Recombinant botulinum toxin and method for manufacturing the same

Abstract

The present invention provides a recombinant botulinum toxin and a method for producing the same. A nucleic acid encoding the recombinant botulinum toxin comprises: a first nucleic acid fragment, a second nucleic acid fragment, a first modified tag, and a second modified tag; wherein the first nucleic acid fragment codes for the heavy chain of the botulinum toxin, and the second nucleic acid fragment codes for the light chain of the botulinum toxin; the first modified tag codes for a His tag; and the second modified tag codes for a GST tag or an MBP tag.

Description

Technology Field

[0001] The present invention relates to the field of biotechnology, and specifically, the present invention relates to recombinant botulinum toxin and a method for manufacturing the same. Background Technology

[0002] The formal name for Botox (botulinum neurotoxin) is botulinum toxin. It is a neurotoxin secreted and produced by Clostridium botulinum, which can be classified into a total of seven serotypes based on antigenicity. Botulinum toxin possesses potent neurotoxicity and can induce nerve paralysis even at minute doses. If food containing botulinum toxin is ingested, the incubation period ranges from 6 hours to 12 days; clinical symptoms generally appear after 3 to 4 days, and the patient ultimately dies from respiratory failure. The toxicity of botulinum toxin is extremely potent, equivalent to approximately 10,000 times that of potassium cyanide. Therefore, strict requirements are necessary for the production of botulinum toxin, making it difficult to obtain in general laboratories.

[0003] The method of directly extracting botulinum toxin from Clostridium botulinum is a relatively traditional method, and its theory is relatively mature. However, the fermentation of Clostridium botulinum requires a very strict anaerobic environment and is highly sensitive to temperature. Therefore, large-scale production is only possible with dedicated fermentation facilities. Furthermore, since the expressed botulinum toxin does not possess an affinity chromatography tag, a relatively pure protein can only be obtained by undergoing several purification steps during the extraction process. In addition, due to the strong toxicity of botulinum toxin, the longer the manufacturing process, the higher the probability of unintended contamination occurring.

[0004] Therefore, there is an urgent need to develop a manufacturing method that is simple, has a short production process, and allows for the acquisition of botulinum toxin in the laboratory. The problem to be solved

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.

[0006] The inventors discovered that when purification is performed using a single tag during the experimental process, the final material may contain incomplete target proteins (WB experimental verification confirmed the presence of incomplete target proteins, which may have been enzymatically degraded by specific proteases), and that it is very difficult to separate incomplete and complete target proteins, resulting in a relatively impure final protein. After numerous experiments, the inventors discovered that applying two tags to the front and back of the target nucleic acid fragment allows for the acquisition of relatively pure and complete target proteins with only two purification steps, while also improving the yield of the target protein. Therefore, by adopting the method of the present invention, the purity of the target protein can be significantly improved, the entire process can be made faster and more efficient, and time and resource costs in the laboratory and production stages can be reduced by eliminating the need for cumbersome purification processes. Compared to traditional protein purification methods, this method significantly shortens purification time and drastically reduces the risk of unintended contamination. Since the acquired target protein possesses substantial purity and yield while reducing costs, it offers significant advantages for further experimental research, application development, and industrial production.

[0007] Specifically, the present invention provides the following technical solution:

[0008] In a first aspect of the present invention, the present invention provides isolated nucleic acids. According to an embodiment of the present invention, the nucleic acid comprises: a first nucleic acid fragment, a second nucleic acid fragment, a first modified tag, and a second modified tag, wherein the first nucleic acid fragment, the second nucleic acid fragment, the first modified tag, and the second modified tag are connected to each other; wherein the first nucleic acid fragment codes for a heavy chain of botulinum toxin; the second nucleic acid fragment codes for a light chain of botulinum toxin; the first modified tag codes for a His tag; and the second modified tag codes for a GST tag or an MBP tag.

[0009] It must be explained that the “first modified tag” or “second modified tag” described herein refers to a nucleotide sequence encoding a His tag, a GST tag, or an MBP tag. The “first modified tag” or “second modified tag” is not limited to the nucleotide sequences listed in the present invention, but further includes other nucleotide sequences capable of encoding amino acids corresponding to the His tag, GST tag, or MBP tag.

[0010] Through numerous experiments, the inventors discovered that when a first modified tag codes for a His tag and a second modified tag codes for a GST tag or an MBP tag, the subsequently obtained recombinant protein botulinum toxin has high activity and purity, and also has a high yield, which can reduce costs.

[0011] According to an embodiment of the present invention, the isolated nucleic acid may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the nucleic acid further comprises a first enzyme cleavage site, a second enzyme cleavage site, and a third enzyme cleavage site.

[0013] According to an embodiment of the present invention, the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site are identical.

[0014] According to an embodiment of the present invention, the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site code for a 3C enzyme cleavage site, a TEV enzyme cleavage site, a Thrombin enzyme cleavage site, an Fxa enzyme cleavage site, or an Enterokinase enzyme cleavage site. When the above-described enzyme cleavage sites are adopted, the modified tag sequence can be completely removed after cleavage with the corresponding protease, and no amino acid residues that are not present in the original sequence remain.

[0015] It must be explained that the “first enzyme cleavage site,” “second enzyme cleavage site,” and “third enzyme cleavage site” described herein refer to nucleotide sequences encoding a 3C enzyme cleavage site, a TEV enzyme cleavage site, a Thrombin enzyme cleavage site, an Fxa enzyme cleavage site, or an Enterokinase enzyme cleavage site. The “first enzyme cleavage site,” “second enzyme cleavage site,” and “third enzyme cleavage site” are not limited only to the nucleotide sequences listed in the present invention, and further include other nucleotide sequences capable of encoding amino acids corresponding to the 3C enzyme cleavage site, the TEV enzyme cleavage site, the Thrombin enzyme cleavage site, the Fxa enzyme cleavage site, or the Enterokinase enzyme cleavage site.

[0016] According to an embodiment of the present invention, the first modified tag codes for a His tag, the second modified tag codes for a GST tag, and the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site code for a 3C enzyme cleavage site or a Thrombin enzyme cleavage site. Through a number of screening experiments, the inventors discovered that when the above-described combination is adopted, the subsequently produced recombinant protein botulinum toxin has relatively high activity and purity.

[0017] According to an embodiment of the present invention, the first modified tag codes for a His tag, the second modified tag codes for an MBP tag, and the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site code for a 3C enzyme cleavage site or a Thrombin enzyme cleavage site. Through a number of screening experiments, the inventors discovered that when the above-described combination is adopted, the subsequently produced recombinant protein botulinum toxin has relatively high activity and purity.

[0018] According to an embodiment of the present invention, the 5' end of the first modified tag is connected to the 3' end of the first enzyme cleavage site, the 5' end of the first enzyme cleavage site is connected to the 3' end of the first nucleic acid fragment, the 5' end of the first nucleic acid fragment is connected to the 3' end of the second enzyme cleavage site, the 5' end of the second enzyme cleavage site is connected to the 3' end of the second nucleic acid fragment, the 5' end of the second nucleic acid fragment is connected to the 3' end of the third enzyme cleavage site, and the 5' end of the third enzyme cleavage site is connected to the 3' end of the second modified tag.

[0019] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence as presented in SEQ ID NO:1.

[0020]

[0021] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence as presented in SEQ ID NO:2.

[0022]

[0023] According to an embodiment of the present invention, the His tag has an amino acid sequence as presented in SEQ ID NO:73.

[0024] HHHHHHHHHHH(SEQ ID NO:73)

[0025] According to an embodiment of the present invention, the sequence encoding the His tag (first variant tag) has a nucleotide sequence as presented in SEQ ID NO:3.

[0026] CATCACCATCACCATCACCATCACCATCAC(SEQ ID NO:3)

[0027] According to an embodiment of the present invention, the GST tag has an amino acid sequence as presented in SEQ ID NO:74.

[0028] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAY SKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK(SEQ ID NO:74)

[0029] According to an embodiment of the present invention, the sequence encoding the GST tag (second variant tag) has a nucleotide sequence as presented in SEQ ID NO:4.

[0030] (SEQ ID NO:4)

[0031] According to an embodiment of the present invention, the MBP tag has an amino acid sequence as presented in SEQ ID NO:75.

[0032] MKIEEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGL TFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLG(SEQ ID NO:75 )

[0033] According to an embodiment of the present invention, the sequence encoding the MBP tag (second variant tag) has a nucleotide sequence as presented in SEQ ID NO:5.

[0034]

[0035] According to an embodiment of the present invention, the 3C enzyme cleavage site has an amino acid sequence as presented in SEQ ID NO:76.

[0036] GSLEVLFQGPGS(SEQ ID NO:76)

[0037] According to an embodiment of the present invention, the sequences encoding the 3C enzyme cleavage sites (first enzyme cleavage site, second enzyme cleavage site and third enzyme cleavage site) have nucleotide sequences as presented in SEQ ID NO:6.

[0038] GGGTCTTTAGAAGTCTTGTTTCAAGGTCCGGGCTCT(SEQ ID NO:6)

[0039] According to an embodiment of the present invention, the TEV enzyme cleavage site has an amino acid sequence as presented in SEQ ID NO:77.

[0040] GSENLYFOSGSS(SEQ ID NO:77)

[0041] According to an embodiment of the present invention, the sequences encoding the TEV enzyme cleavage sites (first enzyme cleavage site, second enzyme cleavage site and third enzyme cleavage site) have nucleotide sequences as presented in SEQ ID NO:7.

[0042] GGGTCTGAAAATCTATACTTCCAGAGCGGGCTCTTCT(SEQ ID NO:7)

[0043] According to an embodiment of the present invention, the Thrombin enzyme cleavage site has an amino acid sequence as presented in SEQ ID NO:78.

[0044] GSSSLVPRGSSS(SEQ ID NO:78)

[0045] According to an embodiment of the present invention, the sequences encoding the Thrombin enzyme cleavage sites (first enzyme cleavage site, second enzyme cleavage site and third enzyme cleavage site) have nucleotide sequences as presented in SEQ ID NO:8.

