CN116970093A - Recombinant protein of membrane-penetrating peptide botulinum toxin, and preparation method and application thereof - Google Patents
Recombinant protein of membrane-penetrating peptide botulinum toxin, and preparation method and application thereof Download PDFInfo
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- CN116970093A CN116970093A CN202311154016.3A CN202311154016A CN116970093A CN 116970093 A CN116970093 A CN 116970093A CN 202311154016 A CN202311154016 A CN 202311154016A CN 116970093 A CN116970093 A CN 116970093A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/10—Anti-acne agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Abstract
The application discloses a recombinant protein of a membrane-penetrating peptide botulinum toxin, wherein the sequence of the recombinant protein is selected from one of the following amino acid sequences: a) The recombinant protein consists of an amino acid sequence of SEQ ID NO. 1; b) A recombinant protein is a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence of SEQ NO. 1; c) The amino acid sequence of the recombinant protein differs from the amino acid sequence of SEQ No. 1 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1. The membrane-penetrating peptide botulinum toxin recombinant protein provided by the application has high safety, and is lethal only when used in a large dosage.
Description
Technical Field
The application belongs to the field of botulinum toxin preparations, and particularly relates to a membrane-penetrating peptide botulinum toxin recombinant protein, a preparation method and application thereof.
Background
Skin cells are damaged by various factors such as contaminants, intense ultraviolet rays, stress, malnutrition, etc. with age, resulting in skin wrinkles, loss of skin elasticity and skin keratinization. With the increase of the pursuit desire of people for beauty, the consumption of products with specific beauty effects (reducing wrinkles) is increased year by year, and the value of functional cosmetics and medical products is further improved.
The stratum corneum cells of the skin are a natural barrier of the skin, can prevent water evaporation, keep the skin moist and full, and resist various external stimuli due to invasion of bacteria outside tissues. However, at the same time the stratum corneum prevents compounds with a molecular weight of 500Da or less from reaching the cells.
Botulinum toxins are neurotoxins produced by the gram positive bacterium clostridium botulinum, which paralyzes muscles by preventing synaptic transmission or release of acetylcholine through neuromuscular junctions, and are therefore useful in the treatment of spasticity, rigidity, and the like. The use of botulinum toxin in the cosmetic field was since 1992 reported that botulinum toxin has an effect of eliminating wrinkles when treating ocular muscle spasms. The FDA approved botulinum toxin for use in cosmetics in month 4 2002. Currently, the main routes of administration of botulinum toxins are subcutaneous or intramuscular injections, which provide therapeutic effects of removing wrinkles by intramuscular injection into the face. When used by injection, botulinum toxin is a problem in clinical safety, and small doses may be fatal.
Patent document CN 114989271a discloses a recombinant botulinum toxin type a which is highly toxic, and in a specific protein virulence assay, the half-lethal dose (LD 50 ) 4.023 pg/min. Botulinum neurotoxin is disclosed in patent document CN 106414759a as the most potent toxin known, the median lethal dose (LD 50 ) Values range from 0.5ng/kg to 5ng/kg. As can be seen from the prior art disclosures, conventional botulinum toxins are highly toxic, less lethal to half, and less safe for clinical use.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a membrane-penetrating peptide botulinum toxin recombinant protein, a preparation method and application thereof, wherein the membrane-penetrating peptide botulinum toxin recombinant protein is lethal only when used in a large dosage, and has high safety.
The application comprises the following technical scheme:
in a first aspect, the present application provides a recombinant protein of the transmembrane peptide botulinum toxin, the sequence of which is selected from one of the following amino acid sequences:
a) The recombinant protein consists of an amino acid sequence of SEQ ID NO. 1;
b) The recombinant protein is a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence of SEQ No. 1;
c) The amino acid sequence of the recombinant protein differs from the amino acid sequence of SEQ No. 1 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1.
The amino acid sequence of SEQ ID NO. 1 is as follows:
MHHHHHHPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD
TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLL
TSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHE
VLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHR
LYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF
KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY
TEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEI
NNMNFTKLKNFTGLFEALGPCMLLLLLLLGLRLPGVWAPPRRRRRRRRR
in a second aspect, the application provides a nucleotide sequence encoding the recombinant transmembrane peptide botulinum toxin protein.
