Recombinant botulinum neurotoxin type A and its preparation method

Abstract

The present invention relates to a recombinant botulinum neurotoxin type A and a preparation method thereof, the recombinant botulinum neurotoxin type A is a botulinum neurotoxin variant, the botulinum neurotoxin variant comprises a first peptide segment and a second peptide segment, the first peptide segment is linked to the second peptide segment by an interchain disulfide bond, the first peptide segment has a mutation at position 134 and / or 165 compared to the light chain of wild-type botulinum neurotoxin type A, and / or the second peptide segment has at least one of the mutation sites at positions 791, 967 and 1060 compared to the heavy chain of wild-type botulinum neurotoxin type A. The method includes the steps of subjecting a first peptide segment to a first denaturation treatment to obtain a first denaturation product, subjecting a second peptide segment to a second denaturation treatment to obtain a second denaturation product, and refolding and assembling the first denaturation product and the second denaturation product to obtain the botulinum neurotoxin type A variant.

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceutical technology, in particular, the present invention relates to recombinant botulinum neurotoxin type A and its preparation method, in particular, the present invention relates to botulinum neurotoxin type A, its variants and preparation method. [Background technology]

[0002] Botulinum neurotoxin (BoNT) is a neurotoxin produced by the anaerobic bacterium Clostridium botulinum (abbreviated as "Clothian botulinum") and is one of the most toxic substances in the world. Botulinum neurotoxin is divided into seven serotypes (types A-G), with type A being the most common, and is called botulinum neurotoxin type A (BoNT / A).

[0003] BoNT / A is divided into two parts: a light chain (LC, 50 kD) and a heavy chain (HC, 100 kD), which are connected by a pair of interchain disulfide bonds (C430-C454) and a non-amide bond. The light chain is the active domain, possessing zinc-dependent metalloendopeptidase activity and the toxic moiety of botulinum neurotoxin. The heavy chain contains two domains: a binding domain and a translocation domain. The binding domain binds to the corresponding receptor on the neuronal membrane and forms an ion channel on the inner membrane. The translocation domain translocates the light chain, transporting it into the cell. The light chain recognizes and specifically cleaves the Q197-R198 site on SNAP-25 (synaptic vesicle-associated protein).

[0004] Numerous studies have shown that BoNT / A has many applications, with broad prospects for application not only in medical cosmetology but also in the treatment of many diseases, and its range of disease treatment is expanding with its clinical application in areas such as various muscle tension disorders, palmar hyperhidrosis, pain, and many other intractable diseases. The therapeutic mechanism of BoNT / A is that at the neuromuscular junction, BoNT / A binds to the corresponding receptor on the surface of nerve cells, penetrates the membrane via the N-terminus of its heavy chain, transports the light chain into the cell, and cleaves SNAP-25, blocking the release of acetylcholine, causing continuous flaccid paralysis of the muscles.

[0005] The native active BoNT / A protein has one pair of disulfide bonds (C1235-C1280) within the heavy chain and one pair of disulfide bonds (C430-C454) between the light and heavy chains. In addition to the four cysteines that form intrachain and interchain disulfide bonds, there are five free cysteines (unpaired disulfide bonds, hereinafter the same) in the molecular structure of botulinum neurotoxin type A. During the preparation of recombinant botulinum neurotoxin type A, disulfide bond mismatches are likely to occur, resulting in the formation of botulinum neurotoxin type A proteins with incorrect structures, which will reduce the activity of botulinum neurotoxin type A and may even cause an immune response after administration to the human body.

[0006] Therefore, it is urgent to develop a type A botulinum neurotoxin with precise spatial structure and high biological activity. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention solves at least to a certain extent one of the technical problems in the related art, and therefore provides a type A botulinum neurotoxin variant that has advantages such as a low disulfide bond mismatch rate and high biological activity (toxicity).

[0008] The present invention has been completed based on the following findings of the inventors.

[0009] The active BoNT / A molecule in its native structure consists of 1,296 amino acids, divided into a light chain and a heavy chain. The light chain contains 448 amino acids (1-448), and the heavy chain contains 848 amino acids (449-1296). A pair of disulfide bonds (C1235-C1280) form within the heavy chain, located near the C-terminus of the heavy chain amino acid sequence. A pair of disulfide bonds (C430-C454) form between the light and heavy chains, and the cysteines that make up these disulfide bonds are located near the C-terminus of the light chain amino acid sequence and near the N-terminus of the heavy chain amino acid sequence, respectively. These disulfide bonds form the correct architecture of the complete BoNT / A molecule between the light and heavy chains, allowing the BoNT / A molecule to bind to receptors, be transported across membranes, and exert its biological effects, such as toxicity.

[0010] In addition to the four cysteines that make up the disulfide bond within the heavy chain (C1235-C1280) and the disulfide bond between the light and heavy chains (C430-C454), other free cysteines remain unpaired in the primary structure of the light and heavy chains. The production of botulinum type A neurotoxin by Clostridium botulinum in nature is a slow process, and the molecular mechanisms underlying the formation of the native structure of botulinum neurotoxin tend to result in the correct conformation of the native type A botulinum neurotoxin. However, the artificial cultivation of Clostridium botulinum or the use of other expression methods (e.g., expression in Escherichia coli) to prepare BoNT / A artificially alters the natural growth pattern of Clostridium botulinum or the rate of BoNT / A formation in other host cells, resulting in some intracellular BoNT / A failing to form the correct conformation consistent with its natural state, thereby affecting the toxicity of BoNT / A. The reason for this is that, in addition to the action of intermolecular forces (e.g., hydrogen bonds, van der Waals forces, hydrophobic, interionic forces, etc.), spatial mismatches between free cysteines form incorrect disulfide bonds, which changes the spatial structure conformation of BoNT / A to a certain extent, so that the heavy chain C-terminal receptor binding region, heavy chain N-terminal transition region, or light chain enzymatic active region of the complete BoNT / A molecule no longer have the higher-order spatial conformation of the subunit structure of the natural BoNT / A molecule, thereby reducing or weakening the biological effect of the BoNT / A molecule in vivo.Finally, the change in the higher-order structure or spatial conformation of the complete molecule or subunit will lead to the production of anti-protein antibodies (APA) against these non-native higher-order structures or spatial conformations after administration to the human body.

[0011] The formation of intramolecular forces is closely related to the transcription rate, translation efficiency, and redox environment within the host cell during the formation of the BoNT / A molecule. A fast or inconsistent transcription rate, translation efficiency, and an inappropriate intracellular environment can easily lead to inaccurate BoNT / A molecule structure. During the regeneration and assembly process of the molecule in vivo, the presence of free cysteines can easily cause disulfide bond mismatches between cysteine ​​molecules. This can occur between cysteines free at their native positions, between cysteines free at their native positions and those with pairing sites at their native positions, or between cysteines with different pairing sites at their native positions. In either case, this can cause a certain degree of change in the conformation of the BoNT / A molecule, affecting its biological effects in vivo and increasing the APA for these "non-native" structures after administration to the human body, resulting in reduced efficacy. [Means for solving the problem]

[0012] Based on this, in one aspect, the present invention provides a type A botulinum neurotoxin variant. According to an embodiment of the present invention, the type A botulinum neurotoxin variant comprises a first peptide segment and a second peptide segment, the first peptide segment is linked to the second peptide segment by an interchain disulfide bond, and the first peptide segment has a mutation at positions 134 and / or 165 compared to the light chain of wild-type type A botulinum neurotoxin, and / or the second peptide segment has at least one mutation at positions 791, 967, and 1060 compared to the heavy chain of wild-type type A botulinum neurotoxin. The inventors analyzed the molecular structure of natural BoNT / A and conducted extensive experiments, and found that by mutating the above amino acids of type A botulinum neurotoxin, disulfide bond mismatches can be reduced and its biological activity can be improved.

[0013] In another aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes a first peptide segment and / or a second peptide segment of the botulinum neurotoxin type A variant. The nucleic acid molecule according to an embodiment of the present invention can encode the botulinum neurotoxin type A variant.

[0014] In yet another aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule. The inventors have found through experiments that, after transforming the expression vector into a suitable host bacterium, the expression of a botulinum neurotoxin type A mutant can be effectively achieved under the mediation of a regulatory system, and the botulinum neurotoxin type A mutant can be obtained in large quantities.

[0015] In yet another aspect, the present invention provides a genetically engineered bacterium. According to an embodiment of the present invention, the genetically engineered bacterium carries the nucleic acid molecule or the expression vector, or expresses the botulinum neurotoxin type A mutant. The inventors have experimentally discovered that the genetically engineered bacterium can efficiently express the botulinum neurotoxin type A mutant by culturing the genetically engineered bacterium under appropriate conditions.

[0016] In yet another aspect, the present invention provides a method for preparing botulinum neurotoxin type A by genetic recombination, which includes the steps of: subjecting a light chain protein to a first denaturation treatment to obtain a first denatured product; subjecting a heavy chain protein to a second denaturation treatment to obtain a second denatured product; and refolding and assembling the first and second denatured products to obtain the botulinum neurotoxin type A.

[0017] The preparation method of the present invention enables the preparation of a single-subtype, single-component BoNT / A protein with the correct conformation, free of sequence heterologs, which is free of non-BoNT / A components such as HA and NTNH contained in Clostridium botulinum extract, and enzyme residues generated when the complete BoNT / A protein is expressed and the botulinum neurotoxin is activated with proteases, thereby offering advantages such as consistent quality between batches and making it suitable for commercial-scale production. Furthermore, the preparation method is simple, efficient, and has a short preparation flow, with the entire process completed in just eight steps and 4-5 days.

[0018] In yet another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition contains the above-mentioned botulinum neurotoxin type A variant. The pharmaceutical composition of the present invention has high biological activity (toxicity) and can be used not only for medical cosmetics but also for the treatment or improvement of diseases such as muscle tension disorders, hyperhidrosis of the hands and feet, or pain.