[0046] GGGTCTAGTTCACTGGTGCCACGCGGTAGCTCCTCT(SEQ ID NO:8)

[0047] According to an embodiment of the present invention, the Fxa enzyme cleavage site has an amino acid sequence as presented in SEQ ID NO:79.

[0048] GSSSIEGRGSSS(SEQ ID NO:79)

[0049] According to an embodiment of the present invention, the sequences encoding the Fxa enzyme cleavage site (first enzyme cleavage site, second enzyme cleavage site and third enzyme cleavage site) have nucleotide sequences as presented in SEQ ID NO:9.

[0050] GGGTTAGTTCAATCGAAGGTAGGGGTAGCTCCTCT(SEQ ID NO:9)

[0051] According to an embodiment of the present invention, the enterokinase enzyme cleavage site has an amino acid sequence as presented in SEQ ID NO:80. GSSDDDDKGSSS(SEQ ID NO:80) According to an embodiment of the present invention, the sequence encoding the enterokinase enzyme cleavage site (first enzyme cleavage site, second enzyme cleavage site and third enzyme cleavage site) has a nucleotide sequence as presented in SEQ ID NO:10.

[0052] GGGTCTAGTGATGACGACGATAAGGGTAGCTCCTCT(SEQ ID NO:10)

[0053] In a second aspect of the present invention, the present invention provides a vector. According to an embodiment of the present invention, the vector carries the nucleic acid described in the first aspect. The expression vector may include a selective control sequence, said control sequence being functionally linked to said nucleic acid molecule. Herein, said control sequence is one or more control sequences capable of directing the expression of said nucleic acid molecule within a host.

[0054] According to an embodiment of the present invention, the vector is selected from a plasmid.

[0055] In this document, “functional linkage” means linking a foreign gene to a vector so that control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform the expected function of regulating the transcription and translation of the foreign gene. When linking the aforementioned nucleic acid molecule to the vector, the nucleic acid molecule may be linked directly or indirectly to a control element on the vector, and it is sufficient that such a control element can control the translation and expression of the nucleic acid molecule. Of course, such a control element may originate directly from the vector itself, may be foreign, or, that is, may not originate from the vector itself.

[0056] In a third aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises the nucleic acid described in the first aspect or the vector described in the second aspect.

[0057] According to an embodiment of the present invention, the cell is selected from a prokaryotic cell or a eukaryotic cell.

[0058] According to an embodiment of the present invention, the cell is selected from E. coli, yeast, cyanobacteria or mammalian cell lines, insect cells, plant cells or amphibian cells.

[0059] According to an embodiment of the present invention, the cell is selected from E. coli.

[0060] In a fourth aspect of the present invention, the present invention provides a method for obtaining recombinant protein botulinum toxin. According to an embodiment of the present invention, the method comprises: a step of culturing the recombinant cells described in the third aspect under conditions suitable for protein expression; a step of purifying the culture treatment product; and a step of enzymatically cleaving the purification treatment product to obtain the recombinant protein botulinum toxin. The method described in the present invention can be easily implemented, features simple and clear steps, and has a rapid manufacturing process. Since it does not require complex equipment or strict experimental conditions, it is suitable for ordinary laboratory operations, and thus possesses greater feasibility and flexibility in actual applications. Furthermore, the recombinant protein botulinum toxin obtained by this method has relatively high purity and activity, thereby laying the foundation for subsequent development of uses and applications of recombinant protein botulinum toxin.

[0061] According to an embodiment of the present invention, the method for obtaining the recombinant protein botulinum toxin may further include at least one of the following additional technical features:

[0062] According to an embodiment of the present invention, the purification process is performed in the following manner: the culture treatment product is subjected to a first purification process, and the first purification process is performed in a second modified tag purification column; the culture treatment product subjected to the first purification process is subjected to a second purification process, and the second purification process is performed in a first modified tag purification column.

[0063] It must be explained that the “second modified tag purification column” has the function of maintaining the biological activity and chemical integrity of the target protein while separating the second modified tag from the total protein; and the “first modified tag purification column” has the function of maintaining the biological activity and chemical integrity of the target protein while separating the first modified tag from the total protein.

[0064] According to an embodiment of the present invention, the purification process is performed in the following manner: the culture treatment product is subjected to a first purification process, and the first purification process is performed in a GST tag purification column; the culture treatment product subjected to the first purification process is subjected to a second purification process, and the second purification process is performed in a His tag purification column, and the site of the enzyme cleavage process is a 3C enzyme cleavage site. By adopting the above method, the purity and activity of the obtained recombinant protein botulinum toxin are relatively high, thereby laying the foundation for the subsequent development of uses and applications of the recombinant protein botulinum toxin.

[0065] According to an embodiment of the present invention, the purification process is performed in the following manner: the culture treatment product is subjected to a first purification process, and the first purification process is performed in a GST-tagged purification column; the culture treatment product subjected to the first purification process is subjected to a second purification process, and the second purification process is performed in a His-tagged purification column, wherein the site of the enzyme cleavage process is a thrombin enzyme cleavage site. By adopting the above method, the purity and activity of the obtained recombinant protein botulinum toxin are relatively high, thereby laying the foundation for the subsequent development of uses and applications of the recombinant protein botulinum toxin.

[0066] According to an embodiment of the present invention, the purification process is performed in the following manner: the culture treatment product is subjected to a first purification process, and the first purification process is performed in an MBP tag purification column; the culture treatment product subjected to the first purification process is subjected to a second purification process, and the second purification process is performed in a His tag purification column, and the site of the enzyme cleavage process is a 3C enzyme cleavage site. By adopting the above method, the purity and activity of the obtained recombinant protein botulinum toxin are relatively high, thereby laying the foundation for the subsequent development of uses and applications of the recombinant protein botulinum toxin.

[0067] According to an embodiment of the present invention, the purification process is performed in the following manner: the culture treatment product is subjected to a first purification process, and the first purification process is performed in an MBP-tagged purification column; the culture treatment product subjected to the first purification process is subjected to a second purification process, and the second purification process is performed in a His-tagged purification column, wherein the site of the enzyme cleavage process is a thrombin enzyme cleavage site. By adopting the above method, the purity and activity of the obtained recombinant protein botulinum toxin are relatively high, thereby laying the foundation for the subsequent development of uses and applications of the recombinant protein botulinum toxin.

[0068] According to an embodiment of the present invention, the purification process may also be performed in the following manner: the culture treatment product is subjected to a third purification process, and the third purification process is performed in a His tag purification column; the culture treatment product subjected to the third purification process is subjected to a fourth purification process, and the fourth purification process is performed in a strong anion exchange chromatography column. Even by adopting the purification method, an equally high-quality recombinant protein botulinum toxin can be obtained.

[0069] Through experiments, the inventor discovered that when the third purification treatment is performed in a His tag purification column, the fourth purification treatment involves passing the culture product purified in the third stage through a strong anion exchange chromatography column (HiTrap Q HP, formally named Q Sepharose High Performance) to isolate the target protein. In this case, the modified tags GST or MBP act to increase the expression level of the target protein.

[0070] In a fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant protein botulinum toxin is prepared and obtained by the method described in the fourth aspect. The recombinant protein botulinum toxin prepared by the method described in the present invention has relatively high purity and activity and can lay the foundation for subsequent medical applications, scientific research, and drug development.

[0071] In a sixth aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises a nucleic acid described in a first aspect, a vector described in a second aspect, a recombinant cell described in a third aspect, or a recombinant protein botulinum toxin described in a fifth aspect.

[0072] In a seventh aspect of the present invention, the present invention provides a use in the manufacture of a drug comprising the nucleic acid described in the first aspect, the vector described in the second aspect, the recombinant cell described in the third aspect, the recombinant protein botulinum toxin described in the fifth aspect, or the composition described in the sixth aspect, said drug being used for the prevention or treatment of a disease.

[0073] According to an embodiment of the present invention, the disease includes a neuromuscular disease, and the symptoms of the disease include: spastic dysphonia, spastic torticollis, laryngeal dystonia, oromandibular dysphonia, tongue dystonia, cervical dystonia, focal dystonia, blepharospasm, strabismus, unilateral facial spasm, blepharoplasty, cerebral palsy, focal spasm, spastic colitis, neurogenic bladder overactivity, idiopathic bladder overactivity, pelvic floor relaxation delay, limb spasm, motor tic, primary hand tremor, head tremor, detrusor-sphincter incoordination, bruxism, dentition, achalasia, dysphagia, hyperhidrosis, hypotonia, excessive gastrointestinal secretion, secretory disorder, pain due to muscle spasms, chronic migraine, or dermatological disease.

[0074] In the eighth aspect of the present invention, the present invention provides a use in the preparation of a reagent of the nucleic acid described in the first aspect, the vector described in the second aspect, the recombinant cell described in the third aspect, the recombinant protein botulinum toxin described in the fifth aspect, or the composition described in the sixth aspect, said reagent is used to interfere with the release of neurotransmitters from isolated nerve cells or to interfere with the release of neurotransmitters in the body to improve related symptoms in the body.

[0075] According to an embodiment of the present invention, the reagent is used for cosmetic purposes.

[0076] In a ninth aspect of the present invention, the present invention provides a drug. According to an embodiment of the present invention, the drug comprises: a nucleic acid described in the first aspect, a vector described in the second aspect, a recombinant cell described in the third aspect, a recombinant protein botulinum toxin described in the fifth aspect, or a composition described in the sixth aspect.

[0077] According to an embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0078] The “pharmaceuticalally acceptable” ingredients used herein are substances applicable to humans and / or mammals that are free from excessive adverse effects (e.g., toxicity, irritation, and allergic reactions), i.e., have a reasonable efficacy-to-risk ratio. The term “pharmaceuticalally acceptable carrier” refers to a carrier for administering therapeutic agents and includes various excipients and diluents.

[0079] The drug of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, physiological saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. In general, the drug formulation must correspond to the method of administration, wherein the method of administration may be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, or intravenous administration, or intraperitoneal administration, and the formulations of the drug of the present invention are injectables, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. It is preferable that the drug be prepared under sterile conditions.

