Botulinum toxin type c1 light chain mutant and use thereof

Abstract

This invention relates to the optimization and application of C1 type botulinum toxin light chain (BoNT / C1LC) mutants. Using the selectively Syntaxin-1 cleaving mutant BoNT / C1α-51 as a template, this invention employs PCR site-directed mutagenesis to replace amino acids at positions 53-51 and evaluates enzyme activity at the cellular level. The results show that mutants 53Y, 52H53Y, 51D52H53Y, 51S52H53Y, 51G52H53Y, 51A52H53Y, and 51Y52H53Y exhibit no cleavage activity against the substrate SNAP-25, but retain cleavage activity against Syntaxin-1. Among these, 52H53Y, 51S52H53Y, and 51G52H53Y show superior Syntaxin-1 cleavage activity compared to BoNT / C1α-51. The mutants provided by this invention, which replace full-length BoNT / C1LC or are fused with a membrane-penetrating peptide and are active only for Syntaxin-1, can be used in the preparation of medical aesthetic products and related drugs that supplement clinical responses to or non-responses to botulinum toxin type A, and whether or not resistance has been developed. Mutants with Syntaxin-1 cleavage activity superior to BoNT / C1α-51 can be used in the preparation of drugs for Syntaxin-1-mediated hypersecretion diseases. Mutants such as 51F52H53Y have no cleavage activity against both SNAP-25 and Syntaxin-1 and can be used in the preparation of targeted delivery drugs for the nervous system using full-length botulinum toxin as a carrier.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and provides C1 botulinum toxin light chain mutants: 53F, 53L, 52H53Y, 51D52H53Y, 51S52H53Y, 51G52H53Y, 51A52H52Y, 51Y52H53Y, 51F52H53Y and the like, and the application of the mutants in replacing part of C1 botulinum toxin light chain or fusing with a cell-penetrating peptide. The mutants can be used in the preparation of a drug for targeted delivery of a drug to a nervous system by taking full-length botulinum toxin as a carrier, or in the preparation of a medical and beauty product responding to or not responding to A-type botulinum toxin and a related drug, or in the preparation of a drug for a Syntaxin-mediated high-secretion disease. BACKGROUND

[0002] Botulinum neurotoxins (BoNTs) are bacterial exotoxins produced by Clostridium botulinum during reproduction, and can be divided into seven serotypes A-G according to immunogenicity. The seven serotypes are highly similar in sequence and structure, and the active form is a 150kDa dimer composed of a 50kDa light chain and a 100kDa heavy chain connected by a disulfide bond. The dimer has three important functional domains: a heavy chain receptor domain (HC C ) that recognizes and binds to the receptor on the surface of a neuron cell and enters the cell through receptor-mediated endocytosis; a heavy chain translocation domain (HC N ) that changes the conformation of the botulinum toxin under the acidic environment of an endosome; and a light chain catalytic domain (LC NA protein translocation channel is formed on the endosomal membrane to deliver the light chain to the cytoplasm; the light chain (LC) domain has zinc metalloprotease activity, and LC specifically cleaves the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex in the cytoplasm of target neuronal cells. The SNARE protein complex can promote the fusion of synaptic vesicle membranes and cell membranes, and after the botulinum LC cleaves the SNAREs, it will block the fusion of synaptic vesicle membranes and cell membranes, thereby inhibiting the release of neurotransmitters, and ultimately leading to muscle flaccid paralysis[1]. The SNARE protein complex is composed of two 25kDa synaptosomal-associated protein (SNAP-25) proteins, one Syntaxin-1 protein, and one VAMP2 protein. Different serotypes of botulinum toxin have different substrates: BoNT / A, BoNT / C, and BoNT / E cleave SNAP-25; among them, BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave VAMP (also known as synaptobrevin) / vesicle-associated membrane protein isoforms 1, 2, and 3; BoNT / C can cleave Syntaxin-1, 2, and 3 in addition to SNAP-25[2].

