Precursor molecule of botulinum toxin a1, nucleic acid molecule, engineered recombinant baculovirus and preparation method therefor, and method for using precursor molecule to prepare natural botulinum toxin a

Abstract

Provided are a precursor molecule of botulinum toxin A1, a nucleic acid molecule, engineered recombinant baculovirus and a preparation method therefor, and a method for using the precursor molecule to prepare natural botulinum toxin A. The precursor molecule comprises a light chain and a heavy chain, and a linking region linking the light chain and the heavy chain, wherein the amino acid sequence of the precursor molecule is as shown in SEQ ID NO: 2; the light chain is an amino acid sequence of positions 1 to 444 of natural botulinum toxin A, and the amino acid sequence thereof is as shown in SEQ ID NO: 5; the heavy chain sequence is an amino acid sequence of positions 449 to 1296 of natural botulinum toxin A, and the amino acid sequence thereof is as shown in SEQ ID NO: 6; and the amino acid sequence of the linking region is as shown in SEQ ID NO: 11.

Description

Botulinum toxin A1 precursor molecule, nucleic acid molecule, engineered recombinant baculovirus and its preparation method, and method for preparing natural botulinum toxin A using the precursor molecule. Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to botulinum toxin A1 precursor molecules, nucleic acid molecules, engineered recombinant baculoviruses for the preparation of natural botulinum toxin A, methods for their preparation, and methods for preparing natural botulinum toxin A using the precursor molecules. Background Technology

[0002] Botulinum neurotoxins (BoNTs), commonly known as botulinum toxin, are neurotoxins produced by Clostridium botulinum. They possess potent neurotoxicity, being 600 million times more toxic than sodium cyanide and 30 million times more toxic than cobra venom for the same molecular weight, making them the most toxic compound known in nature. Based on serotype, botulinum toxin can be classified into seven types, A through G. Currently, the vast majority of commercially available drugs are type A, such as natural botulinum toxin A, whose amino acid sequence is shown in SEQ ID NO. 8, and its precursor amino acid sequence in SEQ ID NO. 7. The amino acid sequence of mature natural botulinum toxin A is shown in SEQ ID NO. 8.

[0003] Type A botulinum toxin, in its natural state, is a complex of approximately 900 kDa composed of multiple proteins. The core is a 150 kDa toxin protein, while the remaining proteins do not exhibit toxicity and primarily protect the toxin protein from the low pH and various digestive enzymes in the digestive system. Most commercially available botulinum toxins are purified from the 900 kDa complex. A few products, such as Xeomin from Merz (Germany), have removed the accessory proteins, retaining only the 150 kDa core protein. This reduces the risk of immune responses and antibody production, and avoids the gradual decline in efficacy caused by repeated administration.

[0004] The world's first commercially available botulinum toxin product was Botox, manufactured by Allergan in the United States. Approved by the FDA in 1989, it was used to temporarily improve moderate to severe frown lines caused by activity of the corrugator supercilii and / or depressor supercilii muscles in adults aged 65 and under. Botox, and most subsequent commercially available botulinum toxin products, including the only approved domestically produced botulinum toxin, Hengli, are derived from the fermentation of Clostridium botulinum Hall strain. The production process requires a very strict anaerobic environment and is temperature-sensitive. Combined with the extremely high toxicity of botulinum toxin itself, the control of the fermentation and purification processes is extremely stringent. Therefore, there is a need to develop a botulinum toxin preparation method that does not exhibit extremely high toxicity in the early stages of fermentation and purification, which would greatly benefit the industry's development.

[0005] Baculovirus expression vector systems (BEVS) are among the most common eukaryotic expression systems. Since their introduction in 1983, BEVS have been widely used to express a wide variety of proteins. The most attractive feature of BEVS compared to other eukaryotic expression systems is its ability to express and produce large quantities of desired proteins within cells, and most importantly, the expressed proteins exhibit correct folding, targeting, and post-translational modifications; therefore, proteins produced by BEVS are more likely to exhibit the full characteristics of the original protein in its natural host environment compared to proteins expressed in bacterial systems. Among the many protein characteristics found to be precisely generalized by BEVS are tertiary structure, disulfide bonds, formation of homologous and heterologous multimeric protein complexes, phosphorylation, membrane targeting and insertion, and cellular localization. Baculoviruses have a limited host range, confined to specific invertebrate species; therefore, they are safer than most mammalian viruses, posing no harm to operators and causing no environmental contamination. Compared to prokaryotes or yeast, insect cells are safer because they cannot reproduce and expand after harvest. Insect cells grow rapidly and are easily mass-produced, thus facilitating large-scale production. Among various BEVS methods, Bac-to-Bac technology is a rapid, efficient, and widely used approach. Bac-to-Bac is more effective for large-scale production and has been widely used for recombinant proteins in mammals. By using site-specific transposition in Bac-to-Bac technology, the requirement for plaque isolation can be overcome, and the recombination efficiency in this system is close to 100%. Therefore, using BEVS as an expression system is suitable.

