Toxicity-enhanced neurotoxin recombinant protein and application thereof

Abstract

The application provides a toxicity-enhanced neurotoxin recombinant protein and application thereof, and belongs to the technical field of biological medicine. The recombinant protein comprises a botulinum toxin type A receptor binding domain and at least one GM1 binding peptide inserted into the botulinum toxin type A receptor binding domain, and the recombinin protein can recognize and bind to ganglioside GM1. The short peptide sequence capable of binding to ganglioside GM1 is introduced into the botulinum toxin type A receptor binding domain to obtain the recombinant protein, the binding capacity of the recombinant protein to ganglioside GM1 is enhanced, the target recognition and binding capacity of the recombinant protein to the nerve cell membrane are improved, the overall affinity and endocytosis efficiency of the recombinant protein to the nerve cell are improved, and the neurotoxicity of the botulinum toxin is enhanced. The application not only improves the binding efficiency of BoNT / A in the in-vitro nerve cell model, but also shows a higher toxicity level in the functional verification, and shows a good clinical conversion prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to recombinant proteins related to botulinum toxin type A, specifically to a toxicity-enhancing neurotoxin recombinant protein and its applications. Background Technology

[0002] Botulinum neurotoxin type A (BoNT / A) is a highly effective neurotoxin secreted by the Gram-positive anaerobic bacterium Clostridium botulinum. It specifically inhibits the release of acetylcholine from cholinergic neurons. Due to its significant neuromuscular blocking ability, it is currently widely used in the treatment of dystonia, migraines, facial muscle spasms, and other diseases, and has promising market prospects and clinical value.

[0003] Gangliosides are a class of compounds derived from glycosyl residues and sphingosine, among which GM1, GD1a, and GT1b are the main gangliosides found in the nervous system. BoNT / A's binding ability to nerve cells depends on a dual receptor recognition mechanism: it first interacts with low-affinity gangliosides, and then binds to the high-affinity protein receptor SV2. Under natural conditions, BoNT / A exhibits some selectivity in binding to gangliosides, preferentially binding to GT1b and GD1a, with lower affinity for GM1. However, the expression levels of GT1b and GD1a vary significantly among different nerve cell types, developmental stages, and under external stress conditions. This uncertainty in endogenous expression limits the targeting stability and consistency of BoNT / A. In contrast, GM1, as a relatively simple and widely expressed monosialotetarian ganglioside, is widely distributed in various nerve cells and nerve tissues.

[0004] Therefore, increasing the affinity of BoNT / A for GM1 could further enhance its binding capacity to gangliosides, thereby improving its overall affinity and endocytosis efficiency with nerve cells, ultimately increasing its neurotoxicity and neurospecificity. Greater neurotoxicity means lower injection doses and fewer immune responses in practical applications, thus extending the frequency of botulinum toxin use. However, there are few existing research reports on enhancing botulinum toxin neurotoxicity by increasing the affinity of BoNT / A for GM1. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a recombinant neurotoxin protein with enhanced toxicity and its application, thereby solving the technical problem that the affinity of botulinum toxin type A to ganglioside GM1 is low in the existing technology.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A recombinant neurotoxin with enhanced toxicity includes a protein domain (i.e., the C-terminus of the botulinum toxin type A receptor-binding domain) shown at positions 1088 to 1293 of Seq NO.1 or a protein domain with sequence identity greater than or equal to 90%; it also includes at least one GM1 binding peptide inserted into the aforementioned protein domain.

[0008] The present invention also includes the following technical features:

[0009] Specifically, the insertion site of the GM1 binding peptide is between any two amino acids from position 1253 to position 1261 of Seq NO.1 (HQFNNIAKL, i.e., histidine-glutamine-phenylalanine-asparagine-asparagine-isoleucine-alanine-lysine-leucine).

[0010] Specifically and preferably, the insertion methods of the GM1 binding peptide include: direct insertion into the protein fragment HQFNNIAKL; and insertion after deletion, addition, or mutation of any amino acid in or near the protein fragment HQFNNIAKL.

[0011] Optionally and preferably, the insertion site of the GM1 binding peptide is between the 1256th and 1257th asparagine positions in Seq NO.1, and the insertion method is direct insertion.