[0003] Currently, botulinum toxins BoNT / A and BoNT / B have been approved for clinical use. Among them, BoNT / A is the most widely used, and BoNT / A was initially approved for the treatment of eyelid spasm, facial spasm, and strabismus, and is now being used in a wider range of applications, including various neuromuscular diseases, autonomic nervous and other non-neuronal diseases[3]. As the clinical application of BoNT / A becomes more and more extensive, some patients do not respond to this serum type of toxin or develop resistance due to specific immune response after long-term treatment, resulting in the ineffectiveness of BoNT / A[4]. BoNT / B is superior to BoNT / A in some aspects, but the affinity of BoNT / B to the target cell receptor is low, resulting in poor absorption of BoNT / B at the injection site, easy diffusion, and the need to use higher doses of toxin to achieve the therapeutic effect obtained when using low doses of BoNT / A, which leads to a significant increase in non-target toxicity, and its incidence of adverse reactions is higher than that of BoNT / A. In fact, the clinical application of BoNT / B is extremely limited[5].

[0004] After BoNT / A-induced paralysis of the neuromuscular junction, the muscle can fully recover as the intracellular toxin light chain is degraded and intracellular SNARE proteins are resynthesized. BoNT / A is generally long lasting, lasting 2-4 months, and is therefore a long-acting toxin. Other serotypes of the toxin can effectively paralyze the neuromuscular junction, but generally fully recover much more quickly. BoNT / C is another long-acting BoNT in addition to BoNT / A that is similarly long lasting [6]. Furthermore, BoNT / C is the only BoNT that cleaves two different SNARE proteins. BoNT / C is a very promising alternative to BoNT / A for patients who have developed resistance to repeated applications of BoNT / A. However, BoNT / C also cleaves the BoNT / A substrate SNAP-25, which limits the implementation of a BoNT / C replacement for BoNT / A strategy.

[0005] BoNT / C has three subtypes, referred to as serotypes CI, C2, and C3. CI neurotoxin paralyzes humans and animals at low doses by blocking acetylcholine release from neurons, and recovery is slow - treatment can require weeks of mechanical ventilation to enable a person to breathe again. C2 toxin is not neurologically active, but causes necrosis and hemorrhage, and C3 toxin is less well studied [7]. Therefore, BoNT / C retrieved from the literature is generally understood to be BoNT / CI unless the BoNT / C subtype is specifically indicated.

[0006] Jackson and Wang in 2014 disclosed in a patent that mutating amino acids in the S1 pocket of the BoNT / CI light chain catalytic active center or mutating amino acids 53-51 in the light chain and substrate binding region can change the specificity and cleavage activity of BoNT / CI LC for the substrates syntaxin-1 or SNAP-25. The BoNT / CI light chain mutant BoNT / CI alpha-51 disclosed in the patent with significantly improved substrate specificity retained only cleavage activity for syntaxin-1 and no detectable cleavage activity for SNAP-25 in their disclosed invention [8]. Unfortunately, BoNT / CI alpha-51 was shown to have reduced cleavage activity for syntaxin-1 and retained 50th to 100th of the SNAP-25 cleavage activity of the wild-type BoNT / CI light chain [9]. Studies have shown that cleavage activity for the substrates SNAP-25 and syntaxin-1 is the cause of BoNT / CI-induced neuronal apoptosis degeneration and muscle paralysis

[10] . For BoNT / CI, lower concentrations can cleave syntaxin-1 and higher concentrations are required to begin cleaving SNAP-25 [9]. It is obvious that obtaining a mutant with greater cleavage activity than BoNT / CI alpha-51 can significantly improve the clinical application potential of BoNT / CI mutants.

[0007] The present application adopts site-directed mutagenesis method to mutate and restructure BoNT / C1 α-51, aiming to provide BoNT / C1 LC mutant with significantly improved Syntaxin-1 cleavage activity, so as to overcome the defects of BoNT / C1 α-51.