[0006] The 150kD core protein of botulinum toxin consists of two subunits, 50kD and 100kD. It is synthesized as a complete precursor protein, which is inactive. Then, a protease cleaves several amino acids between the two subunits to form the active form. The 50kD subunit is the light chain (LC), and the 100kD subunit is the heavy chain (HC), linked by a disulfide bond. HC specifically binds to presynaptic membrane receptors to target nerve cells, and then the disulfide bond is broken, allowing the LC to enter the nerve cell. The LC contains Zn. 2+ Through its metalloproteinase-dependent activity, botulinum toxin type A can specifically degrade the SNAP25 protein, a signaling molecule in cells that transmits nerve impulses, thereby blocking neuromuscular transmission.

[0007] There are currently several strategies for expressing recombinant botulinum toxin. Chinese patent CN115785237A constructs HC and LC into two read frames on a single expression vector, achieving co-expression in *E. coli* and intracellular assembly into active botulinum toxin. While this method is simple, the active botulinum toxin is produced during fermentation, and the host bacteria can easily spread into the natural world, posing significant environmental and safety risks. Therefore, it does not solve the problem of safe botulinum toxin production. Chinese patent CN114989271A expresses LC and HC separately, then denatures and refolds them together in vitro. During refolding, some HC and LC can assemble into active botulinum toxin, but the efficiency of producing active botulinum toxin is low, only about 30%. Furthermore, multiple purification steps are required after refolding, increasing the possibility of contamination. Therefore, using an insect cell baculovirus expression system to express inactive or low-activity full-length botulinum toxin precursor protein, purifying it to a high purity, and then using a specific protease to cleave the linker peptide between LC and HC to activate botulinum toxin activity is a relatively safe production method. Currently, several patents involve this protease cleavage method for botulinum toxin development. Chinese patent CN114957482A uses a 3C protease as the cleavage enzyme and fuses a GST tag to the N-terminus of the protein. A 3C cleavage site is also added between the GST tag and the botulinum toxin. While activating the protein with the 3C protease can cleave the tag, several amino acids remain at both ends of LC and the N-terminus of HC. Another Chinese patent, CN 117925581 A, investigated more tags and enzyme cleavage sites, including GST, MBP, and His tags. The enzyme cleavage sites included 3C protease, TEV protease, thrombin, and enterokinase. Tags were fused to both ends of the entire length of the botulinum toxin. This resulted in more amino acid residues after enzyme cleavage and activation than the previous patent. Furthermore, Chinese patents CN118126143 A and CN118006523 A are similar, all showing amino acid residues. These residual amino acids may cause additional immune responses, and according to the patent report, their LD50 is approximately 0.5 ng / kg, lower than the reported natural protein activity of approximately 0.25-0.45 ng / kg (Toxins 2019, 11, 686). Because these methods cannot completely remove the introduced protease sites, the products cannot be used in humans. The Chinese publication CN117561075A discloses a baculovirus system that expresses botulinum toxin with a membrane-penetrating peptide. The authors were surprised that the insect baculovirus expression system could not directly produce botulinum toxin with the natural sequence and structure. After adding the membrane-penetrating peptide, the LD50 was greatly increased, which is not conducive to the biological activity of botulinum toxin and thus affects the drug activity.

[0008] Therefore, in order to produce genetically engineered botulinum toxin products for human use, it is necessary to establish an expression sequence that is completely identical to the natural sequence after cleavage and activation, thereby obtaining a highly active botulinum toxin product. To this end, this invention reports a method for producing natural botulinum toxin in BEVS. Summary of the Invention

[0009] To address the above problems, this invention constructs a recombinant baculovirus expressing type A botulinum toxin using genetic engineering technology, aiming to obtain a recombinant botulinum toxin with a natural sequence and high activity.

