Preparation method for botulinum toxin type a and use of same

Abstract

Provided is a preparation method for recombinant botulinum toxin type A (BoNT / A), the method comprising: designing a BoNT / A amino acid sequence in a P0DP10 protein database; by using a homologous recombination method, inserting an His tag and an SUMO tag sequence into the N terminal of the BoNT / A amino acid sequence; and then performing cleavage between GG-M by means of a SUMO tag protease. A result shows that no amino acid residue of any tag and the SUMO sequence is present, so that the safety is remarkably improved. Comparative experiments and animal experiments show that the recombinant botulinum toxin type A polypeptide which is prepared, purified and activated on the basis of a bacterium body centrifugal supernatant has higher virulence, so that the effective dose during practical use is reduced, thus remarkably reducing side effects and stimulation and achieving higher safety.

Description

Preparation method of botulinum toxin type A and its application

[0001] Cross-references

[0002] The present invention claims priority to application number 202310681461.9 filed with the Patent Office of China on June 8, 2023, entitled “A method for preparing botulinum toxin type A and its application”, the full text of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the technical field of polypeptide preparation, in particular to the efficient preparation of botulinum toxin type A and its use in preparing medicines. Background Art

[0004] Clostridium botulinum and Clostridium tetani produce highly potent neurotoxins, namely botulinum toxin (BoNT) and tetanus neurotoxin (TeNT), respectively. These clostridial toxins (CNTs) bind specifically to neuronal cells and interfere with the release of neurotransmitters. Clostridium botulinum secretes seven different antigenic serotypes of botulinum toxin (BoNT), named A to G. All serotypes block synaptic exocytosis of Zn 2+ - endoproteases, which do this by cleaving proteins involved in the formation of the SNARE complex that controls cell membrane fusion.

[0005] In 1989, botulinum toxin serotype A (BoNT / A) was approved for human use in the United States for the treatment of strabismus, blepharospasm, and other conditions. It is commercially available as a botulinum toxin A protein formulation, for example, under the trade names BOTOX (Allergan) and DYSPORT (Ipsen Ltd.). The efficient preparation and application of BoNT / A products have demonstrated significant commercial value in the medical field.

[0006] Each of the naturally occurring Clostridial toxins is translated as a single-chain polypeptide of approximately 150 kDa, the disulfide loop of which is subsequently cleaved by proteolytic cleavage by a naturally occurring protease (e.g., such as an endogenous Clostridial toxin protease or a naturally occurring protease produced in the environment). Post-translational processing produces a mature two-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC), which are held together by a single interchain disulfide bond and non-covalent interactions. Between the participating cysteine ​​residues lies a linker region or loop region, the length of which varies greatly among serotypes of fusintoxins. This loop is released from the clostridium at the latest during cell lysis, where the ratio of cleaved and uncleaved species varies among serotypes. Protease cleavage (at the loop region and the terminus of the heavy chain) is random, resulting in the generation of numerous undesirable intermediates, which increases the difficulty of purification and reduces product yield. A method for separately expressing and purifying the heavy and light chains of TeTx and BoNTs in Escherichia coli is available (see Li, et al., Biochemistry, 33:7014-7020 (1994); Zhou, et al., Biochemistry, 34:15175-15181 (1995)). However, in the absence of an L chain, the isolated H chain is poorly water soluble and highly susceptible to proteolytic degradation, making it impractical. Patent US7709228 discloses enzyme cleavage sites that can be modified and proteases that can specifically recognize these sites, but the toxicity of its recombinant product is equivalent to two-fifths of natural TeTx, and the reduction is not very significant. At the same time, existing technicians generally use restriction endonuclease construction methods to construct the target protein into the target region, which will introduce 2-3 amino acid residues, which will pose a risk of immunogenicity. Therefore, there is an urgent need in the field for an efficient and safe preparation method for botulinum neurotoxin to obtain a highly safe and high-yield polypeptide product, so that it can be used more safely and effectively in medical treatment.

[0007] Summary of the Invention

[0008] In view of the deficiencies of the prior art, the present invention aims to provide a method for preparing and applying botulinum toxin.

[0009] Technical Solution of the Present Invention One of the objectives of the present invention is a recombinant botulinum toxin type A (BoNT / A), the amino acid sequence of which is shown in SEQ ID NO.7.