[0080] In the tenth aspect of the present invention, the present invention provides a method for preventing or treating neuromuscular diseases. According to an embodiment of the present invention, the method comprises the step of administering to a subject an effective amount of the nucleic acid described in the first aspect, the vector described in the second aspect, the recombinant cell described in the third aspect, the recombinant protein botulinum toxin described in the fifth aspect, the composition described in the sixth aspect, or the drug described in the ninth aspect.

[0081] As used herein, the terms “effective amount” or “effective dose” refer to an amount that can produce function or activity in humans and / or animals and is acceptable to humans and / or animals. The effective amount described in the present invention may vary depending on the method of administration and the severity of the disease to be treated. The selection of a preferred effective amount may be determined by a person skilled in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, e.g., bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient’s body weight, the patient’s immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, the dose may be divided into multiple daily doses or proportionally reduced.

[0082] In the eleventh aspect of the present invention, the present invention provides the use of the nucleic acid described in the first aspect, the vector described in the second aspect, the recombinant cell described in the third aspect, the recombinant protein botulinum toxin described in the fifth aspect, the composition described in the sixth aspect, or the drug described in the ninth aspect in the prevention or treatment of neuromuscular diseases. Effects of the invention

[0083] The method for manufacturing botulinum toxin provided by the present invention enables the rapid and efficient production of botulinum toxin, has a short host culture cycle and prevents easy contamination by bacteriophages, features a simple and easy-to-implement toxin protein purification process, facilitates large-scale production, significantly reduces the cost of manufacturing botulinum toxin, and produces botulinum toxin with expected biological activity comparable to natural botulinum toxin and excellent application prospects.

[0084] Additional aspects and advantages of the present invention will be presented in part in the description below, and will become apparent in part from the description below or understood through the practice of the invention. Brief explanation of the drawing

[0085] The above-described aspects and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in combination with the accompanying drawings, where: FIG. 1 is a gel electrophoresis of pQe30 after double enzymatic cleavage according to an embodiment of the present invention; FIG. 2 is a gel electrophoresis diagram after a start codon mutation in plasmid 1 according to an embodiment of the present invention; FIG. 3 is a sequencing identification result of plasmid 2 according to an embodiment of the present invention; FIG. 4 is a gel electrophoresis diagram after removal of 6×His from plasmid 2 according to an embodiment of the present invention; FIG. 5 is a sequencing identification result of plasmid 3 according to an embodiment of the present invention; FIG. 6 is a gel electrophoretic graph of a target fragment of a light chain and a heavy chain according to an embodiment of the present invention; FIG. 7 is a gel electrophoresis of plasmid 3 after double enzymatic cleavage according to an embodiment of the present invention; FIG. 8 is a PCR amplification of a GST tag having a 3C enzyme cleavage site and a single enzyme cleavage linearization electrophoresis of plasmid 4 according to an embodiment of the present invention; FIG. 9 is a PCR amplification electrophoresis of the MBP tag sequence according to an embodiment of the present invention and the remaining sequence portion excluding the GST tag sequence in Plasmid 5; FIG. 10 is a gel electrophoresis graph after purification of a single His tag (plasmid 4) according to an embodiment of the present invention (wherein M is a protein marker (MARK); FT1 is a sample after passing the culture treatment product through a His tag column; FT2 is a sample after passing F1 through a His tag column once again; 0 mM is a sample after washing the His tag column with a buffer; 25 mM is a sample after washing the His tag column with a buffer containing 25 mM imidazole; and Elute is a sample after eluting the target protein from the His tag column with a buffer containing 200 mM imidazole); FIG. 11 is a gel electrophoresis graph after the first GST purification according to an embodiment of the present invention (wherein FT is a sample after passing the culture treatment product through a GST tag column; the first W is a sample after washing the GST tag column with a small amount of buffer; the second W is a sample after washing the GST tag column with a large amount of buffer; 1 / 3 / 5 / 8 / 10 / 13 / 15 / 17 are tubes 1, 3, 5, 8, 10, 13, 15, and 17 collected when eluting the target protein from the GST tag column with 20 mM reduced glutathione; beads refers to GST tag beads, intended to check whether there is any target protein that has not yet eluted on the GST tag); FIG. 12 is a gel electrophoresis graph after performing a second His purification following a first GST purification according to an embodiment of the present invention (wherein FT is a sample after passing the target protein eluted by the GST tag column in the first step through the His tag column; 0 mM is a sample after washing the His tag column with a buffer; 25 mM is a sample after washing the His tag column with a buffer containing 25 mM imidazole; and E is a sample after eluting the target protein from the His tag column with a buffer containing 200 mM imidazole); FIG. 13 is an SDS-PAGE electrophoresis graph of a target protein passed through a molecular sieve after two-step purification of 3C enzyme cleavage according to an embodiment of the present invention (wherein 9 / 10 / 11 / 12 / 13 are tubes 9, 10, 11, 12, and 13 collected by passing through the molecular sieve); FIG. 14 is an electrophoretic graph after first-step His purification including an MBP tag according to an embodiment of the present invention (wherein NC is an uninduced culture product, a negative control group; FT is a sample in which the culture treatment product was passed through a His tag column; 0 / 25 is a sample in which the His tag column was washed with a buffer containing 0 / 25 mM imidazole; and E is a sample in which the target protein was eluted from the His tag column with a buffer containing 200 mM imidazole); FIG. 15 is an SDS-PAGE electrophoresis graph after the second-stage ion exchange according to an embodiment of the present invention (wherein M is a protein MARK; before loading is a sample before passing through the ion exchange column; FT is a sample after passing through the ion exchange column; and 1 to 24 are tube samples 1 to 24 collected by passing through the ion exchange column); FIG. 16 is an SDS-PAGE electrophoresis graph obtained by passing through a molecular sieve after ion exchange in the second step of 3C enzyme cleavage according to an embodiment of the present invention (wherein M is a protein MARK; 1 to 13 are samples from tubes 1 to 13 collected by molecular sieve); FIG. 17 is a biological activity experiment regarding the SNAP25 substrate enzymatic cleavage of recombinant protein botulinum toxin with different second modified tags GST / MBP and different enzymatic cleavage sites obtained in different ways according to embodiments of the present invention (wherein 036 is a SNAP25 substrate; M is a protein MARK); FIG. 18 is an anatomical view of the intestine of a mouse in a 3.335 ng / kg dose group according to an embodiment of the present invention. FIG. 19 is an anatomical view of a mouse intestine in a 2.000 ng / kg dosage group according to an embodiment of the present invention; FIG. 20 is an anatomical view of a mouse intestine in a 1,200 ng / kg dose group according to an embodiment of the present invention; FIG. 21 is an anatomical view of a mouse intestine in a 0.720 ng / kg dose group according to an embodiment of the present invention; FIG. 22 is an anatomical view of a mouse intestine in a 0.432 ng / kg dose group according to an embodiment of the present invention; FIG. 23 is an anatomical view of a mouse intestine in a 0.259 ng / kg dose group according to an embodiment of the present invention; FIG. 24 is an anatomical view of a mouse intestine in a 0.156 ng / kg dose group according to an embodiment of the present invention; FIG. 25 is a plasmid map of plasmid 4 according to an embodiment of the present invention; FIG. 26 is a plasmid map of plasmid 5 according to an embodiment of the present invention; FIG. 27 is a plasmid map of plasmid 6 according to an embodiment of the present invention; FIG. 28 is a plasmid spectrum of a plasmid constructed to contain a GST tag + His tag + Thrombin enzyme cleavage site according to an embodiment of the present invention; FIG. 29 is a plasmid spectrum of a plasmid constructed to contain an MBP tag + His tag + Thrombin enzyme cleavage site according to an embodiment of the present invention. Specific details for implementing the invention

[0086] The following describes embodiments of the present invention in detail. The embodiments described below are illustrative and are used only to explain the invention and should not be interpreted as limiting the invention.

[0087] It must be noted that the terms “first” and “second” are used merely for illustrative purposes and should not be understood as indicating or implying relative importance, or as implicitly limiting the quantity of the indicated technical features. Accordingly, the features limited to “first” and “second” may explicitly or implicitly include one or more such features. Additionally, in the description of the invention, “plural” means two or more unless otherwise stated.

[0088] In this document, the terms “containing,” “including,” or “equipped” are open expressions, meaning they include the content specified in the invention but do not exclude other aspects.

[0089] In this document, the terms “optional,” “optional,” or “optional” generally mean that the event or situation described below may or may not occur, and that such description includes both cases where the event or situation occurs and cases where it does not occur.

[0090] The following describes embodiments of the present invention in detail. The embodiments described below are illustrative and are used only to explain the invention and should not be interpreted as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature of the art or in the product description. Where the manufacturer of the reagents or equipment used is not specified, they are all ordinary products available on the market.

[0091] Recombination of pQe30 expression plasmid

[0092] 1. Recombination 1

[0093] The pQe30 plasmid was linearized by double enzymatic cleavage using Thermo Fisher Scientific’s EcoRI and HindIII restriction enzymes. The enzymatic cleavage products were collected using nucleic acid gel electrophoresis, the target band was gel-cleaved (see Fig. 1 for the target band), and the products were recovered using the TIANGEN gel recovery kit. After recovery, homologous recombination was performed with DNA sequences containing the 3C enzymatic cleavage site, 10×His, and pQe30 homologous arm synthesized by General Biol (Anhui); the Vazyme homologous recombination kit (ClonExpress II One Step Cloning Kit) was selected. The procedure steps are identical to those in the instructions.

[0094] 10 μl of the reaction product after homologous recombination was taken and added to DH5a competent cells (purchased from Beijing TransGen Biotech), and after applying heat shock at 42°C for 60 seconds, 800 μl of fresh LB medium was added and incubated at 37°C for 1 hour. Subsequently, the mixture was plated onto resistant LB solid plates (containing 50 μg / mL ampicillin) and incubated overnight at 37°C. A single clone was selected and cultured, and plasmid extraction was performed using the TIANGEN Plasmid Small Extraction Kit to obtain recombinant Plasmid 1.

