Large-scale production method and application of recombinant conopeptide

Abstract

The invention discloses a method suitable for large-scale production of recombinant conopeptide. The conopeptide fusion protein containing the HIS tag-TrxA tag protein-enterokinase recognition site-conopeptide is constructed, a redox pair (reduced glutathione / oxidized glutathione) is introduced in vitro in the separation process, a redox reaction system is optimized, the mismatch rate of the recombinant conopeptide can be remarkably reduced, and the yield of the recombinant conopeptide is increased by more than one time. In addition, the enzyme digestion system is further optimized, and the recombinant conopeptide with high purity and correct structure is finally obtained. The invention provides an effective solution for large-scale production of conopeptide, and has huge application value.

Description

A method for large-scale production of recombinant conospirin and its application Technical Field

[0001] This invention relates to the fields of bioengineering and pharmaceutical engineering, specifically to a method for large-scale production of recombinant conotoxin and its application, and particularly to a method for synthesizing conotoxin using microorganisms and for its separation and purification. Background Technology

[0002] Conopeptides, also known as conotoxins, are small-molecule polypeptide neurotoxins secreted by the venom glands of marine gastropods (Conodontidae family). Their most prominent structural feature is the presence of 1-3 pairs of high-density disulfide bonds, a structure that greatly enhances their stability, allowing them to maintain good biological activity in various physicochemical environments. Conopeptides can target multiple ion channels (such as voltage-gated sodium, calcium, and potassium channels) and neurotransmitter receptors with high specificity and affinity. This specificity makes them an important tool in neuroscience and pharmacology research and shows great potential in drug development. μ-conopeptides, in particular, interfere with nerve signal transmission by blocking the influx of sodium ions into voltage-gated sodium channels, thereby producing potent paralyzing and analgesic effects. Conopeptide-based analgesics (such as ziconopeptide, Prialt®) have analgesic potency 1000 times that of morphine, and long-term use is less likely to induce tolerance or addiction, demonstrating significant advantages. In addition, μ-conocin is also used in the cosmetics industry to reduce wrinkles by relaxing facial muscles. Studies have also shown that some conocines have potential in treating depression, Alzheimer's disease, epilepsy, and diabetes.

[0003] Currently, the main methods for preparing conotoxins include direct extraction from biological tissues, chemical synthesis, and biosynthesis. However, direct extraction of conotoxins from natural conotoxins faces challenges such as resource scarcity, complex processes, extraction difficulties, and high costs, severely limiting yield. Chemical synthesis is currently the primary method for obtaining conotoxins. However, existing chemical synthesis methods, especially in large-scale production, still suffer from a series of significant technical defects. Large-scale production of chemical synthesis faces numerous technical challenges, such as cumbersome synthesis steps, numerous byproducts, low efficiency in correct disulfide bond pairing, and environmental unfriendliness. Biosynthesis technology is still immature, making it difficult to ensure the correct folding and conformation of the product, thus hindering industrialization. For example, patent CN116355932B (publication date 20240123) discloses a recombinant vector and a method for preparing μ-conotoxin. High-purity μ-conotoxin can be obtained through purification, cleavage, and separation. However, this method does not optimize enterokinase cleavage, resulting in a low product yield. Patent CN119462880B (publication date 20250527) describes a Pro.ALP conotoxin for immediate anti-wrinkle and wrinkle reduction, its biosynthesis method, and its application. However, it only uses Ni affinity column purification, resulting in low yield and purity.

[0004] Therefore, there is an urgent need in this field to develop a low-cost, simple-to-operate, mild-condition, low-side-reaction, high-disulfide-bond-pairing-efficiency, easy-to-purify, and high-yield and high-purity method for the large-scale preparation of carotenoids, in order to overcome the shortcomings of existing technologies and meet the growing application needs in the fields of medicine, scientific research and cosmetics. Summary of the Invention

[0005] The purpose of this invention is to overcome the difficulties in large-scale production of high-purity conospiropeptides using existing biosynthetic technologies, and to provide a simple and efficient method for the large-scale preparation of recombinant conospiropeptides. This invention provides a recombinant conospiropeptide fusion protein comprising a HIS tag, a TrxA tag protein, an enterokinase recognition site, and conospiropeptide, and further provides a Pichia pastoris engineered strain that highly expresses the recombinant conospiropeptide fusion protein. During the isolation process, this invention optimizes the redox reaction system by introducing a redox pair (reduced glutathione / oxidized glutathione), which significantly reduces the mismatch rate of recombinant conospiropeptides, increasing their yield by more than 100%. Furthermore, this invention further optimizes the enzymatic digestion system, ultimately obtaining recombinant conospiropeptides with high purity and correct structure.