[0010] Preferably, the amino acid sequence of the recombinant botulinum toxin type A (BoNT / A) includes SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5;

[0011] (a) The Lc region encoding BoNT / A, the amino acid sequence of which is shown in SEQ ID NO. 1;

[0012] (b) encoding a thrombin cleavage site, the amino acid sequence of which is shown in SEQ ID NO.3;

[0013] (c) The Hc region encoding BoNT / A, the amino acid sequence of which is shown in SEQ ID NO. 5.

[0014] Preferably, the combined tag is inserted into the N-terminus of the amino acid sequence of BoNT / A by homologous recombination, that is, the amino acid sequence of the designed recombinant botulinum toxin type A (BoNT / A) is as shown in SEQ ID NO.13.

[0015] Preferably, the combined tag is a His tag and a SUMO tag.

[0016] The His tag has an amino acid sequence as shown in SEQ ID NO.9 and an encoding nucleotide sequence as shown in SEQ ID NO.10.

[0017] The amino acid sequence of the SUMO tag is shown in SEQ ID NO.11, and the encoding nucleotide sequence is shown in SEQ ID NO.12.

[0018] A second object of the present invention is to provide a method for preparing recombinant botulinum toxin type A (BoNT / A), comprising the following steps:

[0019] (1) Designing and constructing a recombinant botulinum toxin type A (BoNT / A) having an amino acid sequence as shown in SEQ ID NO. 13;

[0020] (2) Design and ligation of BoNT / A expression vectors;

[0021] (3) Induced expression of BoNT / A protein and bacterial collection;

[0022] (4) Affinity purification of bacterial supernatant;

[0023] (5) Enzymatic cleavage removes the SUMO tag and activates the BoNT / A protein.

[0024] Preferably, the experimental steps of enzymatically removing the SUMO tag and activating the BoNT / A protein are as follows: after enzymatic cleavage of the SUMO tag, 1 U of thrombin is added per 1 mg of protein.

[0025] Preferably, the step of inducing expression of the BoNT / A protein is as follows: 600 After the cell proliferation rate reached 0.6, IPTG was added at a final concentration of 1 mM and induced at 30°C for 12 h.

[0026] Preferably, the bacterial cell collection step is as follows: centrifugation at 4000 rpm to collect bacterial sludge, resuspending it in 40 ml of 20 mM PB + 150 mM Nacl, pH 7.2, and ultrasonically disrupting it in an ice bath at 420W for 3 seconds, with an interval of 5 seconds, for 15 minutes; centrifuging it at 12000 rpm and 4°C for 20 minutes, and collecting the supernatant again;

[0027] The specific steps of affinity purification of the bacterial supernatant are as follows: the supernatant collected by centrifugation is filtered through a 0.22 μm filter membrane and then subjected to Ni column affinity enrichment purification, wherein the equilibration buffer is 20 mM PB + 150 mM NaCl, the washing buffer is 20 mM PB + 150 mM NaCl + 5 mM imidazole, 20 mM PB + 150 mM NaCl + 20 mM, 20 mM PB + 150 mM NaCl + 50 mM imidazole, 20 mM PB + 150 mM NaCl + 80 mM imidazole, the elution buffer is 20 mM PB + 150 mM NaCl + 300 mM imidazole, and the pH of the reagents is all pH 8.0.

[0028] The third object of the present invention is to obtain a recombinant BoNT / A polypeptide prepared by the above method.

[0029] A fourth object of the present invention is to use the recombinant BoNT / A polypeptide prepared by the above method in the preparation of a drug. Beneficial effects

[0030] The present invention provides a method for preparing a recombinant botulinum toxin type A (BoNT / A). The BoNT / A amino acid sequence is designed based on the P0DP10 protein database. A His tag (whose amino acid sequence is shown in SEQ ID NO. 9, and its encoding nucleotide sequence is shown in SEQ ID NO. 10) and a SUMO tag sequence (whose amino acid sequence is shown in SEQ ID NO. 11, and its encoding nucleotide sequence is shown in SEQ ID NO. 12) are inserted into the N-terminus of the BoNT / A amino acid sequence by homologous recombination. SUMO protease is then used to cleave the residue between GG and M, eliminating any residues of the tag or SUMO sequence.

[0031] When the thrombin dosage was 0 U, the target protein did not present two fragments, resulting in inactive BoNT / A protein. When the thrombin dosage was 0.5 U, 1 U, 2 U, and 3 U, respectively, only when 1 U, 2 U, and 3 U of thrombin were added per 1 mg of protein did the target protein present two fragments after thrombin cleavage, and the BoNT / A protein purified from the supernatant was more activated. Considering the increased safety risk associated with using a larger amount of thrombin, this protocol preferably uses 1 U of thrombin per 1 mg of protein.

