Epidermal growth factor-self-replicating RNA molecular vaccine and application thereof

Abstract

The invention provides an epidermal growth factor-self-replicating RNA molecular vaccine and application thereof, a self-replicating RNA molecule comprises a self-replicating core sequence and an antigen sequence, the self-replicating core sequence is used for coding N protein, P protein and L protein or functional fragments thereof, and the antigen sequence is used for coding a modified epidermal growth factor. The self-replicating RNA molecule disclosed by the invention can activate a relatively high level of immune response in cells, generate corresponding antibodies and EGF in antibody combinations, prevent free EGF from being combined with EGFR to transmit cell proliferation signals, inhibit or slow down tumor growth and realize prevention and treatment of EGF-related tumor diseases, and has important significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an epidermal growth factor-self-replicating RNA molecular vaccine and its application. Background Art

[0002] Epidermal growth factor (EGF) is a polypeptide composed of 53 amino acid residues. After EGF specifically recognizes and binds to EGFR (EGF receptor) on target cells, a series of biochemical reactions occur, which can ultimately promote DNA synthesis and mitosis of target cells. EGF has no sugar site, is very stable, heat-resistant and acid-resistant, and is widely present in body fluids and various glands. It is mainly synthesized by the submandibular gland and duodenum. It has been found in most body fluids of the human body. The content in breast milk, urine, and semen is specifically increased, but the concentration in serum is low. EGF has a wide range of effects and is of great significance in assessing tumor prognosis, selecting treatment options, and treating gastric ulcers, liver failure, etc. EGF vaccine therapy uses antibodies to bind to EGF in the body, preventing free EGF from binding to EGFR to transmit cell proliferation signals, thereby inhibiting or slowing tumor growth.

[0003] Self-replicating RNA molecules can use their own RNA sequences as templates to replicate and translate themselves within cells, express target molecules, and exert the relevant functions of target molecules.

[0004] However, there are currently no reports on the use of self-replicating RNA and epidermal growth factor in combination for vaccine preparation, and further research is needed. Summary of the invention

[0005] It should be noted that the present invention is completed based on the following findings of the inventors:

[0006] The inventors of the present invention have previously conducted in-depth research on the translation and self-amplification mechanisms of RNA from various RNA viruses in cells, and found that by using RNA molecules encoding N protein, P protein and L protein from rhabdoviruses as core regions, RNA self-replication and translation in cells can be achieved, and that the core region, as a powerful "engine", can provide the "kinetic energy" for efficient transcription amplification and the initiation of large molecular proteins, and can further carry a "cargo region" to replicate or translate target molecules.

[0007] Based on this, the inventors tried to construct a self-replicating RNA molecule by using the sequences encoding N protein, P protein and L protein or their functional fragments as the core sequence and connecting the RNA sequence encoding wild-type epidermal growth factor. However, after the self-replicating RNA molecule was administered to mice, the titer of the mouse serum was very low, indicating that it did not effectively produce an immune response.

[0008] After in-depth research, the inventors found that the main reason for the above problems is that the wild-type epidermal growth factor has low immunogenicity and is difficult to activate immune response. Furthermore, the inventors obtained a series of self-replicating RNA molecules (named reRNA in this article) by modifying the wild-type epidermal growth factor. TM -EGF), which can increase the binding with B cells, better activate B cells, thereby activating a stronger level of immune response and producing corresponding antibodies. The antibodies bind to EGF in the body, preventing free EGF from binding to EGFR to transmit cell proliferation signals, inhibiting or slowing tumor growth, and achieving the prevention and treatment of EGF-related tumor diseases, which is of great significance.

[0009] To this end, in one aspect of the present invention, the present invention provides a self-replicating RNA molecule. According to an embodiment of the present invention, the self-replicating RNA molecule comprises: a self-replicating core sequence, the self-replicating core sequence is used to encode N protein, P protein and L protein or their functional fragments; and an antigen sequence, the antigen sequence is used to encode a modified epidermal growth factor.

[0010] In another aspect of the present invention, the present invention provides a polypeptide. According to an embodiment of the present invention, the polypeptide is a modified epidermal growth factor having an amino acid sequence as shown in SEQ ID NO: 9 or 10 or an amino acid sequence having at least 80% homology thereto. The polypeptide is a modified epidermal cell factor encoded by the antigen sequence in the aforementioned self-replicating RNA molecule.