[0019] In yet another aspect, the present invention provides use of the type A botulinum neurotoxin variant, the type A botulinum neurotoxin prepared by the method, or the pharmaceutical composition in the preparation of a drug, wherein the drug is used for medical cosmetic purposes or for the treatment or amelioration of at least one of strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0020] In yet another aspect, the present invention provides use of the type A botulinum neurotoxin variant, the type A botulinum neurotoxin prepared by the method, or the pharmaceutical composition in medical cosmetic treatment or the treatment or amelioration of a disease, wherein the disease includes at least one of strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0021] In yet another aspect, the present invention provides the above-mentioned botulinum neurotoxin type A variant, the botulinum neurotoxin type A prepared by the above-mentioned method, or the above-mentioned pharmaceutical composition for use in medical cosmetic treatment or for the treatment or amelioration of a disease, wherein the disease includes at least one of strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0022] In yet another aspect, the present invention provides a method for ameliorating and / or treating a disease, comprising administering to a subject a pharmaceutically acceptable amount of the botulinum neurotoxin type A variant, the botulinum neurotoxin type A prepared by the method, or the pharmaceutical composition, wherein the disease includes at least one of strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0023] In yet another aspect, the present invention provides a medical cosmetic method, which, according to an embodiment of the present invention, comprises administering to a subject a pharmaceutically acceptable amount of the botulinum neurotoxin type A variant, the botulinum neurotoxin type A prepared by the method, or the pharmaceutical composition. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows an SDS-PAGE image of BoNT / A-LC and BoNT / A-HC expression and inclusion bodies in Example 2 of the present invention. [Figure 2] FIG. 10 is an SDS-PAGE image of a sample after regeneration and assembly in Example 2 of the present invention. [Figure 3] FIG. 1 is an SDS-PAGE image of the BoNT / A protein in Example 2 of the present invention. [Figure 4] 1 shows the SEC chromatographic purity of the BoNT / A protein in Example 2 of the present invention. [Figure 5] FIG. 1 is a complete molecular weight data analysis diagram for the structural identification in Example 3 of the present invention. [Figure 6] FIG. 10 is a diagram showing an analysis of reduced molecular weight data in the structural identification of Example 3 of the present invention. [Figure 7] FIG. 1 is a diagram showing the disulfide bond position analysis in the structural identification of Example 3 of the present invention. [Figure 8] 1 shows agarose gel electrophoresis of two enzyme-digested light chain mutant plasmids and two enzyme-digested heavy chain mutant plasmids in Example 6 of the present invention. [Figure 9] 1 shows the positions of cysteines and disulfide bond linkages in a wild-type botulinum neurotoxin type A in Example 7 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] Examples of the present invention will be described in detail below. The examples described below are illustrative and are used only to explain the present invention and should not be understood as limiting the present invention. If specific techniques or conditions are not specified in the examples, they will be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specifications. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products available on the market.

[0026] It should be noted that the terms "first" and "second" are for explanatory purposes only and cannot be considered to indicate or imply relative importance or the number of technical features. Therefore, a feature qualified as "first" or "second" can expressly or imply the inclusion of one or more of the feature, and in the description of the present invention, unless otherwise specified, the concept of "plurality" is two or more than two.

[0027] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. Numerical ranges can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0028] To facilitate understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless expressly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] Here, the terms "comprise" or "include" are non-limiting expressions, that is, they include the contents shown in the present invention but do not exclude the contents of other embodiments.

[0030] As used herein, the terms "optionally," "optionally," or "optionally" generally mean that the described event or circumstance may occur, but does not necessarily occur, and this description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur.

[0031] As used herein, the amino acid numbers of the botulinum neurotoxin type A are numbered according to the EU numbering system, for example, position 134 refers to position 134 according to the EU numbering system, "C134G" means that the cysteine ​​at position 134 according to the EU numbering system is replaced with glycine, and "C791A" means that the cysteine ​​at position 791 according to the EU numbering system is replaced with alanine.

[0032] As used herein, the term "expression vector" generally refers to a vector of a nucleic acid molecule that can be inserted into a suitable host or inserted into a suitable host and self-replicated, thereby transferring the inserted nucleic acid molecule within and / or between host bacteria. Expression vectors may include vectors primarily used to insert DNA or RNA into cells, vectors primarily used to replicate DNA or RNA, and vectors primarily used for transcription and / or translation of DNA or RNA, preferably DNA. Expression vectors also include vectors with multiple functions, such as those described above. Examples of expression vectors include, but are not limited to, plasmids, linear DNA fragments, viruses, bacterial phages, proviruses, phages, transposons, and artificial chromosomes. The expression vector contains a target gene fragment, and when transformed into a suitable host organism, the target gene fragment can be transcribed and translated into amino acids that constitute a polypeptide. Typically, the expression vector can produce a desired expression product by culturing a suitable host bacteria containing the expression vector.

[0033] As used herein, the term "genetically engineered bacteria" generally refers to a bacterium in which a gene of interest is transformed into a host bacterium to express and produce a desired protein. Here, the term "host bacterium" refers to a bacterium or cell, such as E. coli, that can be transformed with a recombinant expression vector.

[0034] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical arts. All methods include combining the active ingredient with a vector that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid vector, a finely divided solid vector, or both.

[0035] As used herein, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, suitable for a particular intended dosage form. To the extent that any conventional excipient is incompatible with the compounds of the present invention, e.g., causing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0036] As used herein, the term "treatment" means the use of a drug to achieve a desired pharmacological and / or physiological effect. The effect may be therapeutic in that it completely or partially treats or ameliorates a disease or its symptoms, and / or in that it partially or completely cures a disease and / or the adverse effects of a disease. As used herein, "treatment" covers mammalian, particularly human, diseases and includes (a) preventing the disease or onset of a disease in an individual who is susceptible to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease, e.g., slowing the progression of the disease, or (c) alleviating the disease, e.g., reducing symptoms associated with the disease. As used herein, "treatment" covers any drug or compound administered to an individual to treat, cure, alleviate, ameliorate, mitigate, or inhibit a disease in an individual, including, but not limited to, administering a drug or compound described herein to an individual in need thereof.

[0037] As used herein, the terms "BoNT / A," "BoNT / A protein," "BoNT / A component," "BoNT / A molecule," and "botulinum neurotoxin type A protein" all refer to botulinum neurotoxin type A.

[0038] As used herein, the terms "LC," "BoNT / A-LC," "BoNT / A-LC protein," "light chain protein," and "light chain" have the same meaning. The terms "HC," "BoNT / A-HC," "BoNT / A-HC protein," "heavy chain protein," and "heavy chain" have the same meaning. The terms "light chain protein" and "light chain" have the same meaning. The terms "heavy chain protein" and "heavy chain" have the same meaning. The terms "light chain mutant protein" and "light chain mutant" have the same meaning, and the terms "heavy chain mutant protein" and "heavy chain mutant" have the same meaning.

[0039] The present invention provides botulinum neurotoxin type A mutants, nucleic acid molecules, expression vectors, genetically engineered bacteria, methods for preparing botulinum neurotoxin type A by genetic recombination, pharmaceutical compositions and uses thereof, each of which is described in detail below.

[0040] Botulinum neurotoxin type A mutants

[0041] In one aspect, the present invention provides a type A botulinum neurotoxin variant. According to an embodiment of the present invention, the type A botulinum neurotoxin variant comprises a first peptide segment and a second peptide segment, the first peptide segment being linked to the second peptide segment by an interchain disulfide bond, the first peptide segment having a mutation at positions 134 and / or 165 compared to the light chain of wild-type type A botulinum neurotoxin, and / or the second peptide segment having at least one mutation at positions 791, 967, and 1060 compared to the heavy chain of wild-type type A botulinum neurotoxin. The inventors analyzed the molecular structure of natural BoNT / A and, through extensive experiments, discovered that by mutating the above amino acids of type A botulinum neurotoxin, the disulfide bond mismatch can be reduced and the type A botulinum neurotoxin variant has high biological activity (toxicity).

[0042] The light chain amino acid sequence of wild-type botulinum neurotoxin type A is SEQ ID NO: 1: MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVR GIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELI HAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFD KLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNK(SEQ ID NO: 1), The heavy chain amino acid sequence of wild-type botulinum neurotoxin type A is SEQ ID NO: 2: (SEQ ID NO: 2).

[0043] The nucleotide sequence encoding the wild-type botulinum neurotoxin type A light chain is SEQ ID NO: 7: The nucleotide sequence encoding the wild-type botulinum neurotoxin type A heavy chain is SEQ ID NO: 8:

[0044] According to an embodiment of the present invention, the interchain disulfide bond is formed by a cysteine ​​at position 430 in the first peptide segment and a cysteine ​​at position 454 in the second peptide segment.

[0045] According to an embodiment of the present invention, the first peptide segment has mutations at positions 134 and 165 compared to the light chain of wild-type botulinum neurotoxin type A, and / or the second peptide segment has mutations at positions 791, 967, and 1060 compared to the heavy chain of wild-type botulinum neurotoxin type A. This reduces the mismatch rate of disulfide bonds in the botulinum neurotoxin type A variant and improves its biological activity.

[0046] According to an embodiment of the present invention, the cysteine ​​mutation at position 134, 165, 791, 967, or 1060 is one of the amino acids G, A, S, E, and P. This can further reduce the disulfide bond mismatch rate in the botulinum neurotoxin type A mutant and improve its biological activity.

[0047] According to an embodiment of the present invention, the C mutation at position 134 of said first peptide segment is G, A or S.

[0048] In one preferred embodiment of the present invention, the C mutation at position 134 of the first peptide segment is G.

[0049] In one preferred embodiment of the present invention, the C mutation at position 134 of the first peptide segment is an A.

[0050] In one preferred embodiment of the invention, the C mutation at position 134 of the first peptide segment is S.

[0051] According to an embodiment of the present invention, the C mutation at position 165 of said first peptide segment is G, A, P or S.

[0052] In one preferred embodiment of the invention, the C mutation at position 165 of the first peptide segment is G.

[0053] In one preferred embodiment of the invention, the C mutation at position 165 of the first peptide segment is an A.

[0054] In one preferred embodiment of the invention, the C mutation at position 165 of the first peptide segment is a P.

[0055] In one preferred embodiment of the invention, the C mutation at position 165 of the first peptide segment is S.

[0056] According to an embodiment of the present invention, the C mutation at position 791 of said second peptide segment is G, A or S.

[0057] In one preferred embodiment of the present invention, the C mutation at position 791 of the second peptide segment is G.

[0058] In one preferred embodiment of the present invention, the C mutation at position 791 of the second peptide segment is an A.

[0059] In one preferred embodiment of the invention, the C mutation at position 791 of the second peptide segment is S.

[0060] According to an embodiment of the present invention, the C mutation at position 967 of said second peptide segment is G, A or S.

[0061] In one preferred embodiment of the invention, the C mutation at position 967 of the second peptide segment is G.

[0062] In one preferred embodiment of the invention, the C mutation at position 967 of the second peptide segment is an A.

[0063] In one preferred embodiment of the invention, the C mutation at position 967 of the second peptide segment is S.

