Botulinum toxin type a precursor polypeptide and preparation method and application thereof

Abstract

The present application relates to botulinum toxin preparation technical field, disclose a kind of botulinum toxin type A precursor polypeptide and its preparation method and application.The botulinum toxin type A precursor polypeptide includes the light chain structure region, first connecting region and heavy chain structure region sequentially connected from N-terminal to C-terminal;The amino acid sequence of the heavy chain structure region is as shown in SEQ ID NO:3;The amino acid sequence of the first connecting region is as follows: in the connecting region amino acid sequence between natural botulinum toxin type A precursor polypeptide light chain and heavy chain, the first protease recognition sequence is inserted in the 440th position.The botulinum toxin type A precursor polypeptide of the application can improve the yield of botulinum toxin type A, and the botulinum toxin type A prepared after enzymatic activation has higher biological activity.

Description

Technical Field

[0001] This invention relates to the field of botulinum toxin preparation technology, and in particular to a type A botulinum toxin precursor polypeptide, its preparation method, and its application. Background Technology

[0002] Botulinum neurotoxins (BoNT), commonly known as botulinum toxin, are exotoxins produced by the anaerobic bacterium Clostridium botulinum. There are seven serotypes, designated A and B. The structure and functional domains of BoNT are highly conserved. Its precursor is a single polypeptide chain with a molecular weight of approximately 150 kDa. The precursor is broken down by endogenous or exogenous proteolytic enzymes into an active double-stranded structure linked by two disulfide bonds: a light chain (Lc, 50 kDa) with a zinc-dependent protease-catalyzing domain; and a heavy chain (Hc, 100 kDa) with a cell-binding and translocation domain. The heavy chain mediates the binding of BoNT to specific receptors on the cell membrane, allowing it to be taken up by endocytosis. Simultaneously, the disulfide bond connecting the light and heavy chains is reduced, allowing the light chain to diffuse freely into the cytoplasm. The light chain exhibits proteolytic activity; after endocytosis, it binds highly specifically to SNARE proteins, subsequently cleaving the SNARE proteins. Different serotypes target different SNARE proteins. Botulinum toxin type A (BoNT / A) prevents the fusion of synaptic vesicles and presynaptic membranes by severing synaptosome membrane-associated protein 25 kDa (SNAP-25), thereby preventing the release of acetylcholine into the synaptic cleft. Depending on the target tissue, BoNT / A can block cholinergic neuromuscular innervation of skeletal and smooth muscles, or cholinergic autonomic innervation of active exocrine glands, relaxing over-contracted muscles and eliminating wrinkles, thus having wide applications in cosmetic medicine. However, under natural conditions, the cleavage of BoNT precursors by proteases is random, resulting in heterogeneous botulinum toxin activity and making its purification difficult.

[0003] Structurally, BoNT / A consists of an N-terminal Lc catalytic domain and a C-terminal Hc translocation and receptor-binding domain. In the activated state, the loop between adjacent β-sheets of the light and heavy chains is cleaved by the protease, and the disulfide bonds (C430 and C454) on the heavy and light chains are linked together. These disulfide bonds are crucial for the neurotoxicity of botulinum toxin: the pH-sensitive amino acids and disulfide bonds in the botulinum toxin protein respond to changes in pH and redox gradients on the cell membrane, causing conformational changes in the two-chain protein. The heavy chain forms a protein transport channel, transporting the light chain protease to the inner cell membrane. Therefore, existing technologies (such as patent CN116813727A) have shown promise in controlling the cleavage site of the BoNT / A precursor peptide by inserting a protease cleavage site between the light and heavy chains, ensuring that the resulting BoNT / A has high biological activity. However, currently, this method has limited yield for preparing highly active botulinum toxin. Summary of the Invention

[0004] To address the technical problem of low yield in the preparation of highly active botulinum toxin using existing technologies, this invention provides a type A botulinum toxin precursor polypeptide, its preparation method, and its applications. Using the type A botulinum toxin precursor polypeptide of this invention can improve the yield of type A botulinum toxin and enable the type A botulinum toxin obtained after enzymatic digestion and activation to possess higher biological activity.

[0005] The specific technical solution of this invention is as follows:

[0006] In a first aspect, the present invention provides a type A botulinum toxin precursor polypeptide, comprising a light chain structural region, a first linker region, and a heavy chain structural region connected sequentially from the N-terminus to the C-terminus; the amino acid sequence of the heavy chain structural region is shown in SEQ ID NO:3; the first linker region contains a first protease recognition sequence; in the light chain structural region, the sequence preceding the first linker region is TSKTK.

[0007] The above-mentioned type A botulinum toxin precursor polypeptide can be activated by enzymatic cleavage at the first protease recognition sequence to obtain BoNT / A, which has biological activity (can enter cells and enzymatically cleave SNAP-25 protein).

[0008] Heavy chains play a crucial role in the entry of botulinum toxin into cells, thereby affecting its biological activity. In the botulinum toxin type A precursor polypeptide of this invention, the heavy chain sequence has been modified using a BoNT / A(1-1102) truncation. Compared to using the full-length heavy chain sequence, the BoNT / A(1-1102) truncation significantly increases the yield of BoNT / A. Furthermore, this modification of the heavy chain sequence does not significantly affect the activity of BoNT / A, and it also helps to avoid an immune response in the body.

[0009] Furthermore, the insertion position of the protease recognition sequence within the first linker region affects the activity of BoNT / A. This invention inserts the first linker region at a specific position (i.e., the sequence preceding the first linker region in the light chain structure region is TSKTK, and the sequence following the first linker region in the heavy chain structure region is SLDKG), enabling BoNT / A formed after enzymatic digestion of the botulinum toxin type A precursor polypeptide to exhibit higher biological activity. Moreover, compared to replacing an entire or partial sequence in the natural botulinum toxin type A precursor polypeptide with a protease recognition sequence, this invention uses the method of inserting a protease recognition sequence into the natural sequence, making it closer to natural conditions and thus beneficial for improving the activity of BoNT / A.

[0010] Preferably, the amino acid sequence of the first linker region is LVPRGS.