[0011] In another aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the above-mentioned polypeptide.

[0012] In another aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule.

[0013] In another aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: the aforementioned nucleic acid molecule and / or the aforementioned expression vector.

[0014] In another aspect of the present invention, the present invention provides a drug. According to an embodiment of the present invention, the drug comprises at least one of the following: the self-replicating RNA molecule, polypeptide, nucleic acid molecule, expression vector and recombinant cell mentioned above.

[0015] In another aspect of the present invention, the present invention provides the use of at least one of the self-replicating RNA molecules, polypeptides, nucleic acid molecules, expression vectors and recombinant cells described above in the preparation of a drug. According to an embodiment of the present invention, the drug is used to prevent or treat epidermal growth factor-related diseases.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0018] Figure 1 and Figure 2 The schematic diagrams of the structure of a self-replicating molecule according to one embodiment of the present invention are shown respectively;

[0019] Figure 3 A schematic diagram showing the immunization results of vaccines modified with different antigens according to one embodiment of the present invention is shown;

[0020] Figure 4 The reRNA according to one embodiment of the present invention is shown TM -Schematic diagram of EGF-4 vaccine immunization results. DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0022] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0025] The present invention provides a self-replicating RNA molecule, a polypeptide, a nucleic acid molecule, an expression vector, a recombinant cell, a drug and applications thereof, which will be described in detail below.

[0026] Self-replicating RNA molecules

[0027] In one aspect of the present invention, the present invention provides a self-replicating RNA molecule. According to an embodiment of the present invention, the self-replicating RNA molecule comprises: a self-replicating core sequence, the self-replicating core sequence is used to encode N protein, P protein and L protein or their functional fragments; and an antigen sequence, the antigen sequence is used to encode a modified epidermal growth factor.

[0028] See also Figure 1 and Figure 2 As shown, the inventors tried to construct a self-replicating RNA molecule by connecting the RNA sequence encoding wild-type epidermal growth factor with the sequence encoding N protein, P protein and L protein or their functional fragments as the core sequence. However, after the self-replicating RNA molecule was administered to mice, the mouse serum titer was very low, indicating that it did not effectively produce an immune response. To this end, the inventors found through in-depth research that the self-replicating RNA molecule constructed by specifically modifying the wild-type epidermal growth factor and connecting it to the core sequence can activate the immune response and produce antibodies. The antibodies bind to EGF in the body, prevent free EGF from binding to EGFR to conduct cell proliferation signals, inhibit or slow tumor growth, and achieve the prevention and treatment of EGF-related tumor diseases.

[0029] According to an embodiment of the present invention, the unmodified epidermal growth factor has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology thereto. The unmodified epidermal growth factor is a wild-type epidermal growth factor, which is modified to obtain a modified epidermal growth factor. The nucleic acid molecule encoding the unmodified epidermal growth factor has a nucleotide sequence as shown in SEQ ID NO: 14.

[0030] MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR (SEQ ID NO: 1)

[0031] ATGAATAGCGACAGCGAGTGCCCTCTGAGCCACGACGGCTACTGCCTGCACGATGGCGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGTAACTGTGTGGTGGGCTACATCGGCGAGAGATGTCAGTACAGGGATCTGAAGTGGTGGGAGCTGAGGTGA (SEQ ID NO: 14)

[0032] According to an embodiment of the present invention, the antigen sequence has a nucleotide sequence as shown in SEQ ID NO: 3 or 4 or a nucleotide sequence with at least 80% homology thereto. The inventors obtained the above-mentioned preferred modified epidermal growth factor coding sequence through in-depth research, analysis and experimental screening. The self-replicating RNA molecule constructed by connecting it with the core sequence enters the cell, increases the binding with B cells, activates B cells, thereby activating a stronger level of immune response and producing corresponding antibodies. The antibody binds to EGF in the body, prevents free EGF from binding to EGFR to transmit cell proliferation signals, inhibits or slows tumor growth, and achieves the purpose of treating EGF-related tumor diseases, which is of great significance. However, other modified epidermal growth factors, such as the nucleotide sequence shown in SEQ ID NO: 2, cannot be effectively immunized and produce corresponding antibodies.

