WO2024046404A1 - 一种重组肉毒杆菌毒素及其制备方法 - Google Patents

一种重组肉毒杆菌毒素及其制备方法 Download PDF

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WO2024046404A1
WO2024046404A1 PCT/CN2023/115999 CN2023115999W WO2024046404A1 WO 2024046404 A1 WO2024046404 A1 WO 2024046404A1 CN 2023115999 W CN2023115999 W CN 2023115999W WO 2024046404 A1 WO2024046404 A1 WO 2024046404A1
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botulinum toxin
sequence
heavy chain
amino acid
recombinant
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French (fr)
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蒋天宇
周小雪
尹进
李欣茹
滕欣
谈畅
李腾
张浩千
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上海蓝晶生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/63—Vibrio
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a recombinant botulinum toxin and a preparation method thereof.
  • Botulinum toxin is a neurotoxin secreted by Clostridium botulinum. According to its antigenicity, it can be divided into 7 serotypes A to G. Botulinum toxin has strong neurotoxicity and can cause nerve paralysis in very small doses. When a person eats food containing botulinum toxin, the incubation period is 6 hours to 12 days, and clinical symptoms usually appear in 3 to 4 days. The patient eventually suffers from Death from respiratory failure. Botulinum toxin is highly toxic, equivalent to 10,000 times more potent than potassium cyanide.
  • Clostridium botulinum itself can produce a toxin protein complex with a molecular weight of 900kD, which is composed of three parts: hemagglutinin protein (HA), non-toxin non-hemagglutinin protein (NTNH) and neurotoxic protein.
  • HA hemagglutinin protein
  • NTNH non-toxin non-hemagglutinin protein
  • the neurotoxin protein has a molecular weight of 150kD and is produced by Clostridium botulinum under anaerobic conditions. It is then cleaved by the protease produced by Clostridium botulinum itself into two peptides, light and heavy. The chain near the N-terminal is the catalytic domain, and the heavy chain is near the C-terminal and is the receptor binding domain.
  • the heavy chain and light chain are connected by disulfide bonds, they become toxic botulinum toxin.
  • the proteases in the digestive tract cannot degrade it, allowing the active ingredients to enter the blood through the intestinal wall; then, through the blood circulation, it is combined with the motor or sympathetic nerves
  • the heavy chain binds to the receptor; botulinum toxin enters the nerve cells relying on the endocytosis of nerve cells. Since the cytoplasmic matrix presents a reducing environment, the double bonds between the heavy chain and the light chain are reduced and opened.
  • the light chain is released from the complex, enzymatically cleaves the snap-25 protein in the postsynaptic membrane, prevents the synthesis of SNARE complex proteins, inhibits the release of acetylcholine, causes severe muscle paralysis, and then inhibits breathing, leading to death.
  • botulinum toxin Because of its nerve-paralyzing effect, botulinum toxin was initially used to treat facial muscle spasms and other muscle movement disorders. Botulinum toxin is used to paralyze muscle nerves to stop muscle spasms. Botulinum toxin is also used in medical cosmetology. It blocks the nerve impulses between nerves and muscles to relax overly contracted small muscles, thereby achieving the effect of wrinkle removal; or it can use its property of temporarily paralyzing muscles to make them It shrinks due to loss of function in order to achieve the purpose of sculpting the lines of the body. In addition, botulinum toxin also has anti-depressant and anti-depressant properties.
  • botulinum toxin mainly expresses the protein through anaerobic fermentation of Clostridium botulinum, and then undergoes multi-stage purification. Simply put, botulinum toxin is purified from the culture solution through dialysis and a series of acid precipitation. For botulinum toxin, this method takes about 5 days from fermentation to finally obtaining the botulinum toxin protein.
  • the active ingredient obtained through this method is a 900kD botulinum toxin complex. Part of the lectin protein of botulinum toxin is further removed through ion exchange to obtain a toxin complex of about 450kD. Other proteins are then removed by changing the pH. , obtaining 150kD botulinum toxin.
  • botulinum toxin In-situ extraction of botulinum toxin by Clostridium botulinum is a relatively traditional method, and its theory is relatively mature. But because of Botox The fermentation of Bacillus requires a very strict anaerobic environment and is very sensitive to temperature. Therefore, specialized fermentation equipment is required for large-scale production. In addition, since the expressed botulinum toxin does not carry an affinity chromatography tag, multiple purification steps are required during extraction to obtain a relatively pure protein. Moreover, due to the strong toxicity of botulinum toxin, the longer the preparation process, the greater the probability of accidental contamination during the process.
  • chassis cells can also be used to heterologously express recombinant botulinum toxin.
  • the expression of recombinant botulinum toxin basically uses Escherichia coli as the chassis cell.
  • the main method for preparing botulinum toxin from Escherichia coli is to use plasmid transfection technology to transfer the plasmid for expressing botulinum toxin into Escherichia coli, and use inducers (such as IPTG) to induce the expression of 150kD botulinum neurotoxin protein .
  • the existing recombinant expression of botulinum toxin is divided into two methods: single-chain and double-chain expression.
  • Double-stranded expression is designed based on the existing protein structure (protein data bank id: 3V0C). In this structure, there are clear interaction sites between the heavy and light chains.
  • the botulinum toxin light and heavy chain complex can be obtained. Due to the presence of cysteine residues at the carboxyl terminus of the light chain and the amino terminus of the heavy chain, disulfide bonds can be formed between the light chain and the heavy chain, thus forming Active protein complex.
  • coli lacks the necessary protease and cannot digest the full-length single-chain protein, it is necessary to introduce an exogenous protease between the light chain and heavy chain for enzymatic cleavage. site, and activate the protein through in vitro enzyme digestion.
  • the activated protein has similar activity to commercial botulinum toxin products.
  • botulinum toxin in E. coli Although recombinant expression of botulinum toxin in E. coli has the advantage of being fast, in order to avoid introducing amino acid residues that do not exist in the sequence of botulinum toxin itself, proteins without affinity tags are expressed in the prior art, and the purification method is still Mainly ammonium sulfate precipitation, the process is complicated, and botulinum toxin is highly toxic, so there is still the possibility of accidental contamination.
  • a protease cleavage site must be introduced between the heavy chain and the light chain, and additional amino acid residues will also be introduced.
  • the introduction of additional sequences may introduce unknown antigenicity, which may trigger unknown immune responses after injection into the body, causing potential adverse effects on the body. Therefore, although the activity of recombinant botulinum toxin is equivalent to that of natural toxins, currently Still no related products have been approved for use.
  • the object of the present invention is to provide a recombinant botulinum toxin and a preparation method thereof.
  • the present invention aims to prepare botulinum toxin protein with natural sequence and biological activity, and to achieve efficient and stable expression of the protein in a biological chassis.
  • the present invention first specifically designed and optimized the sequences of the heavy chain and light chain of recombinant botulinum toxin.
  • the present invention adopts a double-stranded expression method and introduces protein tags. After extensive research, the present invention found that during the double-chain expression of recombinant botulinum toxin, the expression level of the light chain was much higher than the expression level of the heavy chain.
  • the present invention provides the following technical solutions:
  • the present invention provides a recombinant botulinum toxin, which includes a heavy chain and a light chain connected by a disulfide bond; the amino acid sequence of the heavy chain is sequentially from the N-terminus to the C-terminus. It includes a protein tag sequence, an ENLYFQ polypeptide sequence and a heavy chain sequence of botulinum toxin, wherein the N-terminal first amino acid of the heavy chain sequence of botulinum toxin is glycine.
  • the protein tag introduced into the heavy chain sequence of the recombinant botulinum toxin can be any protein tag that facilitates protein purification, including but not limited to His-tag, Gst, MBP, Strep, Flag and other tags.
  • the protein tag is exemplified by His-tag, and the actual application is not limited thereto.
  • the heavy chain sequence of the above-mentioned botulinum toxin can be the complete heavy chain sequence of botulinum toxin or a part of the sequence, but it needs to be ensured that the first amino acid at the N-terminus of the sequence is glycine and is connected to the light chain through a disulfide bond. Has botulinum toxin activity.
  • the heavy chain structure of the recombinant botulinum toxin provided by the present invention is suitable for each serotype of botulinum toxin (for example, type A, type E, etc.).
  • Types A to G of unactivated botulinum toxin are all composed of a polypeptide chain with a relative molecular weight of about 150kD. After being cleaved by bacterial endogenous proteases or exogenous proteases, they are cleaved into a light chain with a molecular weight of about 50kD and a molecular weight of about 100kD. The heavy chain, light chain and heavy chain are connected by a conserved disulfide bond, thus forming a biologically active double-chain toxin.