[0012] Optionally and preferably, the insertion site of the GM1-binding peptide is between histidine at position 1253 and lysine at position 1260 of Seq NO.1, and the insertion method is to insert after the deletion of some amino acids; the deleted amino acids are glutamine at position 1254, phenylalanine at position 1255, asparagine at position 1256, asparagine at position 1257, isoleucine at position 1258, and alanine at position 1259.

[0013] Optionally and preferably, the insertion site of the GM1 binding peptide is between histidine at position 1253 and lysine at position 1260 of the amino acid sequence of botulinum toxin type A, and the insertion method is to insert after deleting a certain amino acid; the deleted amino acids are glutamine at position 1254, phenylalanine at position 1255, asparagine at position 1256, asparagine at position 1257, isoleucine at position 1258, and alanine at position 1259.

[0014] Specifically, the amino acid sequence structure of the GM1-binding peptide is expressed as GM1x and N. i -GM1x-N j ; where GM1x represents the GM1 core binding peptide, with the sequence VWRLLAPPFSNRLLP; N iThis refers to one or more amino acids located upstream of the GM1 core-binding peptide; N j This refers to one or more amino acids located downstream of the GM1 core-binding peptide segment.

[0015] Specifically, either Ni or Nj is selected from common and uncommon protein amino acids; common protein amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; uncommon protein amino acids include 4-hydroxyproline, 5-hydroxylysine, γ-carboxyglutamic acid, selenocysteine, pyrrolidone, N-formylmethionine, 3-methylhistidine, desmosin, isodesmosin, thyroxine, phosphoserine, citrulline, and ornithine.

[0016] Preferably, any amino acid in Ni and Nj is selected from glycine, valine, methionine, serine, threonine, and alanine; most preferably, glycine and serine.

[0017] Specifically, the recombinant protein also includes the protein domain shown at positions 885 to 1079 of Seq NO.1 (i.e., the N-terminus of the type A botulinum toxin receptor binding domain) or a protein domain with sequence identity greater than or equal to 90%.

[0018] Specifically, the recombinant protein also includes the protein domain (i.e., the botulinum toxin type A protease domain) shown at positions 3 to 410 of Seq NO.1 or a protein domain with sequence identity greater than or equal to 90%.

[0019] Specifically, the recombinant protein also includes the protein domains shown at positions 549 to 866 of Seq NO.1 (i.e., the botulinum toxin type A translocation domain) or protein domains with sequence identity greater than or equal to 90%.

[0020] Optionally, any amino acid in the recombinant protein may be modified with one or more of the following: chemical groups, small molecule compounds, peptides, and macromolecular protein tags. The chemical groups are selected from one or more of amino, thiol, carboxyl, hydroxyl, and aldehyde groups. The small molecule compounds are selected from one or more of biotin, glutathione, steroids, small molecule dye ligands, and metal chelating groups. The peptide and macromolecular protein tags are selected from one or more of the following: histidine tags (short peptides composed of several histidines), GST (glutathione S-transferase) tags, fluorescent proteins (such as green fluorescent protein GFP), MBP (maltose-binding protein) tags, FLAG tags (short peptide DYKDDDDK), Strep-tag II tags (short peptide WSHPQFEK), HA tags (short peptide YPYDVPDYA), and c-Myc (short peptide EQKLISEEDL).

[0021] The present invention also protects the use of the toxicity-enhancing neurotoxin recombinant protein described above in interactions with nerve cells, wherein the use is for non-medical purposes.

[0022] Specifically, the applications include: the recombinant neurotoxin protein with enhanced toxicity as described above achieves binding to nerve cells by recognizing and binding to ganglioside GM1; the concentration of the recombinant neurotoxin protein with enhanced toxicity is 0.05-0.5 μm, preferably 0.1-0.3 μm, and most preferably 0.2 μm.

[0023] Specifically, the applications include: the recombinant neurotoxin protein with enhanced toxicity as described above achieves binding to liposomes by recognizing and binding to ganglioside GM1; the concentration of the recombinant neurotoxin protein with enhanced toxicity is 0.05-1 μm, preferably 0.3-0.8 μm, and most preferably 0.5 μm.

