CN117624315A - Type C1 botulinum toxin light chain mutants and their applications - Google Patents

Abstract

The present invention relates to the optimization, transformation and application of C1 type botulinum toxin light chain (BoNT/C1LC) mutants. The present invention uses the BoNT/C1LC mutant BoNT/C1α-51 that selectively cleaves Syntaxin-1 as a template, uses the PCR site-directed mutagenesis method to replace its 53-51 amino acid and evaluates the enzyme activity at the cellular level. The results show that: 53Y, 52H53Y, 51D52H53Y, 51S52H53Y, 51G52H53Y, 51A52H53Y, 51Y52H53Y, etc. have no cleavage activity towards the substrate SNAP‑25, but retain cleavage activity towards Syntaxin‑1. Among them, 52H53Y, 51S52H53Y, 51G52H53Y, etc. have better cleavage activity towards Syntaxin‑1 than Bo NT/C1α ‑51. The mutants provided by the present invention replace the full-length BoNT/C1LC or are fused with the membrane-penetrating peptide, and are only active against Syntaxin-1. They can be used to prepare and supplement the clinical response or non-response to type A botulinum toxin, and produce or not. Used in drug-resistant medical aesthetic products and related drugs; mutants with Syntaxin-1 cleavage activity better than BoNT/C1α-51 can be used in the preparation of drugs for Syntaxin-1-mediated hypersecretion diseases; 51F52H53Y and other mutations It has no cleavage activity against SNAP-25 and Syntaxin-1 and can be used in the preparation of targeted delivery drugs for nervous system administration using full-length botulinum toxin as a carrier.

Description

C1 type botulinum toxin light chain mutant and application thereof

Technical Field

The invention belongs to the technical field of medicines, and provides a C1 type botulinum toxin light chain mutant: 53F, 53L, 52H53Y, 51D52H53Y, 51S52H53Y, 51G52H53Y, 51A52H52Y, 51Y52H53Y, 51F52H53Y, etc. and their substitution of the C1 botulinum toxin light chain moiety or fusion with a transmembrane peptide, can be used in the preparation of a drug targeted for delivery by nervous system administration using full-length botulinum toxin as a carrier, or in the preparation of medical products and related drugs that respond or do not respond to botulinum toxin type A, or in the preparation of drugs for Syntaxin-mediated high secretion diseases.

Background

Botulinum toxins (Botulinum neurotoxins, boNTs) are bacterial exotoxins produced by anaerobic bacillus botulinum during propagation and can be classified into seven serotypes a-G based on immunogenicity. Seven serotypes are highly similar in sequence and structure, with the active form consisting of a disulfide-linked 150kDa dimer of a 50kDa light chain and a 100kDa heavy chain. The dimer has 3 important functional domains: heavy chain acceptor domain (HC) _C ) Recognizing and binding to receptors on the surface of neuronal cells, entering the cells by receptor-mediated endocytosis; heavy chain translocation domain (HC) _N ) Under the acidic environment of endocytosis, the conformation of botulinum toxin is changed, HC _N Forming a protein transport channel on the endocytosis membrane to deliver the light chain to the cytoplasm; the Light Chain (LC) domain has zinc metalloprotease activity, and LC specifically cleaves soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNAREs) complexes in the cytosol of target neuronal cells. SNARE protein complexes promote fusion of synaptic vesicle membranes and cell membranes, and botulinum toxin LC blocks fusion of synaptic vesicle membranes and cell membranes after cleavage of SNAREs, thereby inhibiting neurotransmitter release, ultimately leading to muscle relaxant paralysis [1 ]]. The SNARE protein complex consists of two 25kDa synaptosomal associated proteins (SNAP-25) protein, one Syntaxin-1 protein, one VAMP2 protein. Different substrates for botulinum toxins of different serotypes: boNT/A, boNT/C and BoNT/E cleave SNAP-25; wherein BoNT/B, boNT/D, boNT/F, boNT/G cleaves VAMP (also known as synaptobrevin)/vesicle-associated membrane protein isoforms 1,2, 3; boNT/C can cleave SNAP-25 and simultaneously can cleave Syntaxin-1, 2, 3[ 2]]。

At present, the use of botulinum toxins BoNT/A and BoNT/B is clinically approved. Among them, boNT/A has been most widely used, and BoNT/A was originally approved for the treatment of blepharospasm, facial spasm and strabismus, and has been increasingly used, including various neuromuscular diseases, autonomic and other non-neuronal diseases [3]. With the increasing clinical use of BoNT/A, some patients either do not respond to this serotype of toxin or develop resistance after prolonged treatment due to specific immune responses, resulting in the inefficiency of BoNT/A [4]. BoNT/B is better than BoNT/A in some aspects, however, its low affinity for binding to target cell receptors results in poor absorption of BoNT/B at the site of injection, ease of diffusion, and the need to apply higher doses of toxin to achieve the therapeutic effect achieved with lower doses of BoNT/A results in a significant increase in non-target toxicity with a higher incidence of adverse effects than BoNT/A. In fact, the clinical use of BoNT/B is extremely limited [5].