  • the N-terminal amino acid of the heavy chain sequence of the recombinant botulinum toxin of the present invention is glycine, and the light chain and the heavy chain are connected through a disulfide bond, which means that the heavy chain of the recombinant botulinum toxin of the present invention
  • the sequence also contains cysteine residues that can form disulfide bonds, starting with glycine, which also indicates that the heavy chain contains a complete functional domain. When it is connected to the light chain through disulfide bonds When, a double-chain toxin with normal biological activity can be formed.
  • botulinum toxin A the full-length single chain of botulinum toxin A was cut from glycine 445 to the C-terminus as the heavy chain, and the TEV protease enzyme cleavage sequence was introduced at the N-terminus of glycine 445.
  • the above sequence is designed so that after the heavy chain is digested by TEV enzyme, no amino acid residues that do not exist in its own sequence will remain (that is, no artificially introduced protein tag sequences and enzyme cleavage site sequences will remain, and the botulinum toxin heavy chain will not remain). Still retaining its original sequence), and the digested heavy chain has the biological activity of natural botulinum toxin after being linked to the light chain through disulfide bonds.
  • the heavy chain sequence of the above-mentioned botulinum toxin is the 445th to 1296th amino acid sequence of botulinum toxin A.
  • the 445th amino acid is glycine.
  • the 445th to 1296th amino acid sequence of botulinum toxin A can be the sequence of natural botulinum toxin A (for example, the 445th to 1296th position of the sequence shown in SEQ ID NO. 1), or it can be a mutation For the botulinum toxin A sequence, the mutated botulinum toxin sequence still needs to ensure that the 445th amino acid is glycine and can be connected to the light chain to form a biologically active botulinum toxin.
  • the amino acid sequence from 445th to 1296th of the above-mentioned botulinum toxin A is Based on the botulinum toxin A sequence shown in SEQ ID NO.1 (the coding gene sequence of botulinum toxin shown in SEQ ID NO.1 is shown in SEQ ID NO.5, and SEQ ID NO.5 The sequence shown is a sequence optimized and suitable for expression in E. coli).
  • the amino acid sequence from 445th to 1296th of botulinum toxin A can also be a variant of the above amino acid sequence, as long as the 445th amino acid is glycine and can be connected with the light chain to form a biologically active botulinum toxin. Can.
  • botulinum toxin can be digested by TEV protease to produce botulinum toxin with the protein tag sequence completely removed, and no amino acid residues that do not exist in the botulinum toxin sequence itself will remain.
  • the amino acid sequence of the heavy chain of the recombinant botulinum toxin also includes methionine from the N-terminus to the C-terminus and 1 to 3 amino acids other than cysteine at the N-terminus of the protein tag sequence.
  • the amino acid sequence of the heavy chain is from the N-terminus to the C-terminus: M-amino acid sequence composed of 1 to 3 amino acid residues except cysteine-protein Tag sequence-ENLYFQ-Botulinum toxin heavy chain sequence (N-terminal position 1 is glycine G).
  • the amino acid sequence of the heavy chain is from the N-terminus to the C-terminus: M-amino acid sequence composed of 1 to 3 amino acid residues except cysteine-protein Tag sequence - ENLYFQ - amino acids 445 to 1296 of botulinum toxin A.
  • the amino acid sequence of the heavy chain is from the N-terminus to the C-terminus: MK-protein tag sequence-ENLYFQ-amino acids 445 to 1296 of botulinum toxin A.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO. 2.
  • the amino acid sequence of the heavy chain of the above-mentioned recombinant botulinum toxin is a sequence carrying a protein tag.
  • the protein tag can be removed by TEV protease enzyme digestion, resulting in a heavy chain that does not contain the tag and residual amino acid residues at the enzyme cleavage site, and will not produce meat. Antigenicity other than bacilli toxins.
  • the present invention attempts to select and optimize the light chain sequence, and determine the position from the N-terminus of the full-length single chain of botulinum toxin to lysine 438, as the light chain
  • the sequence can well ensure the biological activity of the light chain.
  • the light chain and heavy chain designed above can efficiently form disulfide bonds. After being digested by TEV protease, a biologically active botulinum toxin is produced without introducing any amino acid residues that do not exist in the botulinum toxin sequence itself.
  • the amino acid sequence of the light chain is from position 1 to position 438 of botulinum toxin A.
  • the amino acid sequence from positions 1 to 438 of botulinum toxin A can be the sequence of natural botulinum toxin A (such as positions 1 to 438 of the sequence shown in SEQ ID NO. 1), or it can be mutated As for the sequence of botulinum toxin A, the mutated botulinum toxin A sequence can be connected to the above-mentioned heavy chain to form a biologically active botulinum toxin.
  • amino acid sequence of the light chain is as shown in positions 1 to 438 of the sequence shown in SEQ ID NO.1.
  • the heavy chain and light chain described above still retain the original cysteine at the positions corresponding to the amino acids 430 and 454 of the full-length single chain of botulinum toxin A, so the light chain and the heavy chain can form disulfide bonds, and the use of the above-designed light and heavy chains is beneficial to the efficient formation of disulfide bonds.
  • the present invention provides a nucleic acid molecule encoding the recombinant botulinum toxin described above.
  • nucleotide sequences of the nucleic acid molecules encoding the above heavy chain and light chain Based on the degeneracy of codons, the nucleotide sequence of the above-mentioned nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the above-mentioned heavy chain and light chain are within the protection scope of the present invention.
  • nucleotide sequence of the nucleic acid molecule encoding the heavy chain is shown in SEQ ID NO. 3.
  • nucleotide sequence of the nucleic acid molecule encoding the light chain is shown in SEQ ID NO. 4.
  • nucleic acid molecules can achieve efficient and stable expression of light chains and heavy chains in Escherichia coli and Natriuretic Vibrio.
  • the present invention provides biological materials comprising the nucleic acid molecules described above; the biological materials are expression cassettes, vectors or host cells.
  • an expression cassette containing the nucleic acid molecule is operably linked to a promoter and the nucleic acid molecule.
  • the expression cassette may also contain other transcription and translation regulatory elements such as terminators and enhancers.
  • the vector containing the nucleic acid molecule is a plasmid vector, and these plasmid vectors include replicating vectors and non-replicating vectors.
  • the vector containing the nucleic acid molecule is not limited to plasmid vectors, but can also be phage, virus and other vectors.
  • the plasmid vector contains a nucleic acid molecule encoding the above-mentioned heavy chain and/or a nucleic acid molecule encoding the above-mentioned light chain.
  • the plasmid vector uses PQlink as a backbone vector, into which the nucleic acid molecule encoding the heavy chain and the nucleic acid molecule encoding the light chain are connected in series.
  • the nucleic acid molecule encoding the above-mentioned heavy chain and the nucleic acid molecule encoding the above-mentioned light chain each contain corresponding protein expression genes, and both have independent promoters, regulatory factors, ribosome binding sites and terminators.
  • the host cell is Escherichia coli or Vibrio natriureticus, but the type of host cell is not limited thereto and can be any microbial cell or animal cell that can be used for protein expression.
  • the present invention provides an engineering bacterium for producing recombinant botulinum toxin, the engineering bacterium containing the above-mentioned nucleic acid molecule.
  • the engineered bacterium is Escherichia coli or Vibrio natriureticus.
  • both Escherichia coli and Vibrio natriureticus can be used as chassis bacteria to express recombinant botulinum toxin.
  • the present invention unexpectedly discovered that Vibrio natriureticus can more efficiently promote the formation of disulfide bonds between the light chain and heavy chain of botulinum toxin, thereby successfully synthesizing biologically active substances faster and better. Botulinum toxin.
  • the engineered bacterium contains a plasmid vector carrying the nucleic acid molecule described above.
  • the engineered bacterium is obtained by introducing a plasmid vector carrying the above-described nucleic acid molecule into Vibrio natriureticus.
  • the nucleic acid molecule encoding the heavy chain and the nucleic acid molecule encoding the light chain may be on the same plasmid vector, or they may be on different plasmid vectors.
  • the engineered bacterium is obtained by introducing a plasmid vector carrying the above-described nucleic acid molecule into Escherichia coli.
  • the nucleic acid molecule encoding the heavy chain and the nucleic acid molecule encoding the light chain may be on the same plasmid vector, or they may be on different plasmid vectors.
  • the present invention provides the use of the above-mentioned recombinant botulinum toxin or the nucleic acid molecule or the biological material or the recombinant Vibrio natriuretic in the preparation of botulinum toxin.