[0024] Specifically, the applications include: the use of the toxicity-enhanced recombinant neurotoxin protein as a hydrolytic enzyme to cleave the intracellular SN AP-25 protein of nerve cells; the concentration of the toxicity-enhanced recombinant neurotoxin protein used is 0.03-10 nM, preferably 3-10 nM, and most preferably 10 nM.

[0025] Specifically, the applications include: the use of the toxicity-enhanced neurotoxin recombinant protein as a neurotoxin to induce claw paralysis symptoms in animals; the concentration of the toxicity-enhanced neurotoxin recombinant protein used is 1-100 pg, preferably 6-60 pg, and most preferably 60 pg.

[0026] Compared with the prior art, the present invention has the following technical effects:

[0027] This invention enhances the binding capacity of botulinum toxin type A to the receptor-binding domain by introducing a short peptide sequence capable of binding to ganglioside GM1, thereby improving its targeting recognition and binding ability to nerve cell membranes, enhancing its overall affinity and endocytosis efficiency with nerve cells, and increasing the neurotoxicity of botulinum toxin. This invention not only improves the binding efficiency of BoNT / A in in vitro nerve cell models but also demonstrates higher toxicity levels in functional validation, showing promising prospects for clinical translation. Attached Figure Description

[0028] Figure 1 The amino acid sequences of the C-terminal Loop3 domain of the wild-type botulinum toxin type A heavy chain and the mutated recombinant protein were compared. Figure 1 In the first row (Loop3), the amino acid sequence from position 1253 to 1260 of wild-type botulinum toxin type A is shown; the second row (L3GM1-1) shows the amino acid sequence of the recombinant protein of Example 2 after mutation; the third row (L3GM1-2) shows the amino acid sequence of the recombinant protein of Example 3 after mutation; and the fourth column (L3GM1-3) shows the amino acid sequence of the recombinant protein of Example 4 after mutation.

[0029] Figure 2 This is an SDS-PAGE electrophoresis image of the wild-type botulinum toxin type A receptor-binding domain and the recombinant protein. Figure 2 In the first lane, the markers are labeled 180kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, and 10kDa from top to bottom. The second lane (WT-Bind) contains the electrophoretic band of the wild-type botulinum toxin type A receptor-binding domain. The third lane (R3GM1-1) contains the electrophoretic band of the recombinant protein from Example 5. The fourth lane (R3GM1-2) contains the electrophoretic band of the recombinant protein from Example 6. The fifth lane (R3GM1-3) contains the electrophoretic band of the recombinant protein from Example 7.

[0030] Figure 3 Immunoblot diagram of wild-type botulinum toxin type A receptor-binding domain, recombinant protein and primary nerve cells. Figure 3In the table: the first column (WT-Bind) represents wild-type botulinum toxin type A; the second column (R3GM1-1) represents the recombinant protein of Example 5; the third column (R3GM1-2) represents the recombinant protein of Example 6; the fourth column (R3GM1-3) represents the recombinant protein of Example 7; the first row (Binding) represents the receptor-binding domain protein that ultimately binds to neurons; the second row (Total) represents the total receptor-binding domain protein used in the binding experiment; the third row (Syntaxin) represents Syntaxi n protein, which serves as an internal control for the consistency of neuron numbers in different experimental groups.

[0031] Figure 4 Immunofluorescence image of wild-type botulinum toxin type A receptor-binding domain, recombinant protein, and primary nerve cells. Figure 4 In the first column (WT-Bind), wild-type botulinum toxin type A is represented; the second column (R3GM1-1) represents the recombinant protein of Example 2; the third column (R3GM1-2) represents the recombinant protein of Example 3; the fourth column (R3GM1-3) represents the recombinant protein of Example 4; the green fluorescence in the first row (HcA) shows the protein binding signal (labeled with anti-HA antibody); the red fluorescence in the second row (Syntaxin) shows the synaptic protein in nerve cells (labeled with anti-Syntaxin antibody).