[0064] According to an embodiment of the present invention, the C mutation at position 1060 of said second peptide segment is G, A, E or S.

[0065] In one preferred embodiment of the invention, the C mutation at position 1060 of the second peptide segment is G.

[0066] In one preferred embodiment of the invention, the C mutation at position 1060 of the second peptide segment is an A.

[0067] In one preferred embodiment of the invention, the C mutation at position 1060 of the second peptide segment is E.

[0068] In one preferred embodiment of the invention, the C mutation at position 1060 of the second peptide segment is S.

[0069] According to an embodiment of the present invention, the C mutation at position 165 of said first peptide segment is P, or the C mutation at position 1060 of said second peptide segment is E.

[0070] Through extensive testing, the inventors have discovered that type A botulinum neurotoxin mutants obtained by mutating the above-mentioned sites of the first peptide segment and the second peptide segment to amino acids G, A, or S in the process of mutating the above-mentioned sites have reduced disulfide bond mismatches and improved biological activity (toxicity) compared to wild-type type A botulinum neurotoxin, such as type A botulinum neurotoxin mutant 2 in Tables 12 and 14. Furthermore, the inventors unexpectedly discovered that, by using the above mutation method and taking into full consideration the composition of the peptide chain near the mutation site, such as the size, hydrophobicity, possible hydrogen bond formation, and charge state of the amino acid, by mutating cysteines (C165, C1060) that are likely to cause disulfide bond mismatches in wild-type type A botulinum neurotoxin to P or E, disulfide bond mismatches are more significantly eliminated, and even completely eliminated, as in type A botulinum neurotoxin variant 1 in Tables 11 and 14, and the biological activity is also significantly improved, being more than 1-fold higher than that of the wild-type type A botulinum neurotoxin.

[0071] According to an embodiment of the present invention, the first peptide segment has mutations C134G and C165P compared with the light chain of wild-type botulinum neurotoxin type A, which can further reduce the disulfide bond mismatch rate in the botulinum neurotoxin type A variant and improve its biological activity.

[0072] According to an embodiment of the present invention, the second peptide segment has mutations C791A, C967A, and C1060E compared to the heavy chain of wild-type botulinum neurotoxin type A, which further reduces the mismatch rate of disulfide bonds in the botulinum neurotoxin type A variant and improves its biological activity.

[0073] According to an embodiment of the present invention, the first peptide segment has mutations C134G and C165P compared to the light chain of wild-type botulinum neurotoxin type A, and the second peptide segment has mutations C791A, C967A, and C1060E compared to the heavy chain of wild-type botulinum neurotoxin type A. The above-mentioned botulinum neurotoxin type A mutant has higher toxicity than the wild-type botulinum neurotoxin type A.

[0074] According to an embodiment of the present invention, the first peptide segment has the amino acid sequence shown in SEQ ID NO: 3 or 5. MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVR GIPFWGGSTIDTELKVIDTNGINVIQPDGSYRSEELNLVIIGPSADIIQFEPKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELI HAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFD KLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNK(SEQ ID NO: 3), MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVR GIPFWGGSTIDTELKVIDTNGINVIQPDGSYRSEELNLVIIGPSADIIQFEGKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELI HAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFD KLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNK(SEQ ID NO: 5).

[0075] According to an embodiment of the present invention, the second peptide segment has the amino acid sequence shown in SEQ ID NO:4 or 6. ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQASVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINAMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGERDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL(SEQ ID NO: 4); ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQASVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINAMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGGRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL(SEQ ID NO: 6)。

[0076] In some alternative embodiments of the present invention, the variant botulinum neurotoxin type A comprises a first peptide segment having the amino acid sequence set forth in SEQ ID NO:3 and a second peptide segment having the amino acid sequence set forth in SEQ ID NO:4, or the variant botulinum neurotoxin type A comprises a first peptide segment having the amino acid sequence set forth in SEQ ID NO:5 and a second peptide segment having the amino acid sequence set forth in SEQ ID NO:6.

[0077] Nucleic acid molecules, expression vectors and genetically engineered bacteria

[0078] In a second aspect of the present invention, there is provided a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes a first peptide segment and / or a second peptide segment of a botulinum neurotoxin type A variant according to the first aspect. The nucleic acid molecule of the present invention can encode the botulinum neurotoxin type A variant.

[0079] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0080] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect. After the expression vector of the present invention is transformed into a suitable host bacterium, it can effectively express the botulinum neurotoxin type A mutant under the control of a regulatory system, thereby enabling the production of large amounts of the botulinum neurotoxin type A mutant of the first aspect.

[0081] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0082] In a fourth aspect, the present invention provides a genetically engineered bacterium. According to an embodiment of the present invention, the genetically engineered bacterium carries the nucleic acid molecule of the second aspect or the expression vector of the third aspect, or expresses the botulinum neurotoxin type A mutant of the first aspect. Under appropriate conditions, the genetically engineered bacterium of the present invention can efficiently express the botulinum neurotoxin type A mutant of the first aspect.

[0083] According to an embodiment of the present invention, the genetically engineered bacterium is obtained by transforming an expression vector according to the third aspect into a host bacterium.

[0084] According to an embodiment of the present invention, the host bacterium is Escherichia coli.

[0085] Method for preparing botulinum neurotoxin type A by genetic recombination

[0086] In a fifth aspect, the present invention provides a method for preparing a botulinum neurotoxin type A by genetic recombination. According to an embodiment of the present invention, the method includes the steps of: subjecting a light chain protein to a first denaturation treatment to obtain a first denatured product; subjecting a heavy chain protein to a second denaturation treatment to obtain a second denatured product; and refolding and assembling the first denatured product and the second denatured product to obtain the botulinum neurotoxin type A.

[0087] The inventors have studied and analyzed the higher-order structure and protein properties of BoNT / A protein, and through extensive experiments, discovered that a fully active type A botulinum neurotoxin can be finally formed by denaturing the light and heavy chain proteins and then refolding and assembling them in vitro. This method does not require the use of additional proteases for toxic activation, thereby avoiding the residue of exogenous tool enzymes and the generation of inactive isomerized impurities due to incomplete or nonspecific cleavage. At the same time, it does not require the expression of the entire BoNT / A protein molecule alone, thereby avoiding the tendency and risk of the protein molecule being too large to form an incorrect higher-order structure. Therefore, this method can prepare a single-subtype, single-component BoNT / A, which is free from non-BoNT / A components such as HA, NTNH, and enzyme residues generated when the entire BoNT / A protein is expressed and activated with proteases, resulting in consistent quality between batches.

[0088] According to an embodiment of the invention, the light chain protein comprises a first peptide segment according to the first aspect or has the amino acid sequence shown in SEQ ID NO:1, and / or the heavy chain protein comprises a second peptide segment according to the first aspect or has the amino acid sequence shown in SEQ ID NO:2.

[0089] In this context, the term "botulinum neurotoxin type A" may include wild-type botulinum neurotoxin type A, or may include a botulinum neurotoxin type A mutant.

[0090] According to an embodiment of the present invention, the light chain protein has the amino acid sequence shown in SEQ ID NO:1, and the heavy chain protein has the amino acid sequence shown in SEQ ID NO:2.

[0091] According to an embodiment of the present invention, the light or heavy chain protein can be obtained by transforming Escherichia coli with a plasmid carrying a gene encoding the light or heavy chain protein, culturing the Escherichia coli transformed with the plasmid under conditions suitable for protein expression, and obtaining the light or heavy chain protein through processes such as induction, cell collection, cell disruption, and centrifugation, thereby obtaining the light or heavy chain protein that constitutes the BoNT / A protein molecule.

[0092] According to an embodiment of the present invention, the first denaturation treatment or the second denaturation treatment is carried out in a denaturation buffer containing 5-10 M urea, 5-15 mM dithiothreitol, and 10-30 mM Tris or Tris-HCl. The inventors have obtained the above preferred blend ratio through extensive experiments, which allows the amino acid chains of the light chain protein and the heavy chain protein to be fully extended.

[0093] According to an embodiment of the present invention, the pH value of the denaturing buffer solution is 9.5 to 10.5, which is more advantageous for extending the amino acid chains of the light and heavy chain proteins.

[0094] According to an embodiment of the present invention, before the renaturation and assembly processes, the first renatured product and the second renatured product are mixed to obtain a renatured mixture. Through extensive experiments, the inventors have found that by mixing the first renatured product and the second renatured product and then renaturing them simultaneously, single-strand renaturation and assembly between heavy and light chains can be performed simultaneously.

[0095] According to an embodiment of the present invention, the volume ratio of the first modified product to the second modified product in the mixing process is 1:(1-10).

[0096] According to an embodiment of the present invention, the regeneration and assembly process is carried out in a regeneration and assembly buffer, which contains 50-150 mM NaCl, 0.1-1.0 mM ZnCl, 0.1-1.0 mM CaCl, 1.0-10.0 mM reduced glutathione (GSH), 1.0-10.0 mM oxidized glutathione (GSSG), 40-50 mM Tris-HCl, and 0.4-0.6% (w / v) Tween-20. After extensive screening experiments, the inventors obtained the above-mentioned preferred mixing ratio, and by using this refolding and assembly buffer to fold and assemble the denatured, extended light chain protein and heavy chain protein, the mismatch rate of disulfide bonds within the light chain or heavy chain protein and between the light chain protein and heavy chain protein can be reduced during the folding and assembly process, resulting in a high correct refolding rate of single chains (light chain, heavy chain) and an assembly efficiency between two chains, and a high content of the target protein (BoNT / A) in the assembly solution. The subsequently prepared BoNT / A protein with correct intrachain disulfide bonds and interchain disulfide bonds has higher purity and better activity (toxicity).

[0097] According to an embodiment of the present invention, the pH value of the renaturation and assembly buffer is 9.5 to 10.5, which can improve the accurate pairing rate of disulfide bonds within the heavy chain and between the light and heavy chains, and increase the content of the target protein in the assembly solution.

[0098] According to an embodiment of the present invention, the volume ratio of the denaturation mixture to the renaturation and assembly buffer is 1:(1 to 10). The inventors have obtained this preferred mixture ratio through extensive experiments, which can improve the accurate pairing rate of disulfide bonds within the heavy chain and between the light and heavy chains during the renaturation and assembly process, and increase the content of the target protein in the assembly solution.