[0032] In addition, comparative experiments have shown that when labeled with the SUMO tag, the selection of the bacterial centrifugation supernatant for enzymatic cleavage of the tag and activation of the botulinum toxin type A protein polypeptide is more efficient in both tag removal and polypeptide activation; due to limitations in animal experiments, the recombinant botulinum toxin type A polypeptide prepared, purified and activated by the above supernatant is more toxic, so in actual applications, side effects and irritation are significantly reduced, and it has higher safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 shows a schematic diagram of the SUMO-BoNT / A expression vector.

[0034] FIG2 shows the extraction and identification results of the pKMD-SUMO blank plasmid.

[0035] FIG3 shows the results of BoNT / A fragment PCR product identification.

[0036] Figure 4 shows the results of PCR product identification of BoNT / A transformants. Figure 5 shows the enzyme digestion identification of the pKMD-SUMO-BoNT / A expression vector.

[0037] FIG6 shows the transformation and identification of expression strains.

[0038] FIG7 shows the results of supernatant purification of BoNT / A target protein.

[0039] FIG8 shows the results of inclusion body purification of the BoNT / A target protein.

[0040] FIG9 shows the result of enzyme digestion of the SUMO tag of the target protein.

[0041] FIG10 shows the results of thrombin enzymatic activation of the supernatant target protein.

[0042] FIG11 shows the results of thrombin activation of the target protein. DETAILED DESCRIPTION

[0043] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0044] Example 1: Design and Construction of BoNT / A Amino Acid Sequence

[0045] (1) Designing and constructing a BoNT / A having an amino acid sequence as shown in SEQ ID NO. 7 and a nucleic acid molecule as shown in SEQ ID NO. 8, wherein the BoNT / A molecule comprises, from the 5' end, the following:

[0046] (a) The Lc region encoding BoNT / A, the amino acid sequence of which is shown in SEQ ID NO. 1, and the corresponding nucleotide sequence of which is shown in SEQ ID NO. 2;

[0047] (b) encoding a thrombin cleavage site, the amino acid sequence of which is shown in SEQ ID NO.3, and the corresponding nucleotide sequence of which is shown in SEQ ID NO.4;

[0048] (c) Hc region encoding BoNT / A, the amino acid sequence of which is shown in SEQ ID NO. 5, and the corresponding nucleotide sequence of which is shown in SEQ ID NO. 6;

[0049] The BoNT / A protein information was obtained from the Uniprot website. Its unique protein database number is P0DP10. For details, see https: / / www.uniprot.org / uniprotkb / P0DPI0 / entry.

[0050] (2) The combined tag was inserted into the N-terminus of the amino acid sequence of BoNT / A by homologous recombination. The amino acid sequence of the designed recombinant botulinum toxin type A (BoNT / A) is shown in SEQ ID NO. 13, and the corresponding nucleotide sequence is shown in SEQ ID NO. 14.

[0051] The combined tag is a His tag and a SUMO tag.

[0052] The His tag has an amino acid sequence as shown in SEQ ID NO.9 and an encoding nucleotide sequence as shown in SEQ ID NO.10.

[0053] The amino acid sequence of the SUMO tag is shown in SEQ ID NO.11, and the encoding nucleotide sequence is shown in SEQ ID NO.12.

[0054] Example 2: Design and ligation of BoNT / A expression vectors

[0055] (1) Selection of expression vector: A blank plasmid was used as the backbone vector of the BoNT / A expression vector. After codon optimization of the obtained amino acids, a gene codon sequence that could be beneficially expressed in a prokaryotic host was obtained. The plasmid construction design software Snapgene was then used to perform theoretical sequence splicing, i.e., construct and design the vector schematic diagram. The complete expression vector construction schematic diagram is shown in Figure 1.

[0056] (2) Preparation of expression vector: First, the blank plasmid was extracted and identified. The identification results are shown in Figure 2. The left lane shows that the plasmid was extracted.