After the paralysis of the neuromuscular junction is disabled due to reduced synaptic transmission of botulinum toxin, the muscle can fully resume function with degradation of the intracellular light chain of the incoming endotoxin and re-synthesis of intracellular SNARE proteins. BoNT/A generally lasts for 2-4 months and is therefore a long-acting toxin. Toxins of other serotypes, while effective in causing paralysis of the neuromuscular junction, generally resume function completely more rapidly. BoNT/C is another long-acting botulinum toxin, in addition to BoNT/A, which is persistent in comparison to its action [6]. Furthermore, boNT/C is also the only botulinum toxin that can cleave two different SNARE proteins. BoNT/C is a very potential choice for nonresponsive drug resistance that results from repeated use of BoNT/A in patients. However, boNT/C also cleaves substrate SNAP-25 of BoNT/A, which limits the implementation of the BoNT/C replacement BoNT/A strategy.

BoNT/C has 3 subtypes, designated serotypes Cl, C2 and C3. C1 neurotoxin paralyzes humans and animals at low doses by blocking acetylcholine released from neurons, recovery is slow-treatment may require weeks of mechanical ventilation to enable the person to breathe again. C2 toxins have no neural activity, leading to necrosis and hemorrhage, and C3 toxins have been rarely studied [7]. Thus, unless a BoNT/C subtype is specifically indicated, boNT/C found in the literature is generally understood to be BoNT/C1.

Jackson and Wang in 2014 disclose that mutation of the amino acid at the S1 pocket of the BoNT/C1 light chain catalytic active center or mutation of amino acids 53-51 of the light chain and substrate binding region can alter the specificity and cleavage activity of BoNT/C1LC for cleavage of substrate syntaxin-1 or SNAP-25. The BoNT/C1 light chain mutant BoNT/C1 alpha-51, which has significantly improved substrate specificity, retains only the cleavage activity for syntaxin-1, and the cleavage activity for SNAP-25 has not been detected in their disclosed invention [8]. Unfortunately, boNT/C1. Alpha. -51 demonstrated a decrease in synthaxin-1 cleavage activity, leaving about 50-100-fold of the SNAP-25 cleavage activity of the wild-type light chain of BoNT/C1 [9]. Studies have shown that cleavage activity on substrates SNAP-25 and synthaxin-1 is responsible for muscle paralysis by the induction of neuronal apoptosis degradation by BoNT/C1 [10]. For BoNT/C1, syntaxin-1 was cleaved at lower concentrations and SNAP-25 was cleaved at higher concentrations [9]. Obviously, the mutant with stronger cutting activity than the BoNT/C1 alpha-51 is obtained, and the clinical application potential of the BoNT/C1 mutant can be obviously improved.

The invention adopts a site-directed mutagenesis method to mutate BoNT/C1 alpha-51, and aims to provide a BoNT/C1LC mutant with obviously improved Syntaxin-1 cleavage activity, thereby overcoming the defects of BoNT/C1 alpha-51.

Furthermore, studies report Syntaxin-1 in Ca ²⁺ The cleavage of Syntaxin-1a significantly reduces exocytosis, which is crucial in exocytosis of triggered neuronal synaptic vesicles and endocrinocyte dense nuclear vesicles. In addition to exocytosis, syntaxin-1 has many of its usesHe functions, including regulating K ⁺ Channel, ca ²⁺ Channel and K-ATP channel, wherein K-ATP channel is important in the treatment of type II diabetes, and cleavage of Syntaxin-1 increases the activity of K-ATP channel, thereby decreasing beta cell activation and restoring normal insulin secretion [11 ]]. BoNT/C1 has activity in cleaving Syntaxin-1, and after mutation of its light chain, its SNAP-25 cleavage activity is removed, and can be used for the treatment of high secretion diseases mediated by Syntaxin. The invention adopts genetic engineering means to mutate and reform the C1 type botulinum toxin light chain, so that the BoNT/C1 only has cutting activity on Syntaxin-1, and finally the BoNT/C1 is expected to be applied to the preparation of Syntaxin-mediated high secretion disease drugs.