[0099] According to an embodiment of the present invention, the refolding and reassembly treatment time is 12 to 16 hours. The inventors have obtained the above-mentioned preferable refolding and reassembly treatment conditions through extensive experiments, which are advantageous for accurate connection of disulfide bonds within the heavy chain protein or between the light chain protein and the heavy chain protein, and form a botulinum neurotoxin type A with a correct higher-order structure.

[0100] According to an embodiment of the present invention, the stirring speed during the renaturation and assembly treatment is 50 to 200 rpm. The inventors have obtained the above-mentioned preferable renaturation and assembly treatment conditions through extensive experiments, which are advantageous for accurate connection of disulfide bonds within the heavy chain protein or between the light chain protein and the heavy chain protein, and form a type A botulinum neurotoxin with a precise higher-order structure during assembly.

[0101] According to an embodiment of the present invention, the method further includes sequentially subjecting the assembly solution obtained from the regeneration and assembly processes to hydrophobic chromatography, ammonium sulfate salting-out, dialysis, anion chromatography, and molecular sieve chromatography to obtain the type A botulinum neurotoxin. The inventors have conducted extensive experiments to determine the above purification steps and found that the above purification steps and purification system are effective in removing impurities from the assembly solution, and that the purity of the resulting type A botulinum neurotoxin can reach 98.0% or more. The inventors have also found that changing the order of the above five purification processes or eliminating one or more of them can significantly reduce the purity of the final type A botulinum neurotoxin.

[0102] According to an embodiment of the present invention, mobile phase A1 in the hydrophobic chromatography treatment contains 10-30 mmol / L Tris-HCl, 2-8 mmol / L EDTA, and 1-3 mol / L NaCl, and mobile phase B1 contains 10-30 mmol / L Tris and 2-8 mmol / L EDTA, with the pH values ​​of both mobile phase A1 and mobile phase B1 being 8.0-9.0. The inventors have obtained the above preferred purification conditions through extensive experiments, which result in good purification efficiency for an assembly solution containing type A botulinum neurotoxin.

[0103] According to an embodiment of the present invention, the ammonium sulfate salting-out treatment includes slowly adding ammonium sulfate to the eluate obtained by hydrophobic chromatography until the ammonium sulfate saturation concentration reaches 80%, stirring the mixture at 2-8°C for 12-24 hours to obtain a salting-out solution, and then centrifuging the salting-out solution at 2-8°C and 10,000-15,000 rpm for 20-40 minutes to discard the supernatant and obtain a precipitate. The inventors have determined the above-mentioned preferable purification conditions through extensive experiments, which can further increase the purity of botulinum neurotoxin type A.

[0104] According to an embodiment of the present invention, the dialysis treatment includes dissolving the precipitate in a first buffer solution to obtain a dialysis target solution, performing a first dialysis treatment on the dialysis target solution and the first dialysis target solution, and performing a second dialysis treatment on the obtained first dialysis target solution and a second dialysis target solution, wherein the first buffer solution is selected from 40 to 60 mM Tris-HCl buffer solutions, the first dialysis target solution is selected from 40 to 60 mM Tris-HCl salt-containing dialysis targets, and the second dialysis target solution is selected from 40 to 60 mM Tris-HCl dialysis targets. The inventors have determined the above preferred purification conditions through extensive experiments, which result in high purity of the obtained type A botulinum neurotoxin. Furthermore, through extensive experiments, the inventors further discovered that the presence of salt in the first dialysis process can ensure the sustained solubility of the dissolved protein while simultaneously preventing non-specific adsorption of impurities onto the surface of the target protein, and therefore selecting a salt-containing dialysate as the first dialysis solution can further increase the purity of the botulinum neurotoxin type A obtained by dialysis.

[0105] The term "dialysis solution containing 40 to 60 mM Tris-HCl salt" refers to a Tris-HCl dialysis solution containing salt, and the concentration of Tris-HCl in the dialysis solution is 40 to 60 mM.

[0106] According to an embodiment of the present invention, the ratio of the weight of the precipitate (g) to the volume of the first buffer solution (ml) is 1:(5 to 15). The inventors have obtained this preferable blending ratio through extensive experiments, which can further increase the purity of botulinum neurotoxin type A.

[0107] According to an embodiment of the present invention, the dialysis time of the first dialysis treatment is 2 to 5 hours, and the dialysis time of the second dialysis treatment is 12 to 24 hours, thereby improving the effect of the dialysis treatment.

[0108] According to an embodiment of the present invention, the Tris-HCl salt-containing dialysis solution further comprises 200-300 mM NaCl.

[0109] According to an embodiment of the present invention, the cut-off molecular weight of the dialysis bags for the first dialysis treatment and the second dialysis treatment is 80 to 120 kDa.

[0110] According to an embodiment of the present invention, the anionic chromatography treatment is selected from DEAE-cellulose anionic chromatography.

[0111] According to an embodiment of the present invention, the mobile phase A2 of the DEAE cellulose anion chromatography contains 10-30 mmol / L Tris-HCl, and the mobile phase B2 contains 10-30 mmol / L Tris-HCl and 0.5-1.5 mol / L NaCl, and the pH values ​​of the mobile phases A2 and B2 are 8.5. The inventors have obtained the above preferred purification conditions through extensive experiments, which can further improve the purity of botulinum neurotoxin type A.

[0112] According to an embodiment of the present invention, the molecular sieve chromatography treatment is carried out using G-25M molecular sieve chromatography.

[0113] According to an embodiment of the present invention, the mobile phase C of the G-25M molecular sieve chromatography contains 10 to 30 mmol / L Tris-HCl, and the pH value of the mobile phase C is 8.0 to 9.0. The inventors have obtained the above preferred purification conditions through extensive experiments, which can further improve the purity of botulinum neurotoxin type A.

[0114] According to an embodiment of the present invention, the loading of the G-25M molecular sieve chromatography is ≦30% column volume / cycle, and the linear flow rate is 250-350 cm / h.

[0115] In a sixth aspect, the present invention provides a type A botulinum neurotoxin or a type A botulinum neurotoxin variant. According to an embodiment of the present invention, the type A botulinum neurotoxin is prepared by the method described in the fifth aspect. The inventors have experimentally discovered that the method described in the fifth aspect can be used to prepare a single-subtype, single-component BoNT / A with the correct conformation, which is free of non-BoNT / A components such as HA, NTNH, and enzyme residues, and has advantages such as lot-to-lot quality stability.

[0116] In a seventh aspect of the present invention, the present invention provides a botulinum neurotoxin type A. According to an embodiment of the present invention, the LD of the botulinum neurotoxin type A is 50 The purity of the botulinum neurotoxin type A is 98% or more.

[0117] According to an embodiment of the present invention, the light chain protein of the botulinum neurotoxin type A has the amino acid sequence shown in SEQ ID NO:1.

[0118] According to an embodiment of the present invention, the heavy chain protein of the botulinum neurotoxin type A has the amino acid sequence shown in SEQ ID NO:2.

[0119] Pharmaceutical Composition

[0120] In an eighth aspect of the present invention, a pharmaceutical composition is provided. According to an embodiment of the present invention, the pharmaceutical composition comprises a type A botulinum neurotoxin variant according to the first aspect or a type A botulinum neurotoxin prepared by the method according to the fifth aspect. The pharmaceutical composition of the present invention has high biological activity (toxicity) and is used not only for medical cosmetic purposes, but also for the treatment or amelioration of at least one of strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0121] According to an embodiment of the present invention, the medical cosmetic treatment includes improving and / or treating at least one of the symptoms of frown lines, crow's feet, and forehead wrinkles.

[0122] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable adjuvant.

[0123] According to an embodiment of the present invention, the pharmaceutically acceptable auxiliary agent comprises at least one selected from a buffer, a protective agent, an active agent, and an excipient.

[0124] The term "buffer" generally refers to a liquid solution capable of buffering, and should be understood in a broad sense herein. Exemplary buffers may be physiologically compatible buffer systems and / or buffer system compositions, including, but not limited to, acetate, succinate, citric acid, histidine, glutamate, citrate / acetate, citrate / histidine, succinate / histidine, phosphate, and trimethylolaminomethane buffer systems.

[0125] The protective agent is generally a reagent that exerts a protective effect on the pharmaceutical composition, and should be understood in a broad sense here. Examples thereof include, but are not limited to, non-reducing sugars such as trehalose, sucrose, and human blood albumin.

[0126] According to an embodiment of the present invention, the surfactant is a non-ionic surfactant.

[0127] According to an embodiment of the present invention, the non-ionic surfactant comprises at least one selected from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0128] According to an embodiment of the present invention, the pharmaceutical composition is a liquid composition, the protective agent comprises a non-reducing sugar selected from trehalose and / or sucrose, and the non-ionic surfactant comprises at least one selected from polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

[0129] According to an embodiment of the present invention, the pharmaceutical composition is a freeze-dried composition, the protective agent comprises at least one selected from non-reducing sugars trehalose, sucrose, and human blood albumin, and the excipient is a polyhydric alcohol-based excipient.

[0130] use

[0131] In a ninth aspect of the present invention, there is provided use of the type A botulinum neurotoxin variant according to the first aspect, the type A botulinum neurotoxin prepared by the method according to the fifth aspect, or the pharmaceutical composition according to the eighth aspect in the preparation of a medicament, wherein the medicament is used for medical cosmetic purposes or for the treatment or amelioration of at least one of strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0132] According to an embodiment of the present invention, the medical cosmetic treatment includes wrinkle removal and / or facial slimming.

[0133] According to an embodiment of the present invention, the medical cosmetic treatment includes improving and / or treating at least one of the following conditions: glabellar lines, crow's feet, and forehead wrinkles.

[0134] In a tenth aspect of the present invention, the present invention provides a use of the type A botulinum neurotoxin variant, the type A botulinum neurotoxin prepared by the method, or the pharmaceutical composition for medical cosmetic or disease treatment or amelioration, wherein the disease includes at least one of strabismus, cervical dystonia, laryngeal dystonia, localized dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0135] According to an embodiment of the present invention, the medical cosmetic treatment includes wrinkle removal and / or facial slimming.

[0136] According to an embodiment of the present invention, the medical cosmetic treatment comprises improving and / or treating at least one of the symptoms of glabellar lines, crow's feet, and forehead wrinkles.

[0137] In an eleventh aspect of the present invention, the present invention provides the type A botulinum neurotoxin variant, the type A botulinum neurotoxin prepared by the method, or the pharmaceutical composition, which is used for medical cosmetic purposes or for the treatment or amelioration of diseases, including at least one of strabismus, cervical dystonia, laryngeal dystonia, localized dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0138] According to an embodiment of the present invention, the medical cosmetic treatment includes wrinkle removal and / or facial slimming.