[0033] AUGAAGUGCCUGCUGUACCUGGCCUUCCUGUUCAUCGGCGUGAAUUGCAAUAGCGACAGCGAGUGCCCUCUGAGCCACGACGGCUACUGCCUGCACGAUGGCGUGUGCAUCAUCGAGGCCCUGGACAAGUACGCCUGUAACUGUGUGGUGGGCUACAUCGGCGAGAGAUGUCAGUACAGGGAUCUGAAGUGGUGGGAGCUGAGGUGA (SEQ ID NO: 2)

[0034] AUGAAGUGCCUGCUGUACCUGGCCUUCCUGUUCAUCGGCGUGAAUUGCAAUAGCGACAGCGAGUGCCCUCUGAGCCACGACGGCUACUGCCUGCACGAUGGCGUGUGCAUGUACAUCGAGGCCCUGGACAAGUACGCCUGUAACUGUGUGGUGGGC UACAUCGGCGAGAGAUGUCAGUACAGGGAUCUGAAGUGGUGGGAGCUGAGGGGAUGGUUUUCCUCCUGGAAGAGCUCCAUCGCCUCCUUUUUCUUCAUCAUCGGCCUGAUCAUCGGCCUCUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGA(SEQ ID NO:3)

[0035] AUGAAGUGCCUGCUGUACCUGGCCUUCCUGUUCAUCGGCGUGAAUUGCAAUAGCGACAGCGAGUGCCCUCUGAGCCACGACGGCUACUGCCUGCACGAUGGCGUGUGCAUGUACAUCGAGGCCCUGGACAAGUACGCCUGUAACUGUGUGGUGGGCUACAUCGGCGAGAGAUGUCAGUACAGGGAUCUGAA GUGGUGGGAGCUGAGGGGAUGGUUUUCCUCCUGGAAGAGCUCCAUCGCCUCCUUUUUCUUCAUCAUCGGCCUGAUCAUCGGCCUCUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGCAUCAAGCUGAAGCACACAAAGAAGAGACAGAUCUACACCGACAUCGAGAUGAAUAGACUGGGCAAGUGA(SEQ ID NO:4)

[0036] See also Figure 1 and Figure 2 , the self-replicating core sequence includes:

[0037] A first RNA sequence, wherein the first RNA sequence encodes the N protein or a functional fragment thereof;

[0038] A second RNA sequence, wherein the second RNA sequence encodes a P protein or a functional fragment thereof; and

[0039] A third RNA sequence, wherein the third RNA sequence encodes the L protein or a functional fragment thereof.

[0040] Therefore, these first, second and third RNA sequences constitute the core region of the self-replicating RNA molecule, also called the "core life zone", which can serve as the minimum region capable of self-replication and translation in animal cells, and can further carry other coding sequences to express or replicate target molecules, thereby exerting the function of a vaccine.

[0041] The term "functional fragment" used in this article refers to a part of the full-length sequence of a protein, but it can still play a role in the self-replication of RNA molecules. For example, it can be a truncated version of the full-length sequence, or it can be a protein after the amino acid sequence of the full-length sequence of the protein is replaced, mutated, or deleted. According to the embodiments of the present application, for the functional fragment of the N protein, it can bind to the RNA molecule to protect the RNA from the influence of nucleases. For the functional fragment of the P protein, it can bind to the N protein and position the L polymerase on the template. At the same time, it can also serve as a basic component of the RNA polymerase transcription and replication complex. Further, the functional fragment of the L protein can play the function of RNA polymerase and is related to the transcription and replication of RNA.

[0042] According to an embodiment of the present invention, the sequence of the N protein includes the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the N protein has the nucleotide sequence shown in SEQ ID NO: 11 or a nucleotide sequence having at least 80% homology thereto.

[0043] According to an embodiment of the present invention, the sequence of the P protein includes the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the P protein has the nucleotide sequence shown in SEQ ID NO: 12 or a nucleotide sequence having at least 80% homology thereto.

[0044] According to an embodiment of the present invention, the sequence of the L protein includes the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the L protein has the nucleotide sequence shown in SEQ ID NO: 13 or a nucleotide sequence having at least 80% homology thereto.

[0045] MSVTVKRIIDNTVVVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSSKSPYSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDK (SEQ ID NO: 5).