Botulinum toxins BoNTs are capable of targeting motor neurons and transporting their light chains into neurons. They can therefore act as drug carriers, transporting them into neurons. However, boNTs must first be "detoxified" in order to be a delivery vehicle. LC usually results in reduced toxin solubility if simply deleted, whereas LC protease activity is eliminated by mutating key amino acid residues [12]. The invention obtains the corresponding inactivated mutant by mutating the 53-51 amino acid of the BoNT/C1 alpha-51 light chain, and can be used for preparing the drug application of nervous system administration targeted delivery as a carrier. Furthermore, since the receptor on the neuronal cell membrane of BoNT/A is SV2, the receptor of BoNT/C1 has not been found so far. The BoNT/C1 inactivated mutants obtained by the present invention have a different binding receptor relative to BoNT/A, and it is apparent that the BoNT/C1 inactivated mutants obtained by the present invention are useful as drug carriers and have the potential to deliver drugs to cells and tissues different from those which can be reached when the BoNT/A inactivated mutants are used as drug carriers [7].

The invention utilizes the C1 type botulinum toxin mutant obtained by the site-directed mutagenesis method, and detects the cleavage activity of the mutant on substrates SNAP-25 and Syntaxin-1 at the cellular level, and the invention obtains the C1 type botulinum toxin light chain mutant which has activity on only Syntaxin-1 and has no activity on SNAP-25 and the C1 type botulinum toxin light chain mutant which has no activity on both the Syntaxin-1 and the SNAP-25. The C1 type botulinum toxin light chain mutant disclosed by the invention can replace a C1 type botulinum toxin light chain part and is applied to preparing a nervous system administration targeting delivery medicament by taking full-length botulinum toxin as a carrier, or preparing medical products and related medicaments which do not respond to A type botulinum toxin, or preparing a Syntaxin-mediated high secretion disease medicament.

Reference to the literature

[1]Vazquez-Cintron E J,Beske P H,Tenezaca L,et al.Engineering Botulinum Neurotoxin C1as a Molecular Vehicle for Intra-Neuronal Drug Delivery[J].Scientific reports,2017,7:42923.

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

[3] Hu Ping, chen Ping. Effects of botulinum toxin type A in prostate disease and mechanism research progress [ J ]. Medical review 2021,27 (08): 1556-61.

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

[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.

[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.

[7] Adapted for delivery of molecules into selected cells, engineered clostridium botulinum toxin [ P ]. CN201680041056.9 2016-05-14.

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

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

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

[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.

[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.

Disclosure of Invention

The invention aims to provide a BoNT/C1 mutant and application thereof in preparing related disease medicines.

The invention adopts the site-directed mutagenesis technology to replace 53-51 amino acids of BoNT/C1 alpha-51 with other 19 amino acids one by one from 53, selects target active mutants from each round of mutants, and then carries out the next round of mutation. 3-5 clones growing on a transformation plate after site-directed mutagenesis are randomly selected in each round of mutagenesis, plasmids are extracted, the company Jin Weizhi is responsible for sequencing, after sequencing results are compared and matched with theoretical mutation sequences, preserved strains are resuscitated and cultured, endotoxin-free plasmids are extracted by using a large extraction kit, HEK293T is used as packaging cells, virus packaging is carried out for 48 hours, virus is collected by using an ultracentrifugation method, the packaged virus is used for infecting PC12 cells, 48 hours, cell protein extraction is carried out, and activities of cleavage substrates SNAP-25 and Syntaxin-1 of BoNT/C1LC mutants obtained by the invention are respectively detected by using Western Blot. Meanwhile, a comparative study was performed using BoNT/C1 alpha-51 and wild-type light chain (BoNT/C1 LC wt) as positive controls.

The invention relates to optimization transformation and application of a BoNT/C1LC mutant. The invention uses BoNT/C1 alpha-51 as a template, adopts a site-directed mutagenesis mode to sequentially replace amino acids 53 to 51 of BoNT/C1 alpha-51 with other amino acids, obtains 44 mutants, carries out cell level enzyme digestion activity detection on the obtained mutants, evaluates the cleavage activity of the obtained mutants on substrates SNAP-25 and Syntaxin-1, and results show that: boNT/C1LC mutants 53T, 53A, 53F, 53L, 53H, 53G, 53E and 52Y53Y retain weaker cleavage activity for SNAP-25 and significantly better cleavage activity for synthaxin-1 than BoNT/C1 alpha-51. Preferably, the BoNT/C1LC mutants 52H53Y, 52Q53Y, 51K52H53Y, 51W52H53Y, 51P52H53Y, 51D52H53Y, 51R52H53Y, 51A52H53Y, 51E52H53Y, 51G52H53Y, 51Y52H53Y and 51S52H53Y have no cleavage activity for SNAP-25, and have significantly better cleavage activity for synthaxin-1 than BoNT/C1 alpha-51. Finally, boNT/C1LC mutants 53W, 52I53Y and 51F52H53Y had no cleavage activity on both substrates SNAP-25 and synthaxin-1.