[0006] One aspect of the present invention provides a conopod fusion protein, characterized in that the amino acid sequence of the conopod fusion protein is shown in SEQ ID NO. 2.

[0007] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes the conostipin fusion protein of the present invention; preferably, the nucleic acid sequence encoding the conostipin fusion protein is shown in SEQ ID NO.1.

[0008] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention; preferably, the vector is a yeast expression vector; more preferably, the yeast expression vector is selected from any one of pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector; most preferably, the recombinant vector is the recombinant vector ReConotoxin in pPICZaA, the nucleic acid sequence of which is shown in SEQ ID NO. 3.

[0009] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule or the recombinant vector described in the present invention; preferably, the host cell is yeast; more preferably, the yeast is Pichia pastoris; most preferably, the Pichia pastoris is selected from any one of GS115, X-33, KM71 / KM71H, and SMD1168 / SMD1168H.

[0010] Another aspect of the present invention provides a method for preparing recombinant conostin, characterized in that a recombinant yeast strain is constructed to express a conostin fusion protein with an amino acid sequence as shown in SEQ ID NO. 2, and the recombinant conostin is obtained after purification by affinity chromatography, redox reaction, enterokinase digestion and ion exchange chromatography; wherein the redox reaction system is as follows: EDTA final concentration 1 mM, GSH (reduced glutathione) final concentration 1 mM, GSSG (oxidized glutathione) final concentration 1 mM, and the reaction conditions are 25℃ for 18 h.

[0011] Furthermore, the affinity chromatography is Ni-NTA affinity chromatography, and the ion exchange chromatography is cation chromatography.

[0012] Furthermore, the enterokinase digestion step was optimized. The optimized digestion conditions were as follows: the concentration of the conospirin fusion protein was 1 mg / ml, 5 Units of enterokinase were added to each 1 mg of the conospirin fusion protein, and the mixture was incubated at 10°C for 30 h.

[0013] Furthermore, Narrative In the redox reaction, the final concentration of the conospirin fusion protein is 1-20 mg / mL, and the reaction buffer is 20 mM Tris pH 8.0; more preferably, the final concentration of the conospirin fusion protein is 5 mg / mL.

[0014] Further, the preparation method includes centrifuging and filtering the fermentation broth of the engineered bacteria expressing the conopeptide fusion protein to obtain a primary purified solution; the primary purified solution is further purified by any one or more of Ni-NTA affinity chromatography, redox reaction, enterokinase digestion and ion exchange chromatography; preferably, the purification includes any one of the following: (1) NTA affinity chromatography-redox reaction-enterokinase digestion-DEAE anion exchange chromatography; or (2) NTA affinity chromatography-redox reaction-enterokinase digestion-DEAE anion exchange chromatography-SP cation exchange chromatography; or (3) NTA affinity chromatography-redox reaction-enterokinase digestion-SP cation exchange chromatography.

[0015] Another aspect of the present invention provides the application of the conostopeptide fusion protein of the present invention, or the recombinant nucleic acid molecule of the present invention, or the recombinant vector of the present invention, or the recombinant host cell of the present invention, or the preparation method of any one of the present invention, or the product prepared by the preparation method of any one of the present invention, in the preparation of medical aesthetic products or clinical pharmaceuticals.

[0016] Furthermore, the medical aesthetic products are selected from anti-wrinkle medical aesthetic products and repair medical aesthetic products; the clinical drugs are selected from pain treatment drugs, muscle relaxants, neuroprotective and repair drugs, and hormone regulation and metabolic disease drugs.

[0017] The conospirin fusion protein and the method for purifying and separating recombinant conospirin of the present invention have the following excellent technical effects.

