Botulinum toxin A1 type recombinant protein as well as preparation method and application thereof

Abstract

The invention provides a botulinum toxin A1 type recombinant protein as well as a preparation method and application thereof, and belongs to the technical field of recombinant proteins. The amino acid sequence of the botulinum toxin A1 type recombinant protein is as shown in SEQ ID NO. 1. The invention designs a novel botulinum toxin A1 type recombinant protein with 10 His tags and Strep tags, and the botulinum toxin A1 type recombinant protein can realize soluble expression in escherichia coli. The invention also provides a preparation method and a purification method of the botulinum toxin A1 type recombinant protein, and large-scale production of the botulinum toxin A1 type recombinant protein can be realized. The invention provides technical support for the application of the botulinum toxin A1 type.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein technology, and in particular to a botulinum toxin A1 type recombinant protein, its preparation method, and its application. Background Technology

[0002] Botulinum toxin type A1 (BIT) is a highly active neurotoxin secreted by Clostridium botulinum. It consists of a 100kDa heavy chain and a 50kDa light chain covalently linked by disulfide bonds, forming a unique heterodimeric structure. BIT specifically acts on the presynaptic membrane of the neuromuscular junction, precisely inhibiting the exocytotic release of acetylcholine by blocking the fusion of vesicles with the cell membrane, resulting in flaccid paralysis of the muscles due to lack of nerve signal stimulation. In clinical practice, BIT, with its precise muscle relaxation effect, has become a first-line drug for treating dystonia such as blepharospasm and hemifacial spasm, and also occupies a core position in the field of cosmetic medicine, widely used for smoothing dynamic wrinkles and reshaping facial contours. In recent years, with the increasing application of botulinum toxin in the medical and cosmetic fields, cases of botulism have also increased year by year. Reference materials (RMs) for botulinum toxin are crucial for establishing botulinum toxin detection and typing techniques and for evaluating its performance.

[0003] Currently, the industrial production of botulinum toxin type A1 still relies on the deep fermentation process of Clostridium botulinum. This process involves complex fermentation regulation, multi-step chromatographic purification, and toxin concentration, resulting in a long production cycle and a yield of only about 0.3~0.5 mg / L. With the development of biotechnology, recombinant expression strategies are highly anticipated, but they face multiple technical bottlenecks: the complex spatial structure of botulinum toxin type A1, containing 7 pairs of disulfide bonds, easily forms misfolded inclusion bodies in prokaryotic expression systems such as E. coli, resulting in a refolding success rate of less than 15%; while eukaryotic expression systems can achieve correct folding, they suffer from low expression levels (<1 mg / L) and high culture costs.

[0004] To enhance targeted therapy efficacy, botulinum toxin type A1 often requires covalent coupling with carrier molecules such as liposomes and nanoparticles. Currently, this is mainly achieved through chemical cross-linking, but this method suffers from drawbacks such as demanding reaction conditions and significant loss of toxin activity. In the exploration of novel expression technologies, the secretory expression system based on Pichia pastoris has shown some potential, utilizing its endoplasmic reticulum-Golgi pathway to achieve correct disulfide bond folding. However, the uneven protein degradation and glycosylation in large-scale production remain unresolved, becoming a key technological barrier restricting the wider application of botulinum toxin type A1. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant botulinum toxin A1 protein, its preparation method and application. This invention designs a new recombinant expression scheme with 10 His tags and a Strep tag, and constructs a full-length fusion expression plasmid of botulinum toxin A1 (His10-A1-Strep), achieving soluble expression of the full-length botulinum toxin A1 in Escherichia coli.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a recombinant botulinum toxin A1 type protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007]

[0008] This invention provides a gene encoding a recombinant botulinum toxin type A1 protein.

[0009] Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010]

[0011] The present invention provides an expression vector containing the nucleotide sequence of the gene.

[0012] The present invention provides a prokaryotic expression system obtained by transformation of the expression vector, wherein the prokaryotic expression system includes Escherichia coli.

[0013] This invention provides a method for preparing the recombinant botulinum toxin A1 type protein, comprising the following steps: (1) The expression vector is introduced into the prokaryotic expression system, positive clones are screened and sequenced to obtain a prokaryotic expression system containing the recombinant vector; (2) Inducing the expression of recombinant protein in the prokaryotic expression system containing the recombinant vector obtained in step (1) to obtain a prokaryotic expression system for inducing recombinant protein expression; (3) The prokaryotic expression system for inducing recombinant protein expression obtained in step (2) is isolated and purified to obtain botulinum toxin A1 type recombinant protein.