  • the above-mentioned applications include: modifying a host cell so that the host cell expresses the above-described recombinant botulinum toxin or contains the nucleic acid molecule, culturing the host cell, and collecting the recombinant botulinum toxin expressed by the host cell.
  • Botulinum toxin is purified by affinity chromatography and digested by TEV protease to obtain botulinum toxin with the protein tag removed.
  • the above-mentioned applications include: cultivating the recombinant Vibrio natriureticus, collecting the recombinant botulinum toxin expressed by it, and purifying by affinity chromatography and enzymatically digesting it with TEV protease to prepare meat with the protein tag removed. Toxins.
  • the present invention provides a method for preparing botulinum toxin, which method includes: modifying a host cell so that the host cell expresses the above-mentioned recombinant botulinum toxin, cultivating the host cell, and using a biochemical method.
  • the recombinant botulinum toxin is purified from the culture by chromatography, and the purified recombinant botulinum toxin is digested by TEV protease.
  • the column material used in the affinity chromatography can be selected according to the protein tag contained in the heavy chain.
  • a His-tag affinity chromatography column is used corresponding to the His-tag protein tag used.
  • the corresponding purification method can be selected and used according to different protein tag sequences.
  • host cells are cultured to obtain a culture, and the supernatant is separated after lysing the cells for affinity chromatography purification.
  • the method further includes the steps of sequentially performing anion exchange chromatography and molecular sieve chromatography purification after TEV protease digestion.
  • anion exchange chromatography can be performed using an anion exchange column (such as Source Q).
  • Molecular sieve chromatography can be performed using molecular sieve column materials such as SuperDex200.
  • Using the above purification method can significantly improve the purification efficiency and purity of recombinant botulinum toxin.
  • the entire purification process can be completed in less than one day.
  • the purification process is greatly simplified, manpower and materials are saved, and accidental pollution to the environment such as instruments is effectively reduced. possibility.
  • the host cell in some embodiments of the invention, is Vibrio natriureticus.
  • Vibrio natriureticus can more efficiently promote the formation of disulfide bonds between the light chain and heavy chain of botulinum toxin, thereby successfully synthesizing biologically active botulinum toxin faster and better.
  • Bacilli toxins Bacilli toxins.
  • Vibrio natriali as the base bacteria for the preparation of botulinum toxin can also reduce the risk of phage infection and effectively reduce the environmental impact of toxic proteins. The risk of contamination is reduced, and the bacterial culture cycle is shortened.
  • the beneficial effects of the present invention are that: by optimizing the design of the recombinant botulinum toxin heavy chain and light chain sequences, the present invention does not introduce amino acid residues that do not exist in the botulinum toxin sequence itself due to the addition and cutting of protein tags. At the same time, it can perform efficient and stable expression of botulinum toxin and prepare active botulinum toxin. It can also greatly simplify the purification process of toxin protein and achieve efficient preparation of active botulinum toxin.
  • the botulinum toxin preparation method provided by the invention can prepare botulinum toxin quickly and efficiently.
  • the host culture cycle is short and it is not easy to contaminate the phage.
  • the toxin protein purification process is simple and easy to scale up production, which greatly reduces the cost of botulinum toxin.
  • the cost of preparing botulinum toxin, the prepared botulinum toxin has the expected biological activity, is equivalent to that of natural botulinum toxin, and has good application prospects.
  • Figure 1 shows the PCR bands of products P1, P2 and P3 in Example 1 of the present invention.
  • Figure 2 shows the PCR screening bands in Example 2 of the present invention, of which 2 and 4 are in line with expectations.
  • Figure 3 is a structural diagram of the tandem plasmid in Example 2 of the present invention.
  • Figure 4 shows the purification results of His-tag affinity chromatography in Example 5 of the present invention.
  • Figure 5 shows the anion exchange column purification results in Example 5 of the present invention.
  • Figure 6 is the SDS-Page verification result of anion exchange column purification in Example 5 of the present invention.
  • FT is the flow-through liquid
  • numbers 7-14 are the numbers of the anion exchange column collection tube in Figure 5 respectively.
  • Figure 7 shows the purification results of molecular sieve SUPERDEX200 in Example 5 of the present invention.
  • Figure 8 is the SDS-PAGE verification result of purification of molecular sieve SUPERDEX200 in Example 5 of the present invention, in which numbers 11-18 are the collection tube numbers of molecular sieve SUPERDEX200 in Figure 7 respectively.
  • Figure 9 is the SDS-PAGE verification result of molecular sieve SUPERDEX200 purification in Example 6 of the present invention.
  • Figure 10 is a working curve diagram of botulinum toxin protein concentration in Example 7 of the present invention.
  • Figure 11 shows the results of the mouse gastrocnemius muscle injection experiment 12 hours later in Example 7 of the present invention.
  • PQLink homology arms were introduced into the upstream and downstream of the light and heavy chains, and His tag and TEV restriction site were introduced into the upstream of the heavy chain.
  • KOD Mix system perform 20 ⁇ L system reactions in each case.
  • the amplification program was: denaturation at 95°C for 15 seconds; annealing at 56°C for 10 seconds, extension at 68°C for 30 seconds, and the entire reaction lasted 34 cycles.
  • the corresponding primers of PQlink were used to PCR amplify the vector, linearize the vector, and use nucleic acid gel electrophoresis to collect the reaction fragments, marked as P3.
  • the reaction system was 50 ⁇ L.
  • the amplification program was: 95°C, 15s denaturation; 56°C, 10s annealing. , extension at 68°C for 60 s, and the entire reaction was repeated for 34 cycles.
  • the results are shown in Figure 1.
  • the P1 band size is about 1200bp
  • the P2 band size is about 2600bp
  • the P3 band size is about 5000bp. Sizes are as expected.
  • the primers used were as follows.
  • BotLc-3 5'- GTCCTAGGCGGCCGCTTA TTTAGAGGTGATGATACCCACG-3'
  • Primer PQlink-5v 5’-TAAGCGGCCGCCTAGGACCCAGC-3’
  • the operating steps are the same as the instructions.
  • the light and heavy chain recovery fragments P1 and P2 were homologously recombined with P3 respectively.
  • the kit selected was the Novartis homologous recombination kit (ClonExpress II One Step Cloning Kit). The operating steps were the same as the instructions.
  • Primer PQlink-F 5’-TATAAAAATAGGCGTATCACGAGG-3’
  • the coding gene sequence of the heavy chain of botulinum toxin amplified by the above PCR is shown in SEQ ID NO.3, the amino acid sequence coding for the heavy chain is shown in SEQ ID NO.2, and the coding gene sequence of the light chain is shown in SEQ ID As shown in NO.4, the amino acid sequence encoding the light chain is shown in positions 1-438 of SEQ ID NO.1.
  • Plasmids for expressing the light chain and the heavy chain alone were obtained from Example 1, and were designated as B1 and B2 in sequence.
  • B2 was digested with SwaI enzyme (both SwaI and buffer3.1 were purchased from NEB Company).
  • the enzyme digestion system was: 2 ⁇ L plasmid, 0.5 ⁇ L enzyme, 1 ⁇ L Buffer3.1, and water to 10 ⁇ L. Enzyme digestion at room temperature for 2 hours. Afterwards, the system was placed at 65°C for 30 minutes, and the product was recorded as P4.
  • the reaction system is 20 ⁇ L.
  • the amplification program is: 95°C, 15s denaturation; 56°C, 10s annealing, 68°C extension for 30s.
  • the entire reaction is 34 cycles.
  • the primers are as follows:
  • Primer PQlink-5ter 5’-TAACAACACCATTTGTCGAGAA-3’;
  • Primer PQlink-3ter 5’-CCATTTGTAACCACTCCATTT-3’.
  • Primer PQlink-F 5’-TATAAAAATAGGCGTATCACGAGG-3’;
  • Bonta HC is the botulinum toxin heavy chain carrying a His tag at the N terminus
  • Bonta LC is the botulinum toxin light chain
  • the promoters of the heavy chain and light chain genes are both t5.
  • the promoter, the regulatory element is lac operator, and the terminator is lambda t0 terminator.
  • the required Natrivibrio competent strain was purchased from synthetic genomics under the trade name Vmax TM Express. Take 2 ⁇ L of the co-expression plasmid prepared in Example 2 and add it to the Natrivibrio competent cells, and electroporate the cells at 1500V, 5ms.
  • the electric shock conversion cup was purchased from biorad, item number 165-2086 2mm.
  • Add 1 mL of high-salt LB medium (components per liter, NaCl 30g, tryptone 10g, and yeast extract 5g) to the electroporated transformation product. Incubate at 37°C with shaking for 1 hour. Spread onto a high-salt LB plate (components per liter, NaCl 30g, tryptone 10g, yeast extract 5g, agar 10g, containing 50 ⁇ g/mL ampicillin).