[0010] According to a first aspect of the present invention, the present invention provides a botulinum toxin A1 precursor molecule for preparing natural botulinum toxin A, the botulinum toxin A1 precursor molecule comprising a light chain and a heavy chain, and a linker region connecting the light chain and the heavy chain, the amino acid sequence of the botulinum toxin A1 precursor molecule being shown in SEQ ID NO.2; wherein, the light chain (LC) is the amino acid sequence from position 1 to position 444 of natural botulinum toxin A, the amino acid sequence of which is shown in SEQ ID NO.5, the heavy chain (HC) sequence is the amino acid sequence from position 449 to position 1296 of natural botulinum toxin A, the amino acid sequence of which is shown in SEQ ID NO.6, the amino acid sequence of the linker region (LLH) being shown in SEQ ID NO.11, and the amino acid sequence of natural botulinum toxin A being shown in SEQ ID NO.7.

[0011] In the botulinum toxin A1 precursor molecule of the present invention, preferably, the botulinum toxin A1 precursor molecule has two protease Kex2 cleavage sites, the two protease Kex2 cleavage sites being located at KR at positions 444 and 445, and KR at positions 452 and 453 of the amino acid sequence shown in SEQ ID NO.2, respectively.

[0012] Here, the inventors experimentally discovered that introducing a 6×His(HHHHHH) sequence into the sequence facilitates purification; introducing two Kex2 restriction enzyme sites into the sequence facilitates tag removal, protein activation, and protein purification. The resulting nucleic acid fragment was then assembled and inserted into a eukaryotic expression vector.

[0013] It should be noted that, given the homology of sequence structures among various serum botulinum toxin types, the natural botulinum toxin protein covered by this invention is selected from any one of type A, B, C1, C2, D, E, F, G, or H botulinum toxin proteins. Preferably, the natural botulinum toxin protein is natural botulinum toxin A protein with the amino acid sequence shown in SEQ ID NO.7.

[0014] According to a second aspect of the present invention, the present invention provides a nucleic acid molecule encoding the botulinum toxin A1 precursor molecule, the nucleic acid molecule being shown in SEQ ID NO.1, wherein the nucleic acid molecule encoding the light chain is shown in SEQ ID NO.3, the nucleic acid molecule encoding the heavy chain is shown in SEQ ID NO.4, and the nucleic acid molecule encoding the linker region is shown in SEQ ID NO.10.

[0015] According to a third aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleic acid molecule encoding the botulinum toxin A1 precursor molecule, the nucleic acid molecule being shown in SEQ ID NO. 9.

[0016] According to a fourth aspect of the invention, the invention provides an engineered recombinant baculovirus for producing the precursor molecule, the engineered recombinant baculovirus containing the nucleic acid molecule.

[0017] In the engineered recombinant baculovirus of the present invention, preferably, the engineered recombinant baculovirus is alfalfa silver-striped moth nucleopolyhedrovirus (AcNPV), whose host is insect cells Sf9, Sf21, ExpiSf9, ​​and High Five.

[0018] According to a fifth aspect of the present invention, the present invention provides a method for preparing the precursor molecule of the natural botulinum toxin A, the preparation method comprising the following steps:

[0019] 1) The 000C'(2KR) nucleic acid sequence shown in SEQ ID NO.1 was inserted into the baculovirus expression vector to obtain the 000C'(2KR) recombinant vector shown in SEQ ID NO.9;

[0020] 2) The 000C'(2KR) recombinant vector shown in SEQ ID NO.9 was then transformed into competent E. coli cells to obtain the recombinant 000C'(2KR) baculovirus genome via recombinant transposition;

[0021] 3) Extract the genomic DNA of the recombinant 000C'(2KR) baculovirus and transfect it into prepared insect cells to obtain the primary recombinant 000C'(2KR) baculovirus P0; and further amplify it sequentially to obtain P1 generation recombinant 000C'(2KR) baculovirus and P2 generation recombinant 000C'(2KR) baculovirus; and

[0022] 4) Insect cells were infected with the P2 generation recombinant 000C'(2KR) baculovirus. Cells were harvested after 48-96 hours and purified to obtain botulinum toxin A1 precursor protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0023] In the method for preparing botulinum toxin A1 precursor molecules of the present invention, preferably, in step 1), the baculovirus expression vector is pFastBacI, pFastBac dual, pVL1394, pIEx-1, pIEx-2, pIEx-3, pIEx-4, pIEx-5 or pIEx-6.

[0024] In the method for preparing botulinum toxin A1 precursor molecules of the present invention, preferably, in step 2), the competent Escherichia coli cells are DH10Bac, which contain baculovirus genomic DNA.

[0025] In the method for preparing the botulinum toxin A1 precursor molecule of the present invention, preferably, in step 3), the insect cells are ExpiSf9, ​​Sf9, Sf21, or High Five, and the MOI value is 0.01-10. Preferably, the MOI value is 0.1-10, more preferably, the MOI value is 0.2-5.0.