[0095] 2. Recombination 2

[0096] Based on Recombination 1, to inactivate the N-terminal 6×His of Plasmid 1, the inventor mutated the initiation codon ATG at the front of Plasmid 1 to CTG, and the mutant primers are as follows:

[0097] Mut-F:5'-GAGGAGAAATTAACTCTGAGAGGATCGCATC-3'(SEQ ID NO:11)

[0098] Mut-R:5'-GATGGCATCCTCTCCAGAGTTAATTTCTCCTC-3'(SEQ ID NO:12)

[0099] A 20 μl system reaction was performed using the PrimeSTAR Mix system (recombinant plasmid 1 template: 1 μl, approx. 28 ng; mutant primer: Mut-F, 1 μl; mutant primer: Mut-R, 1 μl; sterile water: 7 μl; 2×PrimeSTAR Mix: 10 μl). The reaction program was as follows: 98°C, 10 s denaturation; 58°C, 10 s annealing; 72°C, 4 min extension; a total of 32 reaction cycles. Reaction fragments were collected using nucleic acid gel electrophoresis (see Figure 2 for the target band). The PCR reaction bands were gel-cleaved, and the target bands were recovered using the TIANGEN gel recovery kit. The recovered bands were transformed into DH5a (purchased from Beijing TransGen Biotech), subjected to heat shock at 42°C for 60 seconds, and then incubated at 37°C for 1 hour with the addition of 800 μl of fresh LB medium. The samples were then plated onto resistant LB solid plates (containing 50 μg / mL ampicillin) and incubated overnight at 37°C. A single clone was selected for expansion culture, and plasmid extraction was performed using the TIANGEN Plasmid Small Extraction Kit to obtain recombinant Plasmid 2.

[0100] Plasmid 2 was sent to Sangon Biotech (Shanghai) for sequencing identification, and the identified sequencing results were compared using SnapGene software. As shown in Figure 3, the results confirmed that the start codon ATG was successfully mutated to CTG. 3. To bring the recombination 3-coding region and the ribosome binding site (RBS) closer together, the inventor deleted 18 bases of the 6×His sequence at the N-terminus of Plasmid 2, and the deletion primers are as follows:

[0101] Del-His-F:5'-AAAGAGGAGAAATTAACTCTGAGAGGATCGGGATCCGCATGCGAGCTCC-3'(SEQ ID NO:13)

[0102] Del-His-R:5'-GGAGCTCGCATGCGGATCCCGATCCTCTCAGAGTTAATTTCTCCTCTTT-3'(SEQ ID NO:14)

[0103] A 20 μl system reaction was performed using the PrimeSTAR Mix system (Plasmid 2 template: 1 μl, approx. 30 ng; Primer Del-His-F: 1 μl; Primer Del-His-R: 1 μl; Sterile water: 7 μl; 2×PrimeSTAR Mix: 10 μl). The reaction program was as follows: 98°C, 10 s denaturation; 58°C, 10 s annealing; 72°C, 4 min extension; a total of 32 reaction cycles. Reaction fragments were collected using nucleic acid gel electrophoresis (refer to Figure 4 for the 6×His removal fragment results). PCR reaction bands were gel-cleaved and recovered using the TIANGEN gel recovery kit. The recovered bands were transformed into DH5a (purchased from Beijing TransGen Biotech), subjected to heat shock at 42°C for 60 seconds, and then incubated at 37°C for 1 hour with the addition of 800 μl of fresh LB medium. The samples were then plated onto resistant LB solid plates (containing 50 μg / mL ampicillin) and incubated overnight at 37°C. A single clone was selected for expansion culture, and plasmid extraction was performed using the TIANGEN Plasmid Small Extraction Kit to obtain recombinant Plasmid 3.

[0104] Plasmid 3 was sent to Sangon Biotech (Shanghai) for sequencing identification, and the identified sequencing results were compared in SnapGene software. As shown in Figure 5, it was confirmed that 18 bases of 6×His were successfully deleted.

[0105] Objective: Construction of a plasmid

[0106] Based on the botulinum protein sequence expressed by Clostridium botulinum (UniProtKB / Swiss-Prot: PODPI0.1) disclosed on the NCBI (National Center for Biotechnology Information, URL: https: / / www.ncbi.nlm.nih.gov / ), the design was based on the botulinum protein sequence, and the botulinum toxin-coding gene was optimized and synthesized by General Biol (Anhui).

[0107] Using the PCR amplification method, a 3-times recombined pQe30 homologous arm (i.e., Plasmid 3) was introduced upstream and downstream of the light and heavy chains of the botulinum toxin-coding gene, respectively, and a 3C enzyme cleavage site was introduced between the light and heavy chains. The required primers are as follows:

[0108] pQe-BTXL-F:5'-ATTAACTCTGAGAGGATCGGGATCCATGCCATTTGTTAATAAGCA-3'(SEQ ID NO:15)

[0109] pQe-BTXL-R:5'-CCTTGAAACAAGACTTCTAAAGACCCACTAGTGATGATACCTCT-3'(SEQ ID NO:16)

[0110] pQe-BTXH-F:5'-TTAGAAGTCTTGTTTCAAGGTCCGGGCTCTTTAAACGACCTTTGTATT-3'(SEQ ID NO:17)

[0111] pQe-BTXH-R:5'-CTGGAACAGAACTTCCAGGGTACCAAGGGGCCGCTCTCCCCACC-3'(SEQ ID NO:18)

[0112] Light and heavy chain amplification were performed using a 50 μl system reaction each with the PrimeSTAR Mix system (heavy / light chain template DNA (provided by General Biol): 1 μl, approx. 20-50 ng; primers pQe-BTXL-F / pQe-BTXH-F: 1 μl; primers pQe-BTXL-R / pQe-BTXH-R: 1 μl; sterile water: 22 μl; 2×PrimeSTAR Mix: 25 μl). The reaction program was as follows: denaturation at 98°C for 10 s; annealing at 58°C for 10 s; extension at 72°C (light chain 90 s, heavy chain 180 s); a total of 32 reaction cycles. Reaction fragments were collected using nucleic acid gel electrophoresis (refer to Figure 6 for the target fragments of the light and heavy chains). The PCR reaction bands were gel-cut and recovered using a TIANGEN gel recovery kit to obtain the PCR-amplified light and heavy chains.

[0113] The triple-recombined pQe30 plasmid (plasmid 3) was linearized by double enzymatic cleavage using Thermo Fisher Scientific's BamHI and KpnI restriction enzymes. The enzymatic cleavage products were collected using nucleic acid gel electrophoresis, the target band was gel-cleaved (see Fig. 7 for the target band), and the product was recovered using the TIANGEN gel recovery kit to obtain the double enzymatically cleaved linearized vector.

[0114] Homologous recombination was performed on the PCR-amplified light and heavy chains and the BamHI and KpnI double-enzyme-cleaved linearized vectors after gel recovery, and the Vazyme Homologous Recombination Kit (ClonExpress II One Step Cloning Kit) was selected. The operation steps were identical to those in the instructions. 10 μl of the reaction product was taken and added to DH5a competent cells (purchased from Beijing TransGen Biotech), heat shock was applied at 42°C for 60 seconds, 800 μl of fresh LB medium was added and incubated at 37°C for 1 hour, then plated onto resistant LB solid plates (containing 50 μg / mL ampicillin) and incubated overnight at 37°C. A single clone was selected and expanded cultured, and then plasmid extraction was performed using the TIANGEN Plasmid Small Extraction Kit to obtain Plasmid 4, the plasmid map of which is shown in Figure 25.

[0115] Introduction of tags

[0116] 1. Introduction of GST Tags:

[0117] After successfully linking the target sequence of Example 2 (PCR-amplified light and heavy chains) with the recombinant vector (double-enzyme-cleaved linearized vector), the inventor linearized the linkage plasmid 4 by single-enzyme cleavage with Thermo Fisher Scientific's BamHI restriction enzyme, recovered the vector using a TIANGEN gel recovery kit, and obtained a BamHI single-enzyme-cleaved linearized vector, the band of said vector is shown in the right figure of FIG. 8.

[0118] Simultaneously, primers containing the homologous arm of this linearization plasmid were designed to amplify the GST tag sequence having a 3C enzyme cleavage site at the end (the template plasmid was pGEX-6P-1), with the 3C enzyme cleavage site following the GST tag. The primers are as follows:

[0119] GST-F:5’-GAGAAATTAACTCTGAGAGGATCGGGATCCATGTCCCCTATACTAGGTTATTG-3’(SEQ ID NO:19)

[0120] GST-R:5’-CTGCTTATTAACAAATGGCATGGGCCCCTGGAACAGAACTTCC-3’(SEQ ID NO:20)

[0121] A 50 μl system reaction was performed using the PrimeSTAR Mix system (template DNA (pGEX-6P-1): 1 μl, approx. 20-50 ng; primer GST-F: 1 μl; primer GST-R: 1 μl; sterile water: 22 μl; 2×PrimeSTAR Mix: 25 μl). The reaction program was as follows: 98°C, 10 s denaturation; 58°C, 10 s annealing; 72°C, 60 s extension; a total of 34 reaction cycles. Reaction fragments were collected using nucleic acid gel electrophoresis (refer to the left figure in Fig. 8 for the target reaction fragment band). The PCR reaction bands were gel-cleaved and recovered using the TIANGEN gel recovery kit. After gel recovery, homologous recombination was performed using a BamHI single-enzyme-cleaved linearized vector and a GST tag sequence containing a PCR-amplified 3C enzyme-cleaved site. The Vazyme homologous recombination kit (ClonExpress II One Step Cloning Kit) was selected. The procedure steps were identical to those in the instructions. 10 μl of the reaction product after homologous recombination was taken and added to DH5a competent cells (purchased from Beijing TransGen Biotech). After applying heat shock at 42°C for 60 seconds, 800 μl of fresh LB medium was added and incubated at 37°C for 1 hour. Subsequently, the mixture was plated onto resistant LB solid plates (containing 50 μg / mL ampicillin) and incubated overnight at 37°C. A single clone was selected and cultured, and then plasmid extraction was performed using the TIANGEN plasmid small-batch extraction kit to obtain plasmid 5, and the plasmid map of plasmid 5 is shown in Figure 26.