[0046] MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSDTESEPEIEDNQGLYAPDPEAEQVEGFIQGPLDDYADEEVDVVFTSDWKQPELESDEHGKTLRLTSPEGLSGEQ KSQWLSTIKAVVQSAKYWNLAECTFEASGEGVIMKERQITPDVYKVTPVMNTHPSQSEAVSDVWSLSKTSMTFQPKKASLQPLTISLDELFSSRGEFISVGGDGRMSHKEAILLGLRYKKLYNQARVKYSL(SEQ ID NO:6)。

[0047]

[0048]

[0049] AUGGAUAAUCUCACAAAAGUUCGUGAGUAUCUCAAGUCCUAUUCUCGUCUGGAUCAGGCGGUAGGAGAGAUAGAUGAGAUCGAAGCACAACGAGCUGAAAAGUCCAAUUAUGAGUUGUUCCAAGAGGAUGGAGUGGAAGAGCAUACUAAGCCCUCUUAUUUUCAGGCAGCAGAUGAUUCUGACACAGAAUCUGAACCAGAAAUUGAAGACAAUCAAGGCUUGUAUGCACCAGAUCCAGAAGCUGAGCAAGUUGAAGGCUUUAUACAGGGGCCUUUAGAUGACUAUGCAGAUGAGGAAGUGGAUGUUGUAUUUACUUCGGACUGGAAACAGCCUGAGCUUGAAUCUGACGAGCAUGGAAAGACCUUACGGUUGACAUCGCCAGAGGGUUUAAGUGGAGAGCAGAAAUCCCAGUGGCUUUCGACGAUUAAAGCAGUCGUGCAAAGUGCCAAAUACUGGAAUCUGGCAGAGUGCACAUUUGAAGCAUCGGGAGAAGGGGUCAUUAUGAAGGAGCGCCAGAUAACUCCGGAUGUAUAUAAGGUCACUCCAGUGAUGAACACACAUCCGUCCCAAUCAGAAGCAGUAUCAGAUGUUUGGUCUCUCUCAAAGACAUCCAUGACUUUCCAACCCAAGAAAGCAAGUCUUCAGCCUCUCACCAUAUCCUUGGAUGAAUUGUUCUCAUCUAGAGGAGAGUUCAUCUCUGUCGGAGGUGACGGACGAAUGUCUCAUAAAGAGGCCAUCCUGCUCGGCCUGAGAUACAAAAAGUUGUACAAUCAGGCGAGAGUCAAAUAUUCUCUGUAG(SEQ ID NO:12)。

[0050]

[0051] As used herein, the term "at least 80% homology" refers to at least 80%, and may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% similarity to each reference sequence.

[0052] Peptides

[0053] In another aspect of the present invention, the present invention proposes a polypeptide. According to an embodiment of the present invention, the polypeptide is a modified epidermal growth factor having an amino acid sequence as shown in SEQ ID NO: 9 or 10 or an amino acid sequence having at least 80% homology thereto. The above polypeptide can increase binding with B cells in cells, activate B cells, thereby activating a stronger level of immune response and producing corresponding antibodies. The antibody binds to EGF in the body, preventing free EGF from binding to EGFR to transmit cell proliferation signals, inhibiting or slowing tumor growth. The above polypeptide is particularly suitable for constructing self-replicating RNA. The RNA molecule encoded by the polypeptide is connected with the above self-replicating core sequence to construct a self-replicating RNA that can achieve self-replication and translation when entering the cell. The expressed modified EGF can activate the immune response, promote the body to efficiently, stably and sustainably produce EGF antibodies, and exert a tumor killing effect. However, other modified epidermal growth factors, such as the amino acid sequence shown in SEQ ID NO: 8, cannot be effectively immunized and produce corresponding antibodies.

[0054] MKCLLYLAFLFIGVNCNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR (SEQ ID NO: 8, encoded by SEQ ID NO: 2)

[0055] MKCLLYLAFLFIGVNCNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHL (SEQ ID NO: 9, encoded by SEQ ID NO: 3)

[0056] MKCLLYLAFLFIGVNCNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGE RCQYRDLKWWELRGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDI EMNRLGK (SEQ ID NO: 10, can be encoded by SEQ ID NO: 4)

[0057] Nucleic acid molecules, expression vectors and recombinant cells

[0058] In another aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned polypeptide. Thus, the aforementioned modified epidermal growth factor can be encoded using the nucleic acid molecule of the present invention, which is used to better activate immune response and produce EGF antibodies.

[0059] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 80% homology thereto.

[0060] In another aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. Thus, the modified epidermal growth factor described above can be expressed using the expression vector of the present invention to better activate immune response and produce EGF antibodies.