[0014] Preferably, the prokaryotic expression system is Escherichia coli, and the Escherichia coli is Trans BL21(DE3); In step (2), the induction is performed by IPTG, with a final concentration of IPTG of 0.8~1.2 mmol / L, a temperature of 25~30℃, and a rate of 150~200 r / min.

[0015] This invention provides a method for purifying recombinant botulinum toxin A1 protein. The method involves solid-liquid separation of the prokaryotic expression system used to induce recombinant protein expression, collecting the solid fraction, resuspending it, and then sonicating it. The sonicated prokaryotic expression system is then subjected to solid-liquid separation again, and the liquid fraction is collected. A first purification is performed using the AKTA protein purification system and a HisTrap™ HP column. Finally, the AKTA protein purification system and HiPrep SepHacryl are used for purification. TM A second purification was performed using an S-100 HR column to obtain purified botulinum toxin type A1 recombinant protein.

[0016] Preferably, the solid-liquid separation rate is 7000~9000 g and the time is 10~20 min. The ultrasonic fragmentation power is 65-85%, with a pause of 1-3 seconds every 2-4 seconds of ultrasonic fragmentation, and the ultrasonic fragmentation time is 50-70 minutes. Before the first purification, the liquid portion was filtered through a 0.45 μm filter membrane; The equilibration buffer used in the first purification was a PBS solution containing 18-22 mM imidazole, and the elution buffer was a PBS solution containing 450-550 mM imidazole.

[0017] This invention provides the application of the recombinant botulinum toxin A1 protein described above, or the recombinant botulinum toxin A1 protein prepared by the method described above, or the recombinant botulinum toxin A1 protein obtained by the purification method described above, in any of the following: (a) To prepare drugs for the prevention or treatment of dystonia; (b) To prepare medicines that improve or treat wrinkles; (c) Preparation of botulinum toxin reference material.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention designs a novel recombinant botulinum toxin type A1 protein with 10 His tags and a Strep tag, which can be solublely expressed in *E. coli*. This invention also provides methods for preparing and purifying the recombinant botulinum toxin type A1 protein, enabling large-scale production. This invention provides technical support for the application of botulinum toxin type A1. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 Design diagram of the amino acid sequence of recombinant botulinum toxin A1 type; Figure 2 The results of SDS-PAGE analysis of the low-dose induced expression of recombinant botulinum toxin type A1 protein in TB culture medium in Example 1 are shown. Figure 3 The results of SDS-PAGE analysis of the low-dose induced expression of recombinant botulinum toxin type A1 protein in LB medium in Example 1 are shown. Figure 4 The results are from the botulinum toxin type B test strip in Example 2. Figure 5 The results of SDS-PAGE analysis of the sample purified by HisTrap™ HP column in Example 3; Figure 6 The results of SDS-PAGE analysis of the sample purified by HiPrep SepHacryl™ S-100 HR column in Example 3; Figure 7The results are from simultaneous SDS-PAGE and Western blot analysis of the purified sample in Example 4. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] Example 1: Design and synthesis of recombinant botulinum toxin type A1 protein fused with His and Strep tags (His10-A1-Strep)

[0023] The amino acid sequence of the recombinant botulinum toxin A1 type designed in this invention consists of the following parts in sequence: starting amino acid M-10 × His tag - botulinum toxin A1 type light chain - thrombin cleavage site - botulinum toxin A1 type heavy chain - thrombin cleavage site - Strep tag, as shown in SEQ ID NO.1 and Figure 1 As shown.

[0024] The amino acid sequence of the botulinum toxin A1 recombinant protein, as shown in SEQ ID NO.1, was translated into a nucleotide sequence. Enzyme cleavage sites and stop codons were added, and the nucleotide sequence was optimized according to the codon preference of E. coli. The optimized sequence is shown in SEQ ID NO.2 and was then sent to GenScript Biotech for gene synthesis.

[0025] The preparation method of the botulinum toxin A1 recombinant protein (His10-A1-Strep) is as follows: 1. The synthesized nucleotide sequence as shown in SEQ ID NO.2 is used... Nco I and Xho I. Restriction sites were constructed into the pET-28a vector (GenScript Biotech, Nanjing, China), successfully constructing the recombinant expression vector pET-28a-His10-A1-Strep. The above construction process was completed by GenScript Biotech.

[0026] 2. The recombinant expression vector pET-28a-His10-A1-Strep was introduced into the body via a thermal shock method. Trans In BL21(DE3) (Bomaide Biotechnology Co., Ltd., Beijing, China), after activation in a shaker at 37°C and 180 r / min for 1 h, the bacterial culture was evenly spread on LB plates and incubated at 37°C for 15 h.