The nucleotide sequence may be RNA or DNA, and the DNA includes cDNA and synthetic DNA. The DNA may be single-stranded or double-stranded. The single stranded DNA may be the coding strand or the non-coding (antisense) strand. The coding sequence can code recombinant protein composed of SEQ ID NO. 1 amino acid sequence.
In a specific embodiment of the application, the nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO. 2.
The nucleotide sequence of SEQ ID NO. 2 is as follows:
GATCCCATGCATCATCATCATCACCATCCGTTTGTTAATAAGCAGTTTAATTACAAGGACCCGGTTAATGGTGTTGATATTGCCTATATTAAGATCCCGAATGCCGGTCAGATGCAGCCGGTGAAAGCCTTTAAAATTCATAATAAGATCTGGGTTATTCCGGAACGCGATACCTTTACCAATCCGGAAGAAGGTGACCTGAATCCGCCGCCGGAAGCCAAACAGGTGCCGGTGAGCTATTATGATAGTACCTATCTGAGTACCGATAATGAAAAAGATAACTATCTGAAGGGTGTTACCAAACTGTTTGAACGCATCTATAGTACCGATCTGGGTCGCATGCTGCTGACCAGTATTGTTCGTGGTATTCCGTTTTGGGGCGGCAGCACCATTGATACCGAACTGAAAGTTATTGATACCAATTGCATTAACGTGATTCAGCCGGATGGCAGTTATCGCAGTGAAGAACTGAATCTGGTTATTATTGGTCCGAGCGCAGATATTATTCAGTTTGAATGCAAAAGTTTCGGCCATGAAGTTCTGAATCTGACCCGCAATGGCTATGGCAGCACCCAGTATATTCGCTTTAGCCCGGATTTTACCTTTGGCTTTGAAGAAAGCCTGGAAGTTGATACCAATCCGCTGCTGGGTGCCGGTAAATTTGCCACCGATCCGGCAGTGACCCTGGCACATGAACTGATTCATGCAGGTCATCGCCTGTATGGTATTGCCATTAATCCGAATCGTGTGTTTAAAGTTAACACCAATGCCTATTATGAGATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGTGGCCATGATGCAAAATTCATTGATAGCCTGCAGGAAAATGAATTTCGTCTGTATTATTACAACAAGTTCAAGGATATCGCCAGTACCCTGAATAAGGCCAAAAGCATTGTGGGTACCACCGCAAGTCTGCAGTATATGAAAAATGTTTTTAAGGAGAAGTACCTGCTGAGCGAAGATACCAGTGGTAAATTTTCTGTTGATAAACTGAAGTTCGACAAACTGTATAAAATGCTGACCGAAATCTATACCGAAGATAATTTTGTTAAGTTCTTCAAGGTGCTGAATCGCAAAACCTATCTGAATTTTGATAAGGCAGTGTTTAAGATTAACATCGTGCCGAAAGTGAATTATACCATCTATGATGGCTTTAATCTGCGCAATACCAATCTGGCAGCCAATTTTAATGGCCAGAATACCGAAATTAACAATATGAATTTCACCAAGCTGAAGAATTTCACCGGTCTGTTTGAAGCACTGGGTCCGTGCATGCTGCTGCTGCTGTTACTGCTGGGTCTGCGCCTGCCGGGTGTGTGGGCACCTCCTCGTCGCCGTCGTCGTCGTCGCCGTAGATAA
in a third aspect, the application provides a recombinant protein expression vector comprising a nucleotide sequence encoding a recombinant protein of the transmembrane peptide botulinum toxin. The recombinant protein expression vector is capable of expressing a transmembrane peptide botulinum toxin recombinant protein in a host cell, which is a genetic construct comprising the necessary regulatory factors operably linked to express a gene insert.
In one embodiment of the application, the recombinant protein expression vector consists of the nucleotide sequence shown in SEQ ID NO. 3.