  • botulinum toxin protein expressed by Vibrio natriensis Vmax and Escherichia coli Origami was simultaneously purified under the same conditions below:
  • Ultrasonic lysis conditions: 10 horns, power 350W, ultrasonic for 2 seconds, pause for 2 seconds, 4 minutes per liter of bacteria (for example, to collect 6L of bacteria, the ultrasonic time should be 24 minutes), ultrasonic until the bacterial solution is clear.
  • Wash buffer 25mM Tris 8.0, 150mM NaCl, 10mM imidazole
  • wash 50mL per liter of bacteria 25mM Tris 8.0, 150mM NaCl, 10mM imidazole
  • Elute buffer 25mM Tris 8.0, 50mM NaCl, 250mM imidazole
  • wash 10mL of bacteria per liter and collect the eluent.
  • Add 100 U of TEV enzyme (purchased from Beyotime, product number: P2307) to 10 mL of eluate for enzyme digestion.
  • 5 ⁇ non-denaturing non-reducing protein loading buffer was purchased from YeasenChina, product number 20317ES. Please refer to the instruction manual for sample preparation methods. Among them, centrifugal precipitation was performed, the centrifugation supernatant was loaded with a sample volume of 2 ⁇ L, and the high imidazole eluate was loaded with a sample volume of 5 ⁇ L, and polyacrylamide gel electrophoresis was performed. The detection results are shown in Figure 4.
  • the high imidazole elution of Natrivibrio is further purified through an anion exchange column/SuperDex200 molecular sieve.
  • the A buffer formula of the anion exchange column is: 25mM Tris 8.0
  • the B buffer formula is: 1M NaCl, 25mM Tris 8.0.
  • Dilute the conductivity of the eluent to 7 mS/cm pass it through the anion exchange column Source Q, and collect the flow-through fluid (FT).
  • the elution program is linearly increased to 60% B buffer, and the total elution volume is 200 mL.
  • the elution UV280nm detection results are shown in Figure 5.
  • the corresponding samples were collected, protein samples were prepared, and the sample volume per lane was 5 ⁇ L, and polyacrylamide gel electrophoresis was performed.
  • the results are shown in Figure 6. Among them, the protein purity of tubes 7 and 8 was higher.
  • Collect 7-8 tubes use a 50kD Millipore protein concentration tube, rotate at 2000g, concentrate to 2mL, load the sample into molecular sieve SuperDex200, and use lysis buffer as the mobile phase.
  • the elution UV280nm detection results are shown in Figure 7.
  • the corresponding samples were collected, protein samples were prepared, and the sample volume per lane was 5 ⁇ L, and polyacrylamide gel electrophoresis was performed. The results are shown in Figure 6. Among them, the 14th tube protein is at the peak.
  • the SDS-PAGE detection results of molecular sieve SUPERDEX200 purification are shown in Figure 8.
  • the precast gels used for polyacrylamide gel electrophoresis (SDS-PAGE) of the above samples were all purchased from Beyotime, and the experimental implementation method was carried out in accordance with its instructions.
  • the loading buffer is a reducing buffer, purchased from Polymer, M5 5 ⁇ SDS-PAGE reducing 5 ⁇ protein loading buffer, Cat. No. MF145-10.
  • the positive control was carried out according to Example 4, and the only difference from the sample was that TEV enzyme digestion was not performed in the middle.
  • the mouse gastrocnemius muscle injection experiment was used to detect the activity of the botulinum toxin prepared in Example 5, as follows:
  • the protein sample obtained in Example 5 was diluted to 0.25ng/mL (the detection method is enzyme-linked immunosorbent assay ELISA, the kit was purchased from Shanghai Qincheng Biotechnology, the working curve is shown in Figure 10, in the regression equation, the Abs value is the absorbance value reading, [Protein] refers to the concentration of botulinum toxin, R value is greater than 0.99, data quality is reliable), the solvent is 0.5 ⁇ BSA, 9 ⁇ sodium chloride solution.