[0026] In the method for preparing botulinum toxin A1 precursor molecules according to the present invention, preferably, in step 4), the purification is carried out by Ni column affinity chromatography, UniGel 30Q anion chromatography, Q anion chromatography, and SP cation chromatography.

[0027] According to a sixth aspect of the present invention, the present invention provides a method for preparing natural botulinum toxin A using the botulinum toxin A1 precursor molecule, the method comprising the following steps: using Kex2 protease to cleave the purified botulinum toxin A1 precursor molecule protein with the amino acid sequence shown in SEQ ID NO.2 to remove the 6×His tag and simultaneously activate it, and then using carboxypeptidase B to remove excess arginine to obtain the target product of the present invention (natural botulinum toxin A).

[0028] In the method for preparing natural botulinum toxin A using the botulinum toxin A1 precursor molecule of the present invention, preferably, the Kex2 protease further includes a Kex2 digestion buffer, wherein the Kex2 digestion buffer is 50 mM Tris-HCl, pH 8.0, 0.1-2 mM CaCl2; or the Kex2 digestion buffer is 200 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 0.1% Tween-20.

[0029] Experiments have shown that since botulinum toxin type A is cleaved after the two lysine residues (K) of the intermediate linker peptide during activation, this invention cleverly utilizes its own amino acid sequence to introduce arginine (R) after the lysine residues. The KR sequence is precisely the cleavage site of the protease Kex2. After Kex2 cleavage, no amino acids remain at the N-terminus of HC, but arginine remains at the C-terminus of LC. Then, by using carboxypeptidase B to remove the excess arginine, botulinum toxin type A with a completely identical sequence to the natural one can be obtained.

[0030] In addition, the tool enzymes used (such as KEX2 enzyme, carboxypeptidase B, etc.) can be removed and further purified by methods such as Ni column affinity chromatography and ion exchange chromatography to obtain the final product.

[0031] The beneficial effects of this invention are as follows: 1) Before activation by the tool enzyme, the target protein is in an inactive precursor protein state, making production safer; 2) After activation, only 1 to 2 purification steps are needed to obtain the final product, minimizing the possibility of contamination; 3) The recombinant botulinum toxin obtained by this invention is not only completely identical to the sequence of the natural botulinum toxin active protein, but also has high activity, thus providing a low-cost, high-activity botulinum toxin product for clinical use; 4) Compared with the shortcomings of existing technologies for preparing botulinum toxin, this invention provides an environmentally friendly technical route.

[0032] It should be noted that the carriers, cells, enzymes, etc. mentioned in the embodiments are for the purpose of illustrating the invention, rather than limiting the scope of protection of the invention. Attached Figure Description

[0033] Figure 1a shows the agarose gel electrophoresis image of the recombinant vector pFBD-000C'(2KR);

[0034] Figure 1b shows the sequencing results of the LC and HC linker regions in 000C'(2KR); Figures 1c, 1d, 1e, 1f, 1g, and 1h show the sequencing results of the full-length gene fragment of 000C'(2KR).

[0035] Figure 2 shows the agarose gel electrophoresis diagram of PCR identification of the recombinant rod particles rBacmid000C'(2KR);

[0036] Figure 3 shows the SDS-PAGE electrophoresis image of purified 000C'(2KR);

[0037] Figure 4 shows the SDS-PAGE electrophoresis image of purified 000C'(2KR) after KEX2 digestion and purification.

[0038] Figure 5 shows the Western Blot electrophoresis image of purified 000C'(2KR) after KEX2 digestion and purification.

[0039] Figure 6 shows the SDS-PAGE electrophoresis image of SNAP25 cleaved after 000C'(2KR) activation;

[0040] Figure 7a shows the peptide sequence analysis (LC) of 000C'(2KR) after digestion with Kex2 and carboxypeptidase B.

[0041] Figure 7b shows the peptide sequence analysis (HC) of 000C'(2KR) after digestion with Kex2 and carboxypeptidase B.

[0042] Figure 8 shows the chromatogram of 000C'(2KR) for SEC detection. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0044] The strains, plasmids, and reagents used in the embodiments of this invention are all commercially available products.