[0018] (1) The conopod fusion protein constructed in this invention contains, from the N-terminus to the C-terminus, a HIS tag, a TrxA tag protein, an enterokinase recognition site, and a conopod peptide, which facilitates subsequent separation and purification. Among them, the HIS tag is used to rapidly capture the fusion protein, the TrxA tag is used to promote the expression of the fusion protein and the correct folding of the conopod peptide disulfide bonds, and the enterokinase recognition site is used to separate the tag protein and the conopod peptide.

[0019] (2) The Pichia pastoris engineered strain containing conopeptide fusion protein constructed in this invention can achieve high expression, with an expression level of 5 g / L.

[0020] (3) In Example 4, the redox reaction system and conditions were optimized. Different parameters were set for the final concentrations of GSH, GSSG, and reaction time. The final optimized redox reaction system was: EDTA final concentration 1 mM, GSH final concentration 1 mM, and GSSG final concentration 1 mM. The optimized reaction conditions were 25℃ and 18h. In addition, Example 4 compared the reaction system without the addition of redox pairs with the above-mentioned optimized redox reaction system. HPLC analysis showed that the final proportion of conospirin was 30.67% in the former and 63.47% in the latter, which increased the yield of recombinant conospirin by more than 100%.

[0021] (4) In Example 5, different parameters were set for the concentration of conopeptide fusion protein, enzyme digestion temperature, enzyme digestion ratio and time. Based on purity and yield, the optimal enzyme digestion conditions were selected as follows: the concentration of conopeptide fusion protein was 1 mg / ml, 5 Units of recombinant enterokinase were added to each 1 mg of fusion protein, and the mixture was incubated at 10°C for 30 h.

[0022] (5) In Example 6, the conospirin was separated by cationic chromatography. The final target protein was analyzed by HPLC and the purity was 98.17%. Further analysis by LC-MS showed that the molecular weight of the target was 2391.89 Da (consistent with the theoretical molecular weight of 2.39 kDa) and contained 3 pairs of disulfide bonds. This indicates that the present invention can obtain recombinant conospirin with high purity and correct structure. Attached Figure Description

[0023] Figure 1. Map of recombinant conopeptide expression plasmid;

[0024] Figure 2. SDS-PAGE spectrum of recombinant conopeptide expression induced by Pichia pastoris engineered strain;

[0025] Figure 3. SDS-PAGE map of the Ni-NTA-captured conopeptide fusion protein;

[0026] Figure 4. HPLC detection of conopeptide content (without redox pair).

[0027] Figure 5. HPLC detection of conopeptide content (with added redox pair);

[0028] Figure 6. Results of SDS-PAGE analysis of conopeptide fusion protease digestion and conopeptide separation;

[0029] Figure 7. Detection of conopeptide purity (HPLC);

[0030] Figure 8. Detection of molecular weight of conopeptide (LC-MS). Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the content described. The preparation, separation, purification, and detection methods of recombinant conotoxin provided by the present invention, etc., all use commercially available raw materials and reagents. Based on the basic knowledge of conventional genetic engineering, protein engineering, and genetic modification methods in the art, those skilled in the art can implement the methods and embodiments of the present invention.

[0032] The main bottleneck in the preparation of recombinant conospirin lies in the correct folding of conospirin, specifically the correct pairing of its high-density disulfide bonds. The inventors of this invention discovered that the TrxA tag not only promotes high expression of conospirin, but the TrxA protein itself also possesses redox activity, providing a more favorable local environment for the formation of the crucial disulfide bonds in conospirin. This reduces the disulfide bond mismatch rate from 80-90% (e.g., in GST / SUMO-conospirin fusion expression) to 60-70%. Although this discovery significantly reduces the disulfide bond mismatch rate, the high mismatch rate still results in a low final yield, hindering large-scale production. Therefore, further reducing the disulfide bond mismatch rate of recombinant conospirin to improve its yield is the main bottleneck for achieving large-scale production of recombinant conospirin.