The BoNT/C1LC mutant with the activity of cutting Syntaxin-1 being obviously higher than that of BoNT/C1 alpha-51 can replace the light chain part of full-length BoNT/C1 botulinum toxin, and can be applied to the preparation of medical products which do not respond to A-type botulinum toxin and related anti-dystonia medicines. The cutting activity of the invention to Syntaxin-1 is obviously higher than that of BoNT/C1 alpha-51, and the BoNT/C1LC mutant without cutting activity to SNAP-25 can replace the light chain part of full-length BoNT/C1 botulinum toxin and can be applied to medicines of Syntaxin-mediated high secretion diseases. The BoNT/C1LC mutant which has no cleavage activity on substrates SNAP-25 and Syntaxin-1 provided by the invention can replace the light chain part of full-length BoNT/C1 botulinum toxin and is applied to preparing nervous system administration targeting delivery medicines by taking the full-length botulinum toxin as a carrier.

In addition, if the gene engineering recombination technology is adopted to introduce a penetrating peptide such as TAT into the N end or the C end of the BoNT/C1LC mutant provided by the invention, the BoNT/C1LC mutant provided by the invention can be guided into corresponding cells or tissues under the action of the corresponding penetrating peptide to play a role in cutting the corresponding substrate. Alternatively, the BoNT/C1LC mutant provided by the present invention may be packaged by means of a liposome or the like so as to be easily introduced into a corresponding cell or tissue, and exert a function of cleaving a corresponding substrate, thereby correcting a disease associated with excessive secretion of intracellular vesicles, as will be understood by those skilled in the art.

The technical scheme of the present invention is described in further detail below in conjunction with specific embodiments, but the scope of the present invention is not limited to the following embodiments.

Drawings

FIGS. 1,2, 3 and 4 show the cleavage activity of BoNT/C1LC wt, boNT/C1 alpha-51, 53F, 53L, 51Y52H53Y, 51F52H53Y light chain mutants on SNAP-25 and Syntaxin-1 expressed by PC12 cells using WB. All controls in the figure represent untreated control groups, 5. Mu.L, 15. Mu.L, 30. Mu.L, 50. Mu.L, 80. Mu.L each represent the addition of the respective mutant virus treatment groups at the respective volumes. FIG. 1 shows the cleavage activity of BoNT/C1LC wt and BoNT/C1 alpha-51 on SNAP-25 and Syntaxin-1 of PC12 cells, FIG. 2 shows the cleavage activity of 53F and 53L on SNAP-25 and Syntaxin-1 of PC12 cells, FIG. 3 shows the cleavage activity of 51Y52H53Y on SNAP-25 and Syntaxin-1 of PC12 cells, and FIG. 4 shows the cleavage activity of 51F52H53Y on SNAP-25 and Syntaxin-1 of PC12 cells.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example one construction of mutants by substitution of amino acid residues at positions 53-51 Using BoNT/C1 alpha-51 as template

1.1 biological Material

BoNT/c1α -51 sequence (Seq ID No:1, gold intelligent total synthesis), cloned into syn-lox lenti vector (see Gascon, s., et al J Neurosci Methods 168 (2008): 104-112); top10 competent cells, ampicillin (Amp) were purchased from Biotechnology (Shanghai) Inc., and the other reagents were all analytically pure.

1.2 reagents

2×HieffGold PCR Master Mix Hi-Fi enzyme premix (Shanghai assist in san Biotech Co., ltd., product No. 10149ES 03)

DpnI methylation template digestive enzyme (NEB, cat# R0176)

1.3 carrying out the steps

1.3.1 site-directed mutagenesis:

(1) Primer design

The primer sequences for constructing the corresponding mutants by replacing P at position 53 with other amino acids and using BoNT/C1 alpha-51 as a template are shown in Table 1, the primer sequences for constructing the corresponding mutants by replacing N at position 52 with other amino acids and using 53Y as a template are shown in Table 2, and the primer sequences for constructing the corresponding mutants by replacing T at position 51 with other amino acids and using 52H53Y as a template are shown in Table 3.