[0045] pFastBacDual vector (Thermofisher Scientific, model: 10712024);

[0046] DH10Bac (Thermofisher Scientific, model: 10361012);

[0047] Insect cell ExpiSf9 (Thermofiser Scientific, model: 10359016);

[0048] Insect cell Sf9 (Thermofisher Scientific, model: B82501);

[0049] Insect cell High Five (Thermofisher Scientific, model: B85502)

[0050] Ni Trap-HP column (Cytiva Life Sciences, model: 10347449);

[0051] Gradient pre-formed adhesive (ACE biotech., model: J73354102X);

[0052] Coomassie Brilliant Blue (Beyotime Biotechnology Co., Ltd., Model: Z968240829);

[0053] Pierce BCA Protein Quantitative Kit (Thermofisher Scientific, Model: ZB395549);

[0054] Kex2 protease (Shanghai Yaxin Biotechnology Co., Ltd., model: Re230301);

[0055] Kex2 protease (Chongqing Aimeidi Biotechnology Co., Ltd., model: IM02P02(BP));

[0056] Carboxypeptidase B (MCE MedChemExpress, model: 323706);

[0057] Anti-His antibody (Proteintech, model: 21012256);

[0058] SNAP25 (Beijing Anruit Biotechnology Co., Ltd., Model: BARTSN01);

[0059] Gelatin (Aladdin, model: G108396-500g).

[0060] Example 1: Construction of Recombinant Vector

[0061] The 000C'(2KR) gene fragment (synthesized by Beijing Qingke Biotechnology Co., Ltd.) was cloned into the pFastBacDual vector. The 000C'(2KR) gene sequence was a codon-optimized sequence, with its nucleotide sequence shown in SEQ ID NO.1 and its amino acid sequence shown in SEQ ID NO.2. This 000C'(2KR) gene fragment was cloned into the pFastBacDual vector to obtain the pFBD-000C'(2KR) recombinant vector, the nucleic acid sequence of which is shown in SEQ ID NO.9. A 6×His tag was added to this fragment, with a Kex2 restriction site at each end. Here, the two Kex2 restriction sites are located at KR positions 444 and 445, and KR positions 452 and 453, respectively, in the amino acid sequence shown in SEQ ID NO.2. The results are shown in Figure 1a, an agarose gel electrophoresis image of the recombinant vector pFBD-000C'(2KR). The sequencing data was sent to Qingke Biotechnology Co., Ltd., and the sequencing results were consistent with the design. The results are shown in Figures 1b, 1c, 1d, 1e, 1f, 1g, and 1h. Figure 1b shows the sequencing results of the LC and HC linker regions in 000C'(2KR), confirming that the LLH linker region between LC and HC is correct. Figures 1c, 1d, 1e, 1f, 1g, and 1h show the sequencing data of the full-length 000C'(2KR) gene fragment.

[0062] Example 2: Transformation of E. coli into DH10Bac (transposon recombination)

[0063] 1 μL of pFBD-000C'(2KR) was inoculated into 50 μL of DH10Bac competent cells. Transposition, identification of positive clones, and extraction of recombinant rod midline rBacmid000C'(2KR) were performed according to the Thermofisher BAC-TO-BAC instruction manual. Positive clones were then added to LB medium containing 10% glycerol, mixed well, and stored at -80°C. Figure 2 shows the agarose gel electrophoresis results of PCR identification of the rBacmid000C'(2KR) recombinant rod midline. Figure 2 shows that the PCR results indicate that the rBacmid000C'(2KR) clone is positive.

[0064] Example 3: Expression of the target protein

[0065] Insect cells ExpiSf9, ​​Sf9, or High Five were cultured according to the Thermofisher manual, using suspension culture. ExpiSf9 cells were transfected with recombinant baculovirus 000C'(2KR) according to the BAC-to-BAC manual. Primary baculovirus P0 (2 ml) containing recombinant 000C'(2KR) was collected after 4-6 days. 2 ml of this primary baculovirus P0 was added to 200 ml of 2x10... 6 In ExpiSf9 cells at / ml, the cells were cultured at 27℃ and 130 rpm for 72 h, followed by centrifugation at 300g for 10 min to remove cell debris, yielding P1 generation virus. 2 ml of P1 generation virus was added to 200 ml of 2 x 10⁶ cells / ml of the culture medium. 6 In ExpiSf9 cells at / ml, the cells were cultured at 27℃ and 130rpm for 72h, and centrifuged at 300g for 10min to remove cell debris, thus obtaining high-titer P2 generation virus.

[0066] High Five cells were cultured and maintained according to the Thermofisher instructions. P2 generation virus was cultured at 2 x 10⁻⁶ cells / cells. 6 Infect High Five cells at a density of 1 / ml and an MOI of 1-5, with a dilution factor of 1:200-1000. After 48 hours, harvest cells by centrifugation at 300g for 10 minutes, and immediately purify or freeze at -80℃.