[0032] Figure 5 Immunoblot diagrams showing the binding of wild-type botulinum toxin type A receptor-binding domain (WT'-Bind) lacking ganglioside binding ability and recombinant receptor-binding domain to GM1 liposomes. Figure 5 In the diagram: the first column (WT'-Bind) represents the loss-of-function wild-type botulinum toxin type A receptor-binding domain; the second column (R3GM1-1'), the third column (R3GM1-2'), and the fourth column (R3GM1-3') represent recombinant proteins with different GM1-binding peptides and loss-of-function receptor-binding domains; the first row (Binding) represents the receptor-binding domain proteins that ultimately bind to liposomes in each experimental group; and the second row (Total) represents the total receptor-binding domain proteins used in liposome binding experiments in each experimental group.

[0033] Figure 6 The study demonstrated the cleavage activity of wild-type botulinum toxin A obtained by transpeptidase linker and the mutated recombinant protein against SNAP-25 in nerve cells. Figure 6In Chinese: WT represents wild-type botulinum toxin type A; L3GM1-1 represents the recombinant protein of Example 2; L3GM1-2 represents the recombinant protein of Example 3; L3GM1-3 represents the recombinant protein of Example 4; SNAP-25 represents SNAP-25 protein; Syntaxin represents Syntaxin protein, used as an internal reference to indicate the number of nerve cells used in each experimental group; the topmost number indicates the different protein concentrations (in nM) corresponding to the bands.

[0034] Figure 7 The graph shows the effect of wild-type botulinum toxin A obtained by transpeptidase linkage and the mutated recombinant protein on the motor function of mice in nerve cells. Figure 7 In the graph: A (WT) is the scoring curve of the effect of wild-type botulinum toxin type A on the motor function of mice; B (L3GM1-1) is the scoring curve of the effect of recombinant protein of Example 2 on the motor function of mice; C (L3GM1-2) is the scoring curve of the effect of recombinant protein of Example 3 on the motor function of mice; D (L3GM1-3) is the scoring curve of the effect of recombinant protein of Example 4 on the motor function of mice; the vertical axis represents the score, and the horizontal axis represents the number of days; the effects of different concentrations of toxin protein on the motor function of mice were also evaluated in each experimental group.

[0035] The specific content of the present invention will be further explained in detail below with reference to the embodiments. Detailed Implementation

[0036]

[0037] It should be noted that all reagents, culture media, competent cells, and vectors used in this invention, unless otherwise specified, are those known in the art. For example, the competent cells used are conventional Escherichia coli BL21(DE3) competent cells known in the prior art. The purification packing material used for protein purification is Ni Bestarose FF packing material, purchased from BorgLon Biotech Ltd.

[0038] The following are specific embodiments of the present invention. It should be noted that the present invention is not limited to the following specific embodiments. All equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0039] Example 1:

[0040] This embodiment provides a GM1-binding peptide, whose amino acid sequence structure is expressed as GM1x and N. i -GM1x-N j Among them, GM1x is the core binding peptide; N i This refers to one or more amino acids located upstream of the GM1 core-binding peptide, i.e., one to two amino acids or a peptide consisting of at least three amino acids that are translated or synthesized prior to GM1x; N j This refers to one or two amino acids or a peptide consisting of at least three amino acids located downstream of the GM1 core-binding peptide segment (i.e., j amino acids that are translated or synthesized after GM1x).

[0041] As a first specific and optional scheme in this embodiment, the sequence structure of the GM1 binding peptide is expressed as GM1x, that is, the sequence of the GM1 binding peptide is VWRLLAPPFSNRLLP (see Seq NO.2).

[0042] As a second specific and optional scheme in this embodiment, the sequence structure of the GM1 binding peptide is expressed as N i -GM1x-N j ; where N i For Gly-Ser; N j It is Ser-Gly; that is, the sequence of the GM1 binding peptide is GSVWRLLAPPFSNRLLPSG (see Seq NO.3).

[0043] Example 2 (L3GM1-1, botulinum toxin + binding peptide):

[0044] This embodiment presents a recombinant neurotoxin protein with enhanced toxicity, named L3GM1-1. This recombinant protein includes the protease domain and translocation domain of wild-type botulinum toxin type A, as well as a receptor-binding domain with GM1x inserted in Example 1. Figure 1 As shown, the insertion site of GM1x is between asparagine at position 1256 and position 1257 of the wild-type botulinum toxin amino acid sequence.