The nucleotide sequence of SEQ ID NO. 3 is as follows:
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGATATCGGAATTAATTCGGATCCCATGCATCATCATCATCACCATCCGTTTGTTAATAAGCAGTTTAATTACAAGGACCCGGTTAATGGTGTTGATATTGCCTATATTAAGATCCCGAATGCCGGTCAGATGCAGCCGGTGAAAGCCTTTAAAATTCATAATAAGATCTGGGTTATTCCGGAACGCGATACCTTTACCAATCCGGAAGAAGGTGACCTGAATCCGCCGCCGGAAGCCAAACAGGTGCCGGTGAGCTATTATGATAGTACCTATCTGAGTACCGATAATGAAAAAGATAACTATCTGAAGGGTGTTACCAAACTGTTTGAACGCATCTATAGTACCGATCTGGGTCGCATGCTGCTGACCAGTATTGTTCGTGGTATTCCGTTTTGGGGCGGCAGCACCATTGATACCGAACTGAAAGTTATTGATACCAATTGCATTAACGTGATTCAGCCGGATGGCAGTTATCGCAGTGAAGAACTGAATCTGGTTATTATTGGTCCGAGCGCAGATATTATTCAGTTTGAATGCAAAAGTTTCGGCCATGAAGTTCTGAATCTGACCCGCAATGGCTATGGCAGCACCCAGTATATTCGCTTTAGCCCGGATTTTACCTTTGGCTTTGAAGAAAGCCTGGAAGTTGATACCAATCCGCTGCTGGGTGCCGGTAAATTTGCCACCGATCCGGCAGTGACCCTGGCACATGAACTGATTCATGCAGGTCATCGCCTGTATGGTATTGCCATTAATCCGAATCGTGTGTTTAAAGTTAACACCAATGCCTATTATGAGATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGTGGCCATGATGCAAAATTCATTGATAGCCTGCAGGAAAATGAATTTCGTCTGTATTATTACAACAAGTTCAAGGATATCGCCAGTACCCTGAATAAGGCCAAAAGCATTGTGGGTACCACCGCAAGTCTGCAGTATATGAAAAATGTTTTTAAGGAGAAGTACCTGCTGAGCGAAGATACCAGTGGTAAATTTTCTGTTGATAAACTGAAGTTCGACAAACTGTATAAAATGCTGACCGAAATCTATACCGAAGATAATTTTGTTAAGTTCTTCAAGGTGCTGAATCGCAAAACCTATCTGAATTTTGATAAGGCAGTGTTTAAGATTAACATCGTGCCGAAAGTGAATTATACCATCTATGATGGCTTTAATCTGCGCAATACCAATCTGGCAGCCAATTTTAATGGCCAGAATACCGAAATTAACAATATGAATTTCACCAAGCTGAAGAATTTCACCGGTCTGTTTGAAGCACTGGGTCCGTGCATGCTGCTGCTGCTGTTACTGCTGGGTCTGCGCCTGCCGGGTGTGTGGGCACCTCCTCGTCGCCGTCGTCGTCGTCGCCGTAGATAAGAGATCAAACGGGCTAGCCAGCCAGAACTCGCCCCGGAAGACCCCGAGGATGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
in a fourth aspect, the present application provides a bacterium transformed with a recombinant protein expression vector, said transformed bacterium being cultured to obtain a recombinant protein of the transmembrane peptide botulinum toxin.
In a fifth aspect, the application provides a composition comprising a recombinant protein of the transmembrane peptide botulinum toxin and a pharmaceutically acceptable carrier.
The composition is selected from the following formulations: emulsions, creams, essences, lotions, liposomes, microcapsules, composite particles, shampoos, lotions.
In a sixth aspect, the present application provides a method of producing a recombinant protein of a transmembrane peptide botulinum toxin, the method comprising the steps of:
(1) Amplifying the gene fragments of the membrane penetrating peptide and the botulinum toxin by adopting a PCR method, and connecting the synthesized target gene with an escherichia coli expression plasmid pET-25 b;
(2) Transforming the recombinant expression plasmid into DH5 alpha competent cells, screening positive clones and sequencing to obtain a recombinant protein expression vector;
(3) Extracting and identifying correct recombinant plasmid by sequencing, converting the recombinant plasmid into Rosetta competent cells, storing the cells in glycerinum, screening out successfully constructed transformants by using chloramphenicol and ampicillin resistance, and obtaining recombinant protein by IPTG induction;
(4) And (3) separating and purifying the recombinant protein to obtain the recombinant protein of the membrane-penetrating peptide botulinum toxin.