[0139] According to an embodiment of the present invention, the medical cosmetic treatment comprises improving and / or treating at least one of the symptoms of glabellar lines, crow's feet, and forehead wrinkles.

[0140] method

[0141] In a twelfth aspect, the present invention provides a method for medical cosmetic treatment. According to some embodiments of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of a botulinum neurotoxin type A variant described in the first aspect, a botulinum neurotoxin type A prepared by the method described in the fifth aspect, or a pharmaceutical composition described in the eighth aspect. The method according to some embodiments of the present invention can be effectively used for medical cosmetic treatment.

[0142] According to an embodiment of the present invention, the medical cosmetic treatment includes wrinkle removal and / or facial slimming.

[0143] According to an embodiment of the present invention, the medical cosmetic treatment comprises improving and / or treating at least one of the symptoms of glabellar lines, crow's feet, and forehead wrinkles.

[0144] According to an embodiment of the present invention, the administration comprises subcutaneous or intramuscular injection.

[0145] In a thirteenth aspect, the present invention provides a method for ameliorating and / or treating a disease, according to an embodiment of the present invention, the method comprising the step of administering to a subject a pharmaceutically acceptable amount of the type A botulinum neurotoxin variant according to the first aspect, the type A botulinum neurotoxin prepared by the method according to the fifth aspect, or the pharmaceutical composition according to the eighth aspect, wherein the disease comprises at least one selected from strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

[0146] As used herein, the term "pharmaceutically acceptable amount" may vary depending on the mode of administration, the severity of the disease being treated, and the like, and is preferably an effective amount. The selection of a pharmaceutically acceptable amount can be determined by those skilled in the art based on various factors (e.g., clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life, the severity of the disease being treated in the patient, the patient's body weight, the patient's immune status, and the route of administration. For example, depending on the exigencies of the therapeutic situation, the active ingredient may be administered in multiple doses per day or the dosage may be proportionally reduced.

[0147] According to an embodiment of the present invention, the administration comprises subcutaneous or intramuscular injection.

[0148] Examples of the present invention will be described in detail below. The examples described below are illustrative and are used only to explain the present invention and should not be understood as limiting the present invention. If specific techniques or conditions are not specified in the examples, they will be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specifications. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products available on the market.

[0149] Example 1: Design and construction of wild-type botulinum neurotoxin type A light chain protein (BoNT / A-LC) and heavy chain protein (BoNT / A-HC) expression plasmids

[0150] 1. Design and synthesis of target genes Based on the amino acid sequence of the target protein, the nucleotide sequence of the target gene was designed and optimized based on the preferred codons of E. coli, and the nucleotide sequences of the light chain protein and heavy chain protein were determined. The designed nucleotide sequences were synthesized by Bao Biotechnology (Dalian) Co., Ltd. The amino acid sequence of the light chain protein is SEQ ID NO:1, the amino acid sequence of the heavy chain protein is SEQ ID NO:2, the nucleotide sequence encoding the light chain protein is SEQ ID NO:7, and the nucleotide sequence encoding the heavy chain protein is SEQ ID NO:8.

[0151] 2. Construction of expression plasmids and acquisition of target genetically engineered bacteria The light chain and heavy chain target genes have XbaI and BamHI enzyme cleavage sites at both ends, respectively. The light chain and heavy chain target gene sequences and the pET-28a(+) vector are cleaved with the XbaI and BamHI enzymes, respectively, and the resulting fragments are then ligated to the longer fragments of the pET-28a(+) vector after cleavage. After transformation and screening, genetically engineered bacteria expressing the light chain and heavy chain target proteins, respectively, are obtained.

[0152] Example 2: Preparation of wild-type botulinum neurotoxin type A

[0153] 1. Expression of light chain proteins and heavy chain proteins The genetically engineered bacteria carrying the light chain and heavy chain genes were cultured in shake flasks containing LB medium, and the OD 600 When the OD reached 1.6 to 2.0, the mixture was transferred to a 5 L fermenter and cultured at an initial culture volume of 2.5 L, a culture temperature of 37°C, and an agitation speed of 800 rpm. 600 Induction begins when the pH is increased to 30. The inducer is isopropyl-β-D-thiogalactoside (IPTG), the concentration is 0.5 mM, and the induction time is 4 to 8 hours.

[0154] The growth of the genetically engineered bacteria in the fermentation broth is examined under a microscope to observe the expression status. When protein expression is observed and the induction time is 4 to 8 hours, the fermentation broth is collected and centrifuged at 4°C and 10,000 rpm for 30 minutes to collect the genetically engineered bacteria.

[0155] The collected genetically engineered bacteria were homogenized under high pressure at 700-800 bar for at least two cycles, and then centrifuged to collect inclusion bodies until no intact cells were found under a microscope. The inclusion bodies were washed twice with ultra-clean water at a ratio of 1:20 (w / v, g / ml). The SDS-PAGE analysis of the expression of the light chain protein (BoNT / A-LC) and heavy chain protein (BoNT / A-HC) and the inclusion bodies is shown in Figure 1.

[0156] 2. Denaturation of BoNT / A-LC protein and BoNT / A-HC protein BoNT / A-LC and BoNT / A-HC inclusion bodies were weighed at a weight ratio of 1:4 at room temperature and dissolved in a denaturing buffer solution at a ratio of 1:20 (w / v, g / ml) at 200 rpm for 60 min until completely dissolved. The denaturing buffer solution contained 8 M urea, 10 mM dithiothreitol (DTT), and 20 mM Tris-HCl, with a pH of 10.0.

[0157] 3. Regeneration and in vitro assembly of BoNT / A protein Mix equal volumes of the dissolved light chain denatured product and heavy chain denatured product obtained in step 2 to obtain a denaturation mixture. Mix the denaturation mixture with the renaturation and assembly buffer in a volume ratio of 1:10, and continue stirring at 200 rpm to allow renaturation and assembly to occur overnight to obtain an assembly solution.

[0158] The composition of the renaturation and assembly buffer was 100 mM NaCl, 0.5 mM ZnCl2, 0.5 mM CaCl2, 5 mM GSH, 5 mM GSSG, 50 mM Tris-HCl and 0.5% Tween-20, with a pH value of 10.0.

[0159] A small amount of the assembly solution was taken and subjected to SDS-PAGE electrophoresis to observe the renaturation and assembly status. The results are shown in Figure 2.

[0160] Simultaneously, the target protein content was scanned and analyzed using Image Lab software on a gel electrophoresis imaging device (BIO-RAD, model: ChemiDoc™ XRS+). The analysis results showed that the target protein content obtained in this system (i.e., BoNT / A protein corresponding to number 4 in Table 1) was 30.2%. During the experimental process, to optimize the composition of the refolding and assembly buffer system, numerous component compositions and concentrations, such as metal ions, redox couples, and system pH, were explored. Furthermore, the same amounts and ratios of BoNT / A-LC protein and BoNT / A-HC protein were used in each experiment to ensure the reliability of the experimental results despite the experimental variables. The experimental results are shown in Table 1.

[0161] [Table 1-1] [Table 1-2]

[0162] 4. Purification of BoNT / A protein The assembled solution obtained in step 3 (assembled solution No. 4) is sequentially subjected to hydrophobic chromatography, ammonium sulfate salting out, dialysis, DEAE anion chromatography, and molecular sieve chromatography. The specific treatment process is as follows.

[0163] Hydrophobic Chromatography: Add 4 mol / L of NaCl to the assembly solution obtained in step 3 until the final NaCl concentration in the assembly solution is 2 mol / L. This is the loading stock solution. Chromatography conditions: The composition of mobile phase A1 is 20 mmol / L Tris + 5 mmol / L EDTA + 2.0 mol / L NaCl, pH 8.5. The composition of mobile phase B1 is 20 mmol / L Tris + 5 mmol / L EDTA, pH 8.5. Chromatography steps: Equilibrate three column volumes (CV) with mobile phase A1, load to the loading capacity, wash with mobile phase A1 for 6 CV, and perform a 10 CV gradient elution with mobile phase B1 from 0% to 100%. Collect and absorb samples above 200 mAU to obtain the hydrophobic chromatography eluent. Ammonium sulfate salting out: Ammonium sulfate is slowly added to the hydrophobic chromatography eluent until the saturation concentration of ammonium sulfate reaches 80%, and the mixture is stirred at 4°C for 20 hours to obtain a salting out solution. The salting out solution is then centrifuged at 4°C and 12,000 rpm for 30 minutes, after which the supernatant is discarded to obtain a precipitate. Dialysis: The precipitate was dissolved in 50 mM Tris-HCl buffer (pH 8.5) at a ratio of 1:10 (w / v, g / ml) to obtain a solution to be dialyzed. The resulting solution to be dialyzed was transferred to a dialysis bag (molecular weight cutoff: 100 kDa) and dialyzed for 3 hours at 4°C with 20 times the volume of the solution, 50 mM Tris-HCl buffer (containing 100 mM NaCl, pH 8.5) and stirred at 100 rpm. The solution was then dialyzed for 24 hours with 20 times the volume of the solution, 50 mM Tris-HCl buffer (pH 8.5) and stirred at 100 rpm. DEAE anion chromatography: The dialysate obtained from the above dialysis treatment was directly loaded and subjected to chromatography. The composition of mobile phase A2 was 20 mmol / L Tris, pH 8.5, and the composition of mobile phase B2 was 20 mmol / L Tris + 1.0 mol / L NaCl, pH 8.5. Chromatography step: Equilibrate with mobile phase A2 for 3 CV, load to the loading capacity, wash with mobile phase A2 for 3 CV, and perform gradient elution with 0%-50% mobile phase B1 for 20 CV to obtain the eluent. G-25M molecular sieve chromatography: The eluate from DEAE anion chromatography was directly loaded onto the molecular sieve column. Mobile phase C: 20 mmol / L Tris, pH 8.5, with a loading of ≤30% column volume per cycle and a linear flow rate of 300 cm / h. Each elution peak was collected, combined, and purified by SDS-PAGE to obtain the BoNT / A sample. The SDS-PAGE results are shown in Figure 3. The SEC chromatographic purity of the target protein (i.e., the BoNT / A protein prepared corresponding to number 6 in Table 2) was 98.8% (Figure 4).

[0164] In order to obtain the above-mentioned optimized purification process, the inventors conducted a large number of experiments on influencing factors to optimize the purification process and obtain high-purity BoNT / A samples, focusing on the operation composition of the purification unit and the operation sequence of the purification unit, where each unit operation was carried out under almost the same experimental conditions during each experimental process, including but not limited to the composition of the loading buffer solution, elution conditions, pH value, packing load, loading flow rate, column height, column diameter, column volume, etc. The experimental results are shown in Table 2.