Table 1: primer sequence for 53-site fixed point mutation with BoNT/C1 alpha-51 as template

Table 2: site-directed mutagenesis primer sequence of 52-position mutant with 53Y as template

Table 3: site-directed mutagenesis primer sequence of 51-site mutant with 52H53Y as template

(2) PCR amplification

The first round of mutation is to construct a corresponding mutant by replacing the P at the 53 th site with other amino acids, and constructing different mutants by using the existing Lenti-BoNT/C1 alpha-51 plasmid in the laboratory as a template and adopting different corresponding primers in Table 1. The second round of mutation is to construct a corresponding mutant by replacing N at 52 with other amino acids, and the 53Y obtained by the first round of mutation is used as a template, and different mutants adopt corresponding different primers in the table 2. The third round of mutation was performed using 52H53Y obtained from the second round of mutation as a template, and the different mutants used the corresponding different primers in Table 3. The reaction system and PCR procedure were the same for all mutants.

a. Reaction system

b. Reaction procedure

(3) DpnI degradation template

2. Mu.L of DpnI restriction enzyme (10U/. Mu.L) and 6. Mu.L of enzyme digestion Buffer were added to each tube, the system was made up to 60. Mu.L with water, gently blown, and then placed in a 37℃metal bath for enzyme digestion for 2h.

(4) Purification and concentration of degradation products

(1) Adding 6 mu L of sodium acetate (3M, pH 5.2) into the DNA solution, and fully and uniformly mixing;

(2) adding 130 mu L of ice-precooled ethanol, fully and uniformly mixing, and placing in a refrigerator at the temperature of minus 20 ℃ for 30 minutes;

(3) centrifuge 12000g for 5min, carefully remove supernatant, suck all droplets off the tube wall;

(4) 750 μl of 70% ethanol was added, and 12000g was centrifuged for 2 minutes, carefully removing the supernatant, and sucking off all droplets on the tube wall;

(5) the uncapped EP tube was placed on an ultra clean bench for 8-10 minutes at room temperature to remove ethanol smell.

(6) Add 10. Mu.L of ddH ₂ O dissolves DNA precipitate.

(5) Transformation of Top10 competent cells by thermal shock

(1) Taking 1 tube of Top10 competent cells, and putting the competent cells into ice for melting;

(2) adding all the DNA dissolved in water into 100 mu L of competent cells, gently mixing, and standing on ice for 30min;

(3) heat shock: placing the centrifuge tube at 42 ℃ and thermally striking for 90sec;

(4) rapidly transferring the centrifuge tube onto ice, and standing for 2min;

(5) resuscitating: adding 200uL of LB culture medium into each tube, and incubating for 45min by shaking gently at 37 ℃ to revive bacteria;

(6) uniformly coating the recovered bacteria on an Amp-resistant LB solid culture plate;

(7) culturing in incubator at 37deg.C for 12-16hr.

(6) Sequencing of recombinant plasmids

(1) Culturing bacterial liquid: 3 single clones are selected respectively from the transformation plate and put into 10mL of sterilized LB liquid culture medium, amp antibiotics with corresponding volumes are added, the temperature is 37 ℃, the rpm is 180, and the shaking culture is carried out for 12-16 hours;

(2) extracting plasmid: the plasmid miniprep kit is operated according to the specification of the plasmid miniprep kit.

(3) Sequencing: the plasmid is sent to Tianjin Jin Weizhi limited company for sequencing, and the sequencing result is compared with the theoretical sequence, and the comparison is consistent, so that the cloning construction is successful and the method can be used for subsequent experiments.

EXAMPLE two cellular level detection of cleavage Activity of BoNT/C1LC mutant on substrates SNAP-25 and Syntaxin-1

2.1 biological Material

PC12 and HEK293T cells were purchased from biological wind; lentiviral packaging vectors pSPAX2 and PMD2.G were purchased from Biyun.

2.2 reagents

SNAP-25 antibody (GeneTex, cat# GTX 113839);

syntaxin-1 antibody (Santa Cruz, cat. No. SC-12736);

beta-actin antibody (Immunoway, cat No. YM 3028);

GAPDH antibody (Immunoway, cat No. YM 3215);

RIPA lysate (solebao, cat No. R0010).