[0045]

[0046] Example 3 (L3GM1-2, botulinum toxin + binding peptide):

[0047] This embodiment presents a recombinant neurotoxin protein with enhanced toxicity, named L3GM1-2. This recombinant protein includes the protease domain, translocation domain, and N-type botulinum toxin inserted in Example 1. i -GM1x-N j The receptor-binding domain has some amino acids missing upstream and downstream of the insertion site. For example... Figure 1 As shown, the insertion site is between histidine at position 1253 and lysine at position 1260 in the amino acid sequence of wild-type botulinum toxin A. The missing amino acids are glutamine at position 1254, phenylalanine at position 1255, asparagine at position 1256, asparagine at position 1257, isoleucine at position 1258, and alanine at position 1259.

[0048]

[0049] Example 4 (L3GM1-3, botulinum toxin + binding peptide):

[0050] This embodiment presents a recombinant neurotoxin protein with enhanced toxicity, named L3GM1-3. This recombinant protein includes the protease domain and translocation domain of wild-type botulinum toxin type A, as well as the receptor-binding domain of GM1x inserted in Example 1, with some amino acids deleted upstream and downstream of the insertion site. Figure 1 As shown, the insertion site is between histidine at position 1253 and lysine at position 1260 in the amino acid sequence of wild-type botulinum toxin A. The missing amino acids are glutamine at position 1254, phenylalanine at position 1255, asparagine at position 1256, asparagine at position 1257, isoleucine at position 1258, and alanine at position 1259.

[0051]

[0052] Example 5 (R3GM1-1, receptor domain + binding peptide):

[0053] This embodiment provides a recombinant neurotoxin protein with enhanced toxicity, named R3GM1-1. This recombinant protein includes a wild-type botulinum toxin type A receptor-binding domain with GM1x inserted in Example 1. The insertion site of GM1x is between asparagine at position 1256 and position 1257 of the wild-type botulinum toxin type A amino acid sequence.

[0054] The amino acid sequence of R3GM1-1 is shown in Seq NO.7, specifically as follows: TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDL YDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQ AGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNVWRLLAPPFSNRLLPNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL.

[0055] Example 6 (R3GM1-2, receptor domain + binding peptide):

[0056] This embodiment presents a recombinant neurotoxin protein with enhanced toxicity, named R3GM1-2. This recombinant protein includes N inserted from Example 1. i -GM1x-N j The wild-type botulinum toxin type A receptor-binding domain has some amino acids missing upstream and downstream of the insertion site. The insertion site is between histidine at position 1253 and lysine at position 1260 of the wild-type botulinum toxin type A amino acid sequence. The missing amino acids are glutamine at position 1254, phenylalanine at position 1255, asparagine at position 1256, asparagine at position 1257, isoleucine at position 1258, and alanine at position 1259.

[0057] The amino acid sequence of R3GM1-2 is shown in Seq NO.8, specifically as follows: TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKD LYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNAS QAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHGSVWRLLAPPFSNRLLPSGKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL.

[0058] Example 7 (R3GM1-3, receptor domain + binding peptide):

[0059] This embodiment presents a recombinant neurotoxin protein with enhanced toxicity, named R3GM1-3. This recombinant protein includes a wild-type botulinum toxin type A receptor-binding domain with GM1x inserted in Example 1, and some amino acids are deleted upstream and downstream of the insertion site. The insertion site is between histidine at position 1253 and lysine at position 1260 of the wild-type botulinum toxin type A amino acid sequence. The deleted amino acids are glutamine at position 1254, phenylalanine at position 1255, asparagine at position 1256, asparagine at position 1257, isoleucine at position 1258, and alanine at position 1259.

[0060] The amino acid sequence of R3GM1-3 is shown in Seq NO.9, specifically as follows: TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEI KDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLAT NASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHVWRLLAPPFSNRLLPKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL.

