Recombinant botulinum toxin and preparation method thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to recombinant botulinum toxin and a preparation method thereof. The nucleotide sequence of the coding gene of the recombinant botulinum neurotoxin is as shown in SEQ ID NO: 1; the amino acid sequence of the toxin is as shown in SEQ ID NO: 2. The clostridium botulinum neurotoxin sequence optimization method realizes soluble expression of clostridium botulinum neurotoxin protein by escherichia coli; meanwhile, the purity of the botulinum neurotoxin protein is improved by a novel purification process technology, and the double-chain botulinum neurotoxin with relatively high biological activity is obtained; according to the method, the process operation steps of the botulinum neurotoxin are reduced, the biological safety risk is remarkably reduced, industrial production amplification of the botulinum neurotoxin is facilitated, a solid foundation is provided for large-scale production of the botulinum neurotoxin, and the method can be effectively applied to preparation of medical, beauty and health-care products.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to recombinant botulinum toxin and a preparation method thereof. Background Art

[0002] Botulinum neurotoxin, also known as botulinum toxin, is a neurotoxin secreted by a type of Clostridium botulinum. According to antigenicity, it can be divided into 7 serotypes from A to G. Among them, type A is the most toxic to humans. When a person ingests food containing botulinum toxin, clinical symptoms generally appear after 3 to 4 days. Eventually, the patient dies due to respiratory muscle failure caused by nerve paralysis induced by botulinum toxin. Botulinum toxin is one of the most potent poisons known. 1 mg of purified crystalline botulinum can kill 200 million mice, and the lethal dose for humans is about 0.1 μg, which is 10,000 times that of potassium cyanide. Therefore, there are strict requirements for the production of botulinum toxin, and it is generally difficult to obtain in an ordinary laboratory.

[0003] There are currently two methods for the extraction of botulinum toxin. One is the in-situ extraction of botulinum toxin from Clostridium botulinum. Compared with traditional methods, its theory is relatively mature and it is the main production method chosen by global botulinum toxin production companies. However, since the fermentation of Clostridium botulinum requires a very strict anaerobic environment and is very sensitive to temperature. Therefore, specialized fermentation equipment is needed for large-scale production. In addition, since the expressed botulinum toxin does not carry an affinity chromatography tag, multiple purification steps are required during extraction to obtain relatively pure protein. Moreover, due to the strong toxicity of botulinum toxin, the longer the preparation process, the greater the probability of accidental contamination during the period; The other method is to express botulinum toxin protein using recombinant strains or cells, and obtain bioactive botulinum toxin through in vitro enzymatic cleavage. This method does not use Clostridium botulinum and has relatively simple requirements for equipment and purification processes. However, in the currently published literature, patents, and clinically developed recombinant botulinum toxins, they are basically expressed in the form of inclusion bodies. Therefore, during extraction, protein renaturation is required to restore the protein activity and function, which is complex and increases the difficulty of quality control. In addition, in order to obtain a more pure botulinum neurotoxin protein more quickly, whether the botulinum neurotoxin sequence is expressed as a single chain or a double chain, tag proteins and / or enzymatic cleavage sites are introduced into the botulinum neurotoxin sequence. Therefore, during the enzymatic cleavage process, there will be residual enzymatic cleavage site proteins of non-botulinum neurotoxin proteins, and additional purification steps need to be outsourced to remove the enzymes introduced by enzymatic cleavage, and attention should be paid to whether the sequences left by enzymatic cleavage affect the activity of botulinum neurotoxin protein. Researchers mainly express the botulinum neurotoxin protein in two segments separately according to its function. In summary, whether it is the in-situ extraction of botulinum toxin from traditional Clostridium botulinum or the expression of botulinum neurotoxin protein in Escherichia coli, there are certain limitations. Therefore, there is an urgent need to develop a simple, short preparation process and a preparation method that can obtain botulinum toxin in the laboratory for the extraction of botulinum toxin.

