%;

a (dosing): absorbance of DC2.4 cells, prescription solution and CCK-8 solution added to each well;

a (blank): the absorbance of the CCK-8 solution is added to each well;

a (no drug addition): absorbance of the solution containing DC2.4 cells and CCK-8 was added to each well;

cell viability: cell proliferation activity or cytotoxic activity.

The results are shown in FIG. 4.

And (4) conclusion: the results show that the survival rate of the cells is over 90 percent, which indicates that the polypeptide compound nanoparticle formula has no obvious cytotoxicity and good biocompatibility and can be used for subsequent in vivo animal experiments. Cell transfection DNA (model Luc-pDNA DN A): DC2.4 cell suspension in logarithmic growth phase at 4X 10⁴The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO₂And (5) standing and culturing in an incubator. After 24h, 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. Luc-pDNA was used to prepare polypeptide complex nanoparticles according to the preparation methods described in example two with different recipes, and then diluted to 88. mu.l of polypeptide nanoparticle composition mixture containing 15 ng/. mu.l Luc-pDNA with nuclease-free ultrapure water, and after standing for 30min, the mixture was added to a 96-well plate containing 180. mu.l lopti-MEM medium in a volume of 20. mu.l per well, and 4 wells were repeated for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. And (3) continuing culturing for 24 hours, sucking out the complete culture medium, adding 100 mu l of D-Luciferin solution with the working concentration of 250 mu g/ml into each 96-well plate, continuing culturing in an incubator at 37 ℃ for 5min, imaging by using an Omega-Fluostar enzyme-linked immunosorbent assay, testing the fluorescence expression intensity of the Luc-pDNA, repeating the test once every 24 hours, sucking out the culture medium containing the D-Luciferin after each test is finished, adding a fresh complete culture medium, continuing culturing for 24 hours, adding the D-Luciferin for testing, and repeating for three days. The results are shown in FIG. 5, with the abscissa representing the different prescriptions and the ordinate representing the same dose 24h, 48h, 72h after transfectionRelative fluorescence intensity of Luc-pDNA expression of (a). The results are shown in FIG. 5.

And (4) conclusion: as shown in FIG. 5, the Luc-pDNA-entrapped polypeptide complex nanoparticles show better expression level at the cellular level, the expression level is the highest on the next day and decreases from the third day, wherein Rp.01, Rp.27 and Rp.34 are better than those of other prescriptions.

Example six: transfection of polypeptide compound nanoparticles in mice through fluorescence imaging detection of mice

Each group of three female BALB/c mice takes FLuc-mRNA as model mRNA, and the polypeptide compound nanoparticle formula containing the FLuc-mRNA is prepared by the method. Experimental groups 50 μ l of nanoparticles of polypeptide complex containing 5 μ g fluc-mRNA was injected into each mouse using an insulin needle. Wherein the administration mode of the prescription groups Rp.01, Rp.07, Rp.11, Pp.12, Rp.13, Rp.17, Rp.20, Rp.22 and Rp.24 is subcutaneous injection, and the injection part is subcutaneous on the back of the mouse; the administration mode of the prescription Rp.33, Rp.36, Rp.37 and Rp.42 groups is intraperitoneal injection; the other treatment groups were administered intramuscularly at the site of the mouse thigh muscle. Blank control was denoted NC and insulin needles were 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, kept in the dark for later use, 125 mu l of substrate is injected into the abdominal cavity of each mouse, the mouse is placed in a small animal anesthesia box, and a vent valve is opened to release isoflurane to anesthetize the mouse. 5min after substrate injection, 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). Wherein the prescription groups Rp.33, Rp.36, Rp.37 and Rp.42 take the bioluminescence image of the abdomen of the mouse, and the other prescription groups take the bioluminescence image of the back of the mouse. The results are shown in fig. 6, one representative mouse is taken from each group, the experimental group polypeptide compound nanoparticle formula shows the expression of luciferase in the whole body in vivo imaging, and the higher the fluorescence intensity is, the more the luciferase is expressed.

