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WO2024113643A1 - Neurotoxine botulique recombinante, son procédé de préparation et son utilisation - Google Patents

Neurotoxine botulique recombinante, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2024113643A1
WO2024113643A1 PCT/CN2023/088980 CN2023088980W WO2024113643A1 WO 2024113643 A1 WO2024113643 A1 WO 2024113643A1 CN 2023088980 W CN2023088980 W CN 2023088980W WO 2024113643 A1 WO2024113643 A1 WO 2024113643A1
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Prior art keywords
botulinum neurotoxin
recombinant
chain
double
bont
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Ceased
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PCT/CN2023/088980
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English (en)
Chinese (zh)
Inventor
王师亮
王江田
张彦军
刘应康
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Hyamed Biotechnology Zhuhai Co Ltd
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Hyamed Biotechnology Zhuhai Co Ltd
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of bioengineering technology, and specifically relates to a recombinant botulinum neurotoxin and a preparation method and application thereof.
  • Botulinum neurotoxin is a class of protein toxins produced by Clostridium Botulinum. It is highly specific to target nerve cells and is the most toxic toxin known in nature. There are currently seven known subtypes of this toxin family, of which type A is widely used in disease treatment and medical aesthetics.
  • BoNT/A Botulinum neurotoxin type A
  • Clostridium botulinum which is hydrolyzed and cleaved by proteolytic enzymes at specific sites to form two polypeptides linked together by disulfide bonds, and this double-chain form is the active form of the target drug.
  • the production of BoNT in the prior art is carried out by culturing Clostridium botulinum and then classifying and purifying the clostridial neurotoxin complex.
  • the production of BoNT in this way is inefficient and the protein yield is low.
  • Clostridium botulinum is a spore-forming bacterium and therefore requires specialized culture equipment and devices, which are not required in the culture of bacteria (such as Escherichia coli). Therefore, the increasing application of BoNT has led to the need for alternative and/or improved methods for producing and purifying BoNT.
  • US 20080103098 describes a method for producing a double-chain form of a recombinant BoNT protein, which comprises expressing a recombinant nucleic acid construct in an Escherichia coli host cell.
  • the method requires the insertion of a specific, non-natural (i.e., non-clostridial) pentapeptide sequence into the loop domain of the neurotoxin; the inserted pentapeptide sequence forms an activation cleavage site) that is cleaved by endogenous E. coli proteases after cell lysis. Therefore, the method of US20080103098 states that in order to achieve optimized BoNT expression, it is necessary to insert a non-natural US 7132259 describes a recombinant nucleic acid molecule encoding a BoNT protein. However, the nucleic acid molecule of US 7132259 is modified to use a non-natural cleavage site instead of a natural cleavage site.
  • US 6495143 describes a method for expressing and purifying individual H and L chains in E. coli and then combining the H and L chain subunits through oxidative disulfide bonds under strictly controlled conditions to form an active double-chain BoNT.
  • this method has several shortcomings. Specifically, (1) it is difficult to express and isolate individual H and L chains in large quantities. When the H chain is not present, the isolated L chain is completely insoluble in aqueous solution and is easily hydrolyzed and degraded by proteases. (2) The efficiency of in vitro oxidation of individual H and L chains to generate active double chains is not high, resulting in low yields of active toxins, and most products are inactive, incorrectly folded or oxidized forms of BoNT. (3) It is difficult to purify toxins containing correctly folded and oxidized H and L chains, and it is also difficult to separate them from these inactive forms and unreacted individual H and L chains.
  • one of the objectives of the present invention is to provide a recombinant botulinum neurotoxin, which has an exogenous protease recognition site inserted to effectively improve the cleavage efficiency of the single-chain polypeptide to obtain a double-chain form of BoNT/A with higher biological activity.
  • a second object of the present invention is to provide a method for preparing a recombinant botulinum neurotoxin.
  • a third object of the present invention is to provide a method for preparing a double-chain botulinum neurotoxin.
  • a fourth object of the present invention is to provide a double-chain botulinum neurotoxin.
  • a fifth object of the present invention is to provide an application of a double-chain botulinum neurotoxin.