[0011] Preferably, the amino acid sequence of the light chain structural region is shown in SEQ ID NO:2.

[0012] Preferably, the N-terminus of the light chain structure region is sequentially connected to a second linker region and a His tag; the second linker region contains a second protease recognition sequence, which is a 3C protease recognition sequence.

[0013] This invention discovers that, compared to using the same thrombin cleavage site as in the first linker region, using the 3C protease cleavage site between the His tag and the light chain structure region allows for more thorough removal of the His tag, thereby better avoiding the risk of stimulating the body's immune system to produce antibodies.

[0014] Preferably, the type A botulinum toxin precursor polypeptide comprises the amino acid sequence shown in SEQ ID NO:1.

[0015] Secondly, the present invention provides a nucleic acid molecule for preparing a type A botulinum toxin precursor polypeptide, the nucleic acid molecule comprising a DNA sequence encoding the type A botulinum toxin precursor polypeptide.

[0016] Preferably, the DNA sequence of the nucleic acid molecule is shown in SEQ ID NO:4.

[0017] Thirdly, the present invention provides a recombinant plasmid, wherein the recombinant plasmid is a cloning vector or expression vector carrying the nucleic acid molecule.

[0018] Preferably, the expression vector is the pQE80L plasmid.

[0019] Fourthly, the present invention provides a method for preparing the type A botulinum toxin precursor polypeptide, comprising the following steps: cloning the DNA sequence encoding the type A botulinum toxin precursor polypeptide into an expression vector, transforming it into competent cells, culturing and inducing expression in the transformed competent cells, and then lysing the cells to extract the type A botulinum toxin precursor polypeptide.

[0020] Preferably, the competent cells are Escherichia coli competent cells.

[0021] Preferably, the culture medium used in the induction expression process contains Zn at a concentration of 0.1-0.4 mmol / L. 2+ .

[0022] Botulinum toxin is Zn 2+ The protease, under natural conditions, binds a Zn group to its LC catalytic domain. 2+ However, culture media typically contain large amounts of divalent metal ions (such as Fe). 2+ Cu 2+This substance competitively binds to botulinum toxin, thus affecting its activity. Therefore, this invention adds Zn to the bacterial culture medium at a final concentration of 0.1-0.4 mmol / L. 2+ This allows the final BoNT / A to be coupled with Zn to the maximum extent. 2+ This makes it closer to natural conditions, thereby maximizing its enzyme activity.

[0023] Preferably, the induction process includes: adding isopropyl-β-D-thiogalactoside (IPTG) and Zn to a culture medium containing transformed competent cells. 2+ Incubate at 15-20℃ for 12-14 hours.

[0024] Fifthly, the present invention provides the application of the type A botulinum toxin precursor polypeptide in the preparation of type A botulinum toxin, comprising the following steps:

[0025] S1: The initial product of botulinum toxin type A precursor peptide was obtained by His tag affinity purification, and the botulinum toxin type A precursor peptide was obtained by ion exchange purification.

[0026] S2: The type A botulinum toxin precursor polypeptide was digested with 3C protease, and the product was purified by a secondary affinity method to obtain the polypeptide without the His tag.

[0027] S3: The polypeptide with the His tag removed is digested by the protease corresponding to the first protease recognition sequence, and the product is purified by molecular sieve to remove the protease, thus obtaining the active form of botulinum toxin type A.

[0028] In the active form of botulinum toxin type A prepared by the above method, the disulfide bonds between the heavy chain and the light chain are not broken.

[0029] Preferably, in step S2, the 3C protease is a Prescission protease.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) The type A botulinum toxin precursor polypeptide of the present invention can improve the yield of type A botulinum toxin, help avoid immune response in the body, and enable type A botulinum toxin obtained after enzymatic digestion and activation to have higher biological activity.

[0032] (2) In the type A botulinum toxin precursor polypeptide of the present invention, the removal of the His tag can be more thorough by using a 3C protease cleavage site between the His tag and the light chain structural region. Attached Figure Description

[0033] Figure 1The results are based on the in vitro determination of BoNT / A activity using luciferase.

[0034] Figure 2 The results of SDS-PAGE analysis of the products at each stage in the preparation of the BoNT / A precursor peptide in Example 1 are shown.

[0035] Figure 3 The results are SDS-PAGE analysis of the products at each stage in the preparation of BoNT / A from the BoNT / A precursor peptide in Example 1.

[0036] Figure 4 The results are SDS-PAGE analysis of BoNT / A obtained in Example 1.

[0037] Figure 5 The results of SDS-PAGE analysis of the products at each stage in the preparation of the BoNT / A precursor peptide in Example 2 are shown.

[0038] Figure 6 The results are SDS-PAGE analysis of BoNT / A obtained in Example 2.

[0039] Figure 7 This is the verification result of the BoNT / A prepared in Example 2.

[0040] Figure 8 The results are SDS-PAGE analysis of the products at each stage in the preparation of BoNT / A from the BoNT / A precursor peptide in Example 3.

[0041] Figure 9 The results of Western blot detection of the activity of exogenous SNAP25 digested by BoNT / A enzyme.

[0042] Figure 10 Results of detecting the activity of endogenous SNAP25 cleaved by BoNT / A enzyme in mouse neuroblastoma N2A. Detailed Implementation

[0043] The present invention will be further described below with reference to embodiments.

[0044] General Implementation Examples

[0045] First, the present invention relates to a type A botulinum toxin precursor polypeptide, comprising a light chain structural region, a first linker region, and a heavy chain structural region connected sequentially from the N-terminus to the C-terminus; the amino acid sequence of the heavy chain structural region is shown in SEQ ID NO:3; the amino acid sequence of the first linker region is as follows: in the amino acid sequence of the linker region between the light chain and the heavy chain of the natural type A botulinum toxin precursor polypeptide, a first protease recognition sequence is inserted at position 440.

[0046] In some specific embodiments, the amino acid sequence of the first linker region is LVPRGS.

[0047] In some specific embodiments, the amino acid sequence of the light chain structural region is shown in SEQ ID NO:2.