[0008] In addition, research reports that Syntaxin-1 is essential for triggering the exocytosis of neuronal synaptic vesicles and dense core vesicles of endocrine cells, and the cleavage of Syntaxin-1a significantly reduces exocytosis. 2+ In addition to exocytosis, Syntaxin-1 has many other functions, including regulating K + channels, Ca 2+ channels and K-ATP channels, among which K-ATP channels are very important in the treatment of type II diabetes, and the cleavage of Syntaxin-1 can increase the activity of K-ATP channels, thereby reducing the activation of β cells and restoring normal insulin secretion

[11] . BoNT / C1 has the activity of cleaving Syntaxin-1, and after mutating its light chain and removing its SNAP-25 cleavage activity, it can be used for the treatment of Syntaxin-mediated hypersecretion diseases. The present application intends to use genetic engineering means to mutate and restructure the light chain of C1 botulinum toxin, so that BoNT / C1 only has cleavage activity on Syntaxin-1, and ultimately it is expected to be used in the preparation of Syntaxin-mediated hypersecretion disease drugs.

[0009] Botulinum toxins BoNTs can target motor neurons and deliver their light chains into neurons. Therefore, they can act as drug carriers to deliver them into neurons. However, in order for BoNTs to become a delivery tool, they must be "detoxified". If the toxin LC is simply deleted, it will usually cause a decrease in the solubility of the toxin, and the activity of the LC protease can be eliminated by mutating key amino acid residues

[12] . The present application obtains the corresponding inactivated mutant by mutating the amino acids at positions 53-51 of the light chain of BoNT / C1 α-51, which can be used as a carrier for the preparation of nervous system drug targeting delivery. Moreover, since the receptor of BoNT / A on the membrane of neuronal cells is SV2, and the receptor of BoNT / C1 has not yet been found. Compared with BoNT / A, BoNT / C1 has different binding receptors, and obviously, the inactivated mutant of BoNT / C1 obtained by the present application has the potential to deliver drugs to different cells and tissues that can be reached when the inactivated mutant of BoNT / A is used as a drug carrier [7].

[0010] The application utilizes the method of site-directed mutation to obtain C1 botulinum toxin mutants, and detects the cleavage activity of these mutants on substrates SNAP-25 and Syntaxin-1 at the cell level, and the application obtains C1 botulinum toxin light chain mutants that are only active on Syntaxin-1 and have no activity on SNAP-25, and C1 botulinum toxin light chain mutants that have no activity on Syntaxin-1 and SNAP-25. The C1 botulinum toxin light chain mutant disclosed in the application can replace part of the C1 botulinum toxin light chain, and is applied in the preparation of a drug for nervous system drug targeting delivery by taking full-length botulinum toxin as a carrier, or in the preparation of a medical and beauty product that is not responsive to type A botulinum toxin and related drugs, or in the preparation of a Syntaxin-mediated high secretion disease drug.

[0011] REFERENCES

[0012] [1]Vazquez-Cintron E J,Beske P H,Tenezaca L,et al.Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery[J].Scientific reports,2017,7:42923.

[0013] [2]Dong M,Masuyer G,Stenmark P.Botulinum and Tetanus Neurotoxins[J].Annual review of biochemistry.2019,88:811-837.

[0014] [3]Hu P,Chen P.The role and mechanism of botulinum toxin type A in the treatment of prostate diseases[J].Medical Review,2021,27(08):1556-61.

[0015] [4]Eleopra R,Tugnoli V,Quatrale R,et al.Clinical use of non-Abotulinum toxins:botulinum toxin type C and botulinum toxin type F[J].Neurotoxicity research,2006,9(2-3):127-31.

[0016] [5] Tao L, Peng L, Berntsson RP, et al. Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors [J]. Nature communications. 2017, 3; 8(1): 53.

[0017] [6] Rossetto O, Seveso M, Caccin P, Schiavo G, Montecucco C. Tetanus and botulinum neurotoxins: turning bad guys into good by research [J]. Toxicon. 2001; 39(1): 27-41.

[0018] [7] Benjamin J. Pauwels, Paul Bloom, Kevin Van Cott. Engineered botulinum neurotoxin for use in delivering molecules into selected cells [P]. CN201680041056.9 2016-05-14.

[0019] [8] Jackson MB, Wang DS. Engineered botulinum neurotoxin C1 with selective substrate specificity [P]. US8853360B2 Oct. 7, 2014.

[0020] [9] Zanetti G, Sikorra S, Rummel A, et al. Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission. PLoS Pathog. 2017; 13(8): e1006567.