[0122] 2. Introduction of MBP Tags:

[0123] Using the PCR method, the target plasmid (Plasmid 5) containing the constructed GST tag is used as a template to PCR amplify the MBP tag sequence and replace the GST tag sequence, while simultaneously PCR amplifying the remaining sequence of Plasmid 5 excluding the GST tag sequence. The primers used are as follows:

[0124] MBP-F:5'-CTCTGAGAGGATCGGGATCCATGAAAATCGAAGAAGGTAAAC-3'(SEQ ID NO:21)

[0125] MBP-R:5'-CCGAGGTTGTTGTTATTGTTATTGTTG-3'(SEQ ID NO:22)

[0126] pQe-BTX-F2:5'-ACAATAACAACAACCTCGGGCTGGAAGTTCTTGTTCCAGGGGCCCATGCCATTTGTTAATAAGCAGT-3'(SEQ ID NO:23)

[0127] pQe-BTX-R2:5'-ACCTTCTTCGATTTTCATGGATCCCGATCCTCTCAG-3'(SEQ ID NO:24)

[0128] A 50μl system reaction was performed using the PrimeSTAR Mix system. The 50μl system reaction system for removing the GST sequence of Plasmid 5 is as follows: Template DNA (Plasmid 5), 1μl, approx. 20-50ng; Primers pQe-BTX-F2 / pQe-BTX-R2, 1μl each; Sterile water, 22μl; 2×PrimeSTAR Mix, 25μl. The 50μl system reaction system for amplifying the MBP tag is as follows: Template DNA (Plasmid pGEX-6P-1), 1μl, approx. 20-50ng; Primers MBP-F / MBP-R, 1μl each; Sterile water, 22μl; 2×PrimeSTAR Mix, 25μl). The reaction program is as follows: 98°C, 10s denaturation; 58°C, 10s annealing; 72°C, (MBP tag 90s, pQe30-BTX portion 450s) extension; a total of 34 reaction cycles. Reaction fragments were collected using nucleic acid gel electrophoresis (refer to Fig. 9 for the target reaction fragment band). The PCR reaction bands were gel-cleaved and recovered using the TIANGEN gel recovery kit. The two recovered portions were homologously recombined, and the Vazyme homologous recombination kit (ClonExpress II One Step Cloning Kit) was selected. The procedure steps are identical to those in the instructions. 10 μl of the reaction product after homologous recombination was taken and added to DH5a competent cells (purchased from Beijing TransGen Biotech), heat shock was applied at 42°C for 60 seconds, 800 μl of fresh LB medium was added and incubated at 37°C for 1 hour, then plated onto resistant LB solid plates (containing 50 μg / mL ampicillin) and cultured overnight at 37°C. A single clone was selected and cultured, and then plasmid extraction was performed using the TIANGEN Plasmid Small Extraction Kit to obtain Plasmid 6, and the plasmid map of Plasmid 6 is shown in Figure 27.

[0129] Construction of plasmids containing different enzyme cleavage sites

[0130] 1. The method for constructing other enzyme cleavage sites carrying His tags and GST / MBP tags refers to the method described above, and specifically is as follows.

[0131] (1) First, construct a target protein expression plasmid without GST or MBP tags (e.g., Plasmid 4), introduce a TEV / Fxa / EK / Thrombin enzyme cleavage site between the light chain and the heavy chain using primers, introduce a TEV / Fxa / EK / Thrombin enzyme cleavage site corresponding to the 3' end of the heavy chain and the 5' end of the light chain, and also ensure that two adjacent partial sequences contain homologous arms. Then, a target protein with different enzyme cleavage sites can be obtained through homologous recombination.

[0132] (2) Then, according to the previously described method for constructing Plasmid 5 or Plasmid 6, a GST tag or an MBP tag is introduced into the plasmid of the target protein having different enzyme cleavage sites.

[0133] 2. The primers involved in constructing plasmids containing different tags and enzyme cleavage sites in Step 1 are specifically as follows:

[0134] (1) Construct a target protein expression plasmid without GST or MBP tags (e.g., Plasmid 4)

[0135] 1.1. Primers required for TEV enzyme cleavage site introduction:

[0136] TEV-F:ATTAACTCTGAGAGGATCGGGATCCATGCCATTTGTTAATAAGCAG(SEQ ID NO:25)

[0137] TEV-R:GCTCTGGAAGTATAGATTTTCAGACCCACTAGTGATGATACCTCTAA(SEQ ID NO:26)

[0138] TEV2-F:GAAAATCTATACTTCCAGAGCGGCTTCTTCTTTAAACGACCTTTGTATTAA(SEQ ID NO:27)

[0139] TEV2-R:TGATGGCTCTGGAAGTATAGATTTTCAAGGGGCCGCTCTCCCCACC(SEQ ID NO:28)

[0140] pQe30-TEV-F:TGAAAATCTATACTTCCAGAGCCATCACCATCACCATCACCATC(SEQ ID NO:29)

[0141] pQe30-TEV-R:CTGCTTATTAACAAATGGCATGGATCCCGATCCTCTCAGAGTTAAT(SEQ ID NO:30)

[0142] 1.2. Primers required for introduction of the thrombin enzyme cleavage site:

[0143] Throm-F:TTAACTCTGAGAGGATCGGGATCCATGCCATTTGTTAATAAGCAGTTT(SEQ ID NO:31)

[0144] Throm-R:GCTACCGCGTGGCACCAGTGAACTAGACCCACTAGTGATGATACCTCTAACA(SEQ ID NO:32)

[0145] Throm2-F:GTTCACTGGTGCCACGCGGTAGCTCCTCTTTAAACGACCTTTGTATTAAAG(SEQ ID NO:33)

[0146] Throm2-R:ATGGCTACCGCGTGGCACCAGAAGGGGCCGCTCTCCCCACCCGT(SEQ ID NO:34)

[0147] pQe30-Throm-F:CCTTCTGGTGCCACGCGGTAGCCATCACCATCACCATCACCATCACC(SEQ ID NO:35)

[0148] pQe30-Throm-R:AAACTGCTTATTAACAAATGGCATGGATCCCGATCCTCTCAGAGTTAA(SEQ ID NO:36)

[0149] 1.3. Primers required for introduction of the Fxa enzyme cleavage site:

[0150] Fxa-F:TTAACTCTGAGAGGATCGGGATCCATGCCATTTGTTAATAAGCAGTTT(SEQ ID NO:37)

[0151] Fxa-R:CCTACCTTCGATTGAACTAGACCCACTAGTGATGATACCTCTAACACA(SEQ ID NO:38)

[0152] Fxa2-F:TAGTTCAATCGAAGGTAGGGGTAGCTCCTCTTTAAACGACCTTTGTATTAA(SEQ ID NO:39)

[0153] Fxa2-R:ATGCCTACCTTCGATAAGGGGCCGCTCTCCCCACCCGT(SEQ ID NO:40)

[0154] pQe30-Fxa-F:CCCCTTATCGAAGGTAGGCATCACCATCACCATCACCATCA(SEQ ID NO:41)

[0155] pQe30-Fxa-R:AAACTGCTTATTAACAAATGGCATGGATCCCGATCCTCTCAGAGTTAA(SEQ ID NO:42)

[0156] 1.4. Primers required for EK enzyme cleavage site introduction:

[0157] EK-F:TTAACTCTGAGAGGATCGGGATCCATGCCATTTGTTAATAAGCAGTTT(SEQ ID NO:43)

[0158] EK-R:CCTTATCGTCGTCATCACTAGACCCACTAGTGATGATACCTCTAACA(SEQ ID NO:44)

[0159] EK2-F:TAGTGATGACGACGATAAGGGTAGCTCCTCTTTAAACGACCTTTGTATTAA(SEQ ID NO:45)

[0160] EK2-R:ATGCTTATCGTCGTCATCAAGGGGCCGCTCTCCCCACCC(SEQ ID NO:46)

[0161] pQe30-EK-F:GGCCCCTTGATGACGACGATAAGCATCACCATCACCATCACCATCACC(SEQ ID NO:47)

[0162] pQe30-EK-R:AAACTGCTTATTAACAAATGGCATGGATCCCGATCCTCTCAGAGTTAA(SEQ ID NO:48)

[0163] (2) Introduction of GST tags (e.g., Plasmid 5):

[0164] 2.1. Primers required for introduction of the thrombin enzyme cleavage site:

[0165] GST-Thrombin-F:TTAACTCTGAGAGGATCGGGATCCATGTCCCCTATACTAGGTTATTGG(SEQ ID NO:51)

[0166] GST-Thrombin-R:CTGCTTATTAACAAATGGCATGCTACCGCGTGGCACCAGTTTTGGAGGATGGTCGCCACCAC(SEQ ID NO:52)

[0167] The plasmid spectrum of the constructed plasmid is shown in Fig. 28.

[0168] 2.2. Primers required for TEV enzyme cleavage site introduction:

[0169] GST-TEV-F:TTAACTCTGAGAGGATCGGGATCCATGTCCCCTATACTAGGTTAT(SEQ ID NO:49)

[0170] GST-TEV-R:ACTGCTTATTAACAAATGGCATGCTCTGGAAGTATAGATTTTCTTTTGGAGGATGGTCGCCAC(SEQ ID NO:50)

[0171] The plasmid spectrum of the constructed plasmid is similar to that of Fig. 28, and is simply the same as replacing the Thrombin enzyme cleavage site with the TEV enzyme cleavage site.

[0172] 2.3. Primers required for introduction of the Fxa enzyme cleavage site:

[0173] GST-Fxa-F:TTAACTCTGAGAGGATCGGGATCCATGTCCCCTATACTAGGTTATT(SEQ ID NO:53)

[0174] GST-Fxa-R:TGCTTATTAACAAATGGCATCCTACCTTCGATTTTTGGAGGATGGTCGCCACC(SEQ ID NO:54)

[0175] The plasmid spectrum of the constructed plasmid is similar to that of Fig. 28, and is simply the same as replacing the Thrombin enzyme cleavage site with the Fxa enzyme cleavage site.

[0176] 2.4. Primers required for EK enzyme cleavage site introduction:

[0177] GST-EK-F:TTAACTCTGAGAGGATCGGGATCCATGTCCCCTATACTAGGTTATT(SEQ ID NO:55)

[0178] GST-EK-R:CTGCTTATTAACAAATGGCATCTTATCGTCGTCATCTTTTGGAGGATGGTCGCCACC(SEQ ID NO:56)

[0179] The plasmid spectrum of the constructed plasmid is similar to that of Fig. 28, and is simply the same as replacing the Thrombin enzyme cleavage site with an EK enzyme cleavage site.