[0165] [Table 2]

[0166] Example 3: Structural identification of BoNT / A protein

[0167] 1. Complete molecular weight detection 1.1 Sample treatment: Take 1 ml of the BoNT / A protein sample prepared in Example 2, concentrate it 5 times, and then mix it evenly and load it. 1.2 UPLC conditions: Chromatographic column: BioResolve RP mAb 2.7 μm, 2.1 mm × 100 mm, Waters 01093809916819; column temperature: 50 °C; detection wavelength: 280 nm; flow rate: 0.3 ml / min; loading volume: 10 μl. Mobile phase A: 0.05% TFA·H2O (aqueous trifluoroacetic acid); Mobile phase B: 0.05% TFA·ACN (trifluoroacetic acid·acetonitrile). JPEG0007760716000004.jpg261591.3 MS conditions: Ionization method: ESI positive; quality scan range: 300-4000 Da; capillary voltage: 3.0 KV; source temperature: 100°C; tapered bore voltage: 150 KV; desolvation gas temperature: 450°C; tapered bore reverse blow flow rate: 50 L / H; desolvation gas flow rate: 800 L / H. 1.4 Data collection and processing: Data were collected using Masslynx V4.1 software and analyzed using UNIFI software. The analysis results are shown in Table 3 and Figure 5.

[0168] [Table 3]

[0169] 2. Reduced Molecular Weight Detection 2.1 Sample treatment: Take 150 μl of the BoNT / A protein sample prepared in Example 2, add 150 μl of 7 mol / L guanidine hydrochloride, add 0.1 mol / L Tris (pH 8.0) and 3 μl of 1 mol / L DT, and incubate at 70°C for 30 minutes to mix evenly. 2.2 UPLC conditions are the same as in step 1.2. 2.3 MS conditions were the same as in step 1.3, except that the taper hole voltage was 40 kV. 2.4 Data collection and processing: Data were collected using Masslynx V4.1 software and analyzed using UNIFI software. The analysis results are shown in Table 4 and Figure 6.

[0170] [Table 4]

[0171] 3. Disulfide Bond Analysis 3.1 Sample processing: 1 ml of the BoNT / A protein sample prepared in Example 2 was taken and then concentrated 5-fold. 350 μl of 0.05 mol / L ammonium bicarbonate was added to each tube, mixed uniformly, and the volume was further concentrated to 100 μl. 4 μl of 1 mol / L iodoacetamide solution (IAM) and 350 μl of 0.05 mol / L ammonium bicarbonate were added to each tube, mixed uniformly, and the volume was further concentrated to 100 μl. 180 μl of the concentrated sample was taken, added with 20 μl of 1% RapiGest SF surfactant, and incubated at 60°C for 30 minutes. 8 μg of trypsin was added, and the mixture was incubated at 37°C overnight. After removal, 1 μl of formic acid was added, and the mixture was incubated at 37°C for 45 minutes. After removal, the mixture was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was collected and mixed uniformly for use as a sample. 3.2 UPLC conditions: Chromatographic column: UPLC BEH C18 1.7 μm, 2.1 mm × 150 mm, Waters 01443804318321; column temperature: 60 °C; detection wavelength: 215 nm; flow rate: 0.3 ml / min; loading volume: 10 μl. Mobile phase A: 0.05% TFA·H2O; Mobile phase B: 0.05% TFA·ACN. JPEG0007760716000007.jpg211633.3 MS conditions: Ionization method: ESI positive; quality scan range: 100-2000 Da; capillary voltage: 3.0 KV; source temperature: 100°C; tapered hole voltage: 40 KV; desolvation gas temperature: 450°C; tapered hole reverse blow flow rate: 50 L / H; desolvation gas flow rate: 800 L / H. 3.4 Data collection and processing: Data were collected using Masslynx V4.1 software and analyzed using UNIFI software. The analysis results are shown in Table 5 and Figure 7.

[0172] [Table 5]

[0173] 4. Data analysis results: As can be seen from the analytical data in Tables 3 to 5, the complete molecular weight, reduced molecular weight, and disulfide bond linkage of the BoNT / A protein are consistent with the theoretical values, demonstrating that the purified BoNT / A protein sequence is accurate and has the correct higher-order conformation.

[0174] Example 4: Initial measurement of the in vivo activity (toxicity) of BoNT / A protein Experimental animals: ICR mice, 4-5 weeks old, 17-22 g. Test samples were selected from different lots of BoNT / A samples prepared according to the method of Example 2, numbered 1#, 2#, 3#, and 4#. Each test sample had a volume of 0.5 ml, which was then diluted 10-fold with 4.5 ml of saline, shaken 3-4 times to mix evenly, and placed in an ice-water mixture. Four sets of mice were intraperitoneally inoculated with each of the four test samples, each containing five mice, each receiving 0.1 ml of each. Observations were conducted for three consecutive days, with daily animal mortality records. The analytical results are shown in Table 6.

[0175] [Table 6]

[0176] As can be seen from the experimental results At the given test sample concentrations, each lot of test samples (1#, 2#, 3#, 4#) showed good in vivo activity (toxicity), and test samples 2#, 3#, 4# showed relatively better in vivo activity (toxicity).

[0177] Example 5: Measurement of toxicity of BoNT / A protein Sixty ICR mice (30 males and 30 females) were weighed and randomly divided into 10 groups based on body weight, with 6 mice per group (3 males and 3 females). BoNT / A protein prepared according to the method in Example 2 was used as the test sample. Starting at 300 pg / mouse, ten dose gradients were administered at 2-fold increments: 300, 150, 75, 37.5, 18.75, 9.375, 4.6875, 2.34375, 1.171875, and 0.5859375 pg / mouse. Each mouse in each group was administered a corresponding concentration of the test sample. Each mouse was intraperitoneally injected with 0.1 ml of BoNT / A protein prepared according to the method in Example 2. The number of deaths in each group was observed and recorded daily for 4 consecutive days. Using GraphPad26, a logistic regression fit was performed between the transformed logarithm of the dose and the mortality rate, and the LD was calculated. 50 As the results show, the LD of the test sample was 50 was 4.023 pg / animal, and the converted sample toxicity was 1.06 × 10 8 LD 50 / mg and the analytical results are shown in Table 7.

[0178] [Table 7]

[0179] Example 6: Construction of genetically engineered strains expressing the light and heavy chain variants of botulinum neurotoxin type A variant 1 XbaI and BamHI enzyme cleavage sites were added to both ends of the nucleotide sequence encoding the light chain variant of botulinum neurotoxin type A variant 1 (amino acid sequence is SEQ ID NO: 3, nucleotide sequence is SEQ ID NO: 9) and the nucleotide sequence encoding the heavy chain variant (amino acid sequence is SEQ ID NO: 4, nucleotide sequence is SEQ ID NO: 10), and the sequences were sent to Takara Biotechnology (Dalian) Co., Ltd. to synthesize the target gene nucleotide sequences for the light chain variant and heavy chain variant, respectively.

[0180] The target gene nucleotide sequences for the light chain mutant and heavy chain mutant were double-digested with XbaI and BamHI, respectively. The vector pET-5a(+) was similarly double-digested with XbaI and BamHI, respectively. The resulting target fragments were then collected by cleavage. The double-digested fragments of the light chain mutant and heavy chain mutant were then ligated with the double-digested fragments of the vector. The ligated system was then transformed, subjected to screening and cloning of the target proteins, and validated for plasmid enzyme cleavage. Finally, engineered bacteria expressing botulinum neurotoxin type A light chain mutant proteins and heavy chain mutant proteins were obtained. The validation of the engineered bacterial plasmids for the light chain mutant proteins and heavy chain mutant proteins is shown in Figure 8. The left panel shows agarose gel electrophoresis of the double-digested light chain mutant plasmid, while the right panel shows agarose gel electrophoresis of the double-digested heavy chain mutant plasmid.

[0181] wherein the nucleotide sequence encoding the botulinum neurotoxin type A variant 1 light chain variant is SEQ ID NO: 9: The nucleotide sequence encoding the botulinum neurotoxin type A variant 1 heavy chain variant is SEQ ID NO: 10:

[0182] Example 7: Preparation of botulinum neurotoxin type A variant 1 The genetically engineered bacteria for the expressed light chain mutant proteins prepared in Example 6 were cultivated, and botulinum neurotoxin type A mutant 1 (also referred to as botulinum neurotoxin type A mutant protein 1 or mutant 1 in this example) was obtained through fermentation expression, regeneration, in vitro assembly and purification. Compared with wild-type botulinum neurotoxin type A, the mutation sites of mutant 1 are C134G, C165P, C791A, C967A and C1060E, and the specific steps are as follows:

[0183] (1) Fermentation expression of mutant light and heavy chain proteins The genetically engineered bacteria expressing the mutant light chain protein and the mutant heavy chain protein prepared in Example 6 were inoculated into shake flasks containing LB medium and cultured at OD 600 When the OD reached 1.6-2.0, the mixture was transferred to a 5 L fermenter and cultured at an initial culture volume of 2.5 L, a culture temperature of 37°C, and an agitation speed of 800 rpm. 600 When the temperature reaches 30°C, induction begins. Isopropylthiogalactoside (IPTG) is selected as the inducer, the concentration is 0.5 mM, and the induction time is 4 to 8 hours.

[0184] The growth of the genetically engineered bacteria in the fermentation broth is examined under a microscope to observe the expression status. When protein expression is observed and the induction period is 4 to 8 hours, the fermentation culture is terminated, the fermentation broth is collected, and the bacteria are collected by centrifugation at 10,000 rpm and 4°C. The collected bacteria are observed under an optical microscope to determine whether the morphology is typical of E. coli and whether there is any contamination with other microorganisms.

[0185] The collected cells were homogenized under high pressure at 700-800 bar for at least two cycles until no intact cells were found under a microscope. The cells were then centrifuged to collect the inclusion bodies, which were then washed twice with ultra-clean water at a ratio of 1:20 (w / v, g / ml) to obtain the light chain mutant inclusion body protein (abbreviated as "light chain mutant inclusion body") and the heavy chain mutant inclusion body protein (abbreviated as "heavy chain mutant inclusion body").