[0048] In some specific embodiments, the N-terminus of the light chain structural region is sequentially connected to a second linker region and a His tag; the second linker region contains a second protease recognition sequence, which is a 3C protease recognition sequence.

[0049] In some specific embodiments, the type A botulinum toxin precursor polypeptide comprises the amino acid sequence shown in SEQ ID NO:1.

[0050] Second, the present invention relates to a nucleic acid molecule for preparing a type A botulinum toxin precursor polypeptide, said nucleic acid molecule comprising a DNA sequence encoding the aforementioned type A botulinum toxin precursor polypeptide.

[0051] In some specific embodiments, the DNA sequence of the nucleic acid molecule is shown in SEQ ID NO:4.

[0052] Third, the present invention relates to a recombinant plasmid, wherein the recombinant plasmid is a cloning vector or expression vector carrying the above-mentioned nucleic acid molecules.

[0053] In some specific embodiments, the expression vector is the pQE80L plasmid.

[0054] Fourth, the present invention relates to a method for preparing the above-mentioned type A botulinum toxin precursor polypeptide, comprising the following steps: cloning the DNA sequence encoding the above-mentioned type A botulinum toxin precursor polypeptide into an expression vector, transforming it into competent cells, culturing and inducing expression in the transformed competent cells, and then lysing the cells to extract the type A botulinum toxin precursor polypeptide.

[0055] In some specific embodiments, the competent cells are Escherichia coli competent cells.

[0056] In some specific embodiments, the culture medium used in the induction expression process contains Zn at a concentration of 0.1-0.4 mmol / L. 2+ .

[0057] In some specific embodiments, the induction of expression process includes: adding isopropyl-β-D-thiogalactoside (IPTG) and Zn to a culture medium containing transformed competent cells. 2+ Incubate at 15-20℃ for 12-14 hours.

[0058] Fifth, this invention relates to the application of the above-mentioned type A botulinum toxin precursor polypeptide in the preparation of type A botulinum toxin, comprising the following steps:

[0059] S1: The initial product of botulinum toxin type A precursor peptide was obtained by His tag affinity purification, and the botulinum toxin type A precursor peptide was obtained by ion exchange purification.

[0060] S2: The type A botulinum toxin precursor polypeptide was digested with 3C protease, and the product was purified by a secondary affinity method to obtain the polypeptide without the His tag.

[0061] S3: The polypeptide with the His tag removed is digested by the protease corresponding to the first protease recognition sequence, and the product is purified by molecular sieve to remove the protease, thus obtaining the active form of botulinum toxin type A.

[0062] In some specific embodiments, in step S2, the 3C protease is a Prescission protease. Specific Implementation

[0064] The present invention will now be described through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0065] Example 1: Preparation of BoNT / A precursor peptide (denoted as "pre-BoNT / A-1") and BoNT / A (denoted as "BoNT / A-1")

[0066] 1.1 Preparation of BoNT / A precursor peptide expression bacteria

[0067] 1.1.1 Design of BoNT / A precursor peptide sequence

[0068] The BoNT / A precursor polypeptide of this embodiment consists of, from the N-terminus to the C-terminus, a His tag, a second linker region containing a 3C protease recognition sequence, a light chain region, a first linker region containing a thrombin recognition sequence, and a heavy chain region, connected sequentially. The amino acid sequences are as follows: the full-length sequence of the BoNT / A precursor polypeptide is shown in SEQ ID NO:1; the light chain region sequence is shown in SEQ ID NO:2; the heavy chain region sequence is shown in SEQ ID NO:3; and the first linker region sequence containing the thrombin recognition sequence is LVPRGS. The DNA sequence encoding the above BoNT / A precursor polypeptide is shown in SEQ ID NO:4.

[0069] 1.1.2 Amplification of the Target Fragment and Recovery of PCR Products BoNT / A protein information was obtained from the Uniprot website, with its unique protein database number P0DP10, and the URL is https: / / www.uniprot.org / uniprotkb / P0DPI0 / entry. Based on the amino acid sequence, synonymous codons in the original sequence were replaced with the optimal codons of E. coli using codon degeneracy to ensure that the coding sequence of botulinum toxin protein better matches the codon usage preferences of E. coli and avoid the occurrence of rare codons.

[0070] PCR products were recovered using the TIANGEN agarose gel recovery kit. The procedure was as follows: (1) Prepare a 1% agarose gel using TAE buffer. (2) After taking out the PCR products, add 10× DNA loading buffer, mix well, and load the product into the agarose gel wells. At the same time, add 5 μL of DNA marker to the adjacent wells. Set the power supply to a constant voltage of 140V for 30 min. (3) After the gel run, observe the gel on a blue light gel cutter, compare the band size with the DNA marker, cut the gel block with a blade, and place it in a 1.5 mL centrifuge tube. (4) Add 2 times the volume of PN solution to the centrifuge tube containing the gel block, dissolve it in a 70℃ metal bath, add it to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Add PW solution to the adsorption column, centrifuge, and discard the waste liquid. Repeat the process of adding PW solution once, centrifuging, and discarding the waste liquid. Centrifuge at 12000 rpm for 2 min, and place the adsorption column in a new centrifuge tube. Add deionized water, let stand for 1 min, centrifuge at 12000 rpm for 1 min, and collect the filtrate. Determine the nucleic acid concentration using NanoDrop.

[0071] 1.1.3 Vector linearization (double digestion with restriction endonucleases)

[0072] 1) Prepare the reaction system: Prepare the enzyme digestion reaction system according to the instructions of Thermo Scientific's fast digestion enzyme. The formula is shown in Table 1.

[0073] Table 1. Formulation of the enzyme digestion reaction system

[0074] Components Dosage DNA (expression vector pQE80L) 3μg 10× Rapid digestion buffer 5μL Restriction endonucleases (BamHI and SalI) 3μL sterile water Add to 50 μL

[0075] 2) Enzyme digestion reaction: After mixing the enzyme digestion reaction system evenly, incubate at 37°C for 30 min.

[0076] 3) Enzyme digestion verification: After enzyme digestion, the digestion effect was verified by agarose gel electrophoresis. The digested product was recovered according to the PCR product recovery method to obtain the digested vector.