  • a protein amount of 5pg can cause paralysis of the right leg of mice within 12 hours, but has no effect on the left leg.
  • the recombinant botulinum toxin of the present invention does not introduce amino acid residues that do not exist in the sequence of the botulinum toxin itself due to the addition and cutting of protein tags. At the same time, it can achieve efficient and stable expression and has high activity, and the purification process is simple and easy to implement. It is easy to scale up production, greatly reduces the cost of botulinum toxin preparation, and has good application prospects.

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Abstract

一种重组肉毒杆菌毒素及其制备方法。通过对重组肉毒杆菌毒素重链和轻链序列的设计优化，实现了不因添加和切割蛋白标签引入肉毒杆菌毒素本身序列不存在的氨基酸残基，同时能够进行肉毒杆菌毒素的高效稳定表达和活性肉毒杆菌毒素的制备，还能够极大地简化毒素蛋白的纯化工艺，实现活性肉毒杆菌毒素的高效制备。

Description

一种重组肉毒杆菌毒素及其制备方法 技术领域
本发明涉及生物技术领域，尤其涉及一种重组肉毒杆菌毒素及其制备方法。
背景技术
肉毒杆菌毒素是由一种肉毒梭状芽胞杆菌分泌产生的神经毒素，根据抗原性，可以将其分成A-G共7个血清型。肉毒杆菌毒素具有强烈的神经毒性，可以在极小的剂量下引起神经麻痹，当人食入含有肉毒杆菌毒素的食物后，潜伏期6h到12d，一般3～4d出现临床症状，患者最终因呼吸衰竭而死亡。肉毒杆菌毒素的毒性很强，相当于氰化钾的一万倍。
肉毒梭状芽胞杆菌本身可以产生分子量大小在900kD的毒蛋白复合体，由红细胞凝集素蛋白(HA)、非毒素非血凝素蛋白(NTNH)和神经毒蛋白三部分组成。其中，神经毒蛋白的分子量大小为150kD，由肉毒梭状芽胞杆菌在厌氧条件下产生，之后被肉毒梭状芽胞杆菌自身产生的蛋白酶酶切，成为轻、重两条肽段，轻链靠近N端，为催化结构域，重链靠近C端，为受体结合域。重链、轻链之间由二硫键相连后，成为有毒性的肉毒杆菌毒素。当人类误食含有肉毒杆菌毒素的食品之后，由于保护蛋白的存在，消化道内的蛋白酶无法将其降解，使得有效成分通过肠壁进入血液；之后，通过血液循环，其结合至运动或交感神经细胞的受体部位，重链与受体相结合；肉毒杆菌毒素依靠神经细胞的内吞作用进入神经细胞，由于细胞质基质呈现还原性环境，重链、轻链之间的双键被还原打开，轻链从复合体中释放出来，酶切突触后膜的snap-25蛋白，阻止SNARE复合蛋白的合成，抑制乙酰胆碱的释放，使肌肉出现严重麻痹的症状，进而抑制呼吸，导致死亡。
因具有神经麻痹的作用，肉毒杆菌毒素起初被用于治疗面部肌肉痉挛和其他肌肉运动紊乱症，利用肉毒杆菌毒素麻痹肌肉神经，以达到停止肌肉痉挛的目的。肉毒杆菌毒素也被应用于医学美容，通过阻断神经与肌肉间的神经冲动，使过度收缩的小肌肉放松，进而达到除皱的效果；或者是利用其可以暂时麻痹肌肉的特性，使肌肉因失去功能而萎缩，以达到雕塑机体线条的目的。此外，肉毒杆菌毒素还有抑制肢体痉挛和抗抑郁的效果。
现有的肉毒杆菌毒素的提取方法主要是通过肉毒梭状芽胞杆菌厌氧发酵表达蛋白，之后经过多级纯化，简单来说，是通过透析和一系列酸沉淀从培养液中纯化肉毒杆菌毒素，该方法从发酵到最终得到肉毒杆菌毒素蛋白需要5天左右的时间。通过该方法得到的有效成分是900kD肉毒杆菌毒素复合体，进一步经过离子交换方法将肉毒杆菌毒素的一部分凝集素蛋白去除，得到450kD左右的毒素复合体；再通过改变pH的方法去除其他蛋白，得到150kD的肉毒杆菌毒素。
肉毒梭状芽胞杆菌原位提取肉毒杆菌毒素是相对传统的方法，其理论较为成熟。但是由于肉毒梭 状芽胞杆菌的发酵需要非常严格的厌氧环境，并且对温度非常敏感。因此，需要专门的发酵设备才能进行规模化生产。另外，由于表达的肉毒杆菌毒素没有携带亲和层析标签，在提取中需要多步纯化步骤才能得到相对纯净的蛋白。而且，由于肉毒杆菌毒素的强毒性，制备流程越长，期间造成意外污染的概率越大。
除肉毒梭状芽胞杆菌原位提取肉毒杆菌毒素方法外，还可利用其他底盘细胞异源表达重组肉毒杆菌毒素。目前重组肉毒杆菌毒素的表达基本上都是以大肠杆菌作为底盘细胞。大肠杆菌制备肉毒杆菌毒素的方法主要为：利用质粒转染技术，将用于表达肉毒毒素的质粒转入大肠杆菌中，利用诱导剂(如IPTG)诱导表达150kD的肉毒杆菌神经毒蛋白。
现有的肉毒杆菌毒素的重组表达分为单链和双链表达两种方式。双链表达是依照现有的蛋白结构(protein data bank id：3V0C)设计的。在该结构中，重链和轻链之间有明显的相互作用位点。通过共表达，可以得到肉毒杆菌毒素轻重链复合体，由于轻链的羧基末端和重链的氨基末端存在半胱氨酸残基，轻链、重链之间可以形成二硫键，从而形成有活性的蛋白复合体。另外也有重组表达单链的肉毒杆菌毒素的方法，但由于大肠杆菌缺乏必要的蛋白酶，无法将全长的单链蛋白进行消化，因此需要在轻链、重链之间外源引入蛋白酶酶切位点，通过体外酶切的方式将蛋白活化。经过活化后的蛋白与商业化的肉毒杆菌毒素产品具有相似的活性。
尽管大肠杆菌重组表达肉毒杆菌毒素具有速度快等优势，但是，为了避免引入肉毒杆菌毒素本身序列不存在的氨基酸残基，现有技术中表达的是没有亲和标签的蛋白，提纯方法仍以硫酸铵沉淀为主，工序繁杂，而肉毒杆菌毒素毒性大，仍存在造成意外污染的可能性。而单链融合表达，必须在重链和轻链之间引入蛋白酶酶切位点，也会引入额外的氨基酸残基。而引入额外的序列可能会引入未知的抗原性，注射体内后可能引发未知的免疫反应，会对机体造成潜在的不利影响，因此尽管重组肉毒杆菌毒素活性与天然毒素相比活性相当，但是目前仍然没有相关产品获得使用批准。
发明内容
本发明的目的在于提供一种重组肉毒杆菌毒素及其制备方法。
本发明旨在制备具有天然序列和生物活性的肉毒杆菌毒素蛋白，并实现蛋白在生物底盘中高效稳定的表达。为此，本发明首先对重组肉毒杆菌毒素的重链和轻链的序列进行了特异的设计和优化。为简化后续提纯工艺并尽量避免引入肉毒杆菌毒素本身序列不存在的氨基酸残基，本发明采用双链表达方式，并引入蛋白标签。本发明经大量研究发现，在双链表达重组肉毒杆菌毒素过程中，轻链表达量远远高于重链表达量，因此确定将蛋白标签添加在重链上，以减少多余的轻链对纯化造成的干扰。在引入蛋白标签和去除标签过程中，通常会导致肉毒杆菌毒素本身序列不存在的氨基酸残基的引入。本发明通过序列设计避免了额外氨基酸残基的引入。
具体地，本发明提供以下技术方案：
第一方面，本发明提供一种重组肉毒杆菌毒素，所述重组肉毒杆菌毒素包括由二硫键连接的重链和轻链；所述重链的氨基酸序列从N端到C端的方向依次包含蛋白标签序列、ENLYFQ多肽序列和肉毒杆菌毒素的重链序列，其中，肉毒杆菌毒素的重链序列的N端第1位氨基酸为甘氨酸。
上述重组肉毒杆菌毒素的重链序列中引入的蛋白标签可以为任意便于蛋白纯化的蛋白标签，包括但不限于His-tag、Gst、MBP、Strep、Flag等标签。
在本发明的一些实施方式中，所述蛋白标签以His-tag作为示例性说明，实际应用时并不局限于此。
上述肉毒杆菌毒素的重链序列可为肉毒杆菌毒素的完整重链序列或截取其中的一部分序列，但需要保证序列N端的第1位氨基酸为甘氨酸，且与轻链通过二硫键连接后具有肉毒杆菌毒素的活性。
本发明提供的上述重组肉毒杆菌毒素的重链组成结构适用于肉毒杆菌毒素的各血清型(例如：A型、E型等)。
A～G型未活化肉毒杆菌毒素均由一条相对分子质量约150kD的多肽链组成，经细菌内源蛋白酶或外源蛋白酶切割后，其裂解为一条分子量约50kD的轻链和一条分子量约100kD的重链，轻链和重链之间通过一条保守的二硫键相连，由此构成具有生物活性的双链毒素。本发明所述重组肉毒杆菌毒素其重链序列的N端第1位氨基酸为甘氨酸，同时其轻链和重链通过二硫键相连，这意味着，本发明重组肉毒杆菌毒素的重链序列在以甘氨酸为首的条件下，还包含了可形成二硫键的半胱氨酸残基，这也就表明该重链包含了完整的功能结构域，当其与轻链通过二硫键连接时，即可形成具有正常生物活性的双链毒素。