[0033] The inventors of this invention have discovered that by re-pairing mismatched conospirin disulfide bonds in conospirin fusion proteins through in vitro redox reactions, the mismatch rate of conospirin disulfide bonds can be significantly reduced. More importantly, this invention creatively screened redox reaction conditions and enzymatic digestion conditions, and by optimizing these conditions, ultimately reduced the conospirin disulfide bond mismatch rate from 60-70% to 20-30%, increasing the yield of recombinant conospirin by more than double compared to the original process. This provides an effective solution for the large-scale production of recombinant conospirin.

[0034] Example 1: Construction of Pichia pastoris engineered strain expressing recombinant conotoxin

[0035] The ReConotoxin gene was synthesized by Genewiz Biotechnology Co., Ltd. The nucleic acid sequence of the Pichia pastoris expression codon-optimized ReConotoxin gene is shown in SEQ ID NO. 1, and the amino acid sequence of the ReConotoxin conopod fusion protein is shown in SEQ ID NO. 2, which includes the His tag amino acid sequence as shown in SEQ ID NO. 4, the TrxA tag amino acid sequence as shown in SEQ ID NO. 5, and the enterokinase restriction site amino acid sequence as shown in SEQ ID NO. 6. Specific sequences are shown in Table 1. The ReConotoxin gene was then inserted into the pPICZα A plasmid vector via enzyme digestion ligation or one-step cloning to obtain the recombinant plasmid ReConotoxin in pPICZα A. The recombinant conopod expression plasmid map is shown in Figure 1, and the nucleic acid sequence of pPICZα A containing the ReConotoxin gene is shown in SEQ ID NO. 3. The recombinant plasmid ReConotoxin in pPICZαA was then linearized by digestion with the restriction endonuclease SacⅠ (Quick Cut SacⅠ, TaKaRa). The linearized plasmid was then transformed into P. astoris X-33 competent cells by electroporation, and Pichia pastoris engineered strains with high expression of recombinant conotoxin were obtained by resistance plate selection and expression selection.

[0036] Table 1 Sequence of the present invention

[0037] surface

[0038] Example 2 Expression of recombinant conopodin fusion protein

[0039] The *Pichia pastoris* strain expressing the recombinant conopodione fusion protein was inoculated into 100 mL of BMGY liquid medium at a ratio of 1‰. The medium was then placed in a constant temperature shaker and cultured overnight (~16 h) at 30°C and 250 rpm. The OD of the overnight culture was measured. 600 Collect 1000 OD 600 The bacterial culture was centrifuged at 1500×g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1000 mL of BMMY medium. The culture was placed in a constant temperature shaker and incubated at 30℃ and 250 rpm for 72 hours. Methanol was added every 24 hours to a final concentration of 0.5%. After incubation, the culture was centrifuged at 12000×g for 5 minutes, and the supernatant was collected. The expression product was detected by electrophoresis. The SDS-PAGE spectrum of the recombinant conostipation peptide-expressing Pichia pastoris engineered strain is shown in Figure 2. The H in different lanes represents different induction hours, with the highest expression level (up to 5 g / L) observed after 120 hours of induction. The results indicate that the recombinant Pichia pastoris engineered strain constructed in this invention can achieve efficient expression of the recombinant conostipation peptide fusion protein.

[0040] Example 3 Purification of the expression product

[0041] The fermentation supernatant was clarified by filtration through a 0.45µm filter or a 0.5µm Nernst filter. The conopeptide fusion protein in the clarified filtrate was captured using an AKTApure150 protein purifier and a Nanomicro NW Rose Ni FF chromatography column. The method was as follows: the chromatography column was washed with 2-3 column volumes of purified water, and then equilibrated with 2-3 column volumes of Lysis Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 20 mM imidazole + 10% glycerol, pH 8.0). The clarified filtrate was then loaded at a flow rate of 130 cm / h to ensure that the conopeptide fusion protein was fully incorporated into the chromatography packing material. After loading, the chromatography column was reequilibrated with Lysis Buffer until the baseline stabilized. Finally, the conopeptide fusion protein was obtained by isocratic elution with Elution Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 300 mM imidazole, pH 8.0). The results of the SDS-PAGE experiment are shown in Figure 3. Lane 4 is the target protein, the conopod fusion protein, with a clear target band at approximately 16 kDa, which is the captured conopod fusion protein of the present invention.