[0061] Example 8:

[0062] This embodiment provides a method for preparing the toxicity-enhanced recombinant neurotoxin proteins of Examples 2 to 7, which specifically includes the following steps:

[0063] Step 1, Construction of the recombinant protein expression vector:

[0064] A target fragment containing the encoding nucleotide sequences of the recombinant proteins described in Examples 2 to 7 was artificially synthesized. The target fragment and the pET-28a vector were digested with restriction endonucleases NdeI and XhoI. The digested target fragment and pET-28a vector were then ligated, and the ligation product was transformed into competent *E. coli* cells. The bacterial culture was plated, and single colonies were picked, cultured, and then the plasmid was extracted. The plasmid was sent for sequencing identification. Correct sequencing results confirmed the successful construction of the recombinant protein expression vector. The recombinant protein expression vector was then transformed into competent *E. coli* cells, and the bacterial culture was plated, and single colonies were picked, cultured, and the bacterial culture was stored for later use.

[0065] Step 2, expression and purification of recombinant protein:

[0066] The bacterial culture preserved in step one was taken out and cultured overnight. The next day, the entire overnight culture was inoculated into LB liquid medium containing kanamycin and cultured at 37°C for 3 hours. Then, IPTG was added to a final concentration of 0.4 mM for induction, while simultaneously lowering the culture temperature to 20°C. After 20 hours of induction culture, the bacterial cells were collected by centrifugation and sonicated. The supernatant was purified by column chromatography. For purification, the cells were first washed with 50 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole at pH 7.0, and then eluted with 50 mM Tris-HCl, 150 mM NaCl, and 500 mM imidazole at pH 7.0. The eluent was collected for SDS-PAGE analysis. The results are shown below. Figure 2 As shown. From Figure 2 The results show that the bands of R3GM1-1, R3GM1-2, and R3GM1-3 are located between 40 kDa and 55 kDa, and are all larger than the receptor-binding domain of wild-type botulinum toxin type A; the bands of L3GM1-1, L3GM1-2, and L3GM1-3 are located between 140 kDa and 150 kDa, and are all larger than wild-type botulinum toxin type A. This demonstrates the successful construction of the recombinant protein.

[0067] Effect verification:

[0068] (A) Detection of binding of recombinant botulinum toxin receptor-binding domain to primary nerve cells:

[0069] R3GM1-1, R3GM1-2, and R3GM1-3 were used as experimental groups, while the wild-type botulinum toxin type A receptor-binding domain was used as the control group. Protein samples were added to 200 μl of high-potassium solution to achieve a final concentration of 0.2 μM, and then incubated in primary neural cells. Cellular immunofluorescence and protein immunoblotting experiments were then used to verify the binding ability of the receptor-binding domain-repeated proteins to primary neural cells. Results are as follows: Figure 3 and Figure 4 As shown, it can be seen that R3GM1-1, R3GM1-2 and R3GM1-3 have significantly higher binding capacity to primary nerve cells than the wild-type botulinum toxin type A receptor binding domain.

[0070] (B) Detection of binding between the receptor-binding domain of recombinant botulinum toxin lacking ganglioside-binding ability and GM1-containing liposomes:

[0071] The receptor-binding domain of botulinum toxin originally contains a site that can bind to GM1. To demonstrate that the inserted GM1-binding peptide can enhance the binding ability of the receptor-binding domain to GM1-containing liposomes, the original GM1-binding site in the botulinum toxin receptor-binding domain was mutated, causing it to lose its ability to bind to gangliosides. The constructed proteins are designated as R3GM1-1', R3GM1-2', and R3GM1-3' (loss-of-function receptor domain + binding peptide), and their amino acid sequences are as follows:

[0072] The amino acid sequence of R3GM1-1' is shown in Seq NO.10, specifically as follows: TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDL YDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQ AGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNVWRLLAPPFSNRLLPNIAKLVASNLSNRQIERSSRTLGCSWEFIPVDDGWGERPL.