具体而言，对于肉毒杆菌毒素A，从肉毒杆菌毒素A全长单链的第445位甘氨酸开始截取至C末端作为重链，在第445位甘氨酸的N端引入TEV蛋白酶的酶切序列(ENLYFQ↓G，箭头标注为酶切位置)的N端6个氨基酸残基，并在TEV蛋白酶的酶切序列的N端6个氨基酸残基的N端引入蛋白标签序列。上述序列设计使得重链经TEV酶酶切后，不会残留任何其本身序列不存在的氨基酸残基(即不会残留人工引入的蛋白标签序列和酶切位点序列，肉毒杆菌毒素重链仍保持其原有序列)，而且，酶切后的重链在与轻链通过二硫键链接后，具有天然肉毒杆菌毒素的生物活性。
在本发明的一些实施方式中，上述肉毒杆菌毒素的重链序列为肉毒杆菌毒素A的第445位至第1296位氨基酸序列。
以上所述的肉毒杆菌毒素A的第445位至第1296位氨基酸序列中，第445位氨基酸为甘氨酸。肉毒杆菌毒素A的第445位至第1296位氨基酸序列可为天然肉毒杆菌毒素A的序列(例如SEQ ID NO.1所示的序列的第445位至第1296位)，也可为突变的肉毒杆菌毒素A序列，突变的肉毒杆菌毒素序列仍需要保证第445位氨基酸为甘氨酸，且能够与轻链连接形成有生物活性的肉毒杆菌毒素。
在本发明的一些实施方式中，以上所述的肉毒杆菌毒素A的第445位至第1296位氨基酸序列是 以SEQ ID NO.1所示的肉毒杆菌毒素A序列为依据的(SEQ ID NO.1所示的肉毒杆菌毒素的编码基因序列如SEQ ID NO.5所示，SEQ ID NO.5所示序列为经序列优化的，适于在大肠杆菌中表达的序列)。肉毒杆菌毒素A的第445位至第1296位氨基酸序列也可采用上述氨基酸序列的变体，只需保证第445位氨基酸为甘氨酸且能够与轻链连接形成有生物活性的肉毒杆菌毒素即可。
上述重组肉毒杆菌毒素经TEV蛋白酶酶切后可产生完全去除蛋白标签序列的肉毒杆菌毒素，且不会残留肉毒杆菌毒素本身序列不存在的氨基酸残基。
优选地，所述重组肉毒杆菌毒素的重链的氨基酸序列在蛋白标签序列的N端还包括从N端至C端方向的甲硫氨酸和由1～3个除半胱氨酸以外的氨基酸残基组成的氨基酸序列；
在本发明的一些实施方式中，所述重链的氨基酸序列为从N端至C端方向依次为：M-由1～3个除半胱氨酸以外的氨基酸残基组成的氨基酸序列-蛋白标签序列-ENLYFQ-肉毒杆菌毒素重链序列(N端第1位为甘氨酸G)。
在本发明的一些实施方式中，所述重链的氨基酸序列为从N端至C端方向依次为：M-由1～3个除半胱氨酸以外的氨基酸残基组成的氨基酸序列-蛋白标签序列-ENLYFQ-肉毒杆菌毒素A的第445位至第1296位氨基酸。
在本发明的一些实施方式中，所述重链的氨基酸序列为从N端至C端方向依次为：MK-蛋白标签序列-ENLYFQ-肉毒杆菌毒素A的第445位至第1296位氨基酸。
在本发明的一些实施方式中，所述重链的氨基酸序列如SEQ ID NO.2所示。
上述重组肉毒杆菌毒素的重链的氨基酸序列为携带蛋白标签的序列，可通过TEV蛋白酶酶切去除蛋白标签，产生不含有标签和酶切位点残留氨基酸残基的重链，不会产生肉毒杆菌毒素以外的抗原性。
在上述重链序列的基础上，本发明尝试对轻链序列进行选择和优化，确定从肉毒杆菌毒素全长单链的N末端截取至第438位赖氨酸的位置，以此作为轻链序列能够很好地保证轻链的生物活性。上述设计的轻链和重链能够高效形成二硫键，经TEV蛋白酶酶切后产生不引入任何肉毒杆菌毒素本身序列不存在的氨基酸残基、具有生物活性的肉毒杆菌毒素。
优选地，上述重组肉毒杆菌毒素中，所述轻链的氨基酸序列为肉毒杆菌毒素A的第1位至第438位。
肉毒杆菌毒素A的第1位至第438位氨基酸序列可为天然肉毒杆菌毒素A的序列(例如SEQ ID NO.1所示序列的第1位至第438位)，也可为突变的肉毒杆菌毒素A的序列，突变的肉毒杆菌毒素A序列能够与上述重链连接形成有生物活性的肉毒杆菌毒素即可。
在本发明的一些实施方式中，所述轻链的氨基酸序列如SEQ ID NO.1所示序列的第1位至第438位所示。
以上所述的重链和轻链在对应于肉毒杆菌毒素A全长单链430、454位的氨基酸的位置上仍保留原有的半胱氨酸，因此轻链和重链之间可以形成二硫键，并且采用上述设计的轻链和重链有利于二硫键的高效形成。
第二方面，本发明提供一种核酸分子，所述核酸分子编码以上所述的重组肉毒杆菌毒素。
根据上述重组肉毒杆菌毒素的重链和轻链的氨基酸序列，本领域技术人员可以获得编码上述重链和轻链的核酸分子的核苷酸序列。基于密码子的简并性，上述核酸分子的核苷酸序列并不唯一，所有能够编码上述重链和轻链的核酸分子均在本发明的保护范围内。
在本发明的一些实施方式中，编码重链的核酸分子的核苷酸序列如SEQ ID NO.3所示。
在本发明的一些实施方式中，编码轻链的核酸分子的核苷酸序列如SEQ ID NO.4所示。
上述核酸分子能够实现在大肠杆菌和需钠弧菌中高效稳定表达轻链和重链。
第三方面，本发明提供包含以上所述的核酸分子的生物材料；所述生物材料为表达盒、载体或宿主细胞。
在本发明的一些实施方式中，含有所述核酸分子的表达盒由启动子和所述核酸分子可操作性地连接得到。
根据表达需要以及表达盒上下游序列的不同，表达盒中还可包含终止子、增强子等其他转录、翻译调控元件。
在本发明的一些实施方式中，含有所述核酸分子的载体为质粒载体，这些质粒载体包括复制型载体和非复制型载体。含有所述核酸分子的载体不局限于质粒载体，还可为噬菌体、病毒等载体。
在本发明的一些实施方式中，所述质粒载体含有编码上述重链的核酸分子和/或编码上述轻链的核酸分子。
在本发明的一些实施方式中，所述质粒载体为以PQlink为骨架载体，在其中串联连入编码上述重链的核酸分子和编码上述轻链的核酸分子。编码上述重链的核酸分子和编码上述轻链的核酸分子各自包含相应的蛋白表达基因，并且均拥有独立的启动子，调控因子，核糖体结合位点和终止子。
在本发明的一些实施方式中，所述宿主细胞为大肠杆菌或需钠弧菌，但宿主细胞的种类并不局限于此，可以为任意的微生物细胞或可用于蛋白表达的动物细胞。
第四方面，本发明提供一种用于生产重组肉毒杆菌毒素的工程菌，所述工程菌含有以上所述的核酸分子。
在本发明的一些实施方式中，所述工程菌为大肠杆菌或需钠弧菌。
本发明中，大肠杆菌和需钠弧菌均可作为底盘菌用于表达重组肉毒杆菌毒素。然而，本发明意外地发现，需钠弧菌能够更为高效地促进肉毒杆菌毒素的轻链和重链之间形成二硫键，进而能够更快、更好地成功合成出具有生物活性的肉毒杆菌毒素。
不仅如此，相对于大肠杆菌易被噬菌体感染而发生细胞破裂，导致内容物溶出，以需钠弧菌作为底盘菌进行肉毒杆菌毒素的制备还可以减少噬菌体感染风险，有效降低毒蛋白对环境造成污染的风险，并且缩短菌体培养周期。在本发明的一些实施方式中，所述工程菌含有携带以上所述的核酸分子的质粒载体。
在本发明的一些实施方式中，所述工程菌为在需钠弧菌中导入携带以上所述的核酸分子的质粒载体得到。其中编码重链的核酸分子和编码轻链的核酸分子可以在同一质粒载体上，也可分别在不同的质粒载体上。
在本发明的一些实施方式中，所述工程菌为在大肠杆菌中导入携带以上所述的核酸分子的质粒载体得到。其中编码重链的核酸分子和编码轻链的核酸分子可以在同一质粒载体上，也可分别在不同的质粒载体上。
第五方面，本发明提供以上所述的重组肉毒杆菌毒素或所述核酸分子或所述生物材料或所述重组需钠弧菌在制备肉毒杆菌毒素中的应用。
在本发明的一些实施方式中，上述应用包括：修饰宿主细胞以使得所述宿主细胞表达以上所述的重组肉毒杆菌毒素或含有所述核酸分子，培养宿主细胞，收集其表达的重组肉毒杆菌毒素，经亲和层析纯化和TEV蛋白酶酶切制得去除蛋白标签的肉毒杆菌毒素。
在本发明的一些实施方式中，上述应用包括：培养所述重组需钠弧菌，收集其表达的重组肉毒杆菌毒素，经亲和层析纯化和TEV蛋白酶酶切制得去除蛋白标签的肉毒杆菌毒素。
第六方面，本发明提供一种肉毒杆菌毒素的制备方法，所述方法包括：修饰宿主细胞以使得所述宿主细胞表达以上所述的重组肉毒杆菌毒素，培养所述宿主细胞，采用亲和层析从培养物中纯化所述重组肉毒杆菌毒素，将纯化后的重组肉毒杆菌毒素进行TEV蛋白酶酶切。
上述方法中，所述亲和层析的使用的柱材可根据重链所含有的蛋白标签进行选择。
在本发明的一些实施方式中，对应所采用的His-tag蛋白标签，使用His-tag亲和层析柱进行。实际应用时，可根据不同的蛋白标签序列，选择使用对应的纯化方法。
上述方法中，培养宿主细胞得到培养物，经裂解细胞后分离上清液进行亲和层析纯化。
在本发明的一些实施方式中，所述方法还包括在TEV蛋白酶酶切后依次进行阴离子交换层析和分子筛层析纯化的步骤。