And (4) conclusion: as shown in figure 6, each experimental group of polypeptide compound nanoparticles carrying FLuc-mRNA has better luciferase expression in a mouse body, and simultaneously, the intraperitoneal injection, the subcutaneous injection and the intramuscular injection all effectively express. In the prescription of the experimental group, the luciferase has Rp.27, Rp.33 and Rp.41 superior to other prescriptions.

Example seven: evaluation of humoral immunity effect of polypeptide composite nanoparticles in mouse

New crown S-mRNA is taken as model mRNA, the new crown S-mRNA is provided by Shanghai McBiotech Corporation, and the nucleotide sequence of the new crown S-mRNA (cap1 structure, N1-me-pseudo U modified) is shown as S-mRNA in a sequence table.

The specific information of the S-mRNA stock solution is as follows:

the product name is as follows: COVID-19Spike Protein, Full Length-mRNA;

product description: 4088 nucleotides in length;

modifications (Modifications): fully subsampled with N1-Me-pseudo UTP; (all substituted with N1-Me-pseudo UTP);

concentration: 1.0 mg/ml;

storage environment: 1mM sodium citrate pH 6.4;

the storage requirement is as follows: -40 ℃ or below.

The experimental process comprises the following steps:

step 1: first immunization of mice: on day 0, 5-6 weeks female BALB/c mice were divided into 9 groups (5 per group) and intramuscularly injected with 75 μ l PBS (blank control), 5 μ g naked S-mRNA and 5 μ g S protein combination (positive control) and 5 μ g S-mRNA entrapped polypeptide complex nanoparticle formulations Rp.21, Rp.25, Rp.27, Rp.41, Rp.01, RP.0875 μ l, respectively.

Step 2: first serum collection: on day 28, mice were bled at the outer canthus. After the serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at 4 ℃ at 5000 Xg rotation speed, taking the supernatant, centrifuging for 5 minutes at 4 ℃ at 10000 Xg rotation speed, taking the supernatant, adding the supernatant into eight rows of PCR tubes, subpackaging and preserving for later use at-20 ℃.

And step 3: and (3) carrying out secondary immunization on the mice: on day 28, the mice were bled via the outer canthus, and injected intramuscularly with 75 μ l PBS (blank control), 5 μ g of the combination of naked S-mRNA and 5 μ g S protein (positive control) and the polypeptide complex nanoparticle formulations rp.21, rp.25, rp.27, rp.41, rp.01, rp.0875 μ l, respectively, loaded with 5 μ g S-mRNA. The process of the first immunization is repeated.

And 4, step 4: and (3) collecting serum for the second time: the mice were bled at the outer canthus 21 days after the second immunization. After the serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at 4 ℃ at the rotating speed of 5000 Xg (5000 times of the acceleration of gravity), taking the supernatant, centrifuging for 5 minutes at 4 ℃ at the rotating speed of 10000 Xg, taking the supernatant, adding the supernatant into eight-row PCR tubes, subpackaging and preserving for later use at-20 ℃.

And 5: ELISA detection of serum IgG content: the S protein was diluted in PBS, and the ELISA plate was coated with 100. mu.l of the dilution (containing 1. mu. g S protein) per well and coated for 6h at 4 ℃. The plate was discarded and 200. mu.l of PBST was added to each well for 3 washes, followed by 200. mu.1 PBS containing 5% BSA in each well and shaking-table blocking at 25 ℃ for 2 h. The blocking solution was discarded, 200. mu.l of PBST per well was washed 1 time, 100. mu.l of serum diluted 200-fold with PBS was added, and the mixture was incubated for 2 hours at 25 ℃ in a shaker. Serum was discarded, and after washing the plate 3 times with 200. mu.l PBST per well, 100. mu.l antibody (antibody diluted 1: 1000 in PBS) was added per well and incubated for 1h at 25 ℃ in a shaker. Discarding the antibody, washing the plate for 3 times by 200 mul PBST in each hole, adding 50 mul TMB color development liquid in each hole for reaction in a dark place, adding 50 mul 2M sulfuric acid in each hole to stop the reaction after the positive control hole turns deep blue or reacts for 10 minutes, detecting the optical density at the wavelength of 450nm and 630nm by an enzyme-labeling instrument, and calculating the OD value difference to reflect the level of the anti-S protein IgG in the serum. The results are shown in FIG. 7.