[0180] (3) Introduction of MBP tags (e.g., Plasmid 6):

[0181] 3.1. Primers required for introduction of the thrombin enzyme cleavage site:

[0182] MBP-Thrombin-F:TTAACTCTGAGAGGATCGGGATCCATGAAAATCGAAGAAGGTAAACTGGT (SEQ ID NO:61)

[0183] MBP-Thrombin-R:CTGACGACCGCTGGCGGCGTTGATCACCGCAGTACGCACGGCATACCAGAAAGCGGACAT (SEQ ID NO:62)

[0184] pQe30-Thrombin-F:ATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAG(SEQ ID NO:63)

[0185] pQe30-Thrombin-R:ACCAGTTTACCTTCTTCGATTTTCATGGATCCCGATCCTCTCAGAGTTAA (SEQ ID NO:64)

[0186] The plasmid spectrum of the constructed plasmid is shown in Fig. 29.

[0187] 3.2. Primers required for TEV enzyme cleavage site introduction:

[0188] MPB-TEV-F:TTAACTCTGAGAGGATCGGGATCCATGAAAATCGAAGAAGGTAAACTGG(SEQ ID NO:57)

[0189] MPB-TEV-R:AACTGCTTATTAACAAATGGCATGCTCTGGAAGTATAGATTTTCCCCGAGGTTGTT GTTATTGT(SEQ ID NO:58)

[0190] pQe30-TEV-F:ACAATAACAACAACCTCGGGGAAAATCTATACTTCCAGAGCATGCCATTTGTTAATAAGCAGTT(SEQ ID NO:59)

[0191] pQe30-TEV-R:CCAGTTTACCTTCTTCGATTTTCATGGATCCCGATCCTCTCAGAGTTAA(SEQ ID NO:60)

[0192] The plasmid spectrum of the constructed plasmid is similar to that of Fig. 29, and is simply the same as replacing the Thrombin enzyme cleavage site with the TEV enzyme cleavage site.

[0193] 3.3. Primers required for introduction of the Fxa enzyme cleavage site:

[0194] MBP-Fxa-F:TTAACTCTGAGAGGATCGGGATCCATGAAAATCGAAGAAGGTAAACTGG(SEQ ID NO:65)

[0195] MBP-Fxa-R:CTGCTTATTAACAAATGGCATCCTACCTTCGATCCCGAGGTTGTTGTTATTGT(SEQ ID NO:66)

[0196] pQe30-Fxa-F:ACAATAACAACAACCTCGGGATCGAAGGTAGGATGCCATTTGTTAATAAGCAG(SEQ ID NO:67)

[0197] pQe30-Fxa-R:CCAGTTTACCTTCTTCGATTTTCATGGATCCCGATCCTCTCAGAGTTAA(SEQ ID NO:68)

[0198] The plasmid spectrum of the constructed plasmid is similar to that of Fig. 29, and is simply the same as replacing the Thrombin enzyme cleavage site with the Fxa enzyme cleavage site.

[0199] 3.4. Primers required for EK enzyme cleavage site introduction:

[0200] MBP-EK-F:TTAACTCTGAGAGGATCGGGATCCATGAAAATCGAAGAAGGTAAACTG(SEQ ID NO:69)

[0201] MBP-EK-R:AACAAATGGCATCTTATCGTCGTCATCCCCGAGGTTGTTGTTATTGTTATTGT(SEQ ID NO:70)

[0202] pQe30-EK-F:ACAATAACAATAACAACAACCTCGGGGATGACGACGATAAGATGCCATTTGTT(SEQ ID NO:71)

[0203] pQe30-EK-R:CAGTTTACCTTCTTCGATTTTCATGGATCCCGATCCTCTCAGAGTTAA(SEQ ID NO:72)

[0204] The plasmid spectrum of the constructed plasmid is similar to that of Fig. 29, and is simply the same as replacing the Thrombin enzyme cleavage site with an EK enzyme cleavage site.

[0205] Acquisition of botulinum toxin protein

[0206] 1. Plasmid 6 containing different tags and enzyme cleavage sites was constructed according to the methods described in Examples 1 to 4 above, 1 μl of Plasmid 6 was taken and added to E. coli BL21(DE3) competent cells (purchased from Beijing TransGen Biotech), heat shock was applied at 42°C for 60 seconds, then 800 μl of fresh LB medium was added and incubated at 37°C for 1 hour, then plated onto resistant LB solid plates (containing 50 μg / mL ampicillin) and cultured overnight at 37°C. A single clone was selected from the plate in which the clones were formed, inoculated into 10 mL of LB medium (containing 50 μg / mL ampicillin), and cultured overnight at 37°C. The next day, the cells were inoculated into 1 L of LB medium, and when the OD reached 0.6, IPTG at a final concentration of 0.2 mM was added. The cells were then cultured continuously at 20–37°C, and after the culture was finished, the cells were collected by centrifugation. The collected cells were resuspended in buffer solution and then lysed using a sonicator. The supernatant collected by centrifugation after sonication is the culture treatment product.

[0207] 2. A relatively pure target protein can be obtained by purifying the culture product, and the purification process can be performed in two ways:

[0208] First method: After purifying the culture treatment product using a GST or MBP-tagged purification column, the purified culture treatment product is further purified using a His-tagged purification column, so that a relatively pure botulinum toxin protein can be obtained after the purification is completed.

[0209] Second method: After purifying the culture treatment product using a His-tagged purification column, passing the purified culture treatment product through a strong anion exchange chromatography column (HiTrap Q HP, formally named Q Sepharose High Performance) yields a similarly relatively pure botulinum toxin protein.

[0210] 3. By performing corresponding enzymatic cleavage on the purified target protein and then performing molecular sieve chromatography purification again after enzymatic cleavage, a botulinum toxin protein with relatively high purity can be obtained.

[0211] Botulinum toxin proteins obtained using different tags and enzyme cleavage sites

[0212] The inventor was able to obtain the target protein using different tag combinations (His tag + GST ​​tag or His tag + MBP tag), but when only one His tag was used, the final target protein obtained was not relatively ideal, and as shown in Fig. 10, the experimental results confirmed that the protein obtained after purification was very impure. To purify the purified protein (Fig. 11), the inventor performed molecular sieve filtration on the purified protein. As a result, the target protein could still not be purified further, and the cause of this phenomenon is likely that the complete protein and the incomplete protein were mixed and could not be separated.

[0213] When the inventor used His tags and GST tags, all five aforementioned enzyme cleavage sites (3C, TEV, Thrombin, Fxa, and EK enzyme cleavage sites) can be utilized to obtain the target protein; thus, the inventor can obtain the target protein in five different ways. The inventor obtained the target protein through two-step affinity chromatography purification. In this experimental process, the inventor used the first purification method of Example 5, that is, purification in the order of GST tag first followed by His tag. The electrophoresis results after the first step of GST tag purification are shown in Fig. 11, and the electrophoresis results after the second step of His tag purification are shown in Fig. 12. It can be confirmed that the target protein obtained through two-step tag purification has higher purity than the target protein obtained through single-step tag purification. Subsequently, enzyme cleavage and molecular sieve chromatography purification were performed on the purified target protein to finally obtain a botulinum toxin protein of relatively high purity (electrophoresis is shown in Fig. 13).

[0214] When the inventor replaced the GST tag with an MBP tag, the target protein can be obtained in five additional ways. In this experimental process, the inventor used the second purification method of Example 5; that is, first, His tag purification was performed, and the electrophoresis results after purification are shown in Fig. 14. The figure confirms that the target protein can be obtained approximately well through His tag purification (compared to Fig. 10 for single-tag His). Subsequently, the target protein was passed through a strong anion exchange chromatography column (HiTrap Q HP, formally named Q Sepharose High Performance) to obtain a relatively pure target protein. The electrophoretic graph of the target protein is shown in Fig. 15, confirming that the target protein was isolated relatively purely. After enzymatic cleavage of the target protein, molecular sieve filtration chromatography purification was performed again to obtain a botulinum toxin protein of relatively high purity (electrophoretic graph shown in Fig. 16).

[0215] Verification of the biological activity of botulinum toxin protein through substrate testing

[0216] Although the target protein can be obtained from all 10 aforementioned methods (combinations of His tag + GST ​​tag or His tag + MBP tag with the five enzymatic cleavage sites, respectively), further verification is required to determine whether the target proteins obtained through these 10 methods possess biological activity. To further verify whether the target proteins obtained through these 10 methods possess biological activity, the following experiment was performed: Botulinum toxin proteins obtained in different forms were each prepared as 1 mg / mL stock solutions, and simultaneously, SNAP25 reaction substrates were also prepared as 1 mg / mL stock solutions. 18 μL of each botulinum toxin protein and 1 μL of SNAP25 substrate were taken, 1 μL of PBS was added to make the total volume 20 μL, and the SNAP25 substrates were enzymatically cleaved overnight at room temperature. As shown in Fig. 17, the results show that the botulinum toxin obtained with the combination of the MBP purification tag and the Thrombin enzyme cleavage site can more completely cleave the SNAP25 (036) protein (it can be confirmed that there is almost no band at the original position, i.e., the enzyme cleavage is more complete, and further explains that the activity of the protein obtained with this combination is higher); the botulinum toxin obtained with the combination of the MBP purification tag and the 3C enzyme cleavage site or the combination of the GST purification tag and the two enzyme cleavage sites of Thrombin / 3C also has a relatively good effect in cleaving the SNAP25 protein (compared to 036, the band at the original position is significantly fainter, and explains that the activity of the protein obtained with these two combinations is also relatively good).

[0217] In vivo experiment

[0218] In order to further verify whether the botulinum toxin obtained according to the preparation of the present invention can exert a relatively good effect in the body, the inventor prepared a botulinum toxin according to the method described above in which the purification tags are MBP tag and His tag and the enzyme cleavage site is a 3C combination, named the botulinum toxin prepared with this combination as (030-3c(WT)3(t1)), injected it into a mouse to observe the acute toxicity reaction and calculate the median lethal dose.