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent. Under the condition of not introducing tags and enzymatic cleavage sites, by optimizing the Escherichia coli codon preference for the botulinum neurotoxin protein sequence, the soluble expression of botulinum neurotoxin protein in Escherichia coli is finally achieved; For the fusion botulinum neurotoxin protein induced and expressed by recombinant Escherichia coli, a two-step chromatography purification method can be used to obtain relatively pure and active botulinum neurotoxin protein. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art, and under the condition of ensuring that botulinum neurotoxin does not introduce tags and restriction enzyme cleavage sites, by optimizing the sequence according to the codon preference of Escherichia coli, to construct a recombinant Escherichia coli strain that can express botulinum neurotoxin protein solubly; meanwhile, to develop a new purification technology for the botulinum neurotoxin protein expressed by the recombinant Escherichia coli strain, effectively improve the purity of the botulinum neurotoxin protein, and obtain high biological activity.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a coding gene for recombinant botulinum neurotoxin, and the nucleotide sequence of the gene is as shown in SEQ ID NO:1.

[0008] Furthermore, the gene is obtained by optimizing the nucleotide sequence of the neurotoxin gene region of Clostridium botulinum Hall strain of type A with the accession number AF488749 in the gene bank. The optimization method is: to achieve soluble expression through codon optimization of Escherichia coli without introducing exogenous tags or restriction enzyme cleavage sites.

[0009] The present invention provides a recombinant botulinum toxin, and the amino acid sequence of the toxin is as shown in SEQ ID NO:2.

[0010] The present invention provides a recombinant expression vector, which includes the above-mentioned coding gene, and the gene is inserted into the multiple cloning site of the expression vector pET-28a(+) through NdeI and XhoI restriction enzyme cleavage sites.

[0011] The present invention provides a recombinant Escherichia coli engineering strain, which contains the above-mentioned recombinant expression vector, and the host is Escherichia coli BL21.

[0012] The present invention provides a preparation method for recombinant botulinum neurotoxin, which includes the following steps:

[0013] The above-mentioned coding gene is inserted onto the expression vector pET-28a(+) by using NdeI and XhoI restriction enzyme cleavage sites;

[0014] The recombinant expression vector pET-28a(+) is transformed into Escherichia coli competent cell DH5Q, the plasmid is extracted by fermentation culture, and the plasmid with correct identification by double enzyme digestion and sequencing is named pET28-(rBoNT) recombinant plasmid;

[0015] The pET28-(rBoNT) recombinant plasmid is transformed into Escherichia coli competent cell BL21 to obtain a recombinant botulinum neurotoxin strain, which is fermented and induced in LB(KanR) liquid medium;

[0016] Centrifuge and mix the induced product, and break it through a homogenizer to obtain a crudely purified recombinant botulinum neurotoxin protein;

[0017] Ultrafilter the crudely purified recombinant botulinum neurotoxin protein. After ultrafiltration, perform anion exchange chromatography; after anion exchange chromatography, use a 30kD membrane package for ultrafiltration and buffer exchange again to finally obtain a highly purified recombinant botulinum neurotoxin precursor protein.

[0018] Furthermore, subject the obtained recombinant botulinum neurotoxin precursor protein to a proteolytic cleavage reaction with Trypsin enzyme. After the reaction is terminated, a double-stranded active toxin composed of a 50kD light chain and a 100kD heavy chain linked by a disulfide bond is obtained.

[0019] The application of the above-mentioned coding gene or the recombinant botulinum toxin as described above in the preparation of medical, cosmetic, and health care products.

[0020] The present invention has at least the following beneficial effects:

[0021] The method for optimizing the botulinum neurotoxin sequence of the present invention realizes the soluble expression of botulinum neurotoxin protein in Escherichia coli; at the same time, the new purification process technology improves the purity of the botulinum neurotoxin protein and obtains a double-stranded botulinum neurotoxin with high biological activity. It reduces the process operation steps of botulinum neurotoxin, significantly reduces the biosafety risk, is conducive to the scale-up of industrial production of botulinum neurotoxin, provides a solid foundation for the large-scale production of botulinum neurotoxin, and can be effectively applied in the preparation of medical, cosmetic, and health care products. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 It is the map information of the recombinant plasmid pET28-(rBoNT) of the present invention.

[0024] Figure 2 It is the SDS-Page diagram of the recombinant botulinum neurotoxin strain of the present invention induced to express toxin protein. M is Marker, S is the supernatant after centrifugation of the lysed bacterial solution, and P is the precipitate after centrifugation of the lysed bacterial solution.

[0025] Figure 3This is the SDS-Page diagram of the purified product of the recombinant botulinum neurotoxin protein of the present invention. M is the Marker, FI is the flow-through solution of the supernatant after bacterial liquid lysis and centrifugation and ultrafiltration and loading onto the Q column, E is the eluate of the supernatant after bacterial liquid lysis and centrifugation and ultrafiltration and loading onto the Q column, and the numbers are the numbers of different collection tubes.