[0077] 1.1.4 Homologous recombination

[0078] According to the reaction system shown in Table 2, each component was added on ice, mixed and centrifuged, and then placed in a 37°C water bath for 30 min. The reaction product was taken out and allowed to stand on ice for 5 min to obtain the recombinant product (pQE80L-BoNT / A plasmid).

[0079] Table 2 Recombination Reaction System

[0080] Components Dosage Enzyme digestion vector 50ng gene fragments 0.02 × gene length nt Exnase II recombinase 1μL 5× reaction buffer 2μL Deionized water Add to 10 μL

[0081] 1.1.5 Conversion

[0082] 1) Thawing competent cells: Take a tube of E. coli BL21 (M15) competent cells and thaw them on ice.

[0083] 2) Add the ligation product: Add 100 μL of the recombinant product and gently mix with a pipette tip.

[0084] 3) Place on ice for 15 minutes.

[0085] 4) Heat shock: Heat shock at 42℃ for 1 minute, then quickly transfer to an ice bath for 2 minutes.

[0086] 5) Resuscitation culture: Add 800 μL of LB medium and incubate at 37°C with shaking for 45 min to revive the cells and express the antibiotic resistance gene.

[0087] 6) Centrifuge to collect cells: Centrifuge at 4000 rpm for 3 min, discard most of the culture medium, and resuspend the cells in about 100 μL of culture medium.

[0088] 7) Spreading on plates: Spread the bacterial culture onto LB plates containing the appropriate antibiotics and incubate overnight at 37°C.

[0089] 8) Positive clone identification (colony PCR method): Pick a single colony and mix it into 10 μL of ddH2O. After lysis at 95℃ for 10 min, take 1 μL of the lysis buffer as a template and perform colony PCR identification using appropriate forward and reverse primers (one of which is a universal primer). Cut out the correctly sized band from the agarose gel and verify it by sequencing by Hangzhou Youkang Biotechnology Co., Ltd.

[0090] 1.1.6 Small-scale induction and preservation of expression bacteria

[0091] 1) Transformation and plating: The BL21(M15) strain containing the recombinant product obtained after transformation was plated on an antibiotic-containing plate and incubated at 37°C for 24 hours.

[0092] 2) Pick a single colony: Pick a single colony from the plate and inoculate it into 5 mL of LB medium. Incubate at 37°C until the OD value reaches 0.5-0.8.

[0093] 3) Induction of expression: Add IPTG to a final concentration of 1 mM and induce expression at 37°C for 3 h.

[0094] 4) Bacterial suspension treatment: Take 2 mL of bacterial suspension, centrifuge at 12000 rpm, discard the supernatant, and resuspend in 80 μL of cell lysis buffer.

[0095] 5) Heat treatment: Add 20 μL of 5× loading buffer and heat in a 98℃ metal bath for 10 min.

[0096] 6) Centrifugation and spotting: Centrifuge at 12000 rpm and spot 10 μL of the supernatant.

[0097] 7) Electrophoretic analysis: SDS-PAGE electrophoresis was performed at 240V.

[0098] 8) Preservation of expression bacteria: Take 400 μL of bacterial culture, mix it with 600 μL of sterilized 50% glycerol, and freeze at -80℃.

[0099] 1.1.7 Extensive Induction of Expression

[0100] A small amount of successfully induced colonies were inoculated into 5 mL of 2YT liquid medium containing 50 μg / mL ampicillin resistance and incubated overnight at 37°C with gentle shaking. The next day, the overnight culture was transferred to fresh LB liquid medium containing ampicillin at a ratio of 1:500 and incubated at 37°C with shaking at 220 rpm. After about 3 hours, the OD value of the culture was measured to be between 0.5 and 0.8. IPTG solution was added to a final concentration of 0.1 mM, and ZnCl2 solution was added to a final concentration of 0.25 mmol / L. Induction was carried out at 18°C ​​for 12-14 hours.

[0101] 1.1.8 Collection of bacterial cells

[0102] The induced bacterial culture was transferred to a 1L collection bottle and centrifuged at 4000 rpm for 20 min at 4℃ to collect the bacterial pellet. The supernatant was discarded, the bacterial cells were resuspended in an appropriate amount of water, transferred to a 50mL centrifuge tube, centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the pellet was stored at -80℃ to obtain the BoNT / A precursor polypeptide expression bacteria.

[0103] 1.2 Preparation of protein purification solution

[0104] Prepare the protein purification solution according to the following formula:

[0105] (1) Lysis buffer: 20 mM HEPES, pH 7.4, 150 mM NaCl;

[0106] (2) Washing buffer: 20mM HEPES, pH 7.4, 150mM NaCl, 20mM imidazole;

[0107] (3) Elution buffer: 20 mM HEPES, pH 7.4, 150 mM NaCl, 250 mM imidazole;

[0108] (4) Ion exchange solution A: 20 mM Tris, pH 8.0;

[0109] (5) Ion exchange solution B: 20mM Tris, pH 8.0, 1M NaCl;

[0110] (6) Molecular sieve buffer: PBS pH 7.4.

[0111] 1.3 Preparation of BoNT / A precursor peptide

[0112] 1) Take BoNT / A precursor polypeptide expression bacteria, add 5-10 times the volume of cell lysis buffer, resuspend the cells, add lysozyme at a ratio of 1g / 100mL, stir to dissolve, add protease inhibitor PMSF to a final concentration of 1mM, and incubate on ice for 30min.

[0113] 2) Add 1 mg DNase I to digest genomic DNA and incubate on ice for 5 min.

[0114] 3) Use 100 mL of cell lysis buffer for sonication. Power 35%, sonicate for 3 seconds, pause for 7 seconds, for a total of 10-15 min.

[0115] 4) Transfer the sonicated bacterial culture to a centrifuge tube, balance it, and centrifuge at 18,000 rpm for 60 min at 4°C.

[0116] 5) Take the supernatant of the centrifuged bacterial culture, filter it through a 0.45 μM filter membrane, add nickel affinity packing material equilibrated with lysis buffer to a 50 mL centrifuge tube, and incubate it on a shaker at 4 °C for 1 h.