[0021]

[10] Peng L, Liu H, Ruan H, et al. Cytotoxicity of botulinum neurotoxins reveals a direct role of syntaxin 1 and SNAP-25 in neuron survival. Nat Commun. 2013; 4: 1472.

[0022]

[11] Wang D, Zhang Z, Dong M, et al. Syntaxin requirement for Ca 2+ -triggered exocytosis in neurons and endocrine cells demonstrated with an engineered neurotoxin [J]. Biochemistry, 2011, 50(14): 2711-3.

[0023]

[12] Miyashita S I, Zhang J, Zhang S, et al. Delivery of single-domain antibodies into neurons using a chimeric toxin-based platform is therapeutic in mouse models of botulism [J]. Science translational medicine, 2021, 13(575): eaaz4197. SUMMARY

[0024] The application aims to provide a BoNT / C1 mutant and application thereof in preparation of drugs for related diseases.

[0025] The application adopts site-directed mutagenesis technology to replace the amino acids at positions 53-51 of BoNT / C1 alpha-51 with other 19 kinds of amino acids one by one from position 53, select target active mutants from each round of mutants, and then perform the next round of mutation. In each round of mutation, 3-5 clones growing on the site-directed mutagenesis plate are randomly picked, plasmids are extracted, and sequencing is performed by outsourcing to Goldensunzhi Company. After the sequencing results are compared with the theoretical mutation sequence, the saved bacterial strains are recovered and cultured, endotoxin-free plasmids are extracted by using a large extraction kit, HEK293T is used as packaging cells, virus packaging is performed, after 48 hours of packaging, the virus is collected by ultracentrifugation, PC12 cells are infected with the packaged virus, 48 hours later, the cells are lysed to extract proteins, and the activities of BoNT / C1 LC mutants obtained by the application in cutting substrates SNAP-25 and Syntaxin-1 are detected by Western Blot. At the same time, BoNT / C1 alpha-51 and wild-type light chain (BoNT / C1 LC wt) are used as positive controls for comparative study.

[0026] The present application relates to the optimization of BoNT / C1 LC mutants and their applications. The present application uses BoNT / C1 alpha-51 as a template, and uses site-directed mutagenesis to replace the amino acids at positions 53 to 51 of BoNT / C1 alpha-51 with other amino acids in turn, to obtain 44 mutants, and to detect the cell level enzyme activity of the obtained mutants, to evaluate their cleavage activity on substrates SNAP-25 and Syntaxin-1. The results show that: BoNT / C1 LC mutants 53T, 53A, 53F, 53L, 53H, 53G, 53E and 52Y53Y retain weak cleavage activity on SNAP-25, and the cleavage activity on syntaxin-1 is significantly better than that of BoNT / C1 alpha-51. Preferably, BoNT / C1 LC mutants 52H53Y, 52Q53Y, 51K52H53Y, 51W52H53Y, 51P52H53Y, 51D52H53Y, 51R52H53Y, 51A52H53Y, 51E52H53Y, 51G52H53Y, 51Y52H53Y and 51S52H53Y have no cleavage activity on SNAP-25, and the cleavage activity on syntaxin-1 is significantly better than that of BoNT / C1 alpha-51. Finally, BoNT / C1 LC mutants 53W, 52I53Y and 51F52H53Y have no cleavage activity on substrates SNAP-25 and syntaxin-1.

[0027] The BoNT / C1 LC mutants provided by the present application have significantly higher cleavage activity on Syntaxin-1 than BoNT / C1 alpha-51, which can replace the light chain part of full-length BoNT / C1 botulinum toxin, and can be applied in the preparation of medical and beauty products that are not responsive to type A botulinum toxin and related anti-dystonia drugs. The BoNT / C1 LC mutants provided by the present application have significantly higher cleavage activity on Syntaxin-1 than BoNT / C1 alpha-51, and have no cleavage activity on SNAP-25, which can replace the light chain part of full-length BoNT / C1 botulinum toxin, and can be applied in Syntaxin-mediated high secretion disease drugs. The BoNT / C1 LC mutants provided by the present application have no cleavage activity on substrates SNAP-25 and Syntaxin-1, which can replace the light chain part of full-length BoNT / C1 botulinum toxin, and can be applied in the preparation of nervous system drug target delivery drugs using full-length botulinum toxin as a carrier.