[0026] Figure 4 This is the SDS-Page diagram of the protease cleavage activation of the recombinant botulinum neurotoxin protein of the present invention and the structural diagram of the botulinum neurotoxin protein.

[0027] Figure 5 This is the autopsy result diagram of the mouse toxicity test of the recombinant botulinum neurotoxin protein of the present invention.

[0028] Figure 6 This is the reference diagram for the score of mouse neurotoxicity (mouse abduction score of the hind toe, DAS). The scoring rule is 0-4 points, 0 point represents normal, and 4 points represent maximum muscle denervation.

[0029] Figure 7 This is the test result diagram of the biological activity (DAS) of the recombinant botulinum neurotoxin protein of the present invention. Detailed implementation mode

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the following further details the present invention in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0031] The main purpose of the present invention is to provide a recombinant botulinum toxin and its preparation method, which are specifically as follows:

[0032] Example 1: This example is used to provide a botulinum neurotoxin sequence that can be expressed solubly. The optimized botulinum neurotoxin sequence is shown in SEQ ID NO:1.

[0033] At the same time, the present invention also provides the amino acid fragment of the recombinant botulinum neurotoxin, as shown in SEQ ID NO:2.

[0034] Example 2: This example is used to provide a preparation method of recombinant botulinum toxin. As follows:

[0035] Botulinum neurotoxin type A (BoNT / A) is synthesized as a single-chain polypeptide in Clostridium botulinum, with a molecular weight of approximately 150 kDa. This precursor protein is non-toxic to nerve tissue. However, when it is cleaved by proteases, either exogenous proteases or proteases produced by the target cells themselves, it generates two polypeptide chains linked by a disulfide bond: a light chain (LC) of 50 kDa and a heavy chain (HC) of 100 kDa, which is toxic. The HC serves as a transport carrier, and its carboxyl terminus (HC) is the receptor-binding region, responsible for binding to specific neuronal surface receptors and transporting the LC into the neuron. The LC is the catalytic active center of the toxin, causing neuromuscular paralysis in the body and exerting its toxicity. The codon optimization and purification protocols used in this invention can express the botulinum toxin protein in a soluble form without changing the amino acid sequence of the botulinum toxin and effectively isolate the botulinum toxin protein.

[0036] I. Construction of recombinant Clostridium botulinum neurotoxin strains

[0037] 1. Analyze and optimize the nucleotide sequence of the neurotoxin gene region of Clostridium botulinum Hall strain of type A with the accession number AF488749 in the gene bank. Optimize it using the common codons of Escherichia coli, and at the same time balance the proportion and distribution of the four nucleotides A, T, C, and G, to obtain the nucleic acid sequence of the recombinant botulinum neurotoxin as shown in SEQ ID N0:1, and the amino acid sequence of the recombinant botulinum neurotoxin as shown in SEQ ID NO:2.

[0038] 2. Insert NdeI and XhoI restriction enzyme sites at both ends of the optimized sequence.

[0039] 3. After double-digesting the nucleotide sequence of the neurotoxin gene region of Clostridium botulinum Hall strain of type A after optimized synthesis with NdeI and XhoI, ligate it onto the expression vector pET-28a(+) that has also been double-digested with NdeI and XhoI.

[0040] 4. Transform Escherichia coli competent cells DH5α, heat shock at 42 °C for 60 s, add 800 μl of fresh LB medium, incubate at 37 °C for 1 h, and then spread it on a resistant LB solid plate (containing 50 μg / mL kanamycin) and culture overnight at 37 °C.

[0041] 5. After overnight culture, pick monoclonal colonies on the LB solid plate and amplify them in 5 mL of LB (KanR) liquid medium, and culture overnight at 37 °C.

[0042] 6. After overnight culture in liquid medium, the bacterial solution was used to extract plasmids with the TIANGEN plasmid extraction kit. The plasmids were digested with double enzymes to identify whether the target gene was inserted, and then sent for sequencing to identify whether the nucleotide sequence of the target gene was consistent with the optimized synthetic nucleotide sequence of the Clostridium botulinum (Hall strain) neurotoxin gene region. The plasmids with correct digestion and sequencing were named pET28-(rBoNT). The mass spectrometry information of the recombinant plasmids is as Figure 1 shown.