[0117] 6) After incubation, centrifuge at 1500 rpm for 5 min at 4℃, discard the supernatant, add 40 mL of cell lysis buffer to each tube to resuspend, and incubate on a shaker at 4℃ for 15 min.

[0118] 7) Transfer the nickel affinity packing material to a gravity column, add washing buffer to wash away non-specifically adsorbed proteins, and then elute with elution buffer. Monitor in real time with Nano Drop until the eluted protein reading is below 0.1 mg / mL, and stop collecting to obtain the BoNT / A precursor peptide.

[0119] In this embodiment, the products from each step of the BoNT / A precursor peptide preparation process were analyzed by SDS-PAGE, and the results are shown in the figure. Figure 2 . Figure 2 In the middle, the six lanes from left to right are: Lane 1 ( Figure 2 The marker "M" in the middle is the molecular weight standard (Marker); the second lane ( Figure 2 The middle lane is marked with "-" and the third lane ( Figure 2 The images marked "IPTG induced" show the SDS-PAGE results of the BoNT / A precursor peptide expression products before and after IPTG-induced expression, respectively; the fourth lane ( Figure 2 The middle lane is marked "Supernatant" and the fifth lane ( Figure 2 The results of SDS-PAGE analysis of the supernatant and precipitate obtained by high-speed centrifugation after ultrasonic disruption (labeled "Precipitate") are shown in the sixth lane. Figure 2 The SDS-PAGE results of the 250 mM imidazole elution product after 6-His affinity purification are labeled as “250 mM Mimidazole elution”.

[0120] 1.4 Preparation of BoNT / A1) After elution, the BoNT / A precursor peptide was added with Prescission protease at a ratio of 1:50 (w / w) and digested at 4°C for 2 h.

[0121] 2) The enzyme-digested protein was concentrated in a concentration tube and iteratively diluted with ion exchange buffer A to reduce the salt concentration in the solution to below 50 mM. The solution was then centrifuged at 12,000 rpm for 20 min at 4°C to remove precipitate and air bubbles.

[0122] 3) Wash the Hitrap Q anion exchange column with ion exchange buffers B and A in sequence.

[0123] 4) Load the centrifuged sample onto a 5mL sample loop and set... The procedure involved eluting the ion-exchange chromatography column using a 0-40% NaCl concentration gradient. The location of the target protein was determined by the UV280 absorption peak diagram. Samples were then collected from the corresponding collection tubes, and the target protein was identified by SDS-PAGE. Results are shown below. Figure 3 .

[0124] 5) Based on the SDS-PAGE results, collect protein samples into 50 mL centrifuge tubes, add equilibrated nickel affinity resin, and incubate at 4 °C for 15 min. Then transfer the samples to a gravity column. The 6His tag and some non-specific proteins will adsorb onto the nickel affinity resin, while the target protein will be in the flow-through solution.

[0125] 6) Collect the flow-through solution and quantify it using a Nanodrop. Add 1 μL of thrombin to 1 mg of protein sample and incubate overnight at room temperature. Perform SDS-PAGE analysis after digestion.

[0126] 7) Use a concentration tube to concentrate the protein volume to less than 500 μL.

[0127] 8) Equilibrate the Superdex 200 increase column with 2 column volumes of molecular sieve buffer beforehand, and then inject 0.5 mL of protein solution using the loading loop.

[0128] 9) Settings The program automatically collects samples starting from 0.3 column volumes. The location of the target protein is determined by the UV280 absorption peak diagram. Samples are then taken from the corresponding collection tubes, and the target protein is identified by SDS-PAGE. Results are shown below. Figure 4 .

[0129] Example 2: Preparation of BoNT / A precursor peptide (denoted as "pre-BoNT / A-2") and BoNT / A (denoted as "BoNT / A-2")

[0130] The only difference between the BoNT / A precursor polypeptide of this embodiment and that of Example 1 is that the heavy chain in this embodiment has not been modified and uses the heavy chain sequence from natural BoNT / A; the remaining amino acid sequences are the same as those in Example 1. The heavy chain sequence of the BoNT / A precursor polypeptide of this embodiment is shown in SEQ ID NO:5.

[0131] The BoNT / A precursor polypeptide of this embodiment was prepared according to the method in Example 1, and BoNT / A was further prepared from the BoNT / A precursor polypeptide.

[0132] In this embodiment, the products from each step of the BoNT / A precursor peptide preparation process were analyzed by SDS-PAGE, and the results are shown in the figure. Figure 5 . Figure 5 In the middle, the eight lanes from left to right are: Lane 1 ( Figure 5 The marker "M" in the middle is the molecular weight standard (Marker); the second lane ( Figure 5 The middle lane is marked with "-" and the third lane ( Figure 5 The images marked "IPTG induced" show the SDS-PAGE results of the BoNT / A precursor peptide expression products before and after IPTG-induced expression, respectively; the fourth lane ( Figure 5 The middle is marked "Precipitate" and the fifth lane ( Figure 5The results of SDS-PAGE analysis of the precipitate and supernatant obtained by high-speed centrifugation after ultrasonic disruption (labeled "Supernatant") are shown in the sixth lane. Figure 5 The middle lane is marked "Wash", and the seventh lane is... Figure 5 The middle is marked "20mM imidazole elution" and the eighth lane ( Figure 5 The SDS-PAGE results of the products after 6His affinity purification (labeled as "250mM imidazole elution"), flow-through, 20mM imidazole elution, and 250mM imidazole elution are shown.

[0133] In this embodiment, the final SDS-PAGE analysis results of BoNT / A are shown below. Figure 6 .

[0134] The BoNT / A verification results obtained in this embodiment are shown in [reference needed]. Figure 7 . Figure 7 The left figure shows the results of the 6His secondary affinity assay, which shows that BoNT / A-2 was detected in the flow-through solution, but not detected after elution with 250 mM imidazole. This indicates that BoNT / A-2 no longer binds to the nickel column and the 6His tag has been completely removed. Figure 7 The right figure in the image shows the results of the redox experiment. It shows that after adding 100 mM DTT to break the disulfide bonds between the light and heavy chains of BoNT / A-2, only two bands, light and heavy chains, appeared on the SDS-page gel image of BoNT / A-2, indicating that the light and heavy chains had been completely cut apart. BoNT / A-2 existed in its full-length form without DTT, indicating that the disulfide bonds between the heavy and light chains were not broken.