上述纯化中，阴离子交换层析可采用阴离子交换柱(例如Source Q)进行。分子筛层析可采用SuperDex200等分子筛柱材进行。
采用上述纯化方法能够显著提高重组肉毒杆菌毒素的纯化效率和纯度。
上述方法中，整个纯化过程不到一天的时间即可完成，相比于无标签蛋白的繁琐纯化过程，极大地简化了纯化流程，节省了人力和物料，有效减少了对仪器等环境造成意外污染的可能性。
对于宿主细胞，在本发明的一些实施方式中，所述宿主为需钠弧菌。
本发明意外地发现，需钠弧菌能够更为高效地促进肉毒杆菌毒素的轻链和重链之间形成二硫键，进而能够更快、更好地成功合成出具有生物活性的肉毒杆菌毒素。
不仅如此，相对于大肠杆菌易被噬菌体感染而发生细胞破裂，导致内容物溶出，以需钠弧菌作为底盘菌进行肉毒杆菌毒素的制备还可以减少噬菌体感染风险，有效降低毒蛋白对环境造成污染的风险，并且缩短菌体培养周期。
本发明的有益效果在于：本发明通过对重组肉毒杆菌毒素重链和轻链序列的设计优化，实现了不会因添加和切割蛋白标签引入肉毒杆菌毒素本身序列不存在的氨基酸残基，同时能够进行肉毒杆菌毒素的高效稳定表达和活性肉毒杆菌毒素的制备，还能够极大地简化毒素蛋白的纯化工艺，实现活性肉毒杆菌毒素的高效制备。
本发明提供的肉毒杆菌毒素制备方法能够快速、高效地制备肉毒杆菌毒素，宿主培养周期短、不易污染噬菌体，毒素蛋白纯化过程简单易行，易于规模化放大生产，极大地降低了肉毒杆菌毒素制备的成本，制备肉毒杆菌毒素具有预期的生物活性，与天然肉毒杆菌毒素相当，具有较好的应用前景。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图逐一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例1中产物P1，P2，P3的PCR条带。
图2为本发明实施例2中PCR筛选条带，其中2,4符合预期。
图3为本发明实施例2中串联质粒结构图。
图4为本发明实施例5中His-tag亲和层析纯化结果。
图5为本发明实施例5中阴离子交换柱纯化结果。
图6为本发明实施例5中阴离子交换柱纯化SDS-Page验证结果，FT为流穿液，数字7-14分别为图5中阴离子交换柱收集管编号。
图7为本发明实施例5中分子筛SUPERDEX200纯化结果。
图8为本发明实施例5中分子筛SUPERDEX200纯化SDS-PAGE验证结果，其中，数字11-18分别为图7中分子筛SUPERDEX200收集管编号。
图9为本发明实施例6中分子筛SUPERDEX200纯化SDS-PAGE验证结果。
图10为本发明实施例7中肉毒杆菌毒素蛋白浓度工作曲线图。
图11为本发明实施例7中小鼠腓肠肌注射实验12小时后结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明中的附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1肉毒杆菌轻链重链表达载体的构建
从优宝生物购买PQlink表达载体(http://www.youbio.cn/product/vt8126)，依据NCBI(美国国家生物信息中心，网址：https://www.ncbi.nlm.nih.gov/)公布的肉毒梭状芽胞杆菌表达的肉毒杆菌蛋白序列(UniProtKB/Swiss-Prot:P0DPI0.1)，进行设计，委托金斯瑞生物科技股份有限公司合成肉毒杆菌毒素编码基因。
采用PCR扩增的方法，分别在轻、重链上下游引入PQLink同源臂，并在重链的上游引入His标签和TEV酶切位点。利用KOD Mix体系，各进行20μL体系反应。扩增程序为：95℃，15s变性；56℃，10s退火，68℃延伸30s，整个反应34个循环。利用核酸胶电泳收集反应片段，记为P1，P2。另用PQlink相应引物对载体进行PCR扩增，将载体线性化，利用核酸胶电泳收集反应片段，记为P3，反应体系为50μL，扩增程序为：95℃，15s变性；56℃，10s退火，68℃延伸60s，整个反应34个循环。结果如图1所示，P1条带大小在1200bp左右，P2条带大小在2600bp左右，P3条带在5000bp左右。大小均符合预期。所用引物如下。
引物BotLc-5：5’-GAGGAGAAATTAACTATGCCGTTCGTTAACAAACAGTTC-3’
引物BotLc-3：5’-GTCCTAGGCGGCCGCTTATTTAGAGGTGATGATACCACG-3’
引物BotHc-5：
5’-GAGGAGAAATTAACTATGAAACATCACCATCACCATCACGAGAATCTTTATTTTCAGGGTTACAACAAAGCTCTGAACGA-3’
引物BotHc-3：5’-GTCCTAGGCGGCCGCTTACAGCGGACGTTCACCCCAACCGT-3’(下划线表示PQlink同源臂)
引物PQlink-5v：5’-TAAGCGGCCGCCTAGGACCCAGC-3’
引物PQlink-3v：5’-CATAGTTAATTTCTCCTCTTTAA-3’。
将PCR反应条带切胶，利用TIANGEN胶回收试剂盒回收，操作步骤同说明书。将轻重链回收片段P1，P2分别与P3进行同源重组，试剂盒选择诺唯赞同源重组试剂盒(ClonExpress II One Step Cloning Kit)，操作步骤同说明书。
取反应产物10μL，加入TOP10感受态(市售购买获得)，42℃热激90s；加入200μL新鲜LB培养基孵育30min，然后涂布于抗性LB固体平板板(含50μg/mL氨苄西林)，37℃过夜培养。挑取转 化子进行扩培，然后抽提质粒，先后进行PCR(引物选择轻、重链各自对应的引物)以及测序验证，测序公司为北京睿博兴科生物技术有限公司，测序引物为公司通用引物PQlink-F、PQlink-R。
引物PQlink-F：5’-TATAAAAATAGGCGTATCACGAGG-3’
引物PQlink-R：5’-CCAGTGATTTTTTTCTCCATTTT-3’。
上述PCR扩增得到的肉毒杆菌毒素的重链的编码基因序列如SEQ ID NO.3所示，编码重链的氨基酸序列如SEQ ID NO.2所示，轻链的编码基因序列如SEQ ID NO.4所示，编码轻链的氨基酸序列如SEQ ID NO.1的第1-438位所示。
实施例2肉毒杆菌轻链重链串联共表达载体的构建
由实施例1得到轻链、重链单独表达的质粒，依次记为B1与B2。
将B2进行SwaI酶酶切(SwaI与buffer3.1均购自NEB公司)，酶切体系为：2μL质粒，0.5μL酶，1μL Buffer3.1，水补齐至10μL。室温酶切2h。之后将体系置于65℃，30min，产物记为P4。
以B1为模板，进行PCR反应，反应体系20μL，扩增程序为：95℃，15s变性；56℃，10s退火，68℃延伸30s，整个反应34个循环。引物如下：
引物PQlink-5ter：5’-TAACAACACCATTTGTCGAGAA-3’；
引物PQlink-3ter：5’-CCATTTGTAACCACTCCATTT-3’。
利用核酸胶电泳收集反应片段，将PCR反应条带切胶，利用TIANGEN胶回收试剂盒回收，操作步骤同说明书，产物记为P5。将回收片段P4与P5进行同源重组，试剂盒选择诺唯赞同源重组试剂盒(ClonExpress II One Step Cloning Kit)，操作步骤同说明书。
取反应产物10μL，加入TOP10感受态(市售购买获得)，42℃热激90s；加入200μL新鲜LB培养基孵育30min，然后涂布于抗性LB固体平板板(含50μg/mL氨苄西林)，37℃过夜培养。挑取转化子进行扩培，然后抽提质粒，先后进行PCR(引物选择BotHc-5、BotLc-3)以及测序验证。如图2所示，其中第2道，第4道为4000bp左右，大小符合预期，选取对应菌株进行测序，测序公司为北京睿博兴科生物技术有限公司，测序引物为公司通用引物PQlink-F，PQlink-R。
引物PQlink-F：5’-TATAAAAATAGGCGTATCACGAGG-3’；
引物PQlink-R：5’-CCAGTGATTTTTTTCTCCATTTT-3’。
结果显示，测序结果正确。构建质粒图谱示意图如图3所示，图中Bonta HC为N端携带His标签的肉毒杆菌毒素重链，Bonta LC为肉毒杆菌毒素轻链，重链和轻链基因的启动子均为t5启动子，调控元件为lac operator，终止子为lambda t0 terminator。
实施例3利用大肠杆菌表达重组肉毒杆菌毒素
取实施例2制得的共表达质粒2μL，加入Origami DE3感受态(市售购买获得)，42℃热激90s；加入200μL新鲜LB培养基孵育30min，然后涂布于抗性LB固体平板(每升成分，NaCl 10g， 胰蛋白胨10g，酵母提取物5g，琼脂10g，含50μg/mL氨苄西林)，37℃过夜培养。