Alternatively, ampicillin and chloramphenicol are used in step (2) to screen positive clones.
Optionally, the step (3) specifically includes the following operations:
s1: drawing lines on LB solid culture medium, and culturing overnight at 37 ℃, wherein the LB solid culture medium comprises ampicillin with the concentration of 50 mug/mL and chloramphenicol with the concentration of 15 mug/mL;
s2: randomly picking 3 monoclonals in 3mL of 2YT liquid culture medium, adding a chloramphenicol solution with the concentration of 30mg/mL and an ampicillin sodium solution with the concentration of 100mg/mL, shaking at the rotation speed of 220rpm at 37 ℃, and culturing overnight to obtain a bacterial culture solution;
s3: transferring the bacterial liquid 1:50 cultured overnight into 3mL 2YT liquid culture medium, shake culturing at 37deg.C, and standing until the bacterial liquid OD 600 When the value is 0.4-0.8, adding IPTG with the final concentration of 0.5-1mM, inducing for 12-24 hours at the speed of 220rpm at the temperature of 16-20 ℃, collecting thalli, treating the thalli by loading buffer, and verifying by SDS-PAGE running gel;
s4: bacterial liquid 1 with high protein expression: 50 inoculated in 2YT liquid culture medium, adding 15mg/mL chloramphenicol solution and 50mg/mL ampicillin sodium solution, shaking at 37deg.C and 220rpm, OD 600 Adding IPTG with final concentration of 0.5-1mM at 16-20deg.C and 220rpm at 0.4-0.8, and inducing overnight;
s5: collecting the induced bacterial liquid overnight, centrifuging at 10000rpm for 10-16min, collecting bacterial cells, re-suspending cells with PBS and performing cell lysis by ultrasonic treatment, selecting No. 2 rod or No. 10 rod according to the amount of bacterial cells, performing ultrasonic treatment for 5s with power of 30% and interval of 5s, performing total ultrasonic treatment for 30min, and performing the whole process on ice; until the cells were completely resuspended without cell sinking at the bottom of the tube, the supernatant was collected and centrifuged at 10,000rpm for 30 minutes at 4℃to remove bacterial debris and obtain the supernatant.
In a seventh aspect, the present application provides the use of a recombinant protein of the transmembrane peptide botulinum toxin and/or a composition comprising a recombinant protein of the transmembrane peptide botulinum toxin, comprising the use in any one of the following:
(1) Preventing and/or treating facial spasm, eyelid spasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysarthria, migraine, pruritus ani, hyperhidrosis;
(2) Improving wrinkles, nasal lip lines, fish tail lines, facial lines, head lines, skin injury, skin softening, scar, and acne.
The membrane-penetrating peptide botulinum toxin recombinant protein provided by the application has the advantages of higher safety and lethality only when used in a large dosage.
Drawings
FIG. 1 is a schematic representation of a recombinant protein expression vector for a transmembrane peptide botulinum toxin;
FIG. 2 is a diagram showing SDS-PAGE results of recombinant proteins of the transmembrane peptide botulinum toxin;
FIG. 3 is a graph showing the survival rate results of C57BL/6N mice challenged with the recombinant protein of the transmembrane peptide botulinum toxin.
Detailed Description
The technical solutions of the embodiments of the present application will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present application, but not all. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Botulinum toxins are split into heavy chains (H) by reconstitution after expression C ) And light chain (L) C ) Which are linked by disulfide bonds. The heavy chain is linked to a cellular receptor to allow the botulinum toxin to enter the cell by endocytosis, and the light chain of the botulinum toxin entering the cell is released from the body to function. The heavy and light chains of botulinum toxins are different in their role, each of which plays its own role, however, the light chain alone cannot pass through the cell membrane, and in order to deliver the light chain of botulinum toxin into the cytoplasm of the cell to exhibit a particular botulinum toxin activity, it is necessary that there is involvement of the heavy chain. However, existing botulinum toxins with heavy and light chains are safeLower sex, low doses of botulinum toxin are lethal.