[0073] The amino acid sequence of R3GM1-2’ is shown in Seq NO.11 as follows: TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHGSVWRLLAPPFSNRLLPSGKLVASNLSNRQIERSSRTLGCSWEFIPVDDGWGERPL。

[0074] The amino acid sequence of R3GM1-3’ is shown in Seq NO.12 as follows: TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHVWRLLAPPFSNRLLPKLVASNLSNRQIERSSRTLGCSWEFIPVDDGWGERPL。

[0075] R3GM1-1', R3GM1-2', and R3GM1-3' were used as the experimental groups, while a loss-of-function wild-type botulinum toxin type A receptor-binding domain was used as the control group. The above proteins were mixed with 70 μl of GM1-containing monolayer liposomes to a final protein concentration of 0.5 μm, incubated at room temperature for 30 min, then centrifuged and the uppermost layer was collected. Western blot was used to detect whether the target protein could bind to the liposomes. Results are as follows: Figure 5 As shown, it can be seen that R3GM1-1' and R3GM1-3' have a significantly higher binding capacity to GM1-containing liposomes than the loss-of-function wild-type botulinum toxin receptor-binding domain and R3GM1-2'.

[0076] (C) Recombinant protein activity assay:

[0077] L3GM1-1, L3GM1-2, and L3GM1-3 were used as experimental groups, and wild-type botulinum toxin type A was used as the control group. The protein samples were added to 200 μl of high-potassium solution to achieve final protein concentrations of 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, and 10 nM, respectively. Toxicity was tested using cultured primary neurons, and the cytotoxic cleavage effect of L3GM1-1, L3GM1-2, and L3GM1-3 on intracellular SNAP-25 protein was detected using Western blotting. The results are as follows: Figure 6 As shown, it can be seen that with the increase of recombinant protein concentration, its cleavage effect on SNAP-25 protein also increases. The cleavage effect of L3GM1-3 and L3GM1-1 on SNAP-25 protein is worse than that of wild-type botulinum toxin A. L3GM1-3 has the best cleavage effect, followed by L3GM1-1. The cleavage effect of L3GM1-2 on SNAP-25 protein is not as good as that of wild-type botulinum toxin A.

[0078] (D) Animal-level toxicity testing:

[0079] Wild-type botulinum toxin type A was used as the control group, and L3GM1-1, L3GM1-2, and L3GM1-3 were used as experimental groups. Three concentrations of botulinum toxin were tested in each group, with five replicates per concentration. Sixty SPF-grade male mice were required. Activated full-length botulinum toxin was prepared in 0.2% Gelatin solution, with a series of gradients to ensure that 5 μl systems contained 6 pg, 18 pg, and 60 pg of activated full-length botulinum toxin, respectively, with a preparation volume of 1 mL. Mice were anesthetized for 5 minutes in an anesthesia device, and then injected intramuscularly into the right hind leg using a glass syringe. The injection volume was 5 μl, meaning each mouse received 6 pg, 18 pg, and 60 pg of botulinum toxin.

[0080] Five replicates for each concentration were individually labeled to allow for independent observation of each mouse later. The paralysis of the right hind paw was observed daily in 60 mice, and a scoring system was used to assign scores from 0 to 4. Higher scores indicated more severe paralysis of the right hind paw, indicating stronger toxicity of botulinum toxin at the animal level, while lower scores indicated weaker toxicity. Observations continued for approximately 30-40 days until the paralysis disappeared. The duration of the paralysis indicated the duration of effective action of botulinum toxin in the animal. Detailed experimental results were recorded. Figure 7 As shown, at higher injection doses, the neurotoxicity of the L3GM1-3 mutant is significantly stronger than that of the wild type and other mutants, exhibiting a higher DASAssay score and a longer duration of toxicity at the same time point.

Claims

1. A recombinant protein of a toxicity-enhanced neurotoxin, characterized in that, The recombinant protein comprises a GM1 binding peptide; the amino acid sequence structure of the GM1 binding peptide is expressed as GM1x and GS-GM1x-SG; wherein: GM1x represents a GM1 core binding peptide segment, and the amino acid sequence is VWRLLAPPFSNRLLP. The amino acid sequence of the recombinant protein is shown in any one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO.

9.

2. Use of the toxic enhanced neurotoxin recombinant protein according to claim 1 in interacting with nerve cells, wherein the use is a non-medical purpose.

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