[0042] Example 4: Optimization of Redox Conditions

[0043] Using Cobetter 5 KD, 0.11 m 2 The eluent of the captured conopeptide fusion protein was concentrated and replaced with reaction buffer (20 mM Tris, pH 8.0). Appropriate amounts of EDTA, GSH, and GSSG were added to the conopeptide fusion protein after buffer replacement to prepare a redox reaction system. The reaction temperature was 25℃, and the reaction time was 18 h. Samples with different reaction times were concentrated and replaced with reaction buffer (20 mM Tris, pH 8.0), and then enterokinase (Shanghai Yaxin, catalog number REK08, 2 Units of recombinant enterokinase per 1 mg of conopeptide fusion protein) was added and digested at 25℃ for 16 h. The digested samples were then analyzed by HPLC. In this example, different parameter settings were implemented for the final concentrations of GSH and GSSG, as well as the reaction time. Combinations were screened to select the redox reaction system and conditions with the highest proportion of conopeptide fusion protein. The results are shown in Table 2. The final optimized redox reaction system was: 1 mM EDTA, 1 mM GSH, and 1 mM GSSG, with the optimal reaction conditions of 25℃ and 18 h.

[0044] Furthermore, this embodiment also compares the final conopeptide content in the reaction system without the addition of a redox pair with that in the preferred redox reaction system described above. Figure 4 shows the HPLC detection of the conopeptide content in the reaction system without the addition of a redox pair, which is 30.67%. Figure 5 shows the HPLC detection of the conopeptide content in the preferred redox reaction system with the addition of a redox pair, which is 63.47%.

[0045] Table 2 Optimization of Redox Reaction System

[0046]

[0047] Example 5: Optimization of Enzyme Digestion Conditions

[0048] Using Cobetter 5 KD, 0.11 m 2The conopod peptide fusion protein redox reaction solution was concentrated and replaced with reaction buffer (20 mM Tris, pH 8.0) using a membrane. An appropriate amount of enterokinase (Shanghai Yaxin, catalog number REK08) was added to the replaced conopod peptide fusion protein. In this embodiment, different parameters were set for the conopod peptide fusion protein concentration, digestion temperature, digestion ratio, and time. The results were screened based on purity and yield, as shown in Tables 3, 4, and 5. The optimal digestion conditions were: a conopod peptide fusion protein concentration of 1 mg / ml, 5 Units of recombinant enterokinase added to each 1 mg of fusion protein, and incubation at 10°C for 30 h. The results showed a digestion efficiency of 95%, a purity of 80%, and a yield of 76%. After digestion under the above-optimized conditions, the conopod peptide fusion protein in the reaction solution had its corresponding tag removed, yielding recombinant conopod peptides. SDS-PAGE analysis showed the results, as shown in lanes 2 and 3 of Figure 6, indicating that the present invention yields tag-removed conopod peptides after digestion.

[0049] Table 3 Optimization of protein digestion concentration

[0050] Table 4 Optimization of Enzyme Digestion Temperature

[0051]

[0052] Table 5 Optimization of enzyme digestion ratio and time

[0053]

[0054] Example 6 Isolation of recombinant conospirin

[0055] The conopeptides in the above-mentioned enzyme digestion reaction solution were captured using an AKTA pure150 protein purification system and a NanoGel-50SP-HP cationic chromatography column. The method was as follows: the chromatography column was washed with 2-3 column volumes of purified water, and then equilibrated with 2-3 column volumes of cation equilibration buffer (20 mM Tris, pH 8.0). The enzyme digestion reaction solution with the optimized digestion conditions was then loaded at 130 cm / h to ensure sufficient binding of the recombinant conopeptides to the chromatographic packing material. After loading, the chromatography column was reequilibrated with cation equilibration buffer until the baseline stabilized. Then, isocratic elution was performed with 15% cationic elution buffer (20 mM Tris + 1 M NaCl, pH 8.0) to remove impurities until the baseline stabilized (~20 CV). Finally, isocratic elution was performed with 35% cationic elution buffer to obtain high-purity recombinant conopeptides. The results were analyzed by SDS-PAGE, as shown in Figure 6, where lanes 8-10 represent the eluted conopeptides. Samples from lanes 8-10 were combined and analyzed by HPLC. The results are shown in Figure 7, with a purity of 98.13%. Further LC-MS analysis revealed that the target analyte had a molecular weight of 2391.89 Da (consistent with the theoretical molecular weight of 2.39 kDa) and contained three disulfide bonds. This indicates that the technique of this invention can yield recombinant conospiropeptide with high purity and correct structure.