[0067] Example 4: Purification and Quantification of Target Protein

[0068] 1) Ni column chromatography process parameters:

[0069] Ni Trap-HP column

[0070] A. Equilibrium solution: 20mM Tris + 2M NaCl + 10mM ZnCl2 + 10% glycerol, pH 8.0

[0071] B. Sample preparation: The bacterial cells were resuspended in equilibration buffer at a ratio of 1:20 (w:v), lysed at 800 bar, centrifuged at 15000g for 15 min, and the supernatant was filtered and loaded onto the sample. The retention time was greater than 2 min.

[0072] C. Eluent: 20mM Tris + 2M NaCl + 10mM ZnCl2 + 10% glycerol, pH 8.0

[0073] 20mM Tris + 0.1M NaCl + 10mM ZnCl2 + 10% glycerol, pH 8.0

[0074] D. Eluent: 20mM Tris + 0.1M NaCl + 10mM ZnCl2 + 0.5M imidazole + 10% glycerol, pH 8.5

[0075] E. Cleaning: Rinse with 6M guanidine hydrochloride for 5-10 CV, then rinse with 2M NaCl for 5-10 CV, rinse with water, and store in 20% EtOH.

[0076] This method is applicable to 000C'(2KR) samples expressing insect baculovirus.

[0077] 2) SP cation chromatography:

[0078] A. Equilibrium solution: 20mM PB + 10% glycerol, pH 6.8

[0079] B. Sample preparation: Elute the sample with a Ni column, dilute the sample with equilibration buffer, and then load the sample.

[0080] C. Eluent: 20mM PB + 10% glycerol + 1M NaCl, pH 6.8

[0081] D. Cleaning: Rinse with 0.5M NaOH + 2M NaCl for 5CV, then rinse with water and store in 20% ethanol.

[0082] SDS-PAGE electrophoresis was used to check the accuracy of protein molecular weight. A 4-12% gradient precast gel was used for detection. Electrophoresis was performed at 160V for about 45-50 minutes. After electrophoresis, Coomassie Brilliant Blue rapid staining solution was used. The electrophoresis results are shown in Figure 3. Figure 3 is the SDS-PAGE electrophoresis image of purified 000C'(2KR).

[0083] Protein concentration was determined using the BCA method with the Pierce BCA protein quantification kit, following the manufacturer's instructions. The protein concentration was approximately 50 mg / L.

[0084] Protein purity was determined by high-performance liquid chromatography (HPLC) using the SEC method (results shown in Figure 8). Column type: BioCore SEC-300 3μm, 4.6*300mm, Nanospectrum Analytical Technology (Suzhou) Co., Ltd.; column temperature: 30℃; detector: 280nm; mobile phase composition: 90% 50mM phosphate buffer (pH 6.8, 300mM NaCl), 10% isopropanol; flow rate: 0.35mL / min; injection volume: 10μL. Protein purity reached over 95%.

[0085] Example 5: Kex2 enzyme digestion of target protein and purification with magnetic beads

[0086] Add Kex2 digestion buffer (final concentration 50mM Tris-HCl, pH 8.0, 2mM CaCl2) to the purified protein, and add Kex2 protease at a mass ratio of 1:250-1000 (Kex2 protease: botulinum toxin). Incubate at 25℃ for 10-40 minutes to complete activation. Then add carboxypeptidase B at a mass ratio of 1:335-1000 (carboxypeptidase B: botulinum toxin), and incubate at 37℃ for 10-30 minutes. Add equilibrated Ni magnetic beads to the digested protein, incubate on a rotary mixer for 0.5-1 hour, and adsorb the magnetic beads onto a magnetic rack. The supernatant is the de-labeled double-stranded protein sample, which is the recombinant botulinum toxin of this invention.

[0087] An optimized reaction condition involves adding Kex2 digestion buffer (final concentration 200mM Tris-HCl, pH 8.0, 1mM CaCl2, 0.1% Tween-20) to the purified protein, then adding Kex2 protease (Yaxin Biotechnology), and digesting at 37℃ for 40 min.

[0088] Ni column loading buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5% glycerol.

[0089] Q column loading buffer: 20 mM Tris-HCl (pH 8.0), 5% glycerol.

[0090] Q column elution buffer: 20 mM Tris-HCl (pH 8.0), 1 M NaCl, 5% glycerol.

[0091] Equilibrate 5 column volumes of 1 mL Ni column with Ni column loading buffer, add supernatant of enzyme digested sample, collect flow-through, dilute with Q column loading buffer, and purify using Q column.

[0092] The enzyme digestion products were sampled and analyzed by SDS-PAGE and Western Blot. The results are shown in Figures 4 and 5. Figure 4 is the SDS-PAGE electrophoresis image of purified 000C'(2KR) after KEX2 digestion and purification; Figure 5 is the Western Blot electrophoresis image of purified 000C'(2KR) after KEX2 digestion and purification.