[0057] (3) Synthesis and pre-amplification of target fragments: The chemically synthesized BoNT / A amino acid sequences, i.e., the amino acid sequences including SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, and the corresponding nucleotide sequences are SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6 respectively; first, these three nucleotide sequences are pre-amplified by PCR segmentation, and the primer sequences are the company's internal primers. Specifically

[0058] Upstream primer G0268322-1F: SEQ ID NO.15

[0059] Downstream primer G0268322-1R1: SEQ ID NO.16

[0060] The expected length of the amplified fragment was 2100 bp, and two amplified fragments were identified by gel electrophoresis as shown in FIG3 .

[0061] (4) Self-ligation of the target fragment and its ligation with the vector:

[0062] After the synthesized BoNT / A nucleotide fragments were ligated, the reaction system for ligation with the vector was as shown in Table 1. The ligation principle of the homologous recombinase was used, and the ligation was carried out at a constant temperature of 52°C for 30 minutes, followed by transformation.

[0063] Table 1. Ligation reaction system of homologous recombination enzyme

[0064] (5) Transformation of recombinant plasmid:

[0065] (a) Pipette 1-3 μl of plasmid at a concentration of approximately 100 ng / μl into approximately 100 μl of BL21 competent cells. Gently swirl to mix thoroughly and place on ice for 3 minutes. If the plasmid concentration is high, add less plasmid; if the concentration is low, add more plasmid.

[0066] (b) Place in a 42°C water bath for 90 seconds without shaking, then place in an ice bath for about 3 minutes;

[0067] (c) Add 500-800 μl of 37°C pre-warmed LB medium to each tube and shake gently at 200 rpm at 37°C for 40 minutes.

[0068] (6) Verification of recombinant plasmid:

[0069] (a) preparing agar plates containing corresponding resistance;

[0070] (b) Take 100 μl of BL21 bacterial suspension and spread it on an agar plate containing the corresponding resistance. Use a sterile glass spreader to gently spread the bacteria on the plate surface and incubate the plate at 37°C for 15 minutes.

[0071] (c) Incubate the plate inverted at 37°C for 12-16 hours until colonies appear;

[0072] (d) Bacteria were picked from the plate and shaken at 37°C, 250 rpm for 14 h. The bacterial suspension was used for PCR identification and the positive clones were sequenced.

[0073] Upstream primer: SEQ ID NO.15

[0074] Downstream primer: SEQ ID NO.16

[0075] (7) PCR identification reaction system:

[0076] PCR product identification of BoNT / A transformants: Primers were designed to amplify a fragment of 2100 bp (partial sequence of the target gene). A 20 μL PCR reaction was performed: 0.5 μL of primers, 2 μL of template bacterial solution, 0.5 μL of polymerase buffer, 3 μL of buffer, and 14 μL of ddH2O. Cycling parameters were: 96°C initial denaturation for 3 min; 23 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 20 s, followed by a final extension at 72°C for 1 min. As shown in Figure 4, eight BoNT / A transformants were selected for PCR product identification using the primers described above: G0268322-1F and G0268322-1R1. Positive transformants were selected and isolated.

[0077] Enzyme digestion identification: After obtaining positive transformants, the pKMD-SUMO-BoNT / A expression plasmid (gene ID: G0268322-1) was successfully constructed. The plasmid structure was identified by enzyme digestion using EcoRI. It was expected that two fragments, 1851 and 7599, would be obtained. The reaction system is shown in Table 2, and the experimental results are shown in Figure 5.

[0078] Table 2. Enzyme digestion reaction system

[0079] Example 3: Induced expression of BoNT / A protein and bacterial cell collection

[0080] (1) Transformation of expression strains

[0081] Take 100 μl of Rosstta BL21 competent cells, place on ice to thaw, take a 1.5 ml sterile centrifuge tube in the clean bench, and divide equally into 50 μl competent cells;

[0082] Take 20 μl of the vector constructed in Example 2, add the plasmid to the competent cells in a clean bench, mix gently, and incubate on ice for 30 minutes;

[0083] Heat shock at 42°C for 90 seconds and immediately place on ice for 5 minutes. Take 30 μl and spread on TB (containing 50 μg / ml Kana) plates. Incubate at 37°C for 14 hours (overnight culture) and pick single clones for activation culture.

[0084] (2) Expression identification

[0085] Single colonies were picked from the TB plates transformed with the expression strains and added to 2 ml of TB liquid medium (containing 50 μg / ml Kana). After activation to an OD600 of 0.6-0.8, the cells were transferred to 5 ml of TB liquid medium (containing 50 μg / ml Kana). After culturing to an OD600 of 0.6-0.8, 1 mM IPTG was added to induce expression at 37°C for 4 h before harvesting the cells.