And (3) knotting: the result shows that the OD value corresponding to the prescription Rp14 is higher than that of a control group after two times of immunization, which indicates that the prescription nanoparticle has stronger seroconversion efficiency and humoral immune activation function.

Step 6: ELISA detection of serum IgG titers: the S protein was diluted in PBS, and the ELISA plate was coated with 100. mu.l of the dilution (containing 1. mu.g of S protein) per well and coated for 6 hours at 4 ℃. The plate was discarded and 200. mu.l of PBST was added to each well for 1 wash, followed by 200. mu.l of PBS blocking solution containing 5% BSA in each well and shaking-table blocking at 25 ℃ for 2 h. The blocking solution was discarded, and after washing the plate 3 times with 200. mu.l PBST per well, 50, 250, 1250, 6250, 31250, 156250, 781250, 3906250-fold diluted 1: 3 in PBS was added and incubated for 2h at 25 ℃ in a shaker. Serum was discarded, and after washing the plate 3 times with 200. mu.l PBST per well, 100. mu.l antibody (antibody diluted 1: 1000 in PBS) was added per well and incubated for 1h at 25 ℃ in a shaker. Discarding the antibody, washing the plate with 200. mu.l PBST for 3 times in each well, adding 50. mu.l TMB color development solution in each well for reaction in the dark, adding 50. mu.l 2M sulfuric acid in each well after the positive control well turns dark blue or reacts for 10 minutes to stop the reaction, and detecting the optical density at 450nm and 630nm by an enzyme-labeling instrument. The results are shown in FIG. 8.

And (3) knotting: the invention takes 2 times of the average OD value of the PBS group as a baseline, and the OD value of the Rp.08 group is still 2 times higher than the baseline when the group is diluted to 3906250 times, thus prompting that the prescription Rp.08 has stronger seroconversion efficiency and humoral immune activation function.

Example eight: gene transfection kit

The gene transfection kit is a multipurpose transfection reagent, can consist of any one of the formulas of the invention, and can provide high-efficiency transfection in various adherent and suspension cell lines. Suitable for all common cell lines and many difficult to transfect cell lines, and can be used in medium with or without serum. The kit of the invention is used for transfecting mammalian cells by using a 96-well cell culture plate. The method comprises the following specific steps:

1. one day before transfection, 1X 10 cells were added⁴To 10X 10⁴The individual cells were plated 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 transfection sample, the following complexes were prepared:

a. diluting 200ng of DNA or RNA to 15 mu l with sterile nuclease-free water, and gently mixing;

b. the transfection reagents were mixed gently at the prescribed ratio before use, and then diluted to an appropriate amount to 15 μ l with sterile nuclease-free water;

c. the diluted DNA or RNA is mixed with the diluted transfection reagent gently (total volume: 30 μ l) in the prescribed ratio, and incubated at 25 ℃ for 10 minutes to 30 minutes to obtain nucleic acid-polypeptide complex nanoparticles.

3. After the 96-well plate was rinsed with PBS, 170. mu.l of opti-MEM medium was added to each well, and 30. mu.l of nucleic acid-polypeptide complex nanoparticles were added thereto, the final volume of the medium being 200. mu.l.

4. And putting the cells into a carbon dioxide incubator to incubate the cells, replacing the original culture medium with the complete culture medium after 4 hours, continuously incubating the cells in the carbon dioxide incubator for 12 to 72 hours, and finally detecting the expression quantity of the nucleic acid.