[0093] Purity was determined by BCA quantification and mass spectrometry, and then sent to a testing company for mass spectrometry sequencing. The amino acid sequence of the cleaved LC protein is shown in SEQ ID NO:5, and the amino acid sequence of the cleaved HC protein is shown in SEQ ID NO:6. These were then sent to a testing company (Beijing Biotech Biotechnology Co., Ltd.) for mass spectrometry sequencing.

[0094] Mass spectrometry peptide mapping analysis showed that the test results were consistent with the theoretical calculations. The results are shown in Figures 7a and 7b. Figure 7a shows the peptide sequence analysis (LC) of 000C'(2KR) after digestion with Kex2 and carboxypeptidase B; Figure 7b shows the peptide sequence analysis (HC) of 000C'(2KR) after digestion with Kex2 and carboxypeptidase B.

[0095] The methods for determining protein concentration and purity were the same as in Example 4. The results of the protein concentration and purity determination are as follows: protein concentration was 0.1-1 mg / L and protein purity was 95%.

[0096] The SDS-PAGE detection method is the same as in Example 4. The results are shown in Figure 5. As can be seen from Figure 5, the target protein is present in both the transudate and the elution. The transudate contains protein that has been cleaved at both Kex2 sites, while the elution contains protein that has not been completely cleaved and still contains the 6×His sequence, so it can be adsorbed by magnetic beads.

[0097] After SDS-PGAE electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry transfer method at 10V for 10 min, 25V for 30 min. The protein was then detected using an Anti-His antibody, and the results are shown in Figure 5. As can be seen from Figure 5, only a clear 150KD band and an incompletely cleaved band at 50KD were detected in the elution buffer, indicating that the target protein in the flow-through buffer had been completely cleaved.

[0098] Example 6: Validation of SNAP25 cleavage activity in vitro

[0099] Take purified SNAP25 and purified 000C'(2KR), add SNAP25 digestion buffer (final concentration: 50 mM HEPES, pH 7.5, 1% Tween-20, 50 mM ZnCl2, 25 mM DTT). Incubate at 37°C for 30 min. Use undigested SNAP25 as a control, and perform SDS-PAGE analysis.

[0100] The SDS-PAGE assay method is the same as in Example 4. The results are shown in Figure 6. As can be seen from Figure 6, the Snap protein was completely digested and its size was consistent with the expected cleavage of the last 9 amino acids, indicating that the purified protein has the activity to cleave SNAP25, which is in line with expectations.

[0101] Example 7: Animal experiments to verify the in vivo activity of 000C'(2KR) protein

[0102] Using gelatin phosphate buffer (2.0 g gelatin, 4.0 g disodium hydrogen phosphate (Na2HPO4), 1000.0 mL distilled water, pH adjusted to 6.2, autoclaved at 121℃ for 15 min), serially diluted undigested 000C'(2KR) protein (hereinafter referred to as the undigested group) and Kex2-digested and purified 000C'(2KR) protein (hereinafter referred to as the digested group) were performed. Forty-two female Kunming mice were randomly divided into seven groups. After acclimatization for 3 days, 0.5 mL of each protein concentration was administered intraperitoneally to six mice in each group. Mice in the undigested group received 15.3 ng, 10.2 ng, 6.8 ng, 4.5 ng, 3 ng, 2 ng, and 1.3 ng of the drug, respectively. Mice in the digested group received 8 pg, 5.3 pg, 3.5 pg, 2.3 pg, 1.5 pg, 1 pg, and 0.7 pg of the drug, respectively. After administration, mice were fed for 4 days, and the number of deaths and viability were recorded.

[0103] Calculations showed that the LD50 of the enzyme-digested group was 2.5 pg, the LD50 of naturally extracted botulinum toxin was 20-60 pg, and the LD50 of the undigested group was 6.2 ng. Therefore, the LD50 of the enzyme-digested group of this invention is superior to that of naturally extracted botulinum toxin, and the LD50 of the enzyme-digested group is even superior to that of the undigested group.

[0104] The present invention has been illustrated by the above embodiments; however, it should be understood that the present invention is not limited to the specific embodiments and implementations described herein. The purpose of including these specific embodiments and implementations herein is to assist those skilled in the art in practicing the present invention. Any person skilled in the art can easily make further improvements and modifications without departing from the spirit and scope of the present invention; therefore, the present invention is limited only by the content and scope of the claims, and is intended to cover all alternatives and equivalents included within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A botulinum toxin A1 precursor molecule for preparing natural botulinum toxin A, characterized in that, The botulinum toxin A1 precursor molecule includes a light chain and a heavy chain, and a linker region connecting the light chain and the heavy chain. The amino acid sequence of the botulinum toxin A1 precursor molecule is shown in SEQ ID NO.