[0186] (2) Denaturation of mutant light chain proteins and mutant heavy chain proteins At room temperature, the light chain variant inclusion bodies and the heavy chain variant inclusion bodies are weighed out at a weight ratio of 1:4 (g / g), respectively. Next, the light chain variant inclusion bodies or the heavy chain variant inclusion bodies are dissolved in a denaturing buffer (20 mM Tris with 8 M urea and 10 mM DTT) at a weight-to-volume ratio of 1:20 (w / v, g / ml) to the denaturing buffer, respectively, at a pH of 10.0. The solution is stirred at 200 rpm until completely dissolved, to obtain a denatured solution (i.e., the light chain variant denatured product or the heavy chain variant denatured product).

[0187] (3) Renaturation and in vitro assembly of mutant light and heavy chain proteins The dissolved light chain mutant denatured product and heavy chain mutant denatured product obtained in step (2) are mixed in equal volumes to obtain a denaturation mixture. The denaturation mixture is then mixed with the renaturation and assembly buffer in a volume ratio of 1:10, and then stirred at 200 rpm to perform renaturation and assembly overnight to obtain an assembly solution. The composition of the renaturation and assembly buffer was 100 mM NaCl, 0.5 mM ZnCl2, 0.5 mM CaCl2, 5 mM GSH, 5 mM GSSG, 50 mM Tris-HCl and 0.5% Tween-20, with a pH value of 10.0.

[0188] A small amount of the assembly solution is concentrated at a volume ratio of 5:1 (ml / ml), and the assembly status is observed by SDS-PAGE electrophoresis. When SDS-PAGE electrophoresis reveals a single 150 kD target band, the assembly is stopped and downstream purification is carried out.

[0189] (4) Purification of botulinum neurotoxin type A mutant proteins Sequentially, hydrophobic chromatography, ammonium sulfate salting out, dialysis, DEAE anion chromatography, and molecular sieve chromatography are performed to obtain botulinum neurotoxin type A mutant 1. Hydrophobic chromatography: Add 4 mol / L NaCl to the above in vitro assembly solution until the final NaCl concentration is 2 mol / L, which becomes the loading stock solution. Equilibrate with mobile phase A for 3 CV, load to the loading capacity, wash with mobile phase A for 6 CV, and elute with a 10 CV gradient from 0% B to 100% B. Mobile phase A: 20mmol / L Tris + 5mmol / L EDTA + 2.0mol / L NaCl, pH 8.5; Mobile phase B: 20 ​​mmol / L Tris + 5 mmol / L EDTA, pH 8.5. Ammonium sulfate salting out: Weigh out an appropriate amount of ammonium sulfate with an ammonium sulfate saturation concentration of 80% and add it to the hydrophobic chromatography eluent. Stir until the ammonium sulfate is completely dissolved, leave it in a refrigerator at 2-8°C, and stir continuously for 24 hours at 100 rpm. Centrifuge at 4°C and 12,000 rpm for 30 minutes to separate the precipitate. Dialysis: The precipitate was dissolved in 50 mM Tris-HCl buffer (pH 8.5) at a 1:10 ratio (w / v, g / ml) of precipitate to buffer, then transferred to a dialysis bag (molecular weight cutoff: 100 kDa) and dialyzed at 4°C for 3 hours with stirring (100 rpm) against 20 volumes of 50 mM Tris-HCl buffer (containing 250 mM NaCl, pH 8.5), followed by 24 hours with stirring (100 rpm) against 20 volumes of 50 mM Tris-HCl buffer (pH 8.5). DEAE anion chromatography: Loading chromatography was performed directly on the dialysate. Mobile phase A: 20mmol / L Tris, pH 8.5; Mobile phase B: 20mmol / L Tris + 1.0mol / L NaCl, pH 8.5; Chromatography steps: equilibrate 3 CV with A, load to capacity, wash 3 CV with A, and elute with a 20 CV gradient from 0% B to 50% B. G-25M molecular sieve chromatography: DEAE anion chromatography eluent was directly loaded onto the molecular sieve chromatography. Mobile phase: 20mmol / L Tris, pH 8.5, loading ≦30% column volume / cycle, linear flow rate 300cm / h; Each elution peak is collected, and the combined samples are subjected to SDS-PAGE electrophoresis to detect purity, thereby obtaining botulinum neurotoxin type A variant 1.

[0190] Example 8: Structural identification of botulinum neurotoxin type A variant 1 1. Complete molecular weight detection Sample treatment: 1 ml of one sample of the botulinum neurotoxin type A mutant prepared in Example 7 was taken and concentrated 5 times, then mixed uniformly and loaded. UPLC conditions: Chromatographic column: BioResolve RP mAb 2.7 μm, 2.1 mm × 100 mm, Waters 01093809916819; column temperature: 50°C; detection wavelength: 280 nm; flow rate: 0.3 ml / min; loading volume: 10 μl. JPEG0007760716000011.jpg20165MS Conditions: Ionization method: ESI positive; quality scan range: 300-4000 Da; capillary voltage: 3.0 KV; source temperature: 100°C; tapered hole voltage: 150 KV; desolvation gas temperature: 450°C; tapered hole reverse blow flow rate: 50 L / H; desolvation gas flow rate: 800 L / H; Data collection and processing: Data were collected using Masslynx V4.1 software and analyzed using UNIFI software. The complete molecular weight data analysis is shown in Table 8.

[0191] [Table 8]

[0192] 2. Detection of reduced molecular weight Sample treatment: Take 150 μl of a sample of the botulinum neurotoxin type A mutant prepared in Example 7, add 150 μl of 7 mol / L guanidine hydrochloride / 0.1 mol / L Tris (pH 8.0) and 3 μl of 1 mol / L DTT, and incubate at 70°C for 30 minutes to mix evenly. UPLC conditions: Chromatography column: BioResolve RP mAb 2.7 μm, 2.1 mm × 100 mm, Waters 01093809916819; column temperature: 50 °C; detection wavelength: 280 nm; flow rate: 0.3 ml / min; loading volume: 10 μl; JPEG0007760716000013.jpg19164MS Conditions: Ionization method: ESI positive; quality scan range: 300~4000 Da; capillary voltage: 3.0 KV; source temperature: 100℃; taper hole voltage: 40 KV; desolvation gas temperature: 450℃; taper hole reverse blow flow rate: 50 L / H; desolvation gas flow rate: 800 L / H; Data collection and processing: Data were collected using Masslynx V4.1 software and analyzed using UNIFI software. The reduced molecular weight data analysis is shown in Table 9.

[0193] [Table 9]

[0194] 3. Disulfide bond analysis Sample processing: 1 ml of one sample of the botulinum neurotoxin type A mutant prepared in Example 7 was taken and concentrated 5-fold. 350 μl of 0.05 mol / L ammonium bicarbonate was added to each tube and mixed uniformly. The mixture was then concentrated to 100 μl. 4 μl of 1 mol / L iodoacetamide solution (IAM) and 350 μl of 0.05 mol / L ammonium bicarbonate were added to each tube and mixed uniformly. The mixture was then concentrated to 100 μl. 180 μl of the concentrated sample was taken, 20 μl of 1% RapiGest SF was added, and the mixture was incubated at 60°C for 30 minutes. 8 μg of trypsin was added, and the mixture was incubated at 37°C overnight. 1 μl of formic acid was added, and the mixture was incubated at 37°C for 45 minutes. The mixture was then removed and centrifuged at 13,000 rpm for 10 minutes. The supernatant was then collected, mixed uniformly, and used as a sample. UPLC conditions: Chromatographic column: UPLC BEH C18 1.7 μm, 2.1 mm × 150 mm, Waters 01443804318321; column temperature: 60 °C; detection wavelength: 215 nm; flow rate: 0.3 ml / min; loading volume: 10 μl; JPEG0007760716000015.jpg19165MS Conditions: Ionization method: ESI positive; quality scan range: 100-2000 Da; capillary voltage: 3.0 KV; source temperature: 100°C; tapered hole voltage: 40 KV; desolvation gas temperature: 450°C; tapered hole reverse blow flow rate: 50 L / H; desolvation gas flow rate: 800 L / H; Structural identification of wild-type type A botulinum neurotoxin: The content and method of structural identification were the same as in Example 3 (preparation of wild-type type A botulinum neurotoxin sample, specific preparation methods refer to Examples 1 and 2). Data collection and processing: Data were collected using Masslynx V4.1 software and analyzed using UNIFI software. Specific results are shown in Tables 10 and 11. Table 10 shows the detection results for the wild-type botulinum neurotoxin type A sample, and Table 11 shows the detection results for the botulinum neurotoxin type A mutant 1 sample prepared in Example 7. The positions of C1-C9 are shown in Figure 9. nindicates the nth cysteine ​​from the N-terminus to the C-terminus in the wild-type botulinum neurotoxin type A sequence, where C1 is the first cysteine, C2 is the second cysteine, ..., and C9 is the ninth cysteine.

[0195] [Table 10]

[0196] [Table 11]

[0197] As can be seen from the above, the disulfide bond linkage accuracy of type A botulinum neurotoxin variant 1 is 100.00%, with no disulfide bond mismatches. The disulfide bond linkage accuracy of wild-type type A botulinum neurotoxin is 94.80%, with 5.20% mismatches, a C3=C8 mismatch rate of 0.67%, and a C3=C9 mismatch rate of 0.23%. Therefore, compared with wild-type type A botulinum neurotoxin, the disulfide bond mismatch rate of type A botulinum neurotoxin variant 1 of the present invention is significantly lower, and there are no C3=C8 and C3=C9 mismatches.

[0198] Example 9: Preparation of botulinum neurotoxin type A variant 2 and structural identification of disulfide bonds For the preparation of botulinum neurotoxin type A variant 2 (abbreviated as variant 2), see Examples 6 and 7 for specific preparation methods, and see Example 8 for the details and method of structural identification. The difference is that variant 2 has a different amino acid sequence (compared to wild-type botulinum neurotoxin type A, variant 2 has mutation sites C134G, C165G, C791A, C967A, and C1060G). The corresponding amino acid and nucleotide sequences of variant 2's light chain variant (amino acid sequence is SEQ ID NO:5, nucleotide sequence is SEQ ID NO:11) and heavy chain variant (amino acid sequence is SEQ ID NO:6, nucleotide sequence is SEQ ID NO:12) are shown. The disulfide bond of botulinum neurotoxin type A variant 2 was detected; for specific detection methods, see Example 8; the detection data are shown in Table 12.