[0028] In addition, if a transmembrane peptide such as TAT is introduced into the N-terminus or C-terminus of the BoNT / C1 LC mutant provided by the present application by genetic engineering recombination technology, under the action of the corresponding transmembrane peptide, the BoNT / C1 LC mutant provided by the present application can be guided into the corresponding cells or tissues to play a role in cutting the corresponding substrate. Alternatively, the BoNT / C1 LC mutant provided by the present application is packaged by means of liposomes and the like, so that it is easy to enter the corresponding cells or tissues to play a role in cutting the corresponding substrate, thereby correcting the related diseases caused by excessive secretion of intracellular vesicles, which can be understood by those skilled in the art.

[0029] The technical solutions of the present application are described in further detail below in combination with specific examples, but the protection scope of the present application is not limited to the following examples. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 The cutting activity of BoNT / C1 LC wt, BoNT / C1 alpha-51, 53F, 53L, 51Y52H53Y, and 51F52H53Y light chain mutants on the expression of SNAP-25 and Syntaxin-1 in PC12 cells was detected by WB. All controls in the figure represent the control group without virus treatment, and 5 μL, 15 μL, 30 μL, 50 μL, and 80 μL represent the virus treatment groups of the respective mutants added with the corresponding volume. Figure 1 The cutting activity of BoNT / C1 LC wt and BoNT / C1 alpha-51 on the expression of SNAP-25 and Syntaxin-1 in PC12 cells is shown, Figure 2 The cutting activity of 53F and 53L on the expression of SNAP-25 and Syntaxin-1 in PC12 cells is shown, Figure 3 The cutting activity of 51Y52H53Y on the expression of SNAP-25 and Syntaxin-1 in PC12 cells is shown, Figure 4 The cutting activity of 51F52H53Y on the expression of SNAP-25 and Syntaxin-1 in PC12 cells is shown. DETAILED DESCRIPTION

[0031] Example 1: Construction of mutants by replacing amino acid residues at positions 53-51 with BoNT / C1 alpha-51 as a template

[0032] 1.1 Biological materials

[0033] BoNT / C1a-51 sequence (Seq ID No: 1, synthesized by Genway Biotech), cloned into syn-lox lenti vector (see Gascon, S., et al., J Neurosci Methods 168 (2008): 104-112); Top10 competent cells, ampicillin (Amp) purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and other reagents were all of analytical purity.

[0034] 1.2 Reagents

[0035] 2xHieff Gold PCR Master Mix high-fidelity enzyme premix (Shanghai Yisen Biotechnology Co., Ltd., Catalog No. 10149ES03)

[0036] DpnI methylated template digestion enzyme (NEB, Catalog No. R0176)

[0037] 1.3 Implementation steps

[0038] 1.3.1 Site-directed mutagenesis:

[0039] (1) Primer design

[0040] The primer sequence for constructing the corresponding mutant by replacing the 53rd P of BoNT / C1a-51 with other amino acids is shown in Table 1, the primer sequence for constructing the corresponding mutant by replacing the 52nd N of 53Y with other amino acids is shown in Table 2, and the primer sequence for constructing the corresponding mutant by replacing the 51st T of 52H53Y with other amino acids is shown in Table 3.

[0041] Table 1: Primer sequence for site-directed mutagenesis of 53rd site of BoNT / C1a-51 as template

[0042]

[0043]

[0044] Table 2: Primer sequence for site-directed mutagenesis of 52nd site of 53Y as template

[0045]

[0046] Table 3: Primer sequence for site-directed mutagenesis of 51st site of 52H53Y as template

[0047]

[0048]

[0049] (2) PCR amplification

[0050] The first round of mutation constructs 53P replaced by other amino acids to construct the corresponding mutant, the laboratory has Lenti-BoNT / C1 alpha-51 plasmid as a template, different mutants are constructed using the corresponding different primers in Table 1. The second round of mutation is 52N replaced by other amino acids to construct the corresponding mutant, 53Y obtained by the first round of mutation as a template, different mutants are constructed using the corresponding different primers in Table 2. The third round of mutation takes 52H53Y obtained by the second round of mutation as a template, different mutants are constructed using the corresponding different primers in Table 3. The reaction system and PCR program of all mutants are the same.