[0061] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0062] In another aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: the aforementioned nucleic acid molecule and / or the aforementioned expression vector. Thus, the aforementioned modified epidermal growth factor can be expressed in the recombinant cell of the present invention to better activate immune response and produce EGF antibodies.

[0063] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and / or secretion of the target protein. The recombinant cells of the present invention do not include human cells at various stages of formation and development, and specifically do not include human germ cells, fertilized egg cells and embryonic cells.

[0064] It should be noted that the features and advantages described above for the polypeptide are also applicable to the nucleic acid molecule, expression vector and recombinant cell, and will not be repeated here.

[0065] drug

[0066] In another aspect of the present invention, the present invention proposes a drug. According to an embodiment of the present invention, the drug comprises at least one of the following: the self-replicating RNA molecule, polypeptide, nucleic acid molecule, expression vector and recombinant cell described above. Thus, the drug of the present invention can activate a stronger level of immune response, produce corresponding antibodies, and the antibodies bind to EGF in the body to prevent free EGF from binding to EGFR to transmit cell proliferation signals, inhibit or slow tumor growth, and achieve the purpose of preventing and treating EGF-related tumor diseases.

[0067] According to an embodiment of the present invention, the drug is selected from vaccines.

[0068] According to an embodiment of the present invention, the vaccine further includes a vaccine adjuvant.

[0069] According to an embodiment of the present invention, the adjuvant includes at least one of aluminum adjuvant, Poly IC adjuvant, Freund's adjuvant, CpG ODN, DDA, MPLA, IC31, AS01 and AS03, preferably aluminum adjuvant. The inventors have found through experiments that the use of the above adjuvants can improve the immunogenicity of the vaccine.

[0070] In this article, the term "aluminum adjuvant" generally refers to an immune adjuvant containing aluminum, which includes at least but not limited to one or more of aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum and potassium alum. For example, it can be aluminum hydroxide and magnesium hydroxide suspension.

[0071] According to an embodiment of the present invention, the drug further includes a pharmaceutically acceptable excipient.

[0072] In this article, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, the "pharmaceutically acceptable" of the present invention refers to the use in animals, especially humans, approved by the national government or listed in the Chinese Pharmacopoeia or other generally recognized pharmacopoeias.

[0073] In this article, the term "pharmaceutically acceptable excipient" is a pharmaceutically or food-acceptable carrier, solvent, suspending agent or excipient used to deliver the active ingredient (e.g., self-replicating RNA molecule) in the drug of the present invention to an animal or human. Exemplary excipients can be liquid or solid, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in combination with pharmacodynamic compounds. Examples of excipients include various lactose, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrin and starch.

[0074] It should be noted that the characteristics and advantages described above for self-replicating RNA molecules, polypeptides, nucleic acid molecules, expression vectors and recombinant cells are also applicable to this drug and will not be repeated here.

[0075] application

[0076] In another aspect of the present invention, the present invention provides the use of at least one of the self-replicating RNA molecules, polypeptides, nucleic acid molecules, expression vectors and recombinant cells described above in the preparation of a drug. According to an embodiment of the present invention, the drug is used to prevent or treat epidermal growth factor-related diseases.

[0077] In addition, the present invention provides a method for preventing or treating epidermal growth factor-related diseases, the method comprising: administering at least one of the self-replicating RNA molecules, polypeptides, nucleic acid molecules, expression vectors and recombinant cells described above to an individual in need.

[0078] According to an embodiment of the present invention, the disease includes at least one of the following: breast cancer, colorectal cancer, gastric cancer, liver cancer, lung cancer, nasopharyngeal carcinoma, gastric ulcer and liver failure.

[0079] As used herein, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug described herein to an individual in need.

[0080] It should be noted that the features and advantages described above for self-replicating RNA molecules, polypeptides, nucleic acid molecules, expression vectors and recombinant cells are also applicable to this application and will not be repeated here.

[0081] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0082] Example 1

[0083] 1. Insert the EGF antigen nucleotide sequence (RNA sequence corresponding to SEQ ID NO: 14) and the modified EGF antigen nucleotide sequence (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) into reRNA TM Plasmid (containing SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, encoding N protein, P protein and L protein respectively), constructing the corresponding reRNA TM Vaccine master plasmid: reRNA TM -EGF-1, reRNA TM -EGF-2, reRNA TM -EGF-3, reRNA TM -EGF-4. Constructed reRNA TM The vaccine main plasmid and auxiliary plasmid (pCI-VSVG plasmid and T7 RNA polymerase plasmid pCAG-T7pol) were co-transfected into 293T cells. After 48 hours, the supernatant was harvested and purified to obtain reRNA. TM vaccine.