[0086] Samples of the bacteria under various conditions before and after induction were taken for SDS-PAGE analysis; the experimental results are shown in FIG6 , and lanes 2 and 3 both showed clear expression.

[0087] (3) Bacterial cell amplification and collection

[0088] The BL21 strain capable of expressing the target protein was selected from the expression identification and inoculated into 1LTB medium (containing 50 μg / ml kana) and cultured at 220 rpm and 37°C until the OD 600 To 0.6-0.8, then add IPTG with a final concentration of 1mM and induce culture at 30℃ for 12h, and then collect the bacteria by centrifugation at 4000rpm for use.

[0089] The collected bacterial sludge was resuspended in 40 ml of 20 mM PB + 150 mM Nacl, pH 7.2, and ultrasonically disrupted at 420 W in an ice bath for 3 seconds, with an interval of 5 seconds, for 15 minutes; centrifuged at 12000 rpm, 4°C for 20 minutes, and the supernatant and precipitate were collected separately for use.

[0090] Example 4: Affinity purification of bacterial supernatant and precipitated inclusion bodies

[0091] (1) Supernatant purification: The supernatant collected by centrifugation was filtered through a 0.22 μm filter membrane and then purified by Ni column affinity enrichment.

[0092] Equilibration buffer: "20 mM PB + 150 mM NaCl, pH 8.0"

[0093] Wash buffer: 20mM PB + 150mM NaCl + 5mM imidazole, pH 8.0

[0094] 20mM PB + 150mM NaCl + 20mM imidazole, pH 8.0

[0095] 20mM PB + 150mM NaCl + 50mM imidazole, pH 8.0

[0096] 20mM PB + 150mM NaCl + 80mM imidazole, pH 8.0

[0097] Elution buffer (20 mM PB + 150 mM NaCl + 300 mM imidazole, pH 8.0) was used to flow through the purified sample. After adding loading buffer, the sample was boiled at 100°C and analyzed by SDS-PAGE. The experimental results are shown in FIG7 . The BoNT / A target protein was obtained in lanes 2-9.

[0098] (2) Purification of inclusion body protein: The precipitate collected by centrifugation was resuspended in 20mM PB + 150mM NaCl, pH 7.2, and then purified by Ni column affinity enrichment.

[0099] Equilibration buffer: 20 mM PB + 150 mM NaCl, pH 8.0

[0100] Wash buffer: 20mM PB + 150mM NaCl + 5mM imidazole, pH 8.0

[0101] 20mM PB + 150mM NaCl + 20mM imidazole, pH 8.0

[0102] 20mM PB + 150mM NaCl + 50mM imidazole, pH 8.0

[0103] 20mM PB + 150mM NaCl + 80mM imidazole, pH 8.0

[0104] Elution buffer (20 mM PB + 150 mM NaCl + 300 mM imidazole, pH 8.0) was used to flow through the purified sample. After adding loading buffer, the sample was boiled at 100°C and analyzed by SDS-PAGE. The experimental results are shown in FIG8 . The BoNT / A target protein was obtained in lanes 2-10.

[0105] (3) Enzymatic cleavage to remove the SUMO tag and activate the BoNT / A protein: After the SUMO tag was cleaved by enzyme, the SUMO tag was completely cleaved. The experimental results are shown in Figure 9. The supernatant-purified BoNT / A protein was more complete than the inclusion body-purified BoNT / A protein after SUMO cleavage.

[0106] The BoNT / A protein was optimized for thrombin digestion with the following process:

[0107] Thrombin: Shanghai Yuanye Biotechnology Co., Ltd. Product No.: S10117-1ku Thrombin / Thrombin Lot: M25HS179066

[0108] Experimental group 1: 0 U of thrombin was added to 1 mg of protein and the protein was digested for 16 hours.

[0109] Experimental group 2: 0.5 U of thrombin was added to 1 mg of protein and the protein was digested for 16 hours.

[0110] Experimental group 3: 1 U of thrombin was added to 1 mg of protein and the protein was digested for 16 hours.

[0111] Experimental group 4: 2 U of thrombin was added to 1 mg of protein and the protein was digested for 16 hours.

[0112] Experimental group 5: 3 U of thrombin was added to 1 mg of protein and the protein was digested for 16 hours.