[0027] 3. Select single colonies for sequencing and comparison; those with correct sequences are cryopreserved.

[0028] 4. Inoculate the correctly sequenced bacterial strain into 10 mL of LB and TB medium (kanamycin, 50 μg / mL), and incubate at 37°C with shaking at 180 r / min until OD. 600 =Approximately 0.6, add IPTG to make the final concentration 1 mM, culture with shaking at 180 r / min, set 25℃ and 30℃ as induction conditions, and culture overnight.

[0029] 5. Centrifuge the induced bacterial culture at 8000 g for 15 min to enrich it, then resuspend and wash with 1×PBS, mix with 5× protein loading buffer (containing DTT) (Solepro Technology Co., Ltd., Beijing, China), and boil for 10 min.

[0030] The results of SDS-PAGE analysis of the low-dose induced expression of recombinant botulinum toxin type A1 protein in TB medium are as follows: Figure 2 As shown, the identification results of low-dose induced expression in LB medium are as follows: Figure 3 As shown, M is the marker, and 3, 4, 5, 6, 8, and 9 are the single colony numbers with correct sequences after sequencing alignment. It can be seen that recombinant botulinum toxin A1 protein can be induced under the conditions of 25℃, 30℃, and 1mmol / L IPTG.

[0031] Example 2 His10-A1-Strep test strip detection

[0032] The correctly sequenced bacteria No. 8 and No. 9 from Example 1 were inoculated into 10 mL of LB and TB medium (kanamycin, 50 μg / mL) and cultured at 37°C with shaking at 180 rpm for 8 h. When the bacterial culture reached an OD600 of approximately 0.6, IPTG was added to a final concentration of 1 mM, and the culture was incubated overnight at 30°C with shaking at 180 rpm.

[0033] The induced bacterial culture was placed into a 5 mL centrifuge tube, centrifuged at 8000 g for 15 min for enrichment, then resuspended and washed with 5 mL 1×PBS, centrifuged again and discarded the supernatant, resuspended with 2 mL 1×PBS, and then sonicated (power 75% (93.75 W), sonicated for 3 seconds and paused for 2 seconds, sonicated for 2 min).

[0034] Centrifuge the ultrasonically disrupted bacterial culture at 8000 g for 15 min, aspirate the supernatant into a 5 mL centrifuge tube, resuspend the precipitate in 2 mL of 1×PBS, and store the supernatant and precipitate in a 4°C refrigerator for later use.

[0035] Remove the botulinum toxin type A test strip, add 80 μL of liquid to each group, wait 15 minutes, and then read the value. The test results are as follows: Figure 4As shown, it can be seen that recombinant botulinum toxin A1 protein was detectable after induction in LB medium, but not after induction in TB medium; similarly, recombinant botulinum toxin A1 protein was detectable after induction in TB medium, but not after induction in LB medium. This indicates that different bacterial colonies are suited to different expression conditions, with bacterium 8 being more suitable for LB medium and bacterium 9 being more suitable for TB medium.

[0036] Example 3: Purification of His10-A1-Strep

[0037] 1. Inoculate the bacterial strain with the correct sequence into 10 mL of LB medium (kanamycin, 50 μg / mL), and culture at 37℃ with shaking at 180 r / min for 8 h. Then transfer it into 1 L of LB medium (kanamycin, 50 μg / mL).

[0038] 2. When the bacterial culture reaches an OD600 of approximately 0.6, add IPTG to achieve a final concentration of 1 mM, and incubate overnight at 180 rpm and 16°C.

[0039] 3. Centrifuge the induced bacterial culture at 8000 g for 15 min to enrich it, then resuspend and wash with 1×PBS, centrifuge and discard the supernatant, resuspend in 200 mL of equilibration buffer (Solution A of HisTrap™ HP column), and then sonicate to disrupt it (power 75% (93.75W), sonicate for 3 seconds, pause for 2 seconds, sonicate for 1 h).

[0040] 4. After sonication, centrifuge the bacterial culture at 8000g for 15 min, collect the supernatant, filter it through a 0.45μm pore size filter membrane, and store it at 4℃ for later use. Prepare the equilibration buffer as a 20mM imidazole PBS solution (Solution A) and the elution buffer as a 500mM imidazole PBS solution (Solution B), filter them through a 0.45μm pore size filter membrane, and set aside for later use.

[0041] 5. Using the AKTA protein purification system and HisTrap™ HP column, the procedure sequence is as follows: clean the instrument and tubing with deionized water - install the HisTrap™ HP column - rinse the column with deionized water - rinse with solution A using pump A to equilibrate the column - load the sample (bacterial solution after step 4) using pump A - rinse with solution A until equilibration - set up gradient elution (100% B, 20 min) - collect the sample after peak elution - rinse the column - disassemble and store the column.