2.3 carrying out the steps

2.3.1 preparation of BoNT/C1LC mutant viruses

(1) Cell plating: 4X 10 of each dish ⁶ The density of individual cells is about 50%;

(2) Transfection: culturing for 24hr after passage, changing cell culture medium into serum-free and double-antibody-free medium, and transfecting by liposome method;

a. plasmid total 22.5 μg/dish, plasmid of interest: pSPAX2: pmd2.g=4: 3:2, adding 3 plasmids with corresponding volumes and mixing with 1mL of MEM culture medium;

b. plasmid (μg): nucleic acid transfection reagent Liposomal (μl) =1: 1, namely 22.5 mu L liposomal, adding 1mL of MEM culture medium, uniformly mixing, and standing at room temperature for 5min;

c. mixing the plasmid with Liposomal, and standing at room temperature for 20min;

(3) Adding the treated liposome DNA mixture into a culture dish, wherein the volume of the liposome DNA mixture is 2 mL/dish;

(4) Liquid replacement: culturing for 6hr, and changing into medium containing serum and double antibody;

(5) And (3) virus packaging: culturing in a 37 deg.C cell incubator for 48hr;

(6) And (3) virus collection: collecting virus supernatant, filtering with 0.22 μm water filter membrane into ultracentrifuge tube, centrifuging at 25000rpm and 4 ℃ for 2hr;

(7) Split charging and preserving the virus: the supernatant was discarded, 200. Mu.L of serum-free medium was added, and the mixture was stored at 4℃for overnight swelling and at-80℃after packaging.

2.3.2 infection of PC12 cells with Virus

(1) Cell plating:

PC12 cells with good growth state in log phase were obtained according to 2X10 ⁵ Inoculating the cells/hole into 24-hole plate, and culturing in cell incubator for 12hr;

(2) Viral infection:

the virus of each mutant was added to PC12 cells at 0, 5, 15, 30, 50, 80. Mu.L and cultured for 48hr;

(3) Extracting protein: the medium was aspirated and 100. Mu.L of lysate (V _RIPA :V _PMSF =100:1), collecting cell lysate, lysing 20min on ice, centrifuging at 10000rpm/min at 4deg.C for 10min, sucking out supernatant, adding 5×protein loading buffer of corresponding volume, and denaturing at 100deg.C for 10min in a metal bath;

(4) WB assay of cleavage Activity of each mutant virus against PC12 cells SNAP-25, syntaxin-1:

(1) SDS-PAGE electrophoresis: each sample was run at 80V for 2.5hr at 10. Mu.L.

(2) Transferring: and (3) manufacturing a sandwich structure according to the layer cellulose pad-two layers of filter paper-membrane-glue-two layers of filter paper-layer cellulose pad, removing bubbles, adding a membrane transfer buffer solution, and performing 300mA constant flow membrane transfer for 2h under the low-temperature condition.

(7) Closing: 5% skimmed milk powder, and sealing at room temperature for 1hr.

(8) Incubating primary antibodies: SNAP-25 antibody was diluted 1:5000 and Syntaxin-1 antibody was diluted 1:500 and incubated at room temperature for 2hr.

(9) Washing the film: TBST was washed 3 times for 10min each.

(10) Incubating a secondary antibody: the secondary antibody was diluted 1:10000 and incubated at room temperature for 1hr.

(11) Washing the film: TBST was washed 3 times for 10min each.

(10) Exposure: and preparing ECL luminous liquid, uniformly adding the luminous liquid onto the film, and taking a picture by using an exposure meter. The results of BoNT/C1LC wt, boNT/C1 alpha-51, 53F, 53L, 51Y52H53Y, 51F52H53Y are shown in FIGS. 1,2, 3 and 4, respectively, and the results of the remaining BoNT/C1LC mutants are shown in Table 4.

TABLE 4 cleavage Activity results of BoNT/C1LC mutants obtained according to the invention on PC12 cells SNAP-25 and Syntaxin-1

Note that: for SNAP25, the observed cleavage band is considered to be the detection of cleavage activity, for syncaxin, the observed significant cleavage bands or a significant decrease in the protein content of syncaxin is considered to be the detection of cleavage activity, +++++ is shown at 5. Mu.L cleavage activity was detected at dose; ++ + + and is shown in dosage of 15. Mu.L cleavage activity was detected down; ++ is shown in dosage of 30. Mu.L cleavage activity was detected down; ++ indicates that cleavage activity was detected at a dose of 50. Mu.L; + indicates that cleavage activity was detected at a dose of 80 μl; ND indicates that no enzyme cleavage activity was detected at a dose of 80. Mu.L. Each mutant virus was tested for cleavage activity of substrates SNAP-2 and Syntaxin-1 using BoNT/C1LC wt and BoNT/C1 alpha-51 virus as positive controls, as in FIG. 1.