  • a recombinant botulinum neurotoxin the amino acid sequence of which is shown in SEQ ID NO:6.
  • nucleic acid sequence of the gene encoding the recombinant botulinum neurotoxin includes those shown in SEQ ID NO:1-5.
  • a method for preparing a recombinant botulinum neurotoxin comprises the following steps:
  • step 2) the specific operation is: introducing the recombinant pET-15b plasmid into BL21 (DE3) or BL21 (DE3) pLySs competent cells, screening and obtaining a monoclonal strain expressing recombinant botulinum neurotoxin, inoculating the monoclonal strain into LB medium, culturing to an OD value of 0.4-0.6, adding IPTG inducer to induce expression at 25-37°C to obtain recombinant botulinum neurotoxin.
  • a method for preparing a double-chain botulinum neurotoxin is prepared by cutting the recombinant botulinum neurotoxin.
  • the recombinant pCDFDuet-1 plasmid was introduced into Escherichia coli, cultured and induced to express, and the recombinant Group-engineered bacteria;
  • exogenous protease is EV71-3C protein.
  • a double-chain botulinum neurotoxin is prepared by the preparation method of the double-chain botulinum neurotoxin.
  • the present invention has the following beneficial effects:
  • the recombinant botulinum neurotoxin of the present invention is inserted with an exogenous protease recognition site, which effectively improves the cleavage efficiency of single-chain polypeptides and obtains a double-chain BoNT/A with higher biological activity.
  • the present invention provides a method for preparing a double-chain botulinum neurotoxin, wherein the double-chain botulinum neurotoxin is prepared by efficiently cleaving the recombinant botulinum neurotoxin. Furthermore, the single-chain recombinant botulinum neurotoxin and an exogenous protease (EV71-3C protein) are quantitatively expressed in equal proportions in Escherichia coli, and the exogenous protease can cleave the single-chain recombinant botulinum neurotoxin in Escherichia coli to produce active A double-chain botulinum neurotoxin in this form was produced in one step using Escherichia coli.
  • Figure 2 is a gel electrophoresis diagram comparing the induced expression of the seq1 gene of the present invention under different strains; wherein, lane M is a marker group, lane seq1 is a bacterial solution group containing the seq1 gene, lane seq1_S is a supernatant group containing the seq1 gene, and lane seq1_p is a precipitate group containing the seq1 gene.
  • Figure 3 is a gel electrophoresis diagram comparing the induced expression of the seq2 gene of the present invention at different temperatures; wherein, lane M is a marker group, lane seq2 is a bacterial solution group containing the seq2 gene, lane seq1_S is a supernatant group containing the seq2 gene, and lane seq2_p is a precipitate group containing the seq2 gene.
  • Figure 4 is a gel electrophoresis diagram comparing the induced expression of the seq3 gene of the present invention at different temperatures; wherein, lane M is a marker group, lane seq3 is a bacterial solution group containing the seq3 gene, lane seq3+IPTG is a bacterial solution group containing the seq3 gene plus IPTG solution, lane seq1_S is a supernatant group containing the seq3 gene, and lane seq3_p is a precipitate group containing the seq3 gene.
  • FIG5 is a gel electrophoresis diagram of the comparison of the induced expression of the seq4 gene of the present invention at different temperatures.
  • lane M is a marker group
  • lane seq4_LB is a bacterial solution group containing the seq4 gene in LB medium
  • lane seq4_p is a precipitate group containing the seq4 gene at different temperatures.
  • FIG. 6 is an electrophoresis analysis diagram of the strains of each gene of the present invention before and after induction.
  • FIG. 7 is an electrophoretic analysis diagram of single-chain BoNT/A.
  • FIG. 8 is an electrophoretic analysis diagram of double-chain BoNT/A produced by the recombinant botulinum neurotoxin of the present invention.
  • Botulinum neurotoxin type A (BoNT/A) is synthesized as a single-chain polypeptide in Clostridium botulinum, which is hydrolyzed and cleaved at a specific site by a proteolytic enzyme to form two polypeptides linked by a disulfide bond. This double-chain form is the active form of the target drug. As shown in Figure 1, these two chains are called heavy chain (HC, with a molecular weight of about 100 kDa) and light chain (LC, with a molecular weight of about 50 kDa).
  • HC heavy chain
  • LC light chain
  • the present invention replaces the cleavage site in the single-chain polypeptide with a specific recognition site (amino acid sequence RTATVQGPSLDFE) of an exogenous protease (EV71-3C protein), thereby effectively improving the specificity of site cleavage.
  • a specific recognition site amino acid sequence RTATVQGPSLDFE
  • E71-3C protein exogenous protease