[0135] Comparing the experimental results of Example 1 and Example 2, it can be seen that the expression level and yield of BoNT / A precursor peptide in Example 1 are higher than those in Example 2. This indicates that modifying the heavy chain sequence in the BoNT / A precursor peptide using the method of the present invention can increase the expression level of the BoNT / A precursor peptide, which will help improve the yield of BoNT / A.

[0136] Example 3: Preparation of BoNT / A precursor polypeptide (denoted as "pre-BoNT / A-3") and BoNT / A (denoted as "BoNT / A-3") The only difference between the BoNT / A precursor polypeptide in this example and that in Example 2 is that the 3C protease recognition sequence in the second linker region is replaced with a thrombin recognition sequence. The full-length sequence of the BoNT / A precursor polypeptide in this example is shown in SEQ ID NO:6.

[0137] The BoNT / A precursor polypeptide of this embodiment was prepared according to the method in Example 1, and then BoNT / A was prepared according to the following steps:

[0138] 1) Concentrate the target protein using a concentration tube, and iteratively dilute it with ion exchange buffer A to reduce the salt concentration in the solution to below 50 mM. Centrifuge at 12000 rpm for 20 min at 4°C to remove precipitate and air bubbles.

[0139] 2) Wash the Hitrap Q anion exchange column with ion exchange buffers B and A in sequence.

[0140] 3) Load the centrifuged sample onto a 5mL sample loop and set... The procedure involves eluting the ion-exchange chromatography column using a NaCl concentration gradient of 0-40%. The location of the target protein is determined by the UV280 absorption peak diagram, and samples are collected from the corresponding collection tubes for identification of the target protein by SDS-PAGE.

[0141] 4) The collected solution was quantified using a nanodrop. 1 μL of thrombin was added to 1 mg of protein sample, and the solution was digested overnight at room temperature. After digestion, SDS-PAGE analysis was performed.

[0142] 5) Collect protein samples into 50 mL centrifuge tubes, add equilibrated nickel affinity resin, and incubate at 4 °C for 15 min. Then transfer the samples to a gravity column. The 6His tag and some non-specific proteins will adsorb onto the nickel affinity resin, while the target protein will remain in the flow-through. Perform SDS-PAGE analysis after affinity bonding is complete.

[0143] In this embodiment, during the preparation of BoNT / A from the BoNT / A precursor peptide, SDS-PAGE analysis was performed on the products of each step. The results are shown in [Figure number missing]. Figure 8 . Figure 8 In the middle, the five lanes from left to right are: Lane 1 ( Figure 8 The marker "M" in the middle is the molecular weight standard (Marker); the second lane ( Figure 8 The image marked "Hitrap Q fraction" represents the SDS-PAGE result of the ion-exchange purified product of the BoNT / A precursor peptide in step 3); the third lane ( Figure 8 The image labeled "Thrombin Digestion" represents the SDS-PAGE result of the thrombin digestion product from step 4) after overnight digestion at room temperature; the fourth lane ( Figure 8 The image marked "Flow through" represents the SDS-PAGE result of the His secondary affinity flow-through solution in step 5); the fifth lane ( Figure 8The SDS-PAGE results of the elution product of 250mM imidazole (labeled as "250mM imidazole elution") are as follows. Figure 8 The results showed that after thrombin digestion, the cleavage site between the heavy and light chains was completely cleaved, and BoNT / A-3 was activated; however, after secondary affinity, BoNT / A-3 was not detected in the flow-through, and the digested BoNT / A-3 still bound to the nickel column, and the cleavage site between the 6His tag and BonT / A was not cleaved.

[0144] Comparing the experimental results of Example 2 and Example 3, it can be seen that, between the His tag and the light chain structure region, the removal of the His tag is more thorough than that of the thrombin cleavage site.

[0145] Example 4: BoNT / A Activity Detection

[0146] 4.1 In vitro determination of BoNT / A activity based on luciferase

[0147] 1) Prepare the luciferase reaction buffer according to the following formula: 20mM HEPES, 20μM ZnCl2, 2mM DTT, 3% BSA; pH 7.2.

[0148] 2) Take 1 μg of BoNT / A protein sample (prepared according to the method in Example 1) and dilute it with PBS buffer to 100 μL (10 μg / mL). Then, dilute it sequentially according to the 10-fold dilution method to approximately 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL to obtain the BoNT / A protein sample solution.

[0149] 3) Dilute the SNAP25 chimeric luciferase protein sample to 30 μM with PBS buffer. For each BoNT / A dilution gradient, take 200 μL of the SNAP25 chimeric luciferase protein sample into an EP tube and add 2 μL of BoNT / A protein sample solution. Perform three replicates for each dilution gradient and react at 37°C for about 4 hours. At the same time, set up a blank control with an equal amount of PBS.

[0150] 4) After the reaction is complete, transfer the sample from each EP tube to an opaque multi-well plate according to the dilution gradient. Dilute Furimazine to 5 μM with reaction buffer, add 200 μL to the reaction sample, and incubate at room temperature for 5 min.

[0151] 5) Turn on the microplate reader and preheat for at least 15 minutes to ensure instrument stability. Select the full-path (LUM) fluorescence detection mode according to experimental requirements. Set appropriate gain, integration time, and filters based on the characteristics of the fluorescent dye or probe. Place the plate into the microplate reader and start the detection program. The instrument will automatically read the fluorescence signal of each well and record the relative fluorescence units (RFU). When performing data analysis, subtract the RFU value of the blank control well to correct for background signal. Use appropriate statistical methods in GraphPad Prism8 to analyze the data and evaluate the significance of the experimental results.

[0152] Test results are shown Figure 1 .