The results show that: boNT/C1LC mutants 53T, 53F, 53L, 53H, 53G, 53E and 52Y53Y retain weaker cleavage activity for SNAP-25 and significantly better cleavage activity for synthaxin-1 than BoNT/C1 alpha-51. Preferably, the BoNT/C1LC mutants 52H53Y, 52Q53Y, 51K52H53Y, 51W52H53Y, 51P52H53Y, 51D52H53Y, 51R52H53Y, 51A52H53Y, 51E52H53Y, 51G52H53Y, 51Y52H53Y and 51S52H53Y have no cleavage activity for SNAP-25, and have significantly better cleavage activity for synthaxin-1 than BoNT/C1 alpha-51. Finally, boNT/C1LC mutants 53W, 52I53Y and 51F52H53Y had no cleavage activity on both substrates SNAP-25 and synthaxin-1.

The results of the present invention also show that when only amino acid 53 of BoNT/C1 alpha-51 is replaced, high frequency causes the corresponding mutant to resume cleavage activity for SNAP-25, thus the present disclosure suggests that substitution and modification of amino acid 53 of BoNT/C1LC is critical for cleavage activity of BoNT/C1LC for SNAP-25. Amino acid substitutions and modifications may be made by any genetic engineering means or chemical means, as will be appreciated by those skilled in the art.

The BoNT/C1LC mutant nucleotide sequence disclosed by the invention can be fully synthesized by adopting a codon optimization method, so that the BoNT/C1LC is more suitable for expression in different host cells, but the primary structure of the protein amino acid of an expression product is not influenced, and the BoNT/C1LC mutant nucleotide sequence is understood by a person skilled in the art.

The invention has the beneficial effects that the BoNT/C1LC is subjected to mutation transformation, so that the BoNT/C1LC is nontoxic, can replace a wild type light chain in the full-length toxin, is used as a drug carrier when a nervous system drug is targeted for delivery, and can be used for treating diseases related to Syntaxin after transformation, such as exocytosis regulation, type II diabetes treatment and the like for the light chain mutant with the cleavage activity only to Syntaxin-1. Although currently marketed botulinum toxin is only type a and type B, and type a has long-term and broad clinical application. However, botulinum toxin type A has larger side effects and drug resistance, botulinum toxin type B has poor absorption effect in the injection part, the defects limit the use of botulinum toxins type A and botulinum toxin type B, and a novel botulinum toxin mutant capable of cloning the defects is urgently needed clinically, so that the BoNT/C1 mutant provided by the invention has great potential to enter clinic.

The protein sequence related to the invention:

Seq ID No:1

BoNT/C1α-51

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFTNPSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENML

the nucleotide sequence of the invention is as follows:

Seq ID No:2

BoNT/C1α-51

ATGCCCATCACCATTAACAATTTTAATTACAGCGATCCTGTGGATAACAAGAATATCCTGTACCTGGATACCCACCTGAACACCCTGGCCAACGAACCAGAGAAAGCCTTCCGCATCACAGGCAACATTTGGGTGATCCCCGACAGGTTTACCAATCCTTCTAATCCTAACCTGAACAAACCCCCAAGAGTGACCTCCCCCAAGAGCGGGTACTACGATCCCAACTACCTGAGCACCGATTCCGACAAAGACCCCTTTCTGAAGGAGATCATCAAGCTGTTCAAGAGAATCAACAGCCGGGAGATCGGAGAGGAGCTGATCTATAGACTGTCTACAGACATCCCATTCCCTGGGAACAATAACACCCCAATTAACACCTTTGACTTTGATGTGGACTTCAATTCTGTGGACGTGAAAACCAGGCAGGGCAATAATTGGGTGAAGACAGGCAGCATTAACCCTTCCGTGATTATTACCGGACCCAGAGAGAATATCATCGATCCTGAGACCTCTACCTTCAAGCTGACCAACAATACATTCGCCGCCCAGGAAGGCTTCGGCGCCCTGTCTATCATTAGCATTAGCCCTAGGTTTATGCTGACCTACTCCAATGCCACAAATGATGTGGGCGAGGGACGCTTCTCTAAGAGCGAGTTCTGTATGGACCCCATTCTGATTCTGATGCACGAGCTGAATCACGCCATGCACAATCTGTACGGCATCGCCATCCCCAACGACCAGACCATTTCCAGCGTGACAAGCAATATCTTCTATTCCCAGTATAATGTGAAGCTGGAATACGCCGAAATTTACGCCTTCGGCGGCCCCACCATCGACCTGATTCCCAAGTCCGCCAGGAAGTACTTTGAAGAGAAGGCCCTGGATTACTACAGGTCTATCGCCAAGCGGCTGAACAGCATCACAACAGCCAATCCCAGCTCTTTTAATAAGTACATTGGAGAATATAAACAGAAGCTGATCAGAAAGTATAGGTTCGTGGTGGAGTCCAGCGGCGAGGTGACAGTGAACAGAAACAAGTTTGTGGAACTGTATAACGAGCTGACCCAGATTTTCACCGAGTTCAACTACGCCAAGATTTACAATGTGCAGAATCGGAAGATTTACCTGTCTAACGTGTACACCCCTGTGACCGCCAACATCCTGGATGACAACGTGTATGACATTCAGAACGGATTCAATATTCCTAAATCCAACCTGAATGTGCTGTTTATGGGCCAGAACCTGAGCCGGAACCCCGCCCTGAGAAAGGTGAACCCCGAGAACATGCTG