[0051] a. Reaction system

[0052]

[0053] b. Reaction program

[0054]

[0055]

[0056] (3) DpnI degrades template

[0057] Add 2 μL DpnI restriction enzyme (10 U / μL) and 6 μL enzyme digestion buffer to each tube, add water to the system to 60 μL, gently blow evenly, and then place in a 37°C metal bath for 2 h.

[0058] (4) Purification and concentration of degradation products

[0059] ① Add 6 μL of sodium acetate (3M, pH 5.2) to the DNA solution and mix well;

[0060] ② Add 130 μL of ice-cold ethanol, mix well and then place in a -20°C refrigerator for 30 minutes;

[0061] ③ 12,000 g centrifuge for 5 minutes, carefully remove the supernatant, and suck off all the liquid drops on the wall of the tube;

[0062] ④ Add 750 μL of 70% ethanol, centrifuge at 12,000 g for 2 minutes, carefully remove the supernatant, and suck off all the liquid drops on the wall of the tube;

[0063] ⑤ Place the open EP tube on the clean bench at room temperature and blow for 8-10 minutes until there is no ethanol smell.

[0064] ⑥ Add 10 μL of ddH2O to dissolve the DNA precipitate.

[0065] (5) Heat shock Top 10 competent cells

[0066] ① Take 1 tube of Top 10 competent cells, melt on ice;

[0067] ② Add all the DNA dissolved in water to 100 μL of competent cells, mix gently, and place on ice for 30 min;

[0068] ③ Heat shock: place the centrifuge tube at 42℃ for 90 sec;

[0069] ④ Quickly transfer the centrifuge tube to ice and place for 2 min;

[0070] ⑤ Recovery: add 200 uL of LB medium to each tube, and incubate at 37℃ in a shaker for 45 min to recover the bacteria;

[0071] ⑥ Uniformly spread the recovered bacteria on Amp-resistant LB solid culture plates;

[0072] ⑦ Incubate in a 37℃ incubator for 12-16 hr.

[0073] (6) Sequencing of recombinant plasmid

[0074] ① Bacterial liquid culture: when colonies grow on the transformation plate, pick 3 single colonies into 10 mL of sterilized LB liquid medium, add the corresponding volume of Amp antibiotic, and incubate at 37℃ with shaking at 180 rpm for 12-16 hr;

[0075] ② Plasmid extraction: follow the instructions of the plasmid extraction kit.

[0076] ③ Sequencing: send the plasmid to Tianjin Goldensky Co., Ltd. for sequencing. Compare the sequencing results with the theoretical sequence. If the sequences are consistent, it indicates that the cloning is successful and can be used for subsequent experiments.

[0077] Example 2 Cell-level detection of the enzymatic activity of BoNT / C1 LC mutants on substrates SNAP-25 and Syntaxin-1

[0078] 2.1 Biological materials

[0079] PC12 and HEK293T cells were purchased from Biofeng; the lentivirus packaging vectors pSPAX2 and PMD2.G were purchased from Biyun.

[0080] 2.2 Reagents

[0081] SNAP-25 antibody (GeneTex, Cat. No. GTX113839);

[0082] Syntaxin-1 antibody (Santa Cruz, Cat. No. SC-12736);

[0083] β-actin antibody (Immunoway, Item No. YM3028);

[0084] GAPDH antibody (Immunoway, Item No. YM3215);

[0085] RIPA lysis buffer (Solarbio, Item No. R0010).