[0153] 4.2 Western blot detection of BoNT / A enzyme digestion of exogenous SNAP25 activity

[0154] 4.2.1 Construction of pCDNA3.1-3flag-SNAP25 plasmid

[0155] The pCDNA3.1-3flag-SNAP25 plasmid was constructed by PCR amplification and homologous recombination, wherein the DNA sequence of SNAP25 is shown in SEQ ID NO:7 and the amino acid sequence it encodes is shown in SEQ ID NO:8.

[0156] 4.2.2 pcDNA3.1-3flag-SNAP25 transfected HEK293T cells to express SNAP25 protein.

[0157] 1) Preheat OPTI-MEM medium; prepare 8 EP tubes, add 100μL of medium to each tube, label them, and divide them into two groups of 4 EP tubes each, namely the plasmid group and the lipo group.

[0158] 2) Add 2 μg of pCDNA3.1-3flag-SNAP25 plasmid to the plasmid group, add 4 μL of transfection reagent to the lipo group, and mix well; add the mixture of plasmid group to the lipo group, mix well, and let stand for 20 min.

[0159] 3) Add all the solution from each tube to the 12-well plate and shake evenly to spread the cells (the cells cultured in the 12-well plate are HEK293T cells); change the medium after 5 hours (remove all the culture medium from the pump head and add 1 mL of fresh DMEM medium) or add medium (remove half of the culture medium and add half of the culture medium).

[0160] 4.2.3 Enrichment of SNAP25 using Flag antibodies

[0161] 1) Sample preparation:

[0162] Cell lysis: Collect cells expressing Flag-tag proteins and wash three times with pre-chilled PBS. Add lysis buffer (containing protease inhibitors) and lyse on ice for 30 min. Centrifuge at 12,000 rpm for 15 min at 4°C and collect the supernatant (total protein extract).

[0163] 2) Antibody binding to magnetic beads / agarose beads: Take an appropriate amount of protein A / G magnetic beads and wash three times with PBS. Incubate the anti-FLAG antibody with the magnetic beads at 4°C for 1 hour, gently shaking. Wash three times with PBS to remove unbound antibody.

[0164] 3) Immunoprecipitation: Incubate the total protein extract with the antibody-magnetic bead complex at 4°C for 2 hours, gently shaking. Wash the magnetic beads three times with pre-cooled washing buffer to remove non-specifically bound proteins.

[0165] 4) Competitive elution: Add 150 μg / mL FLAG peptide solution, incubate at room temperature for 30 min, and gently shake. Collect the supernatant (containing the target protein).

[0166] 5) Western blot detection: The eluted protein sample is subjected to SDS-PAGE electrophoresis, and after transfer to a membrane, it is detected with anti-FLAG antibody or other target antibody.

[0167] 4.2.4 Detection of BoNT / A enzyme digestion activity

[0168] 1) Prepare the enzyme digestion buffer according to the following formula: 20mM HEPES, 20μM ZnCl2, 2mM DTT, 3% BSA; pH 7.2;

[0169] 2) Take 10 ng of BoNT / A protein sample (prepared according to the methods in Examples 1 and 2, respectively), and dilute it to 200 μL (250 μg / mL) with enzyme digestion reaction buffer. Dilute it to 25 μg / mL and 2.5 μg / mL using the 10-fold dilution method. Take SNAP25 protein extracted from cells via intracellular IP, dilute it with enzyme digestion reaction buffer, and add 100 μL to each dilution gradient.

[0170] 3) Western blot detection of endogenous SNAP25 migration:

[0171] a) Remove the culture medium from the 12-well plate and incubate the cells in 100 μL of loading buffer (56 mM sodium dodecyl sulfate, 0.05 M Tris-HCl pH 6.8, 1.6 mM UltraPure EDTA, 6.25% glycerol and 0.00001% bromophenol blue) for 5 min.

[0172] b) Add one unit of benzoylurease and 1 μL of 1M MgCl2 to each well, and shake for 10 min.

[0173] c) The sample was boiled at 95°C for 1 min and then detected on a 12% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel.

[0174] d) Activate the PVDF membrane with methanol, rinse with ddH2O, and equilibrate the wet transfer buffer; pre-equilibrate the filter paper with the wet transfer buffer; soak the transfer clamp and cotton in the wet transfer buffer, assemble the membrane according to the following steps: anode (red) double-layer filter paper - PVDF membrane - adhesive - (black) double-layer filter paper, and remove air bubbles; add ice box and ice pack, and transfer the membrane at 300mA for 180min.

[0175] e) Cut the converted PVDF membrane according to the sample loading channels; seal it with 5% skim milk (20 mL of TBST, 1 g of skim milk) for 1 h.

[0176] f) Wash once with TBST, shake for 5 minutes, and repeat the washing 3 times; dilute the FIPV antibody at a ratio of 1:5000, add 2.4 μL of the diluted antibody to 12 mL of TBST, and incubate for 1 hour.

[0177] g) Antibody recovery: Wash once with TBST, shake for 5 minutes, repeat 3 times.

[0178] h) Dilute the secondary antibody from the corresponding source at a ratio of 1:5000, incubate for 1 hour, wash once with TBST, shake for 5 minutes, and repeat the washing process 3 times.

[0179] i) Take 0.5 mL TBST, 0.25 mL luminescent solution, and 0.25 mL colorimetric solution, and mix them.

[0180] j) Draw the colorimetric mixture onto the packaging bag, remove air bubbles, immerse the PVDF film face down in the mixture, and react in the dark for 3 minutes; spray water on both sides of the film in the dark clip, wipe it clean with absorbent paper, place the film face up, cover it with the film, and remove air bubbles; develop in the dark room, take 3 X-ray films, crease one corner, press the film for 2 minutes / 5 minutes, and develop by machine.

[0181] Test results are shown Figure 9 .

[0182] 4.3 Detection of BoNT / A restriction enzyme cleavage of endogenous SNAP25 in mouse neuroblastoma N2A

[0183] 4.3.1 Mouse neuroblastoma N2A culture

[0184] 1) N2A cells were cultured in MEM EARLES supplemented with 10% FBS, 1% MEM NEAA and 100 U / mL penicillin / streptomycin, and placed in a fully humidified incubator at 37°C and 5% CO2.