Claims (12)

1. A polypeptide or protein containing a BoNT/C1 light chain mutant, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is the substitution of amino acids 51 to 53 of BoNT/C1α-51 of Seq ID No: 1 is 51X52X53X, where 51X, 52X and 53X are any amino acids. 2. A kind of polypeptide or protein containing BoNT/C1 light chain mutant according to claim 1, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is that of BoNT/C1α-51 of Seq ID No: 1. Amino acid 51X at position 51 can be any one of A, D, E, G, H, K, M, N, P, R, S, V, W, and Y. 3. A kind of polypeptide or protein containing BoNT/C1 light chain mutant according to claim 1, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is that of BoNT/C1α-51 of Seq ID No: 1. The amino acid 52X at position 52 can be any one of D, G, H, M, Q, and Y. 4. A kind of polypeptide or protein containing BoNT/C1 light chain mutant according to claim 1, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is that of BoNT/C1α-51 of Seq ID No: 1. Amino acid 53X at position 53 can be any one of A, E, F, G, H, L, T, and Y. 5. A polypeptide or protein containing a BoNT/C1 light chain mutant according to any one of claims 1 to 4, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is BoNT of Seq ID No: 1 The amino acid positions 51 to 53 of /C1α-51 are replaced by any one of the following (a) to (t): (a)THY (b)TQY (c)KHY (d)WHY (e)PHY (f)DHY (g)RHY (h)AHY (i)EHY (j)GHY (k)YHY (l)SHY (m)TNY (n)TDY (o)TMY (p)TGY (q)VHY (r)HHY (s)MHY (t)NHY. 6. A polypeptide or protein containing a BoNT/C1 light chain mutant according to any one of claims 1 to 5, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is BoNT of Seq ID No: 1 The amino acid positions 51 to 53 of /C1α-51 are replaced by any one of the following (a) to (l): (a)THY (b)TQY (c)KHY (d)WHY (e)PHY (f)DHY (g)RHY (h)AHY (i)EHY (j)GHY (k)YHY (l)SHY. 7. A polypeptide or protein containing a BoNT/C1 light chain mutant, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is the substitution of amino acids 51 to 53 of BoNT/C1α-51 of Seq ID No: 1 It is any one of the following (u) to (w): (u)TNF (v)TNL (w)TYY. 8. A polypeptide or protein containing a BoNT/C1 light chain mutant, characterized in that the amino acid sequence of the BoNT/C1 light chain mutant is the substitution of amino acids 51 to 53 of BoNT/C1α-51 of Seq ID No: 1 It is any one of the following (x) to (z): (x)TNW (y)TIY (z)FHY. 9. A polypeptide or protein containing a BoNT/C1 light chain mutant according to any one of claims 1 to 7, which can be used in the preparation of medical and aesthetic products and related drugs that are responsive or unresponsive to botulinum toxin type A. 10. A polypeptide or protein containing a BoNT/C1 light chain mutant according to any one of claims 1 to 7, which can be used in the preparation of drugs for Syntaxin-mediated hypersecretion diseases. 11. A polypeptide or protein containing a BoNT/C1 light chain mutant according to any one of claims 1 to 7, which can be used in the preparation of drugs for dystonia-related diseases. 12. A polypeptide or protein containing a BoNT/C1 light chain mutant according to claim 8, which can be used as a carrier in the preparation of targeted delivery drugs for nervous system administration.