[0043] 7. The pET28-(rBoNT) plasmid correctly ligated with the optimized nucleotide sequence of the Clostridium botulinum (Hall strain) neurotoxin gene region was transformed into Escherichia coli competent cell BL21 and cultured overnight at 37°C. After correct identification by PCR and sequencing, the bacterial solution was mixed with 50% glycerol solution in a ratio of 1:1, and then aliquoted into sterilized 2 mL cryotubes after mixing. After quick freezing in liquid nitrogen, the cryotubes were stored in an -80°C refrigerator.

[0044] II. Fermentation and purification of recombinant botulinum neurotoxin

[0045] 8. When the correctly identified recombinant botulinum neurotoxin strain was cultured in LB (KanR) liquid medium at 37°C and 220 rpm until OD600 = 0.6, IPTG with a final concentration of 1 mM was added, and the culture continued at 25°C and 220 rpm for 8 h.

[0046] 9. The bacterial solution after induced culture was centrifuged at 4°C and 5000g to collect the bacterial cells. The bacterial cells were resuspended with 50 mM Tris-HCl and 500 mM NaCl (pH = 7.4), and then disrupted by a homogenizer (800 bar). The disrupted bacterial cell lysate was centrifuged at 4°C and 10000g to collect the supernatant, and SDS-PAGE electrophoresis was performed to identify the expression of the recombinant botulinum neurotoxin protein, as Figure 2 shown.

[0047] 10. The supernatant after disruption and centrifugation of the bacterial solution was ultrafiltered using a 30 kD membrane package, and at the same time, the buffer was replaced with 50 mM Tris-HCl 8.0 buffer. After ultrafiltration, anion exchange chromatography was carried out. The anion exchange chromatography column used was QHP, and the anion exchange chromatography equilibration buffer was 50 mM Tris-HCl (pH = 8.0). After equilibration until the conductivity and pH value were stable, the sample was loaded, and the flow-through was collected, as Figure 3 shown. The flow-through was ultrafiltered and the buffer was replaced with 20 mM PB and 150 mM NaCl (pH = 7.5) buffer using a 30 kD membrane package, and the target protein with high purity could be obtained after ultrafiltration.

[0048] III. Proteolytic activation of recombinant botulinum neurotoxin

[0049] 11. Proteolytic cleavage was performed on the purified botulinum toxin precursor protein without neurotoxicity and with a molecular weight of approximately 150 kDa. First, small-scale cleavage with Trypsin was carried out to determine the optimal concentration of the enzyme. Trypsin was serially diluted by a factor of 3, with 9 dilutions, namely 1500 μg / mL, 500 μg / mL, 166 μg / mL, 55 μg / mL, 18 μg / mL, 6 μg / mL, 2 μg / mL, 0.7 μg / mL, and 0 μg / mL. The botulinum toxin precursor protein was diluted to 1 μg / mL and added to different concentrations of Trypsin at a ratio of 1:1. The reaction was carried out at 25 °C for 10 minutes. After the reaction, PMSF was added to terminate the enzymatic cleavage reaction.

[0050] 12. After determining the optimal concentration of Trypsin, large-scale cleavage of the botulinum toxin precursor protein was performed. After cleavage, a 150 kD protein band was visible on the SDS non-reducing gel. On the SDS reducing gel, two protein bands were visible, namely a 50 kD light chain (LC) and a 100 kD heavy chain (HC). This demonstrated that the botulinum toxin precursor protein had been cleaved by Trypsin to form a botulinum toxin protein composed of a light chain and a heavy chain linked by a disulfide bond. The proteolytic activation of recombinant botulinum neurotoxin is shown in Figure 4 as follows.

[0051] Referring to Figures 2 to 4 , it can be seen that an obvious band appeared at 150 kDa after induction of the bacterial solution. After lysis and purification of the bacterial solution, a relatively pure single-chain form of recombinant botulinum neurotoxin could be obtained. After digestion with trypsin, a double-chain recombinant botulinum neurotoxin protein composed of a light chain and a heavy chain linked by a disulfide bond was formed. In this project, the recombinant botulinum neurotoxin protein was expressed by Escherichia coli fusion, and the purification process and enzymatic cleavage process of the recombinant botulinum neurotoxin protein were basically achieved, and the production of recombinant botulinum neurotoxin could be further carried out.