[0042] 6. Collect the samples purified by the HisTrap™ HP column and identify them by SDS-PAGE. The results are as follows: Figure 5 As shown, the sample containing the target protein is concentrated to about 5 mL for later use.

[0043] 7. Perform a second purification using the AKTA protein purification system and the HiPrep SepHacryl™ S-100 HR column. The program sequence is as follows: clean the instrument and tubing with deionized water - install the HiPrep SepHacryl™ S-100 HR column - rinse the column with deionized water - rinse the PBS with pump A - load 5 mL of sample (sample processed in step 6) through the loading loop - select the inject program - collect the sample after peak elution - rinse the column - disassemble and store the column.

[0044] 8. Collect the purified sample from step 6 and identify it by SDS-PAGE. The results are as follows: Figure 6 As shown, the samples are aliquoted and stored.

[0045] Figure 5 and Figure 6 In the diagram, M stands for marker, 1 is the sample before loading (i.e., the supernatant obtained after ultrasonic disruption and centrifugation in step 4), 2 is the sample after penetration (i.e., the waste liquid during sample loading when purifying the protein, used to verify whether the protein binds to the nickel column), 3-6 are samples obtained from different elution steps, and 7 is the sample precipitated by ultrasonication (i.e., the precipitate obtained after ultrasonic disruption and centrifugation in step 4). Figure 5 and Figure 6 It can be seen that the method of the present invention successfully induced and purified His10-A1-Strep protein.

[0046] Example 4: SDS-PAGE and Western blot identification of His10-A1-Strep

[0047] 1. After thawing the samples aliquoted and frozen in Example 3, mix thoroughly with 5× protein loading buffer (non-denaturing, non-reducing) for later use. Perform SDS-PAGE on two 4-20% BT precast gels, 3-color Regular Protein (8-180kDa), electrophoresis settings: 400mA, 160V, 45min.

[0048] 2. After running SDS-PAGE, stain one piece using an Estain L1 protein staining instrument and observe the film using a film viewing lamp. Then, transfer the other piece using a rapid wet transfer instrument.

[0049] 3. Prepare blocking buffer: Mix 2g of skim milk powder with 40mL of 1×PBST and vortex for 1.5min to ensure thorough mixing. After transfer, cut the PVDF membrane and place it in a square culture dish. Pour in the blocking buffer and incubate at 70r / min for 2h at room temperature.

[0050] 4. After blocking, recover the blocking solution, wash three times with 1×PBST, and shake at 70 rpm for 15 min each time at room temperature. After washing, place the mixture into a hybridization bag, add the diluted primary antibody, and block using a sealing machine. Incubate overnight at 4°C with inverted mixing. The primary antibody with the His tag is a rabbit polyclonal antibody; the one without the His tag is rabbit antiserum. The diluent is a 1:100 dilution of the primary antibody with Western blot-specific primary and secondary antibody dilution buffer (Absin).

[0051] 5. After primary antibody incubation, wash three times with 1×PBST, and shake at 70 rpm for 15 min each time. After washing, place in a square culture dish, add the diluted secondary antibody, and shake at 70 rpm for 2 h at room temperature. The secondary antibody with the His tag is goat anti-mouse; the one without the His tag is goat anti-rabbit. The diluent is obtained by diluting the recovered blocking solution at a ratio of 1:10000.

[0052] 6. After the secondary antibody incubation is complete, wash three times with 1×PBST, and shake at 70 rpm for 15 minutes each time. After washing, perform color development and save the results.

[0053] Identification was performed using SDS-PAGE and Western blot, and the results are as follows: Figure 7 As shown, M is the marker, 2× is a 2-fold dilution, and 10× is a 10-fold dilution. The target band is clear.

[0054] Example 5 His10-A1-Strep mouse challenge experiment

[0055] Take 2 mL of purified and frozen His10-A1-Strep protein with a protein concentration of 0.28 mg / mL, add 20 μL of 1M Tris-HCl (pH 9.0) to each 1 mL, add 5 μL of diluted thrombin solution, vortex to mix, and react at 37℃ for 16 h.

[0056] Enzyme-digested mouse challenge experiment: BALB / c mice were injected with enzyme-digested His10-A1-Strep. Five groups of mice (n=3 per group) were formed, with challenge dose gradients of 280 ng / mouse, 28 ng / mouse, 2.8 ng / mouse, and 0.28 ng / mouse. A negative control was 2 U / mL thrombin solution. The mice were administered the enzyme via intraperitoneal injection. Results were recorded after 7 days of observation.