[0219] To observe acute toxic reactions occurring in mice following a single intraperitoneal injection of the drug 030-3c(WT)3(t1), mice were administered a single intraperitoneal injection of 030-3C(WT)3(t1) at doses of 3.335, 2.000, 1.200, 0.720, 0.432, 0.259, 0.156, and 0.093 ng / kg, respectively. The condition of the mice was observed for 7 days after administration, and the median lethal dose (LD50) was calculated. The specific method is as follows:

[0220] 80 Kunming (KM) mice, equal numbers of males and females, 5 females and 5 males per group, body weight 18–28g, purchased from the Animal Research Center of Lanzhou University, Animal Certificate of Approval No.: No.62000800000298.

[0221] 1. Group classification: 80 mice were divided equally into male and female groups. 40 male mice were weighed first and then randomly distributed into 8 groups in descending order of weight, with each group consisting of 5 male mice (numbers 1, 2, 3, 4, 5); 40 female mice were randomly distributed into the aforementioned 8 groups in the same manner, with each group consisting of 5 female mice (numbers 6, 7, 8, 9, 10). It was confirmed that all 8 groups consisted of 10 mice each, with equal numbers of male and female. The aforementioned 8 groups were each designated as a single drug dosage group.

[0222] 2. Administration: The 030-3c(WT)3(t1) drug is a colorless liquid with a concentration of 0.77 mg / ml. Using a 0.01 M PBS solution containing 0.2% gelatin as the drug diluent, the 0.77 mg / ml drug stock solution was prepared at concentrations of 6.6670, 4.0010, 2.4006, 1.4403, 0.8642, 0.5185, 0.3111, and 0.1866 ng / ml, respectively. These concentrations were then administered intraperitoneally to the eight aforementioned groups of mice at a volume of 0.5 μl / g, with the dosages for the eight groups being 3.335, 2.000, 1.200, 0.720, 0.432, 0.259, and 0.156, respectively. It is 0.093 ng / kg.

[0223] The experimental results are as follows:

[0224] 1. Animal responses in each dose group after administration

[0225] (1) 3.335ng / kg capacity group:

[0226] Deaths: 10 Survivors: 0

[0227] Observation after administration: 8 min after intraperitoneal injection, all male mice began to frequently scratch behind their ears and eyes, and after 25 min, the frequency of scratching gradually decreased until it stopped. 15 min after injection, all female mice began to frequently scratch behind their ears and eyes, and after 40 min, the frequency of scratching gradually decreased. After 60 min, both male and female mice completely stopped scratching and returned to normal. Among them, 9 mice were found dead after 20 h, and another mouse died from generalized convulsions after 27 h. In gross examination after dissection, intestinal vesicles were observed in all mice, minor ulcers were present, and minor hemorrhage was found in the intestines of some mice. The dissection photos are shown in Fig. 18.

[0228] (2) 2.000ng / kg capacity group:

[0229] Deaths: 10 Survivors: 0

[0230] Observation after administration: 12 min after intraperitoneal injection, all male mice began frequently scratching behind their ears and eyes, and after 30 min, the frequency of scratching gradually decreased until it stopped. 20 min after injection, all female mice began frequently scratching behind their ears and eyes, and after 40 min, the frequency of scratching gradually decreased. After 60 min, both male and female mice completely stopped scratching and returned to normal. Among these, six mice were found dead after 20 hours, one mouse died from generalized convulsions after 22 hours, and three mice died from generalized convulsions after 24 hours. At the time of death, the mice first showed slowed movements, dizziness, and increased respiratory fluctuations, followed by the onset of generalized convulsions. Upon visual examination after dissection, intestinal vesicles and minor ulcers were observed in all mice, with no intestinal hemorrhage or other organ abnormalities. The dissection photos are shown in Fig. 19.

[0231] (3) 1,200ng / Kg Capacity Group:

[0232] Deaths: 9 Survivors: 1

[0233] Observation after administration: 15 minutes after intraperitoneal injection, all male mice began frequently scratching behind their ears and eyes; after 40 minutes, the frequency of scratching gradually decreased until it stopped. 25 minutes after injection, all female mice began frequently scratching behind their ears and eyes; after 50 minutes, the frequency of scratching gradually decreased, and after 60 minutes, both male and female mice completely stopped scratching and returned to normal. Of these, three mice were found dead after 20 hours, another three died from generalized convulsions after 26 hours, two mice died from generalized convulsions after 29 hours, and one mouse died from generalized convulsions after 32 hours. At the time of death, the mice first exhibited slowed movements, dizziness, and increased respiratory fluctuations, followed by the onset of generalized convulsions. Gross examination after dissection revealed intestinal vesicles and minor ulcers in all mice, with no intestinal hemorrhage or other organ abnormalities. The surviving animal began scratching behind the ears and the abdominal cavity 32 min after observation, and the scratching stopped after 40 min. There were no behavioral abnormalities or distinct phenomena during 7 days of continuous observation, and changes in body weight were also recorded. The autopsy photos are shown in Fig. 20.

[0234] (4) 0.720ng / kg capacity group:

[0235] Deaths: 6 Survivors: 4

[0236] Observation after administration: 20 minutes after intraperitoneal injection, all male mice began frequently scratching behind their ears and eyes; after 40 minutes, the frequency of scratching gradually decreased until it stopped. Similarly, 30 minutes after injection, all female mice began frequently scratching behind their ears and eyes; after 50 minutes, the frequency of scratching gradually decreased, and after 60 minutes, both male and female mice completely stopped scratching and returned to normal. Of these, two mice were found dead after 20 hours, two others died from generalized convulsions after 24 hours, one mouse died from generalized convulsions after 27 hours, and one mouse died from generalized convulsions after 31 hours. At the time of death, the mice first exhibited slowed movements, dizziness, and increased respiratory fluctuations, followed by the onset of generalized convulsions. Gross examination after dissection revealed intestinal blistering and intestinal distension in all mice; no ulcers were observed, there was no intra-intestinal hemorrhage, and no other organ abnormalities were found. All surviving animals showed varying degrees of scratching response during observation, and after the scratching phenomenon disappeared, there were no behavioral abnormalities or distinct phenomena during 7 days of continuous observation, and changes in body weight were also recorded, and the autopsy photos are shown in Fig. 21.

[0237] (5) 0.432ng / kg capacity group:

[0238] Deaths: 4 Survivors: 6

[0239] Observation after administration: 25 minutes after intraperitoneal injection, male mice began frequently scratching behind their ears and eyes; after 50 minutes, the frequency of scratching gradually decreased until it stopped. Similarly, 35 minutes after injection, female mice began frequently scratching behind their ears and eyes; after 60 minutes, the frequency of scratching gradually decreased, and after 65 minutes, both male and female mice completely stopped scratching and returned to normal. Of these, two mice were found dead after 20 hours, and the other two died from generalized convulsions after 26 hours. At the time of death, the mice first exhibited slowed movements, dizziness, and increased respiratory fluctuations, followed by the onset of generalized convulsions. Gross examination after dissection revealed intestinal blistering and intestinal distension in all mice; no ulcers were observed, there was no intra-intestinal hemorrhage, and no other organ abnormalities were found. Four mice among the surviving animals showed varying degrees of scratching response, and after the scratching phenomenon disappeared, there were no behavioral abnormalities or distinct phenomena during 7 consecutive observation days, and changes in body weight were also recorded, and the dissection photos are shown in Fig. 22.

[0240] (6) 0.259ng / kg capacity group:

[0241] Deaths: 2 Survivors: 8

[0242] Observation after administration: 30 minutes after intraperitoneal injection, male mice began frequently scratching behind their ears and eyes; after 50 minutes, the frequency of scratching gradually decreased until it stopped. 40 minutes after injection, female mice began frequently scratching behind their ears and eyes; after 60 minutes, the frequency of scratching gradually decreased, and after 70 minutes, both male and female mice completely stopped scratching and returned to normal. Two mice were found dead after 20 hours; at the time of death, the mice initially exhibited slowed movements, dizziness, and increased respiratory fluctuations, followed by the onset of generalized convulsions. Gross examination after dissection revealed intestinal blistering and intestinal distension in all mice; no ulcers were observed, there was no intra-intestinal hemorrhage, and no other organ abnormalities were found. In the observation of surviving animals, five mice showed varying degrees of scratching response, and after the scratching phenomenon disappeared, there were no behavioral abnormalities during 7 consecutive observation days, while other mice showed no distinct phenomena after administration of the drug, and changes in body weight were also recorded, and the dissection photos are shown in Fig. 23.

[0243] (7) 0.156ng / kg capacity group:

[0244] Deaths: 1 Survivors: 9

[0245] Observation after administration: 30 min after intraperitoneal injection, male mice began frequently scratching behind their ears and eyes; after 50 min, the frequency of scratching gradually decreased until it stopped. 40 min after injection, female mice began frequently scratching behind their ears and eyes; after 60 min, the frequency of scratching gradually decreased, and after 70 min, both male and female mice completely stopped scratching and returned to normal. 43 h after administration, one mouse exhibited slowed movement, dizziness, and increased respiratory fluctuations, followed by generalized convulsive death after 44 h. In gross examination after dissection, intestinal vesicles and intestinal distension were observed in all mice; no ulcers were observed, there was no intra-intestinal hemorrhage, and no other organ abnormalities were found. In observation of surviving animals, three mice exhibited varying degrees of scratching response; after the scratching disappeared, there were no behavioral abnormalities during 7 consecutive days of observation. The other mice showed no distinct symptoms after administration, and changes in body weight were recorded; the dissection photos are shown in Figure 24.

[0246] (8) 0.093ng / Kg Capacity Group:

[0247] Deaths: 0 Survivors: 10

[0248] Observation after administration: 35 min after intraperitoneal injection, two male mice began slightly scratching their faces; after 45 min, the frequency of scratching gradually decreased until it stopped. 45 min after injection, one female mouse began slightly scratching her eyes; after 55 min, the frequency of scratching gradually decreased, and after 60 min, both male and female mice completely stopped scratching and returned to normal. No behavioral abnormalities were observed during the 7 consecutive days of observation, and changes in body weight were also recorded. There were no animal deaths.