[0084] 2. The four vaccines were used to immunize mice. The specific experiment was as follows: 6-8 week old Balb / C female mice were used, and intramuscular immunization was used on day 0 and day 14. Blood was collected on day 0, day 7, day 15, day 21, day 28, and day 35. The indirect ELISA method was used to detect the titer of mouse serum.

[0085] The results show that Figure 3 ), reRNA without antigen modification TM -EGF-1 vaccine group can hardly induce mice to produce antibodies; different antigen modification strategies induce different antibody production situations: reRNA TM -EGF-2 also cannot provide effective immunity, reRNA TM -EGF-3 can induce the production of a certain titer of antibodies, reRNA TM -EGF-4 can induce the production of higher titers of antibodies. TM -The antigen modification strategy of EGF-4 can effectively activate the immune response and produce higher titer antibodies.

[0086] 3. ReRNA TM -EGF-4 vaccine was administered to Balb / C female mice aged 6-8 weeks using intramuscular immunization on days 0, 14, 28 and 56, and blood was collected on days 0, 15, 21, 28, 35, 56 and 63. The first three immunizations were to test the highest level of antibody titer that the vaccine could induce, and the fourth immunization was to test whether the antibody level could be maintained after a longer period of time.

[0087] The indirect ELISA method was used to detect the titer of mouse serum. The specific test results are as follows Figure 4 As shown in the figure: 15 days after the first immunization, the serum titer of EGF vaccine can reach more than 64k, indicating that the vaccine has good immunogenicity and induces a strong specific antibody response in mice; one week after the second immunization, the serum titer is further increased to more than 128K, and it does not increase further after the third immunization. Considering that 128k is a relatively high level of antibody level, two vaccine immunizations can reach an ideal level in animals. Four weeks after the third immunization, that is, blood was collected on the 56th day, and the antibody level in the mouse serum remained above 128K, which shows that the antibody level in the animal body can remain at a high level for a long time without decreasing after this vaccine immunization. At the same time, the fourth immunization was carried out on the 56th day to detect whether the vaccine can further stimulate the animal to produce higher levels of antibodies. According to the serum test results on the 63rd day, the antibody level in the animal body further increased one week after the fourth immunization.

[0088] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A self-replicating RNA molecule, characterized in that The self-replicating RNA molecule comprises: A self-replicating core sequence, wherein the self-replicating core sequence is used to encode N protein, P protein and L protein or a functional fragment thereof; and An antigen sequence encoding a modified epidermal growth factor.

2. The self-replicating RNA molecule according to claim 1, characterized in that The unmodified epidermal growth factor has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology thereto; Optionally, the antigen sequence has a nucleotide sequence as shown in SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 80% homology thereto; Optionally, the sequence of the N protein comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 80% homology thereto; Optionally, the sequence of the P protein comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% homology thereto; Optionally, the sequence of the L protein includes the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto.

3. A polypeptide, characterized in that The polypeptide is a modified epidermal growth factor having an amino acid sequence as shown in SEQ ID NO: 9 or 10 or an amino acid sequence having at least 80% homology thereto.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide according to claim 3.

5. An expression vector, characterized in that: The expression vector carries the nucleic acid molecule according to claim 4.

6. A recombinant cell, characterized in that The recombinant cell comprises: the nucleic acid molecule according to claim 4 and / or the expression vector according to claim 5.

7. A drug, characterized in that The drug comprises at least one of the following: the self-replicating RNA molecule according to claim 1 or 2, the polypeptide according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 and the recombinant cell according to claim 6.

8. The drug according to claim 7, characterized in that: The drug is selected from vaccines; Optionally, the vaccine further comprises a vaccine adjuvant.

9. Use of at least one of the self-replicating RNA molecule according to claim 1 or 2, the polypeptide according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 and the recombinant cell according to claim 6 in the preparation of a drug, characterized in that: The medicine is used for preventing or treating epidermal growth factor-related diseases.

10. The use according to claim 9, characterized in that: The disease includes at least one of the following: breast cancer, colorectal cancer, gastric cancer, liver cancer, lung cancer, nasopharyngeal cancer, gastric ulcer and liver failure.

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