取单克隆转化，接菌于10mL LB培养基中(每升成分，NaCl 10g，胰蛋白胨10g，酵母提取物5g，含50μg/mL氨苄西林)，在摇床中220rpm 37℃过夜培养。之后将菌液接菌于1L LB培养基中(含50μg/mL氨苄西林)，220rpm 37℃培养。培养6小时，OD至1.5，降温至18℃，加入500mM IPTG 400μL诱导。
实施例4利用需钠弧菌表达重组肉毒杆菌毒素
需钠弧菌感受态购置于synthetic genomics，商品名VmaxTM Express。取实施例2制得的共表达质粒2μL加入需钠弧菌感受态细胞，电击转化1500V，5ms。电击转化杯购于biorad，货号165-2086 2mm。电击转化产物加入高盐LB培养基(每升成分，NaCl 30g，胰蛋白胨10g，酵母提取物5g)1mL。37℃振荡培养1小时。涂布至高盐LB平板上(每升成分，NaCl 30g，胰蛋白胨10g，酵母提取物5g，琼脂10g，含50μg/mL氨苄西林)。
取单克隆转化，接菌于10mL高盐LB培养基中(每升成分，NaCl 30g，胰蛋白胨10g，酵母提取物5g，含50μg/mL氨苄西林)，在摇床中220rpm 37℃过夜培养。之后将菌液接菌于1L高盐LB培养基中(每升成分，NaCl 30g，胰蛋白胨10g，酵母提取物5g，含50μg/mL氨苄西林)，220rpm37℃培养。培养3小时，OD至1.5，降温至18℃，加入500mM IPTG 400μL诱导。
实施例5肉毒杆菌毒素的纯化
对实施例3和4中表达的重组肉毒杆菌毒素，同时在以下相同条件下纯化需钠弧菌Vmax和大肠杆菌Origami表达的肉毒毒素蛋白：
收集菌液，低速大容量离心机3800rpm离心十分钟。除去上清，每升菌实体用25mL lysis buffer(25mM Tris 8.0，150mM NaCl)重悬。
超声裂解，条件：变幅杆10,功率350W，超声2s，停2s，每升菌实体用时4min(例如，收集6L菌，超声时间应为24min)，超声至菌液澄清。
50mL离心管为容器，高速离心13000rpm，1小时。取上清。
高速离心上清液，过His-tag柱材，每升菌实体，柱材用量3mL。过两遍。
Wash buffer(25mM Tris 8.0，150mM NaCl，10mM咪唑)，每升菌实体洗50mL。
Elute buffer(25mM Tris 8.0，50mM NaCl，250mM咪唑)，每升菌实体洗10mL，收集洗脱液。10mL洗脱液加入100U的TEV酶(购于碧云天产品编号:P2307)进行酶切处理。
电泳检测，5×非变性非还原蛋白上样缓冲液购于YeasenChina，货号20317ES，制样方法参见说明书。其中，离心沉淀，离心上清上样量为2μL，高咪唑洗脱液上样量为5μL，进行聚丙烯酰胺凝胶电泳。检测结果如图4所示。
将需钠弧菌与大肠杆菌Origami表达重组肉毒杆菌毒素的结果进行对比，大肠杆菌Origami细胞 质二硫化物还原途径在trxB/gor存在两个突变，可以增加大肠杆菌细胞质中二硫键的形成。出乎意料的是，在需钠弧菌中，肉毒杆菌毒素重链、轻链之间也可以形成正常的二硫键，而且需钠弧菌表达的蛋白纯度与Origami表达结果相似。在图4中显示为，高咪唑洗脱过程中，需钠弧菌与大肠杆菌蛋白条带纯度相似。蛋白分子量大小均在135kD以上，几乎不存在重链100kD，轻链50kD的条带。
将需钠弧菌的高咪唑洗脱进一步通过阴离子交换柱/SuperDex200分子筛纯化，阴离子交换柱的A buffer配方为：25mM Tris 8.0，B buffer配方为：1M NaCl，25mM Tris 8.0。将洗脱液电导稀释至7mS/cm，过阴离子交换柱Source Q，收集流穿液(FT)，洗脱程序为线性提升至60％B buffer，总洗脱体积200mL。洗脱UV280nm检测结果如图5所示，并对相应样品进行收集，蛋白制样，每道上样量为5μL，进行聚丙烯酰胺凝胶电泳。结果如图6所示。其中第7管，第8管蛋白纯度较高。
收集7-8管，使用50kD Millipore蛋白浓缩管，2000g转速，浓缩至2mL，上样至分子筛SuperDex200，流动相为lysis buffer。洗脱UV280nm检测结果如图7所示，并对相应样品进行收集，蛋白制样，每道上样量为5μL，进行聚丙烯酰胺凝胶电泳。结果如图6所示。其中第14管蛋白处于峰尖。收集第14管。分子筛SUPERDEX200纯化SDS-PAGE检测结果如图8所示。
以上样品进行聚丙烯酰胺凝胶电泳(SDS-PAGE)的预制凝胶均购置于碧云天，实验实施方法依照其说明书进行。
实施例6利用Western-blot检测肉毒杆菌毒素的组氨酸标签残留
选择实施例5分子筛洗脱第14管以及阳性对照进行SDS-PAGE电泳，上样缓冲液为还原缓冲液，购于聚合美，M5 5×SDS-PAGE还原性5×蛋白上样缓冲液，货号MF145-10。阳性对照依照实施例4进行，与样品的区别仅在于中间未进行TEV酶酶切。
凝胶进行免疫印迹转印至硝酸纤维膜。电压25v，时间30min，转膜液购置于碧云天。抗体购于金斯瑞，THETM His Tag Antibody[HRP],mAb,Mouse，使用参照其说明进行。转膜之后显影液购于thermo，SuperSignalTM。结果如图9所示，经过还原，蛋白在聚丙烯酰胺凝胶电泳中，分离成重链、轻链两条带。其中，重链大小在100kD-135kD之间。TEV酶切的样品没有阳性条带，对照组阳性较强。
实施例7肉毒杆菌毒素的活性检测
利用小鼠腓肠肌注射实验检测实施例5制备的肉毒杆菌毒素的活性，具体如下：
将实施例5得到的蛋白样品稀释至0.25ng/mL(检测方法为酶联免疫吸附剂测定ELISA，试剂盒购于上海钦诚生物，工作曲线如图10所示，回归方程中，Abs值的是吸光值读数，[Protein]指的是肉毒杆菌毒素浓度，R值大于0.99，数据质量可靠)，溶剂为0.5‰BSA，9‰氯化钠溶液。右后腿腓肠肌注射50μL肉毒杆菌毒素试剂、左后腿腓肠肌注射50μL阴性对照(溶剂)。如图11所示，5pg的蛋白量可以在12小时内引起小鼠的右腿瘫痪，而左腿没有影响。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
工业实用性
本发明的重组肉毒杆菌毒素不会因添加和切割蛋白标签引入肉毒杆菌毒素本身序列不存在的氨基酸残基，同时能够实现高效稳定表达并具有较高的活性，而且纯化工艺简单易行，易于规模化放大生产，极大地降低了肉毒杆菌毒素制备的成本，具有较好的应用前景。

Claims (10)

  1. 重组肉毒杆菌毒素，其特征在于，所述重组肉毒杆菌毒素包括由二硫键连接的重链和轻链；所述重链的氨基酸序列从N端到C端的方向依次包含蛋白标签序列、ENLYFQ多肽序列和肉毒杆菌毒素的重链序列，
    其中，肉毒杆菌毒素的重链序列的N端第1位氨基酸为甘氨酸。
  2. 根据权利要求1所述的重组肉毒杆菌毒素，其特征在于，所述肉毒杆菌毒素的重链序列为肉毒杆菌毒素A的第445位至第1296位氨基酸序列；
    优选地，所述重组肉毒杆菌毒素的重链的氨基酸序列在蛋白标签序列的N端还包括从N端至C端方向的甲硫氨酸和由1～3个除半胱氨酸以外的氨基酸残基组成的氨基酸序列；
    更优选地，所述重组肉毒杆菌毒素的重链的氨基酸序列如SEQ ID NO.2所示。
  3. 根据权利要求1或2所述的重组肉毒杆菌毒素，其特征在于，所述轻链的氨基酸序列为肉毒杆菌毒素A的第1位至第438位；
    优选地，所述轻链的氨基酸序列如SEQ ID NO.1所示序列的第1位至第438位所示。
  4. 核酸分子，其特征在于，所述核酸分子编码权利要求1～3任一项所述的重组肉毒杆菌毒素；
    优选地，编码重链的核酸分子如SEQ ID NO.3所示，和/或，编码轻链的核酸分子如SEQ ID NO.4所示。
  5. 生物材料，其特征在于，所述生物材料包含权利要求4所述的核酸分子；所述生物材料为表达盒、载体或宿主细胞。
  6. 一种用于生产重组肉毒杆菌毒素的工程菌，其特征在于，所述工程菌含有权利要求4所述的核酸分子。
  7. 根据权利要求6所述的工程菌，其特征在于，所述工程菌为大肠杆菌或需钠弧菌。
  8. 一种肉毒杆菌毒素的制备方法，其特征在于，所述方法包括：修饰宿主细胞以使得所述宿主细胞表达权利要求1～3任一项所述的重组肉毒杆菌毒素，培养所述宿主细胞，采用亲和层析从培养物中纯化所述重组肉毒杆菌毒素，将纯化后的重组肉毒杆菌毒素进行TEV蛋白酶酶切。
  9. 根据权利要求8所述的方法，其特征在于，所述方法还包括在TEV蛋白酶酶切后依次进行阴离子交换层析和分子筛层析纯化的步骤。
  10. 根据权利要求8或9所述的方法，其特征在于，所述宿主细胞为需钠弧菌。
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