[0052] Example 3: This example provides a study on the mouse toxicity of recombinant botulinum neurotoxin protein

[0053] (1.1) Experimental preparation:

[0054] Experimental animals: Kunming mice (body weight 14 - 16 g), 6 mice in each experimental group, all female; Experimental samples: Recombinant botulinum neurotoxin protein after enzymatic cleavage (1 μg / mL), serially diluted 10-fold with commercial physiological saline, with a total of 6 dilutions, namely 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 pg / mL; Commercially available type A botulinum toxin (100 U / vial), diluted with 1 mL of commercial physiological saline and then serially diluted 10-fold, with a total of 2 dilutions, namely 10 U / mL and 1 U / mL.

[0055] (1.2) Test operation:

[0056] For intraperitoneal injection of mice, each mouse in the positive group was injected with 0.1 mL of commercially available botulinum toxin type A diluted 10-fold serially; each mouse in the negative group was injected with 0.1 mL of commercial physiological saline; each mouse in the experimental group was injected with 0.1 mL of the double-chain botulinum neurotoxin prepared by the method of Example 3, which was commercially available botulinum toxin type A diluted 10-fold serially.

[0057] After the mice were inoculated, they were continuously observed for 96 hours. Record the mental state of each group of mice after inoculation, count the mortality rate, and calculate the median lethal dose of mice according to Reed-Muench. Autopsy the dead mice to observe the pathological changes of organs during the observation period.

[0058] (1.3) Test results:

[0059] After the mice in the experimental group were inoculated, the clinical neurological symptoms (such as scratching the head and back, abdominal collapse, etc.) and the death time were both correlated with the dose of the recombinant botulinum neurotoxin protein. The higher the dose, the faster the neurological symptoms and the death time, showing a dose-dependent relationship. The clinical observation results of the mouse virulence test of the recombinant botulinum neurotoxin protein are shown in Table 1, and the autopsy atlas is shown in Figure 5 .

[0060] Table 1 Clinical observation results of the mouse virulence test of the recombinant botulinum neurotoxin protein

[0061]

[0062]

[0063] Experimental result judgment: When the positive control and negative control are valid, the median lethal dose of the recombinant botulinum neurotoxin protein is 10 pg / mouse.

[0064] The median lethal dose (LD50) represents the minimum number of bacteria or amount of toxin required to kill half of a certain animal of a certain body weight or age within a specified time through a specified infection route. In toxicology, the median lethal dose is a commonly used indicator to describe the toxicity of toxic substances or radiation. It is thus determined in this project that the single-chain recombinant botulinum neurotoxin protein expressed by Escherichia coli fusion, after purification and enzymatic digestion, the double-chain recombinant botulinum neurotoxin protein composed of a light chain and a heavy chain linked by a disulfide bond has neurotoxicity, and the virulence is 10 pg / mouse.

[0065] Example 4: This example provides a study on the neurotoxicity of recombinant botulinum neurotoxin protein in mice

[0066] (1.1) Test preparation:

[0067] Test animals: Kunming mice (body weight 14 - 16 g), 4 mice in each test group, all female; Test samples: Recombinant botulinum neurotoxin protein after enzymatic digestion (1 μg / mL), diluted with commercial saline by 10-fold serial dilution, a total of 6 concentrations were diluted, namely 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 pg / mL; Commercially available type A botulinum toxin (100 U / vial), after dilution with 1 mL of commercial saline, diluted by 10-fold serial dilution, a total of 2 concentrations were diluted, namely 10 U / mL and 1 U / mL.

[0068] (1.2) Test operation:

[0069] For the positive group 1, the mice were in the physical injury group, and the gastrocnemius muscle of the left leg of the mice was cut off surgically; for each mouse in the positive group 1, 0.1 mL of commercially available type A botulinum toxin diluted by 10-fold serial dilution was injected into the gastrocnemius muscle of the left leg; for each mouse in the experimental group, 0.1 mL of the double-chain botulinum neurotoxin prepared in Example 3 diluted by 10-fold serial dilution of commercially available type A botulinum toxin was injected into the gastrocnemius muscle of the left leg.

[0070] After the mice were inoculated, they were continuously observed for 96 hours. Record the condition of toe abduction of each group of mice after inoculation, count the mortality rate, and calculate the median lethal dose of mice according to Reed-Muench.