  • a DNA sequence that can efficiently express recombinant botulinum neurotoxin in Escherichia coli was obtained through codon optimization.
  • the obtained coding genes include: seq1 gene, the nucleic acid sequence is shown in SEQ ID NO: 1; seq2 gene, the nucleic acid sequence is shown in SEQ ID NO: 2; seq3 gene, the nucleic acid sequence is shown in SEQ ID NO: 3; seq4 gene, the nucleic acid sequence is shown in SEQ ID NO: 4; Seq5 gene, the nucleic acid sequence is shown in SEQ ID NO: 5.
  • the seq1 gene, seq2 gene, seq3 gene and The DNA sequence of seq4 gene was introduced into the pET-15b expression vector by restriction endonuclease and T4 DNA ligase to construct the recombinant pET-15b plasmids of four target proteins: seq1-pET15b, seq2-pET15b, seq3-pET15b, and seq4-pET15b;
  • the recombinant pET-15b plasmid obtained in step (3) was introduced into BL21(DE3) competent cells or BL21(DE3)pLySs competent cells by heat shock method, and the monoclonal strain expressing BONT/A was obtained by ampicillin antibiotic screening.
  • the strain containing the seq1 gene was expressed in BL21(DE3) competent cells and BL21(DE3)pLySs competent cells at 25°C, and an obvious color band appeared at 140kDa.
  • the expression effect in BL21(DE3) competent cells was better.
  • the strain containing the seq2 gene was expressed in BL21(DE3)pLySs competent cells at 30°C and 37°C, respectively. The protein expression was better at 30°C.
  • the strain containing the seq3 gene was expressed in BL21(DE3)pLySs competent cells at 30°C and 37°C, respectively, and the protein expression was better at 37°C; referring to Figure 5, the strain containing the seq4 gene was expressed in BL21(DE3)pLySs competent cells at 25°C, 30°C and 37°C, respectively, and the protein expression was better at 25°C.
  • the strains containing each DNA sequence were induced to express in large quantities; specifically: 1.5ml EP tubes were shaken to activate the bacteria for 4h (50ul of the preserved bacterial solution was inoculated into 500ul of the culture medium), and then transferred into LB culture medium containing ampicillin resistance (1:100 transfer (250ml for large-scale induction, or two 100ml). When transferring, 0.2g/100ml glucose was added to the LB culture medium, 180rpm, 37°C for 3h.
  • Figure 6 is the gel electrophoresis images of the bacterial solution of each DNA sequence before and after induction, and the lower part is the grayscale analysis of each gel electrophoresis lane. It can be seen that an obvious color band appears at 140kDa in the bacterial solution after induction, proving that multiple DNA sequences of the present invention can express single-chain recombinant botulinum neurotoxin in Escherichia coli.
  • a method for preparing a double-chain botulinum neurotoxin comprises the following steps:
  • the recombinant pCDFDuet-1 plasmid was introduced into BL21 (DE3) competent cells or BL21 (DE3) pLySs competent cells by heat shock method, and the monoclonal strain expressing BONT/A was obtained by ampicillin antibiotic screening.
  • the monoclonal strain was activated, it was transferred into LB medium containing ampicillin resistance. During the transfer, 0.2 g/100 ml glucose was added to the LB medium. After 3 hours at 180 rpm and 37°C, when the OD value was between 0.4 and 0.6, it was cooled on ice for 10 minutes, 1 mM IPTG (final concentration) was added, and cultured at 150 rpm at an appropriate temperature (seq1: 25°C, seq2: 30°C, seq3: 37°C, seq4: 25°C) for 6 hours, and the bacteria were collected by centrifugation.
  • the double-chain botulinum neurotoxin type A was tested by gel electrophoresis, and the single-chain botulinum neurotoxin type A that had not undergone genetic recombination was used as a control group. The results are shown in Figures 7-8.
  • the botulinum neurotoxin type A supernatant group (BoNT/A+IPTG-S) induced by IPTG showed an obvious band at 140 kDa;
  • the botulinum neurotoxin type A supernatant group (BoNT/A+IPTG-S) and the botulinum neurotoxin type A precipitate group (BoNT/A+IPTG-P) induced by IPTG showed obvious bands at 50 kDa and 100 kDa, indicating that the recombinant botulinum neurotoxin of the present invention can be obtained by specific enzymatic hydrolysis of EV71-3C protein to obtain a double-chain BoNT/A, and it is also proved that the preparation method of the present invention can realize the one-step production of double-chain BoNT/A and simplify the production process.
  • single-chain recombinant botulinum neurotoxin and exogenous protease are quantitatively expressed in equal proportions in Escherichia coli.
  • the exogenous protease can cleave the single-chain recombinant botulinum neurotoxin in Escherichia coli to produce an active double-chain botulinum neurotoxin, thereby achieving one-step production using Escherichia coli and simplifying the production process.
  • the immobilized protease reaction column can be reused to reduce production costs.