[0199] [Table 12] wherein the nucleotide sequence encoding the botulinum neurotoxin type A variant 2 light chain variant is SEQ ID NO: 11: The nucleotide sequence encoding the botulinum neurotoxin type A mutant double chain variant is SEQ ID NO: 12:

[0200] Example 10: Activity measurement of botulinum neurotoxin type A mutant proteins Test sample: Botulinum neurotoxin type A variant 1 prepared in Example 7, botulinum neurotoxin type A variant 2 prepared in Example 9, and wild-type botulinum neurotoxin type A prepared in Example 2, number 6.

[0201] Experimental design and group administration After the 162 mice were adapted and reared, 54 mice from each group were used to test the three test samples. After administration, the toxic reactions of the mice and the mortality of the animals in each group were closely observed for four consecutive days.

[0202] Before the experiment, the experimental mice were weighed and distributed evenly among groups to ensure there were no statistical differences in the average weights of the animals in each group. Each experiment was divided into nine groups, each with six mice, half male and half female. The test samples were administered intraperitoneally, with one person extracting the test sample solution, one person matching, one person holding the animals, and one person completing the animal administration procedures and recording the administration time after each group's injection. The group-by-group dosage information for the test sample, botulinum neurotoxin type A mutant 1, is shown in Table 13.

[0203] [Table 13]

[0204] As shown in Table 13 above, the 50% cumulative mortality rate lies between 45.45455%-83.33333%. Under these experimental conditions, the LD of the test sample, botulinum neurotoxin type A variant 1, was calculated according to the Reed-Muench method. 50 was 1.76381 pg / animal, and the converted sample toxicity was 5.67 × 10 8 LD 50 / mg.

[0205] Under the same experimental conditions, the LD of the test sample, botulinum neurotoxin type A variant 2, was calculated according to the Reed-Muench method. 50 was 3.0770 pg / animal, and the converted sample toxicity was 3.25 × 10 8 LD 50 / mg.

[0206] Under the same experimental conditions, the LD of the test sample wild-type botulinum neurotoxin type A was calculated according to the Reed-Muench method. 50 was 4.5662 pg / animal, and the converted sample toxicity was 2.19 × 10 8 LD 50 / mg. A comparison of the toxicity of the three different test samples is shown in Table 14.

[0207] [Table 14]

[0208] As can be seen from Table 14 above, the botulinum neurotoxin type A variant 1 of the present invention has higher biological activity (toxicity) than the wild-type botulinum neurotoxin type A, and is 2.6 times more toxic than the wild-type botulinum neurotoxin type A.

[0209] Although embodiments of the present invention have been presented and described, the above embodiments are illustrative and should not be construed as limiting the present invention, and those skilled in the art will appreciate that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. A botulinum neurotoxin type A mutant, a first peptide segment and a second peptide segment, said first peptide segment being linked to said second peptide segment by an interchain disulfide bond; the botulinum neurotoxin type A variant comprises a first peptide segment having the amino acid sequence set forth in SEQ ID NO:3 and a second peptide segment having the amino acid sequence set forth in SEQ ID NO:4; or The botulinum neurotoxin type A variant is characterized in that it comprises a first peptide segment having the amino acid sequence shown in SEQ ID NO: 5 and a second peptide segment having the amino acid sequence shown in SEQ ID NO:

6.

2. The botulinum neurotoxin type A mutant according to claim 1, characterized in that the interchain disulfide bond is formed by a cysteine ​​at position 430 in the first peptide segment and a cysteine ​​at position 454 in the second peptide segment.

3. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes a first peptide segment and a second peptide segment in the botulinum neurotoxin type A mutant according to claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule is DNA.

5. An expression vector, characterized in that it carries the nucleic acid molecule of claim 3.

6. The expression vector according to claim 5 , wherein the expression vector is a plasmid expression vector.

7. A genetically engineered fungus, A genetically engineered bacterium carrying the expression vector according to claim 6.

8. The genetically engineered fungus according to claim 7, wherein the genetically engineered fungus is obtained by transforming a host fungus with the expression vector according to claim 6.

9. The genetically engineered bacterium according to claim 8, wherein the host bacterium is Escherichia coli.

10. A method for preparing the botulinum neurotoxin type A mutant according to claim 1 or 2 by genetic recombination, comprising: subjecting the light chain protein to a first denaturation treatment to obtain a first denaturation product; subjecting the heavy chain protein to a second denaturation treatment to obtain a second denaturation product; and refolding and assembling the first denatured product and the second denatured product to obtain the botulinum neurotoxin type A mutant; The light chain protein has a first peptide segment according to claim 1 or 2, and The heavy chain protein has a second peptide segment according to claim 1 or 2. A method for preparing a botulinum neurotoxin type A mutant by genetic recombination, comprising:

11. The light chain protein or the heavy chain protein is transforming Escherichia coli with a plasmid carrying a gene encoding the light chain protein or the heavy chain protein; The method according to claim 10, wherein the light chain protein or the heavy chain protein is obtained by culturing E. coli transformed with the plasmid under conditions suitable for protein expression, inducing the cells, centrifugally collecting the cells, disrupting the cells, and disrupting the product by centrifugation.

12. The first denaturation treatment and the second denaturation treatment are performed in a denaturation buffer, and the denaturation buffer contains 12. The method according to claim 11, comprising 5-10 M urea, 5-15 mM dithiothreitol, and 10-30 mM Tris or Tris-HCl.

13. The preparation method according to claim 12, characterized in that the pH value of the denaturing buffer solution is 9.5-10.

5.

14. The preparation method according to claim 10, characterized in that the first modified product and the second modified product are mixed in advance before the regeneration and assembly treatment to obtain a modified mixed solution.

15. The preparation method according to claim 14, characterized in that in the mixing process, the volume ratio of the first modified product to the second modified product is 1:(1-10).

16. The preparation method according to claim 14, characterized in that the volume ratio of the denaturation mixture to the renaturation and assembly buffer is 1:(1-10).

17. The renaturation and assembly process is carried out in a renaturation and assembly buffer, the renaturation and assembly buffer comprising: 50-150 mM NaCl, 0.1-1.0 mM ZnCl 2 , 0.1-1.0 mM CaCl 2 11. The method of claim 10, comprising: 1.0-10.0 mM reduced glutathione; 1.0-10.0 mM oxidized glutathione; 40-50 mM Tris-HCl; and 0.4-0.6% Twain 20.

18. The preparation method according to claim 17, characterized in that the pH value of the renaturation and assembly buffer is 9.5-10.

5.

19. The preparation method according to claim 17, characterized in that the processing time of the regeneration and assembly process is 12-16 h.

20. The preparation method according to claim 17, characterized in that the stirring rotation speed during the regeneration and assembly process is 50 to 200 rpm.

21. The preparation method described in claim 10 further comprises the step of sequentially subjecting the assembly liquid obtained by the regeneration and assembly process to hydrophobic chromatography, ammonium sulfate salting out, dialysis, anion chromatography and molecular sieve chromatography to obtain the botulinum neurotoxin type A mutant.

22. 22. The method according to claim 21, wherein the mobile phase A1 of the hydrophobic chromatography treatment comprises 10-30 mmol / L Tris-HCl, 2-8 mmol / L EDTA, and 1-3 mol / L NaCl; the mobile phase B1 comprises 10-30 mmol / L Tris-HCl and 2-8 mmol / L EDTA; and the pH values ​​of the mobile phase A1 and the mobile phase B1 are both 8.0-9.

0.

23. The ammonium sulfate salting-out treatment is The method according to claim 21, comprising mixing ammonium sulfate with the eluate obtained by the hydrophobic chromatography at 2 to 8°C for 12 to 24 hours, and then centrifuging the mixture at 2 to 8°C and 10,000 to 15,000 rpm for 20 to 40 minutes to obtain a precipitate.

24. The dialysis treatment is dissolving the precipitate in a first buffer solution to obtain a solution to be dialyzed; performing a first dialysis treatment on the dialysis target liquid and a first dialysis liquid; and subjecting the obtained first dialysis treatment product and a second dialysate to a second dialysis treatment; 24. The method of claim 23, wherein the first buffer solution is selected from 40-60 mM Tris-HCl buffer solutions, the first dialysate is selected from 40-60 mM Tris-HCl salt-containing dialysates, and the second dialysate is selected from 40-60 mM Tris-HCl dialysates.

25. 25. The method according to claim 24, wherein the ratio of the weight (g) of the precipitate to the volume (ml) of the first buffer solution is 1:(5-15).

26. The method of claim 24, wherein the first dialysis treatment has a dialysis time of 2 to 5 hours, and the second dialysis treatment has a dialysis time of 12 to 24 hours.

27. 25. The method of claim 24, wherein the Tris-HCl salt-containing dialysis solution further comprises 200-300 mM NaCl.

28. The preparation method according to claim 24, wherein the cut-off molecular weight of the dialysis bags in the first dialysis treatment and the second dialysis treatment is 80 to 120 kDa.

29. 22. The method of claim 21, wherein the anionic chromatography treatment is selected from DEAE-cellulose anionic chromatography.

30. 30. The method of claim 29, wherein the mobile phase A2 of the DEAE cellulose anion chromatography comprises 10-30 mmol / L Tris, and the mobile phase B2 comprises 10-30 mmol / L Tris and 0.5-1.5 mol / L NaCl, and the pH values ​​of the mobile phase A2 and mobile phase B2 are 8.0-9.

0.

31. The preparation method according to claim 21, characterized in that the molecular sieve chromatography treatment is carried out using G-25M molecular sieve chromatography.

32. A pharmaceutical composition comprising the botulinum neurotoxin type A mutant according to claim 1 or 2.

33. 33. The pharmaceutical composition of claim 32, further comprising a pharmaceutically acceptable adjuvant.

34. 34. The pharmaceutical composition of claim 33, wherein the pharmaceutically acceptable auxiliary agent comprises at least one selected from the group consisting of a buffer, a protectant, an active agent, and an excipient.

35. A therapeutic agent for use in medical cosmetic or disease treatment or amelioration, comprising the botulinum neurotoxin type A mutant according to claim 1 or 2, The disease includes at least one of strabismus, cervical dystonia, laryngeal dystonia, focal dystonia of the upper limbs, primary hand tremor, sialorrhea, blepharospasm, lateral facial spasm, upper / lower limb spasms due to stroke, upper / lower limb spasms due to cerebral palsy, axillary hyperhidrosis, palmar hyperhidrosis, detrusor-sphincter dyssynergia, chronic migraine, and neurogenic and idiopathic bladder overactivity.

36. The medical beauty treatment is 36. The method of claim 35, comprising improving and / or treating at least one of the conditions including frown lines, crow's feet and forehead wrinkles.

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