2. The light chain is the amino acid sequence from position 1 to position 444 of natural botulinum toxin A, as shown in SEQ ID NO.

5. The heavy chain sequence is the amino acid sequence from position 449 to position 1296 of natural botulinum toxin A, as shown in SEQ ID NO.

6. The amino acid sequence of the linker region is shown in SEQ ID NO.

11. The amino acid sequence of natural botulinum toxin A is shown in SEQ ID NO.

7.

2. The botulinum toxin A1 precursor molecule as described in claim 1, characterized in that, The botulinum toxin A1 precursor molecule has two protease Kex2 cleavage sites, which are located at KR positions 444 and 445, and KR positions 452 and 453, respectively, of the amino acid sequence shown in SEQ ID NO.

2.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the botulinum toxin A1 precursor molecule as described in claim 1 or 2, the nucleic acid molecule being shown in SEQ ID NO.1, wherein the nucleic acid molecule encoding the light chain is shown in SEQ ID NO.3, the nucleic acid molecule encoding the heavy chain is shown in SEQ ID NO.4, and the nucleic acid molecule encoding the linker region is shown in SEQ ID NO.

10.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises the nucleic acid molecule encoding the botulinum toxin A1 precursor molecule of claim 3, as shown in SEQ ID NO.

9.

5. An engineered recombinant baculovirus for producing the botulinum toxin A1 precursor molecule as described in claim 1 or 2, characterized in that, The engineered recombinant baculovirus contains the nucleic acid molecule as described in claim 3 or 4.

6. The engineered recombinant baculovirus according to claim 5, characterized in that, The engineered recombinant baculovirus is AcNPV (AcPV), and its host is insect cells Sf9, Sf21, ExpiSf9, ​​and High Five.

7. A method for preparing the botulinum toxin A1 precursor molecule as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: 1) The 000C'(2KR) nucleic acid sequence shown in SEQ ID NO.1 was inserted into the baculovirus expression vector to obtain the 000C'(2KR) recombinant vector shown in SEQ ID NO.9; 2) The 000C'(2KR) recombinant vector shown in SEQ ID NO.9 was then transformed into competent E. coli cells to obtain the recombinant 000C'(2KR) baculovirus genome via recombinant transposition; 3) Extract the genomic DNA of the recombinant 000C'(2KR) baculovirus and transfect it into prepared insect cells to obtain the primary recombinant 000C'(2KR) baculovirus P0; and further amplify it sequentially to obtain P1 generation recombinant 000C'(2KR) baculovirus and P2 generation recombinant 000C'(2KR) baculovirus; and 4) Insect cells were infected with the P2 generation recombinant 000C'(2KR) baculovirus. The cells were harvested after 48-96 hours and purified to obtain botulinum toxin A1 precursor protein, the amino acid sequence of which is shown in SEQ ID NO.

2.

8. The preparation method according to claim 7, characterized in that, In step 1), the baculovirus expression vector is pFastBacI, pFastBac dual, pVL1394, pIEx-1, pIEx-2, pIEx-3, pIEx-4, pIEx-5, or pIEx-6.

9. The preparation method according to claim 7, characterized in that, In step 2), the competent E. coli cells are DH10Bac, which contain baculovirus genomic DNA.

10. The preparation method according to claim 7, characterized in that, In step 3), the insect cells are ExpiSf9, ​​Sf9, Sf21, or High Five, and the MOI value is 0.01-10.

11. The preparation method according to claim 7, characterized in that, In step 4), the purification is performed by Ni column affinity chromatography, UniGel 30Q anion chromatography, Q anion chromatography, and SP cation chromatography.

12. A method for preparing natural botulinum toxin A using the botulinum toxin A1 precursor molecule as described in claim 1 or 2, the method comprising the following steps: using Kex2 protease to cleave the botulinum toxin A1 precursor molecule protein with the amino acid sequence shown in SEQ ID NO.2 to remove the 6×His tag and simultaneously complete the activation; then using carboxypeptidase B to cleave the excess arginine to obtain natural botulinum toxin A as shown in SEQ ID NO.

8.

13. The preparation method according to claim 12, characterized in that, The Kex2 protease also includes a Kex2 digestion buffer, which is 50 mM Tris-HCl, pH 8.0, 0.1-2 mM CaCl2; or 200 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 0.1% Tween-20.

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