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Abstract

L'invention concerne une neurotoxine botulique recombinante (BoNT), son procédé de préparation et son utilisation. La séquence d'acides aminés de la BoNT recombinante est telle que représentée dans SEQ ID NO : 6, et la BoNT est insérée dans un site de reconnaissance de protéase exogène, de façon à améliorer efficacement l'efficacité de clivage d'un polypeptide à chaîne unique, et à obtenir une BoNT/A à double chaîne ayant une activité biologique relativement élevée. Le procédé de préparation de la BoNT à double chaîne permet de préparer une BoNT à double chaîne par clivage efficace d'une BoNT recombinante. Une BoNT recombinante à chaîne unique et une protéase exogène sont exprimées quantitativement dans Escherichia coli à un rapport égal, et la protéase exogène peut cliver la BoNT recombinante à chaîne unique dans l'Escherichia coli pour générer la BoNT à double chaîne sous une forme active, ce qui permet de réaliser une production en une étape à l'aide d'Escherichia coli.
PCT/CN2023/088980 2022-12-02 2023-04-18 Neurotoxine botulique recombinante, son procédé de préparation et son utilisation Ceased WO2024113643A1 (fr)

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CN202211539717.4A CN115819526A (zh) 2022-12-02 2022-12-02 一种重组肉毒杆菌神经毒素及其制备方法和应用
CN202211539717.4 2022-12-02

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CN115819526A (zh) * 2022-12-02 2023-03-21 海雅美生物技术(珠海)有限公司 一种重组肉毒杆菌神经毒素及其制备方法和应用
CN117603323B (zh) * 2023-05-16 2024-08-16 张文康 一种肉毒毒素的制备方法

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WO2009038770A2 (fr) * 2007-09-20 2009-03-26 University Of Massachusetts Cvip Neurotoxine botulique recombinante détoxifiée
CN104755098A (zh) * 2012-10-31 2015-07-01 突触融合蛋白有限公司 重组的肉毒梭菌神经毒素
WO2019081022A1 (fr) * 2017-10-26 2019-05-02 Merz Pharma Gmbh & Co. Kgaa Nouvelles neurotoxines botuliques recombinantes présentant une durée d'effet accrue
CN109715820A (zh) * 2016-09-16 2019-05-03 益普生生物制药有限公司 用于产生双链梭菌神经毒素的方法
CN115819526A (zh) * 2022-12-02 2023-03-21 海雅美生物技术(珠海)有限公司 一种重组肉毒杆菌神经毒素及其制备方法和应用

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CN106474103A (zh) * 2016-10-12 2017-03-08 湖北工业大学 五羟基黄酮类化合物在制备具有3c蛋白酶活性抑制作用的药物中的应用
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WO2009038770A2 (fr) * 2007-09-20 2009-03-26 University Of Massachusetts Cvip Neurotoxine botulique recombinante détoxifiée
CN104755098A (zh) * 2012-10-31 2015-07-01 突触融合蛋白有限公司 重组的肉毒梭菌神经毒素
CN109715820A (zh) * 2016-09-16 2019-05-03 益普生生物制药有限公司 用于产生双链梭菌神经毒素的方法
WO2019081022A1 (fr) * 2017-10-26 2019-05-02 Merz Pharma Gmbh & Co. Kgaa Nouvelles neurotoxines botuliques recombinantes présentant une durée d'effet accrue
CN115819526A (zh) * 2022-12-02 2023-03-21 海雅美生物技术(珠海)有限公司 一种重组肉毒杆菌神经毒素及其制备方法和应用

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