[0249] 2. Calculation of median lethal dose using the Bliss method. The median lethal dose was calculated using the results described above. As can be seen from the results in Table 1, the regression equation is y(Probit) = 5.9644 + 3.174Log(D), the median lethal dose LD50 = 0.49676 ng / kg, the 95% confidence interval of LD50 (Feiller corrected) is 0.36659~0.6715 ng / kg, LD5 = 0.15063 ng / kg, and LD95 = 1.6383 ng / kg.

[0250] The above results explain that the LD50 of a single intraperitoneal injection of the drug 030-3C(WT)3(t1) in mice is 0.49676 ng / kg and the 95% confidence interval is 0.36659–0.6715 ng / kg, which means it has relatively high activity and is comparable to the activity of commercially available botulinum toxins.

[0251] Table 1 Calculation of median lethal dose using the Bliss method

[0252] volume Log capacity Number of animals Number of deaths Death percentage experimental probability units Regression probability units ng / kg ng / mL (x) (number of animals) (number of animals) (%) (Y) (Y) 3.335 6.6670 0.5231 10 10 100.00 --- 7.6247 2.000 4.0010 0.30103 10 10 100.00 --- 6.9199 1.200 2.4006 0.079181 10 9 90.00 6.2817 6.2157 0.720 1.4403 -0.14267 10 6 60.00 5.2529 5.5116 0.432 0.8642 -0.36452 10 4 40.00 4.7471 4.8075 0.259 0.5185 -0.5867 10 2 20.00 4.1585 4.1023 0.156 0.3111 -0.80688 10 1 10.00 3.7183 3.4034 0.093 0.1866 -1.0315 10 0 0.00 --- 2.6904

[0255] In the description herein, the reference terms “one embodiment,” “some embodiment,” “example,” “specific example,” or “some example” mean that specific features, structures, materials, or properties described in combination with such embodiments or examples are included in at least one embodiment or example of the present invention. In this application, general expressions of the terms described above do not necessarily refer to the same embodiment or example. Furthermore, the described specific features, structures, materials, or properties may be combined in a suitable manner in any one or more embodiments or examples. Additionally, those skilled in the art may combine features of different embodiments or examples described herein with features of different embodiments or examples, provided that such combinations are not contradictory.

[0256] Although embodiments of the present invention have been presented and described above, the above-described embodiments are exemplary and should not be understood as a limitation to the present invention, and those skilled in the art will understand that changes, modifications, replacements, and variations can be made to the above-described embodiments within the scope of the present invention.

Claims

Claim 1 Isolated nucleic acid comprising a first nucleic acid fragment, a second nucleic acid fragment, a first modified tag, and a second modified tag, wherein the first nucleic acid fragment, the second nucleic acid fragment, the first modified tag, and the second modified tag are connected to each other; wherein the first nucleic acid fragment codes for a heavy chain of botulinum toxin; the second nucleic acid fragment codes for a light chain of botulinum toxin; the first modified tag codes for a His tag; and the second modified tag codes for a GST tag or an MBP tag. Claim 2 A nucleic acid according to claim 1, further comprising a first enzyme cleavage site, a second enzyme cleavage site, and a third enzyme cleavage site; wherein the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site are identical; and wherein the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site code for a 3C enzyme cleavage site, a TEV enzyme cleavage site, a thrombin enzyme cleavage site, an Fxa enzyme cleavage site, or an enterokinase enzyme cleavage site. Claim 3 A nucleic acid according to claim 1 or 2, wherein the first modified tag codes for a His tag, the second modified tag codes for a GST tag, and the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site code for a 3C enzyme cleavage site or a Thrombin enzyme cleavage site. Claim 4 A nucleic acid according to claim 1 or 2, wherein the first modified tag codes for a His tag, the second modified tag codes for an MBP tag, and the first enzyme cleavage site, the second enzyme cleavage site, and the third enzyme cleavage site code for a 3C enzyme cleavage site or a Thrombin enzyme cleavage site. Claim 5 A nucleic acid according to any one of claims 1 to 4, wherein the 5' end of the first modified tag is connected to the 3' end of the first enzyme cleavage site, the 5' end of the first enzyme cleavage site is connected to the 3' end of the first nucleic acid fragment, the 5' end of the first nucleic acid fragment is connected to the 3' end of the second enzyme cleavage site, the 5' end of the second enzyme cleavage site is connected to the 3' end of the second nucleic acid fragment, the 5' end of the second nucleic acid fragment is connected to the 3' end of the third enzyme cleavage site, and the 5' end of the third enzyme cleavage site is connected to the 3' end of the second modified tag. Claim 6 In any one of claims 1 to 5, the first nucleic acid fragment has a nucleotide sequence as presented in SEQ ID NO:1; the second nucleic acid fragment has a nucleotide sequence as presented in SEQ ID NO:2; the sequence coding for the His tag has a nucleotide sequence as presented in SEQ ID NO:3; the sequence coding for the GST tag has a nucleotide sequence as presented in SEQ ID NO:4; the sequence coding for the MBP tag has a nucleotide sequence as presented in SEQ ID NO:5; the sequence coding for the 3C enzyme cleavage site has a nucleotide sequence as presented in SEQ ID NO:6; the sequence coding for the TEV enzyme cleavage site has a nucleotide sequence as presented in SEQ ID NO:7; the sequence coding for the Thrombin enzyme cleavage site has a nucleotide sequence as presented in SEQ ID NO:8; and the sequence coding for the Fxa enzyme cleavage site is SEQ ID A nucleic acid having a nucleotide sequence as presented in NO:9; wherein the sequence encoding the enterokinase enzyme cleavage site has a nucleotide sequence as presented in SEQ ID NO:

10. Claim 7 A vector wherein the vector carries a nucleic acid according to any one of claims 1 to 6; and wherein the vector is selected from a plasmid. Claim 8 A recombinant cell comprising a nucleic acid according to any one of claims 1 to 6 or a vector according to claim 7; wherein the cell is selected from a prokaryotic cell or a eukaryotic cell; and wherein the cell is selected from an E. coli, yeast, cyanobacteria, or mammalian cell line. Claim 9 A method for obtaining a recombinant protein botulinum toxin, comprising the steps of: culturing a recombinant cell according to claim 8 under conditions suitable for protein expression; purifying the culture-treated product; and enzymatically cleaving the purified product to obtain the recombinant protein botulinum toxin. Claim 10 A method according to claim 9, wherein the purification treatment is performed in the following manner: the culture treatment product is subjected to a first purification treatment, and the first purification treatment is performed in a second modified tag purification column; the culture treatment product subjected to the first purification treatment is subjected to a second purification treatment, and the second purification treatment is performed in a first modified tag purification column. Claim 11 A method according to claim 9, wherein the purification treatment is performed in the following manner: the culture treatment product is subjected to a first purification treatment, and the first purification treatment is performed in a GST tag purification column; the culture treatment product subjected to the first purification treatment is subjected to a second purification treatment, and the second purification treatment is performed in a His tag purification column, wherein the site of the enzyme cleavage treatment is a 3C enzyme cleavage site or a Thrombin enzyme cleavage site. Claim 12 A method according to claim 9, wherein the purification treatment is performed in the following manner: the culture treatment product is subjected to a first purification treatment, and the first purification treatment is performed in an MBP tag purification column; the culture treatment product subjected to the first purification treatment is subjected to a second purification treatment, and the second purification treatment is performed in a His tag purification column, wherein the site of the enzyme cleavage treatment is a 3C enzyme cleavage site or a Thrombin enzyme cleavage site. Claim 13 A method according to claim 9, wherein the purification treatment is performed in the following manner: the culture treatment product is subjected to a third purification treatment, and the third purification treatment is performed in a His tag purification column; the culture treatment product subjected to the third purification treatment is subjected to a fourth purification treatment, and the fourth purification treatment is performed in a strong anion exchange chromatography column; and the site of the enzyme cleavage treatment is a 3C enzyme cleavage site or a Thrombin enzyme cleavage site. Claim 14 A recombinant protein botulinum toxin characterized by being manufactured and obtained by a method according to any one of claims 9 to 13. Claim 15 A composition characterized by comprising, as a composition, a nucleic acid according to any one of claims 1 to 6, a vector according to claim 7, a recombinant cell according to claim 8, or a recombinant protein botulinum toxin according to claim 14. Claim 16 A use in the manufacture of a drug comprising a nucleic acid according to any one of claims 1 to 6, a vector according to claim 7, a recombinant cell according to claim 8, a recombinant protein botulinum toxin according to claim 14, or a composition according to claim 15, wherein the drug is used for the prevention or treatment of a disease. Claim 17 In claim 16, the above disease includes neuromuscular disease, and the symptoms of the above disease include one or more of: spastic dysphonia, spastic torticollis, laryngeal dystonia, oromandibular dysphonia, tongue dystonia, cervical dystonia, focal dystonia, blepharospasm, strabismus, unilateral facial spasm, blepharoplasty, cerebral palsy, focal spasm, spastic colitis, neurogenic bladder overactivity, idiopathic bladder overactivity, pelvic floor relaxation delay, limb spasms, motor tics, primary hand tremor, head tremor, detrusor-sphincter incoordination, bruxism, dentition, achalasia, dysphagia, hyperhidrosis, hypotonia, excessive gastrointestinal secretion, secretory disorder, pain due to muscle spasms, chronic migraine, or dermatological disease. Claim 18 A use in the preparation of a reagent according to any one of claims 1 to 6, a vector according to claim 7, a recombinant cell according to claim 8, a recombinant protein botulinum toxin according to claim 14, or a composition according to claim 15, wherein the reagent is used to improve related symptoms in the body by interfering with the release of neurotransmitters from isolated nerve cells or interfering with the release of neurotransmitters in the body, and preferably, the reagent is used for cosmetic purposes. Claim 19 A method for the prevention or treatment of a neuromuscular disease, characterized by comprising the step of administering to a subject an effective amount of a nucleic acid according to any one of claims 1 to 6, a vector according to claim 7, a recombinant cell according to claim 8, a recombinant protein botulinum toxin according to claim 14, or a composition according to claim 15.

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