[0113] Thrombin is synthesized and does not exist in nature. It is typically used for enzymatic cleavage, cutting protein chains. When using thrombin, dosage is a crucial factor. Excessive dosage can lead to unsafe conditions such as thrombosis, bleeding, and environmental pollution.

[0114] Figure 10 shows the results of thrombin cleavage and activation of the supernatant target protein: when the thrombin dosage was 0 U, the target protein did not present two fragments, resulting in inactive BoNT / A protein. When the thrombin dosage was 0.5 U, 1 U, 2 U, and 3 U, respectively, only when 1 U, 2 U, and 3 U of thrombin were added per 1 mg of protein did the target protein present two fragments after thrombin cleavage, and the BoNT / A protein purified from the supernatant was more activated. Considering the risk that using a larger amount of thrombin may increase safety, this protocol preferably uses 1 U of thrombin per 1 mg of protein.

[0115] Thrombin activation results for the supernatant and inclusion body target proteins: 1 U of thrombin was added to 1 mg of protein and digested for 16 hours. As shown in Figure 11, both the supernatant-purified BoNT / A protein and the inclusion body-purified BoNT / A protein were fully activated after thrombin activation.

[0116] Example 5: Comparative Verification of Peptide Activity and Safety

[0117] (1) Test drug: The test sample is the lyophilized protein powder of the recombinant botulinum toxin type A active polypeptide purified from the supernatant and precipitated inclusion bodies after thrombin activation according to this method. Take one bottle of the test sample (50 μg) and add 1 ml of sterile water for injection to dissolve it.

[0118] Animals: Kunming mice, SPF grade, 26–30 days old, sourced from Guangdong Medical Laboratory Animal Center; Laboratory Animal Production License: SCXK(Guangdong)2022-0002, Certificate of Conformity: No. 44007200115254, purchased on March 23, 2023. Laboratory Animal Use License: SYXK(Guangdong)2021-0253. Quarantine period: March 23–26, 2023.

[0119] Instruments: MS8001TS electronic balance (20213248); 902 ultra-low temperature refrigerator (20141115); HYC-390F medical refrigerator (20212898); DW-25L262 medical low-temperature storage box (20212917).

[0120] (2) Experimental methods:

[0121] (a) Sample preparation: Take 1 bottle of the sample to be tested (50 μg), add 1 ml of sterile water for injection, and dissolve. Dilute 10 times in sequence according to the 10-fold dilution method. 3 , 10 4 , 10 5 , 10 6 .

[0122] (b) Animal injection: dilution 10 3 , 10 4 , 10 5 , 10 6 There are 4 dilutions in total. Each dilution was intraperitoneally injected into 4 SPF Kunming mice aged 26-30 days, with 0.1 ml injected per mouse. The survival and death of mice were counted within 4 days. The toxicity of this product was calculated using the statistical method (Reed-Muench) in units of LD. 50 / ml.

[0123] (3) Experimental results: The protein safety data of the recombinant botulinum toxin type A active polypeptides purified from the supernatant and the recombinant botulinum toxin type A active polypeptides derived from the precipitated inclusion bodies are shown in Table 3.

[0124] Table 3. Protein safety data of recombinant botulinum toxin type A active polypeptide

[0125] LD 50 Distance ratio calculation: Calculated according to the Reed-Muench method: distance ratio = (number of deaths above 50% - 50%) / (percentage of deaths above 50% - percentage of deaths below 50%), that is, distance ratio = (100% - 50%) / (100% - 0%) = 50 / 100 = 0.5, LD 50 Logarithm of = logarithm of sample dilution above 50% + distance ratio × logarithm of dilution factor, i.e., LgLD 50 =-5+0.5×(-1)=-5.5,1×10 5.5 LD 50 It refers to the toxicity unit of the sample under the condition of injection volume of 0.1ml, that is, 1×10 5.5 LD 50 / 0.1ml. The pharmacopoeia takes 0.5ml volume to inject into mice, so 5×10 5.5 LD50 / ml. Pharmacopoeia 3.1 stipulates 1×10 7 LD 50 / mg or more, 1000 / 50=20 times, i.e. 5×10 5.5 LD 50 / ml×20 times=1×10 7.5 LD 50 / mg.

[0126] Therefore, the toxicity of the sample purified from the supernatant of recombinant botulinum toxin type A active polypeptide is 1×10 5.5 LD 50The protein toxicity of the recombinant botulinum toxin type A active polypeptide derived from precipitated inclusion bodies is significantly weaker, that is, in use, the recombinant botulinum toxin type A active polypeptide derived from supernatant purification can be effective at a lower dose, which is significantly safer than the recombinant botulinum toxin type A active polypeptide derived from precipitated inclusion bodies, which requires large doses.