[0071] (1.3) Test results:

[0072] After the mice in the experimental group were inoculated, the clinical appearance of nerve symptoms of toe abduction and the death time were both correlated with the dose of recombinant botulinum neurotoxin protein. The higher the dose, the more obvious the toe abduction condition (the higher the degree of muscle denervation), the faster the death time, and the more deaths, showing a dose-dependent relationship. At the same time, the median lethal dose corresponding to the toe abduction condition of local muscle injection in mice was lower than that of intraperitoneal injection, and the score of toe abduction in mice was greater than 3, and the mortality rate of mice was high. The clinical neurotoxicity observation results of the recombinant botulinum neurotoxin protein mouse neurotoxicity test are shown in Table 2, the clinical lethal dose results are shown in Table 3, and the toe abduction results are shown in Figure 7 .

[0073] Table 2 Observation results of clinical neurotoxicity (toe abduction) of recombinant botulinum neurotoxin protein mouse neurotoxicity test

[0074]

[0075]

[0076] Note: The scoring principle of toe abduction is shown in Figure 6The maximum abduction score is 4 and the minimum is 0. The values in the table are the average scores of digit abduction of each group of animals.

[0077] Table 3 Results of Clinical Lethal Dose in Mouse Neurotoxicity Test of Recombinant Botulinum Neurotoxin Protein

[0078]

[0079] Experimental result determination: When the positive control and negative control are valid, the median lethal dose of recombinant botulinum neurotoxin protein DAS>2 is 10 pg / animal.

[0080] (Digit Abduction Score, DAS) is used to test the degree of muscle denervation and is an important indicator reflecting the effectiveness of botulinum toxin. In this project, it is determined that the single-chain recombinant botulinum neurotoxin protein expressed by E. coli fusion, after purification and enzymatic cleavage, the double-chain recombinant botulinum neurotoxin protein composed of a light chain and a heavy chain linked by a disulfide bond has extremely strong biological activity, and the production of recombinant botulinum neurotoxin can be further carried out.

[0081] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A coding gene for recombinant botulinum neurotoxin, characterized in that, The nucleotide sequence of the said gene is as shown in SEQ ID NO:

1.

2. The coding gene of the recombinant botulinum neurotoxin according to claim 1, characterized in that, The said gene is obtained by optimizing the nucleotide sequence of the neurotoxin gene region of Clostridium botulinum Hall strain of type A with the accession number AF488749 in the gene bank. The optimization method is: achieving soluble expression through codon optimization of Escherichia coli without introducing exogenous tags or restriction enzyme sites.

3. A recombinant botulinum toxin, characterized in that, The amino acid sequence of the said toxin is as shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 1, and the said gene is inserted into the multiple cloning site of the expression vector pET-28a(+) through NdeI and XhoI restriction enzyme sites.

5. A recombinant Escherichia coli engineering strain, characterized in that, It contains the recombinant expression vector as described in claim 4, and the host is Escherichia coli BL21.

6. A method for preparing a recombinant botulinum neurotoxin, characterized in that, It includes the following steps: Using NdeI and XhoI restriction enzyme sites, insert the coding gene as described in claim 1 onto the expression vector pET-28a(+); Transform the recombinant expression vector pET-28a(+) into Escherichia coli competent cell DH5α, ferment and culture to extract the plasmid, and identify the correct plasmid through double enzyme digestion and sequencing, named pET28-(rBoNT) recombinant plasmid; Transform the pET28-(rBoNT) recombinant plasmid into Escherichia coli competent cell BL21 to obtain a recombinant Clostridium botulinum neurotoxin strain, and ferment and culture and induce it in LB(KanR) liquid medium; Centrifuge and mix the induced product, break it through a homogenizer to obtain a crude pure recombinant Clostridium botulinum neurotoxin protein; Ultrafilter the crude pure recombinant Clostridium botulinum neurotoxin protein, and perform anion exchange chromatography after ultrafiltration; after anion exchange chromatography, use a 30kD membrane package for ultrafiltration and buffer exchange again to finally obtain a high-purity recombinant Clostridium botulinum neurotoxin precursor protein.

7. The method for activating the recombinant botulinum toxin obtained as claimed in claim 6, characterized in that, Perform a protease digestion reaction on the obtained recombinant Clostridium botulinum neurotoxin precursor protein with Trypsin enzyme, and after the reaction terminates, obtain a double-chain active toxin in which a 50kD light chain and a 100kD heavy chain are connected by a disulfide bond.

8. The application of the coding gene as described in claim 1 or the recombinant Clostridium botulinum toxin as described in claim 3 in the preparation of medical, beauty, and health care products.

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