[0086] 2.3 Implementation steps

[0087] 2.3.1 Preparation of BoNT / C1 LC mutant virus

[0088] (1) Cell plating: 4 x 10 6 cells per dish, with a density of about 50% or so;

[0089] (2) Transfection: After 24 hr of culture, the cell culture medium was replaced with serum-free and double-antibiotic-free medium, and transfection was performed using the liposome method;

[0090] a. A total of 22.5 μg of plasmid per dish, target plasmid: pSPAX2: PMD2.G = 4:3:2, add the corresponding volume of 3 plasmids mixed with 1 mL of MEM medium;

[0091] b. Plasmid (μg): nucleic acid transfection reagent Liposomal (μL) = 1:1, i.e. 22.5 μL of liposomal, add 1 mL of MEM medium, mix well, and incubate at room temperature for 5 min;

[0092] c. Mix the plasmid with Liposomal and incubate at room temperature for 20 min;

[0093] (3) Add the treated liposome DNA mixture to the culture dish, 2 mL per dish;

[0094] (4) Medium change: After 6 hr of culture, replace with serum-containing and double-antibiotic-containing medium;

[0095] (5) Virus packaging: culture at 37°C in a cell incubator for 48 hr;

[0096] (6) Virus collection: collect the virus supernatant, filter it through a 0.22 μm water system filter membrane into an ultracentrifuge tube, centrifuge at 25000 rpm, 4°C, for 2 hr;

[0097] (7) Virus aliquot storage: discard the supernatant, add 200 μL of serum-free medium, store at 4°C overnight for swelling, and store the aliquots at -80°C after aliquotting.

[0098] 2.3.2 Virus infection of PC12 cells

[0099] (1) Cell plating:

[0100] PC12 cells in the logarithmic growth phase and in good growth condition were selected and processed at a ratio of 2 x 10⁻⁶. 5 Seeds were planted per well in 24-well plates and cultured in a cell culture incubator for 12 hours.

[0101] (2) Viral infection:

[0102] The virus from each mutant was added to PC12 cells at doses of 0, 5, 15, 30, 50, and 80 μL, and cultured for 48 hours.

[0103] (3) Protein extraction: Discard the culture medium and add 100 μL of lysis buffer (V) to each well. RIPA :V PMSF =100:1), collect cell lysis buffer, lyse on ice for 20 min, centrifuge at 10000 rpm / min and 4℃ for 10 min, aspirate supernatant, add the corresponding volume of 5× protein loading buffer, and denature in a metal bath at 100℃ for 10 min;

[0104] (4) Western blot analysis was performed to detect the cleavage activity of each mutant virus against SNAP-25 and Syntaxin-1 in PC12 cells:

[0105] ①SDS-PAGE electrophoresis: Load 10μL of each sample, run at 80V for 2.5hr.

[0106] ② Transfer membrane: Prepare a sandwich structure according to the following steps: cellulose pad-two layers of filter paper-membrane-gel-two layers of filter paper-cellulose pad, remove air bubbles, add transfer buffer, and transfer membrane at a constant current of 300mA for 2 hours under low temperature conditions.

[0107] (7) Sealing: 5% skim milk powder, sealed at room temperature for 1 hour.

[0108] (8) Incubation of primary antibodies: SNAP-25 antibody was diluted at 1:5000 and Syntaxin-1 antibody was diluted at 1:500. Incubate at room temperature for 2 hours.

[0109] (9) Washing membrane: Wash 3 times with TBST, 10 minutes each time.

[0110] (10) Incubation of secondary antibody: The secondary antibody was diluted at 1:10000 and incubated at room temperature for 1 hour.

[0111] (11) Washing membrane: Wash 3 times with TBST, 10 minutes each time.

[0112] (10) Exposure: Prepare the ECL luminescent solution, then evenly add the solution onto the membrane, and take pictures using an exposure device. The results for BoNT / C1LC wt, BoNT / C1α-51, 53F, 53L, 51Y52H53Y, and 51F52H53Y are shown below. Figure 1, Figure 2 , Figure 3 , Figure 4 Results for the rest of the BoNT / C1 LC mutants are shown in Table 4.