All references, including patent documents, disclosed herein are incorporated by reference.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement 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 Shenzheng Nanyao pharmaceutical Co Ltd

<120> polypeptide and polypeptide compound nanoparticle thereof, nucleic acid vaccine and application

<130> PCT002

<141> 2021-09-02

<160> 54

<170> SIPOSequenceListing 1.0

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Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg

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Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg

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Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val

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Arg

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Arg Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile

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Val Arg

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Arg Arg Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser

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Arg Arg Arg Arg Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu

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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

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Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Trp

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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

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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

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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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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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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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Arg Arg Arg Arg Arg Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg

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Arg Arg Arg Arg Arg Cys Tyr Pro Trp Cys Arg Val Gln Pro Thr Glu

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Arg Arg Arg Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg

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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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Arg Arg Arg Arg Arg Pro Phe Cys Arg Val Gln Pro Thr Glu Ser Ile

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Val Arg

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<212> PRT

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Arg Arg Arg Arg Arg Arg Trp Cys Val Gln Pro Thr Glu Ser Ile Val

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Arg Cys

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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

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Arg Arg Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu

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Ser Ile Val Arg

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His Lys Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu

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Ser Ile Val Arg

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Trp Cys Arg Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu

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Trp Cys Lys Leu Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu

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Trp Cys Arg Arg Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg

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Arg Arg Arg Arg Cys Trp

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<212> PRT

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Arg Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile

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Val Arg Cys Trp Arg Arg Cys Trp Arg Arg

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<212> PRT

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Phe Cys Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu Ser

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Ile Val Arg Arg Arg Arg Cys Trp Arg Arg Cys Phe

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Phe Cys Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu Ser

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Arg Arg Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu

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His Lys Arg Trp Cys Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu

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Trp Cys Arg Arg Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu

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Trp Cys Lys Leu Arg Trp Cys Arg Arg Arg Arg Val Gln Pro Thr Glu

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Cys Arg Arg Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val

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Arg Cys Trp Arg Arg Arg Arg Cys

20

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Trp Cys Arg Arg Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg

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Arg Arg Arg Arg Trp Cys

20

<210> 39

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Arg Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile

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Val Arg

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<212> PRT

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His Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile

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Val Arg

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Trp Cys Arg Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val

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Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile Val Arg Cys Trp Arg

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Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg Arg Arg Cys

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Arg Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser Ile

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His Lys Arg Trp Cys Arg Arg Trp Cys Arg Val Gln Pro Thr Glu Ser

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Trp Cys Arg Arg Trp Cys Arg Arg Val Gln Pro Thr Glu Ser Ile Val

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Trp Cys Arg Arg Arg Val Gln Pro Thr Glu Ser Ile Val Arg Arg Arg

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<212> RNA

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<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

1. A polypeptide compound having the structure of formula I:

(Xaa)_x-Arg-Val-Gln-Pro-Thr-Glu-Ser-Ile-Val-Arg-(Yaa)_y(general formula I) is shown in the specification,

wherein: x is an integer of 1 to 25, and y is an integer of 0 to 10;

(Xaa)_xis a polypeptide segment consisting of any amino acid, (Yaa)_yIs a polypeptide segment consisting of any amino acid.

2. The polypeptide compound of claim 1, wherein Xaa is selected from at least one of Arg, Trp, Cys, Lys, Leu, Phe, Pro, or His; and/or the Yaa is selected from at least one of Arg, Trp, Phe or Cys.

3. The polypeptide compound according to any one of claims 1 to 2, wherein (Xaa)_xConsists of Arg; or

The (Xaa)_xIs (Xa 'a')_n(Arg)_1-10(Xa’a’)_nWherein' a in Xa is at least one of Arg, Trp, Cys, Lys, Leu, Phe, Pro or His, and n is an integer of 0-10.