Based on this, the inventors of the present application have attempted to develop a recombinant protein of a transmembrane peptide botulinum toxin capable of delivering a light chain into a cell instead of a heavy chain to exhibit botulinum toxin activity, and which is safer and lethal at a larger dose, and a method for producing the same. The membrane-penetrating peptide botulinum toxin is prepared by the following scheme.
Examples
Reagents used in the examples of the present application
Preparation process of membrane-penetrating peptide botulinum toxin recombinant protein
1. Obtaining recombinant protein expression vectors
(1) PCR method is adopted to amplify gene fragments of the membrane penetrating peptide and the botulinum toxin, the DNA sequence (SEQ ID NO: 2) of LC-PTD is used as a template, and the primer sequence information is shown in the following table:
the PCR reaction conditions included: thermal denaturation at 95℃for 5 min, reaction at 95℃for 30 sec, 58℃for 1 min, and 72℃for 1 min for 30 cycles, and finally amplification at 72℃for 8 min.
(2) After the reaction, electrophoresis was performed using 1% agarose gel to confirm amplified products, and then the amplified recombinant fragments were collected from the agarose gel and extracted and purified using a gel extraction kit, the PCR products, vector pET-25b (+) vector and homologous recombinase were mixed and treated at 37 ℃ for 1 hour, and the synthesized target gene was ligated with escherichia coli expression plasmid pET-25 b;
(3) Transforming the recombinant expression plasmid into DH5 alpha competent cells, screening positive clones with ampicillin, and sequencing to obtain a recombinant protein expression vector, as shown in FIG. 1;
(4) The correct recombinant plasmid was extracted and identified by sequencing and transformed into Rosetta competent cells and stored in glycerol bacteria.
2. Culturing glycerol bacteria to obtain crude recombinant protein
(1) Drawing lines on LB solid culture medium, and culturing overnight at 37 ℃, wherein the LB solid culture medium comprises ampicillin with the concentration of 50 mug/mL and chloramphenicol with the concentration of 15 mug/mL;
(2) Randomly picking 3 monoclonals in 3mL of 2YT liquid culture medium, adding a chloramphenicol solution with the concentration of 30mg/mL and an ampicillin sodium solution with the concentration of 100mg/mL, shaking at the rotation speed of 220rpm at 37 ℃, and culturing overnight to obtain a bacterial culture solution;
(3) Transferring the bacterial liquid 1:50 cultured overnight into 3mL 2YT liquid culture medium, shake culturing at 37deg.C, and standing until the bacterial liquid OD 600 At a value of 0.8, adding IPTG with a final concentration of 1mM, shaking at 220rpm at 20 ℃ for 24 hours, collecting thalli, treating the thalli with loading buffer, and running gel for verification by SDS-PAGE;
(4) Bacterial liquid 1 with high protein expression: 50 inoculated in 2YT liquid culture medium, adding chloramphenicol solution with concentration of 15mg/mL and ampicillin sodium solution with concentration of 50 mug/mL; shaking at 37deg.C and 220rpm, OD 600 At a value of 0.8, 5. Mu.L of IPTG was added at a final concentration of 1mM, and induced overnight at 20℃and 220 rpm;
(5) Collecting induced bacterial liquid overnight, centrifuging at 10000rpm for 10min, collecting bacterial cells, re-suspending cells with PBS and performing cell lysis by ultrasonic treatment, selecting No. 2 rod with power of 30%, ultrasonic treatment for 5s and interval of 5s, and performing total ultrasonic treatment for 30min on ice; until the cells were completely resuspended without cell sinking at the bottom of the tube, the supernatant was collected and centrifuged at 10,000rpm for 30 minutes at 4℃to remove bacterial debris and obtain the supernatant.
3. Recombinant protein purification
The first resin used requires 5mL of equilibration buffer to wash (binding buffer); if the resin is reused, the resin is eluted with 20% ethanol and ddH is used first 2 O10 mL was washed and then 10mL equilibration buffer was used.