[0113] Table 4 Results of the cleavage activity of the BoNT / C1 LC mutants of the application on PC12 cells SNAP-25 and Syntaxin-1

[0114]

[0115]

[0116] Note: For SNAP25, the observation of a cleavage band is considered as detection of cleavage activity, for Syntaxin, the observation of a clear cleavage band or a clear reduction of the Syntaxin protein content is considered as detection of cleavage activity, ++++ means that cleavage activity is detected at a dose of 5 μL; +++ means that cleavage activity is detected at a dose of 15 μL; ++ means that cleavage activity is detected at a dose of 30 μL; + means that cleavage activity is detected at a dose of 50 μL; + means that cleavage activity is detected at a dose of 80 μL; ND means that no cleavage activity is detected at a dose of 80 μL. BoNT / C1 LC wt and BoNT / C1 α-51 viruses are used as positive controls for each mutant virus, and the detection method of the cleavage activity of all mutants on the substrates SNAP-2 and Syntaxin-1 is the same as described in Example 1. Figure 1

[0117] The results show that BoNT / C1 LC mutants 53T, 53F, 53L, 53H, 53G, 53E and 52Y53Y retain weak cleavage activity on SNAP-25 and significantly better cleavage activity on syntaxin-1 than BoNT / C1 α-51. Preferably, BoNT / C1 LC mutants 52H53Y, 52Q53Y, 51K52H53Y, 51W52H53Y, 51P52H53Y, 51D52H53Y, 51R52H53Y, 51A52H53Y, 51E52H53Y, 51G52H53Y, 51Y52H53Y and 51S52H53Y have no cleavage activity on SNAP-25 and significantly better cleavage activity on syntaxin-1 than BoNT / C1 α-51. Finally, BoNT / C1 LC mutants 53W, 52I53Y and 51F52H53Y have no cleavage activity on substrates SNAP-25 and syntaxin-1.

[0118] ​The results of the present application also show that when only the 53rd amino acid of BoNT / C1 alpha-51 is replaced, a high frequency of the corresponding mutant restores the cleavage activity to SNAP-25, thus the disclosure of the present application indicates that the replacement and modification of the 53rd amino acid of BoNT / C1 LC is crucial to the cleavage activity of BoNT / C1 LC to SNAP-25. The amino acid replacement and modification can be achieved by any genetic engineering means or chemical means, which can be understood by those skilled in the art.

[0119] The nucleotide sequence of the disclosed BoNT / C1 LC mutant can be fully synthesized by codon optimization method, so that the BoNT / C1 LC is more suitable for expression in different host cells, but does not affect the primary structure of the expressed product protein amino acid, which can be understood by those skilled in the art.

[0120] The present application has the beneficial effect that after the mutation and modification of BoNT / C1 LC, it is non-toxic, and can replace the wild-type light chain in the full-length toxin, and can be used as a drug carrier for targeted delivery of nervous system drugs. For the light chain mutant which only has cleavage activity to Syntaxin-1, after modification, it can be used for the treatment of diseases involving Syntaxin, such as regulation of exocytosis, treatment of type II diabetes, etc. Although the currently marketed botulinum toxin only has types A and B, and type A has a long time and wide clinical application. However, type A botulinum toxin has a large side effect and drug resistance, and type B botulinum toxin has poor absorption effect in the injection part, which limits the use of type A and type B botulinum toxins, and there is an urgent need in clinic for a new botulinum toxin mutant which can clone the above defects, and therefore the BoNT / C1 mutant provided by the present application has great potential for entering the clinic.

[0121] The protein sequence involved in the present application is:

[0122] Seq ID No:1

[0123] BoNT / C1 alpha-51

[0124] MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFTNPSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENML

[0125] The nucleotide sequence according to the present application is:

[0126] Seq ID No: 2

[0127] BoNT / C1 a-51

[0128]

Claims

1. A polypeptide or protein comprising a BoNT / C1 light chain mutant, wherein, the BoNT / C1 light chain mutant is a BoNT / C1 α-51 having an amino acid sequence of Seq ID No: 1, wherein the amino acid at position 52 is mutated to H, the amino acid at position 53 is mutated to Y, and the amino acid at position 51 is not mutated or is mutated to K, W, P, D, R, A, E, G, Y, S; or the BoNT / C1 light chain mutant is a BoNT / C1 α-51 having an amino acid sequence of Seq ID No: 1, wherein the amino acid at position 52 is mutated to Q, the amino acid at position 53 is mutated to Y, and the amino acid at position 51 is not mutated. ​ ​

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