[0127] In summary, the present invention has verified through comparative experiments that the recombinant botulinum toxin type A active polypeptide derived from the supernatant can efficiently remove the tag when purified using the SUMO tag, is highly activated after enzymatic cleavage, and has significant safety in use despite its outstanding potent toxicity.

[0128] The present invention may be summarized in other specific forms that do not violate the spirit or main features of the present invention. Therefore, no matter from which point of view, the above-mentioned embodiments of the present invention can only be regarded as illustrative of the present invention and not limiting of the present invention. The claims indicate the scope of the present invention, while the above-mentioned description does not indicate the scope of the present invention. Therefore, any changes within the meaning and scope equivalent to the claims of the present invention should be considered as included in the claims of the present invention.

Claims

1. A recombinant botulinum toxin type A (BoNT / A), characterized in that: Its amino acid sequence is shown in SEQ ID NO.

7.

2. The recombinant botulinum toxin type A (BoNT / A) according to claim 1, characterized in that The amino acid sequence of the recombinant botulinum toxin type A (BoNT / A) includes SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5; (a) Lc region encoding BoNT / A, the sequence of which is shown in SEQ ID NO.1; (b) encoding a thrombin cleavage site, the sequence of which is shown in SEQ ID NO.3; (c) The Hc region encoding BoNT / A, the sequence of which is shown in SEQ ID NO.

5.

3. The recombinant botulinum toxin type A (BoNT / A) according to claim 2, characterized in that The combined tag is inserted into the N-terminus of the amino acid sequence of BoNT / A by homologous recombination, that is, the amino acid sequence of the designed recombinant botulinum toxin type A (BoNT / A) is shown in SEQ ID NO.

13.

4. The recombinant botulinum toxin type A (BoNT / A) according to claim 3, characterized in that The combined tag is a His tag and a SUMO tag.

5. The method for preparing recombinant botulinum toxin type A (BoNT / A) according to claim 4, characterized in that: The following steps are involved: (1) Designing and constructing a recombinant botulinum toxin type A (BoNT / A) having an amino acid sequence as shown in SEQ ID NO. 13; (2) Design and ligation of BoNT / A expression vector; (3) Induced expression of BoNT / A protein and bacterial collection; (4) Affinity purification of bacterial supernatant; (5) Enzymatic cleavage removes the SUMO tag and activates the BoNT / A protein.

6. The method for preparing recombinant botulinum toxin type A (BoNT / A) according to claim 5, characterized in that: The experimental steps of enzymatically removing the SUMO tag and activating the BoNT / A protein are as follows: after the SUMO tag is enzymatically cleaved, 1 U of thrombin is added according to 1 mg of protein.

7. The preparation method according to claim 6, characterized in that: The induction expression steps of the BoNT / A protein are as follows: 600 After the concentration of 1 mM IPTG was added, the cells were induced at 30°C for 12 h.

8. The preparation method according to claim 7, characterized in that: The bacterial cell collection step is as follows: centrifuge at 4000rpm to collect bacterial sludge, resuspend with 40ml 20mM PB+150mM Nacl, pH 7.2, and ultrasonically disrupt with 420W ice bath, ultrasonically disrupt for 3S, with an interval of 5S, for 15min; centrifuge at 12000rpm, 4℃ for 20min, and collect the supernatant again; The specific steps of affinity purification of the bacterial supernatant are as follows: the supernatant collected by centrifugation is filtered through a 0.22 μm filter membrane and then purified by Ni column affinity enrichment, wherein the equilibration buffer is 20 mM PB + 150 mM NaCl, and the washing buffer is 20 mM PB+150mM NaCl+5mM imidazole, 20mM PB+150mM NaCl+20mM imidazole, 20mM PB+150mM NaCl+50mM imidazole, 20mM PB+150mM NaCl+80mM imidazole, the elution buffer was 20mM PB+150mM NaCl+300mM imidazole, the pH of the reagents was pH 8.

0.

9. A recombinant BoNT / A polypeptide prepared by the method of any one of claims 1-8.

10. Use of the recombinant BoNT / A polypeptide prepared by the method according to any one of claims 1 to 9 in preparing a drug.

Images