(1) Loading: adding the crude recombinant protein to the balanced Ni column, combining for 2 hours at room temperature, opening an outlet at the lower end of the column, collecting penetrating fluid, and repeating the sample loading to improve the column hanging efficiency;
(2) Balance the Ni post after loading: the Ni column was again equilibrated with 10 column volumes of Binding Buffer (pH 7.4) to remove unbound protein;
(3) Eluting the Ni column by using a gradient mixing solution of an absorption Buffer/Binding Buffer (PH 7.4), adding 20mM imidazole and 50mM imidazole into the Binding Buffer respectively, performing gradient Elution to remove the impurity protein, collecting the impurity-eluting solution, eluting the target protein from the column by using the absorption Buffer, collecting the eluting solution for each Elution, and determining the target protein by SDS-PAGE detection to obtain the purified recombinant protein.
SDS-PAGE detection of recombinant proteins
The penetration solution, the impurity washing solution and the eluent collected in the recombinant protein purification process are respectively subjected to electrophoresis test in SDS-PAGE gel, and the results are shown in FIG. 2. Wherein M represents a Marker;1 represents a penetrating fluid; 2 represents a cleaning solution; 3-9 are all eluents. The 50KD in the SDS-PAGE electrophoresis of FIG. 2 corresponds to the light chain of botulinum toxin, and the presence of 50KD represents a recombinant protein comprising the transmembrane peptide botulinum toxin.
From the results of FIG. 2, it can be seen that the eluent 3-eluent 6 has a larger proportion of recombinant protein of the transmembrane peptide botulinum toxin, which indicates that the recombinant protein is expressed and has better purification effect.
Effect example
Recombinant protein toxicity assay
60C 57BL/6N mice were weighed, and the mice weighed 20+ -2 g were randomly divided into 6 groups of 10 mice each. Wherein, groups 1-5 are respectively injected with the recombinant protein solution (recombinant protein+solvent) prepared by the embodiment of the application in the dosage of 125 mg/dose, 250 mg/dose, 500 mg/dose, 1000 mg/dose and 2000 mg/dose, and group 6 is a control group injected with the same volume of solvent (same as the solvent of groups 1-5). The death of each group of mice was recorded for various time periods and the results are shown in figure 3. In addition, death (180 h) of mice injected with recombinant protein was recorded, and the results are shown in Table 1, and the half-Life (LD) was calculated by SPSS software 50 )。
TABLE 1 death number of the transmembrane peptide botulinum toxin recombinant protein-challenged C57BL/6N mice
As can be seen from the results of FIG. 3, when the injection amount of the recombinant protein was 2000 mg/mouse, mice were all killed in less than 10min, and when the injection amount of the recombinant protein was 125 mg/mouse, 10% of mice were dead at 100h, and 10% of the death rate was maintained until 180h, indicating that the recombinant protein prepared by the present application had in vivo toxicity (activity), but required a large dose to be lethal. Meanwhile, the recorded results of Table 1 were subjected to SPSS software to calculate the median Lethal Dose (LD) of the recombinant protein of the membrane-penetrating peptide botulinum toxin prepared by the present application 50 ) 428.72 mg/dose indicates that the recombinant protein of the transmembrane peptide botulinum toxin prepared according to the application requires a relatively large dose to be lethal.
In the prior art, the common mouse lethal dose of the botulinum toxin consisting of the heavy chain and the light chain is pg (picogram) grade, and the mouse lethal dose of the recombinant protein of the transmembrane peptide botulinum toxin prepared by the application is mg (milligram) grade, so that the safety is higher.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (10)
1. A recombinant protein of the transmembrane peptide botulinum toxin, characterized in that the sequence of the recombinant protein is selected from one of the following amino acid sequences:
a) The recombinant protein consists of an amino acid sequence of SEQ ID NO. 1;
b) The recombinant protein is a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence of SEQ No. 1;
c) The amino acid sequence of the recombinant protein differs from the amino acid sequence of SEQ No. 1 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1.
2. A nucleotide sequence encoding the recombinant protein of the transmembrane peptide botulinum toxin of claim 1.
3. The nucleotide sequence according to claim 2, characterized in that it consists of the nucleotide sequence shown in SEQ ID NO. 2.