[0056] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A conopod peptide fusion protein, characterized in that, The amino acid sequence of the conopod fusion protein is shown in SEQ ID NO.

2.

2. A recombinant nucleic acid molecule, characterized in that, The conopeptide fusion protein of claim 1 is encoded; preferably, the encoding nucleic acid sequence of the conopeptide fusion protein is shown in SEQ ID NO.

1.

3. A recombinant vector, characterized in that, The invention comprises the recombinant nucleic acid molecule of claim 2; preferably, the vector is a yeast expression vector; more preferably, the yeast expression vector is selected from any one of pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector; most preferably, the recombinant vector is the recombinant vector ReConotoxin in pPICZaA, the nucleic acid sequence of which is shown in SEQ ID NO.

3.

4. A recombinant host cell, characterized in that, The formulation comprises the recombinant nucleic acid molecule of claim 2 or the recombinant vector of claim 3; preferably, the host cell is yeast; more preferably, the yeast is Pichia pastoris; most preferably, the Pichia pastoris is selected from any one of GS115, X-33, KM71 / KM71H, and SMD1168 / SMD1168H.

5. A method for preparing recombinant conospirin, characterized in that, A recombinant yeast strain was constructed to express the conospirin fusion protein with the amino acid sequence shown in SEQ ID NO.

2. The recombinant conospirin was purified by affinity chromatography, redox reaction, enterokinase digestion, and ion exchange chromatography. The redox reaction system consisted of 1 mM EDTA, 1 mM GSH (reduced glutathione), and 1 mM GSSG (oxidized glutathione), and the reaction conditions were 25°C for 18 h.

6. The preparation method according to claim 5, characterized in that, The affinity chromatography is Ni-NTA affinity chromatography, and the ion exchange chromatography is cation chromatography.

7. The preparation method according to claim 5 or 6, characterized in that, The enterokinase digestion process was optimized. The optimized digestion conditions were as follows: the concentration of the conospirin fusion protein was 1 mg / ml, 5 Units of enterokinase were added to each 1 mg of the conospirin fusion protein, and the mixture was incubated at 10°C for 30 h.

8. The preparation method according to any one of claims 5-7, characterized in that, Place Narrative In the redox reaction, the final concentration of the conospirin fusion protein is 1-20 mg / mL, and the reaction buffer is 20 mM Tris pH 8.0; more preferably, the final concentration of the conospirin fusion protein is 5 mg / mL.

9. The preparation method according to any one of claims 5-8, characterized in that, The preparation method includes centrifuging and filtering the fermentation broth of the engineered bacteria expressing the conopeptide fusion protein to obtain a primary purified solution; the primary purified solution is further purified by any one or more of Ni-NTA affinity chromatography, redox reaction, enterokinase digestion and ion exchange chromatography; preferably, the purification includes any one of the following: (1) NTA affinity chromatography-redox reaction-enterokinase digestion-DEAE anion exchange chromatography; or (2) NTA affinity chromatography-redox reaction-enterokinase digestion-DEAE anion exchange chromatography-SP cation exchange chromatography; or (3) NTA affinity chromatography-redox reaction-enterokinase digestion-SP cation exchange chromatography.

10. The use of the conopod fusion protein of claim 1, or the recombinant nucleic acid molecule of claim 2, or the recombinant vector of claim 3, or the recombinant host cell of claim 4, or the preparation method of any one of claims 5-9, or the product prepared by the preparation method of any one of claims 5-9, in the preparation of medical aesthetic products or clinical pharmaceuticals.

11. The application according to claim 10, characterized in that, The medical aesthetic products are selected from anti-wrinkle medical aesthetic products and repair medical aesthetic products; the clinical drugs are selected from pain treatment drugs, muscle relaxants, neuroprotective and repair drugs, and hormone regulation and metabolic disease drugs.

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