[0185] 2) Every 3 days, wash the cells with phosphate-buffered saline (PBS), resuspend the cells in the culture medium using flow pressure, and then count them with a hemocytometer.

[0186] 3) Take 15 μg of BoNT / A protein sample (prepared according to the methods in Example 1 and Example 2, respectively) and add it to N2A cells, and incubate for 48 h.

[0187] 5.3.2 Western blot detection of endogenous SNAP25 migration

[0188] 1) Remove the culture medium from the 12-well plate and incubate the cells in 100 μL of loading buffer (56 mM sodium dodecyl sulfate, 0.05 M Tris-HCl pH 6.8, 1.6 mM UltraPure EDTA, 6.25% glycerol and 0.00001% bromophenol blue) for 5 min.

[0189] 2) Add one unit of benzoylurease and 1 μL of 1M MgCl2 to each well, and shake for 10 min.

[0190] 3) The sample was boiled at 95℃ for 1 min and then detected on a 12% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel.

[0191] 4) Activate the PVDF membrane with methanol, rinse with ddH2O, and equilibrate the wet transfer buffer; pre-equilibrate the filter paper with the wet transfer buffer; soak the transfer clamp and cotton in the wet transfer buffer, assemble the membrane according to the following order: anode (red) double-layer filter paper - PVDF membrane - adhesive - (black) double-layer filter paper, and remove air bubbles; add ice box and ice pack, and transfer the membrane at 300mA for 180min.

[0192] 5) Cut the transferred PVDF membrane according to the sample loading channels; seal it with 5% skim milk (20mL TBST, 1g skim milk) for 1h.

[0193] 6) Wash once with TBST, shake for 5 minutes, and repeat the washing 3 times; dilute the FIPV antibody at a ratio of 1:5000, add 2.4 μL of diluted antibody to 12 mL of TBST, and incubate for 1 hour.

[0194] 7) Antibody recovery: Wash once with TBST, shake for 5 minutes, repeat 3 times.

[0195] 8) Dilute the secondary antibody from the corresponding source at a ratio of 1:5000, incubate for 1 hour, wash once with TBST, shake for 5 minutes, and repeat the washing process 3 times.

[0196] 9) Take 0.5 mL of TBST, 0.25 mL of luminescent solution, and 0.25 mL of colorimetric solution, and mix them.

[0197] 10) Draw the color-developing mixture onto the packaging bag, remove air bubbles, immerse the PVDF film face down in the mixture, and react in the dark for 3 minutes; spray water on both sides of the film in the dark clip, wipe it clean with absorbent paper, place the film face up, cover it with the film, and remove air bubbles; develop in the darkroom, take 3 X-ray films, crease one corner, press the film for 2 minutes / 5 minutes, and develop in the machine.

[0198] Test results are shown Figure 10 .

[0199] Example 5: Sample preparation for mouse biosafety experiment: Take 5 μg of BoNT / A protein sample (prepared according to the method in Example 1) and dilute it to 500 μL (2.5 μg / mL) with PBS buffer. Dilute it sequentially to 250 ng / mL (S1), 25 ng / mL (S2), 2.5 ng / mL (S3), 0.25 ng / mL (S4), and 0.025 ng / mL (S5) using the 10-fold dilution method to obtain BoNT / A protein sample solutions.

[0200] Animal injection: Five dilution gradients (S1-S5) of 250 ng / mL, 25 ng / mL, 2.5 ng / mL, 0.25 ng / mL, and 0.025 ng / mL were used to inject 0.05 mL of BoNT / A protein sample solution into two 4-6 week old SPF mice via intraperitoneal injection. A control group was set up, with each mouse injected with an equal volume of PBS. The survival and mortality of mice were recorded at regular intervals. The fractional survival rate is shown in Table 3.

[0201] Table 3 Results of the mouse biosafety experiment

[0202] S1 S2 S3 S4 S5 control group 2 hours 100％ 100％ 100％ 100％ 100％ 100％ 4 hours 100％ 100％ 100％ 100％ 100％ 100％ 12 hours 0％ 0％ 0％ 50％ 100％ 100％

[0203] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used in this invention are conventional in the art and can be obtained through conventional commercial means; unless otherwise specified, the methods used in this invention are conventional methods in the art.

[0204] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A type A botulinum toxin precursor polypeptide, characterized in that, The amino acid sequence of the type A botulinum toxin precursor polypeptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule for preparing a type A botulinum toxin precursor polypeptide, characterized in that, The nucleic acid molecule encodes the type A botulinum toxin precursor polypeptide of claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid is a cloning vector carrying the nucleic acid molecule described in claim 2.

4. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector carrying the nucleic acid molecule described in claim 2.

5. The recombinant plasmid according to claim 4, characterized in that, The expression vector is the pQE80L plasmid.

6. A method for preparing the type A botulinum toxin precursor polypeptide according to claim 1, characterized in that, The process includes the following steps: cloning the DNA sequence encoding the type A botulinum toxin precursor polypeptide into an expression vector, transforming it into competent cells, culturing and inducing expression in the transformed competent cells, and then lysing the cells to extract the type A botulinum toxin precursor polypeptide.

7. The preparation method according to claim 6, characterized in that, During the induction of expression, the culture medium used contains Zn at a concentration of 0.1-0.4 mmol / L. 2+ .

8. The preparation method according to claim 7, characterized in that, During the induction of expression, the culture medium used contained Zn at a concentration of 0.25 mmol / L. 2+ .

9. The application of the type A botulinum toxin precursor polypeptide according to claim 1 in the preparation of type A botulinum toxin, characterized in that, Includes the following steps: S1: The initial product of botulinum toxin type A precursor peptide was obtained by His tag affinity purification, and the botulinum toxin type A precursor peptide was obtained by ion exchange purification. S2: The type A botulinum toxin precursor polypeptide was digested with 3C protease, and the product was purified by a secondary affinity method to obtain the polypeptide without the His tag. S3: The polypeptide with the His tag removed is digested by the protease corresponding to the first protease recognition sequence, and the product is purified by molecular sieve to remove the protease, thus obtaining the active form of botulinum toxin type A.

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