4. A recombinant protein expression vector comprising a nucleotide sequence encoding the recombinant protein of the transmembrane peptide botulinum toxin of claim 1.
5. The recombinant protein expression vector according to claim 4, wherein said recombinant protein expression vector consists of a nucleotide sequence shown in SEQ ID No. 3.
6. A bacterium transformed with the recombinant protein expression vector of claim 4 or 5.
7. A composition comprising the recombinant transmembrane peptide botulinum toxin protein of claim 1 and a pharmaceutically acceptable carrier.
8. A method of producing the recombinant protein of the transmembrane peptide botulinum toxin of claim 1, comprising the steps of:
(1) Amplifying the gene fragments of the membrane penetrating peptide and the botulinum toxin by adopting a PCR method, and connecting the synthesized target gene with an escherichia coli expression plasmid pET-25 b;
(2) Transforming the recombinant expression plasmid into DH5 alpha competent cells, screening positive clones and sequencing to obtain a recombinant protein expression vector;
(3) Extracting and identifying correct recombinant plasmid by sequencing, converting the recombinant plasmid into Rosetta competent cells, storing the cells in glycerinum, screening out successfully constructed transformants by using chloramphenicol and ampicillin resistance, and obtaining recombinant protein by IPTG induction;
(4) And (3) separating and purifying the recombinant protein to obtain the recombinant protein of the membrane-penetrating peptide botulinum toxin.
9. The method according to claim 8, wherein the step (3) comprises the following operations:
s1: drawing lines on LB solid culture medium, and culturing overnight at 37 ℃, wherein the LB solid culture medium comprises ampicillin with the concentration of 50 mug/mL and chloramphenicol with the concentration of 15 mug/mL;
s2: randomly picking 3 monoclonals in 3mL of 2YT liquid culture medium, adding a chloramphenicol solution with the concentration of 30mg/mL and an ampicillin sodium solution with the concentration of 100mg/mL, shaking at the rotation speed of 220rpm at 37 ℃, and culturing overnight to obtain a bacterial culture solution;
s3: transferring the bacterial liquid 1:50 cultured overnight into 3mL 2YT liquid culture medium, shake culturing at 37deg.C, and standing until the bacterial liquid OD 600 When the value is 0.4-0.8, adding IPTG with final concentration of 0.5-1mM, inducing at 220rpm for 12-24 hr at 16-20deg.C, and collecting thallusAfter the thalli are treated by loading buffer, the thalli are verified by SDS-PAGE running gel;
s4: bacterial liquid 1 with high protein expression: 50 inoculated in 2YT liquid culture medium, adding 15mg/mL chloramphenicol solution and 50mg/mL ampicillin sodium solution, shaking at 37deg.C and 220rpm, OD 600 Adding IPTG with final concentration of 0.5-1mM at 16-20deg.C and 220rpm at 0.4-0.8, and inducing overnight;
s5: collecting the induced bacterial liquid overnight, centrifuging at 10000rpm for 10-16min, collecting bacterial cells, re-suspending cells with PBS and performing cell lysis by ultrasonic treatment, selecting No. 2 rod or No. 10 rod according to the amount of bacterial cells, performing ultrasonic treatment for 5s with power of 30% and interval of 5s, performing total ultrasonic treatment for 30min, and performing the whole process on ice; until the cells were completely resuspended without cell sinking at the bottom of the tube, the supernatant was collected and centrifuged at 10,000rpm for 30 minutes at 4℃to remove bacterial debris and obtain the supernatant.
10. Use of the recombinant protein of the transmembrane peptide botulinum toxin of claim 1 and/or the composition of claim 7, comprising use in any one of the following:
(1) Preventing and/or treating facial spasm, eyelid spasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysarthria, migraine, pruritus ani, hyperhidrosis;
(2) Improving wrinkles, nose lip lines, fish tail lines, mouth and corner lines, head lifting lines, skin injury, skin softening and scar.
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| CN202211176049 | 2022-09-26 | ||
| CN2022111760493 | 2022-09-26 |
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