[0057] Undigested His10-A1-Strep challenge experiment in mice: BALB / c mice were injected with undigested His10-A1-Strep in 5 groups of 3 mice each. The challenge dose gradients for each group were 280 ng / mouse, 28 ng / mouse, 2.8 ng / mouse, and 0.28 ng / mouse, respectively. PBS was used as a negative control. The mice were administered the solution intraperitoneally. Results were recorded after 7 days of observation.

[0058] The results showed that in the enzyme digestion groups, 3 animals died in groups 1 (280 ng / animal) and 2 (28 ng / animal), while no animals died in groups 3 (2.8 ng / animal), 4 (0.28 ng / animal), and 5 (control). In the undigested groups, 3 animals died in group 1 (280 ng / animal), while no animals died in groups 2 (28 ng / animal), 3 (2.8 ng / animal), 4 (0.28 ng / animal), and 5 (control), as shown in Table 1. The enzyme digestion groups... The concentration was 0.8855 ng / animal, or 46.6 ng / kg; the undigested group... The dosage was 8.855 ng / mouse, or 466 ng / kg; intraperitoneal injection in natural BoNT / A mice. The concentration is 1 ng / kg. Therefore, the recombinant botulinum toxin A1 type protein provided by this invention has higher safety.

[0059] Table 1. Number of deaths in each treatment group after mouse challenge experiment

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant botulinum toxin type A1 protein, characterized in that, The amino acid sequence of the botulinum toxin A1 recombinant protein is shown in SEQ ID NO.

1.

2. A gene encoding the botulinum toxin A1 type recombinant protein of claim 1.

3. The gene as described in claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. An expression vector comprising the nucleotide sequence of the gene of claim 2 or 3.

5. A prokaryotic expression system obtained by transformation with the expression vector of claim 4, characterized in that, The prokaryotic expression system includes Escherichia coli.

6. A method for preparing the recombinant botulinum toxin type A1 protein according to claim 1, characterized in that, Includes the following steps: (1) The expression vector described in claim 4 is introduced into a prokaryotic expression system, positive clones are screened and sequenced to obtain a prokaryotic expression system containing the recombinant vector; (2) Inducing the expression of recombinant protein in the prokaryotic expression system containing the recombinant vector obtained in step (1) to obtain a prokaryotic expression system for inducing recombinant protein expression; (3) The prokaryotic expression system for inducing recombinant protein expression obtained in step (2) is isolated and purified to obtain botulinum toxin A1 type recombinant protein.

7. The method as described in claim 6, characterized in that, The prokaryotic expression system is Escherichia coli, and the Escherichia coli is Trans BL21(DE3); In step (2), the induction is performed by IPTG, with a final concentration of IPTG of 0.8~1.2 mmol / L, a temperature of 25~30℃, and a rate of 150~200 r / min.

8. A method for purifying recombinant botulinum toxin type A1 protein, characterized in that, The prokaryotic expression system for inducing recombinant protein expression as described in claim 6 or 7 is subjected to solid-liquid separation. The solid fraction is collected, resuspended, and then sonicated. The sonicated prokaryotic expression system is then subjected to solid-liquid separation again, and the liquid fraction is collected. A first purification is performed using the AKTA protein purification system and a HisTrap™ HP column. The final purification is achieved using the AKTA protein purification system and HiPrep SepHacryl. TM A second purification was performed using an S-100 HR column to obtain purified botulinum toxin type A1 recombinant protein.

9. The method as described in claim 8, characterized in that, The rate of solid-liquid separation is independently 7000~9000 g, and the time is independently 10~20 min; The ultrasonic fragmentation power is 65-85%, with a pause of 1-3 seconds every 2-4 seconds of ultrasonic fragmentation, and the ultrasonic fragmentation time is 50-70 minutes. Before the first purification, the liquid portion was filtered through a 0.45 μm filter membrane; The equilibration buffer used in the first purification was a PBS solution containing 18-22 mM imidazole, and the elution buffer was a PBS solution containing 450-550 mM imidazole.

10. The use of the recombinant botulinum toxin type A1 protein according to claim 1, or the recombinant botulinum toxin type A1 protein prepared by the method according to claim 6 or 7, or the recombinant botulinum toxin type A1 protein obtained by the purification method according to claim 8 or 9, in any of the following: (a) To prepare drugs for the prevention or treatment of dystonia; (b) To prepare medicines that improve or treat wrinkles; (c) Preparation of botulinum toxin reference material.

Images