4. The polypeptide compound according to any one of claims 1 to 3, which has an amino acid sequence of: 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.47, seq.48, seq.49, seq.48, seq.47, seq.48.

5. The polypeptide compound of claim 1 or 4, general formula (I) is at least 50% similar to any of seq.01-seq.53 and which improves delivery of the nucleic acid molecule into a cell by at least 20%; or

Formula (I) is at least 75% similar to any of seq.01-seq.53 and which improves delivery of the nucleic acid molecule into a cell by at least 50%; or

Formula (I) is at least 90% similar to any of seq.01-seq.53 and which improves delivery of the nucleic acid molecule into a cell by at least 100%; or

Formula (I) is at least 90% similar to either RRRRRWCRVQPTESIVR, RRRRRWFCRVQPTESIVR, FCRWCRRVQPTESIVRRCWRCF, FCRWCRRVQPTESIVCWRRRCF, HKRWCRRWCRVQPTESIVRC or WCRRRVQPTESIVRRRWC.

6. A polypeptide complex nanoparticle comprising:

a) at least one polypeptide compound according to any one of claims 1 to 5; and

b) a nucleic acid.

7. The polypeptide complex nanoparticle of claim 6, wherein the nucleic acid is chemically modified or chemically unmodified DNA, single-stranded or double-stranded DNA, coding or non-coding DNA; or

The nucleic acid is selected from a plasmid, an oligodeoxynucleotide, genomic DNA, a DNA primer, a DNA probe, an immunostimulatory DNA, an aptamer, or any combination thereof;

or the nucleic acid is chemically modified or chemically unmodified RNA, single-or double-stranded RNA, coding or non-coding RNA; or

The nucleic acid is selected from messenger RNA (mrna), oligoribonucleotides, viral RNA, replicon RNA, transfer RNA (trna), ribosomal RNA (rrna), immunostimulatory RNA (isrna), microrna, small interfering RNA (sirna), small nuclear RNA (snrna), small hairpin RNA (shrna) or riboswitch, RNA aptamer, RNA decoy, antisense RNA, ribozyme, or any combination thereof or the nucleic acid is chemically modified messenger RNA (mrna).

8. The polypeptide complex nanoparticle of claim 7, wherein the nucleic acid is mRNA.

9. The polypeptide complex nanoparticle of claim 6, further comprising at least one auxiliary material.

10. The polypeptide complex nanoparticle according to any one of claims 6 to 9, wherein the mass ratio of the nucleic acid to the polypeptide is less than or equal to 1: 1; and/or the mass ratio of the nucleic acid to the auxiliary material may be less than or equal to 1: 2.

11. The polypeptide complex nanoparticle of claim 9, wherein the auxiliary material is at least one selected from the group consisting of a lipid and a PEG derivative.

12. A polypeptide complex nanoparticle, comprising: nucleic acid, a polypeptide compound with an amino acid sequence of seq.05 and an auxiliary material

90R4 and lecithin; the nucleic acid, the polypeptide compound with the amino acid sequence of seq.05,

The mass ratio of 90R4 to lecithin is 1: 2: 322: 1; or

It includes: nucleic acid, a polypeptide compound with the amino acid sequence of seq.49 and auxiliary materials, wherein the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphocholine and cholesterol; the mass ratio of the nucleic acid to the polypeptide compound with the amino acid sequence of seq.49 to the 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000 to the 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine to the cholesterol is 10: 300: 8: 16: 31; or

It includes: nucleic acid, a polypeptide compound with the amino acid sequence of seq.53 and auxiliary materials, wherein the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and cholesterol; the mass ratio of the nucleic acid to the polypeptide compound with the amino acid sequence of seq.53, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and cholesterol is 10: 40: 8: 16: 5.

13. A nucleic acid vaccine comprising a polypeptide complex nanoparticle according to any one of claims 6 to 12.

14. Use of a nanoparticle of a polypeptide complex according to any one of claims 6 to 12 in the manufacture of a medicament or kit.