Construction method and application of engineering bacteria for producing conopeptide

Abstract

The invention belongs to the field of genetic engineering and microbial pharmacy, and particularly relates to a construction method and application of engineering bacteria for producing conopeptide, and the construction method comprises the following steps: designing a gene segment for coding ketosteroid isomerase-Strep tag-conopeptide fusion protein, and the nucleotide sequence of the gene segment is as shown in SEQ ID NO: 2; the amino acid sequence of the ketosteroid isomerase-Strep tag-conopeptide fusion protein is as shown in SEQ ID NO: 1. Inserting the gene segment into multiple cloning sites of an expression vector to obtain a recombinant expression vector; and introducing the recombinant expression vector into escherichia coli competent cells through a chemical conversion method to obtain the engineering bacteria for producing conopeptide. According to the method, soluble efficient expression of conopeptide can be achieved, high-purity conopeptide is obtained through one-step affinity chromatography purification of the Strep tag, and the problems that in the prior art, conopeptide expression is difficult, and the purification process is complex are solved.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial pharmaceuticals, specifically relating to a method for constructing an engineered bacterium that produces conopeptides and its application. Background Technology

[0002] Conotoxins, also known as cone snail toxins, are a class of small polypeptide toxins composed of 10 to 100 amino acids, secreted by the marine mollusc cone snail. Conotoxins are widely distributed in tropical and subtropical seas, and they secrete these toxins through venom glands during predation, rapidly paralyzing their prey. Conotoxins have complex structures, typically forming stable cyclic structures with disulfide bonds, giving them high stability even under extreme pH and temperature conditions. There are many types of conotoxins, and each type of cone snail produces toxins with unique molecular structures and biological activities. Their mechanisms of action are diverse, specifically binding to and regulating various ion channels and receptors, making them important research tools in neuroscience, pharmacology, and biotechnology. Conotoxins have broad application potential in pharmacology and cosmetic science, particularly showing significant effects in analgesia, muscle relaxation, and anti-wrinkle applications.

[0003] There are three main methods for obtaining conopod peptides: extraction from the venom glands of cone snails, chemical synthesis, and biosynthesis. Natural extraction faces challenges such as resource scarcity and low extraction efficiency. Conopods are limited in number, and the venom gland extraction process is complex, resulting in low yields and high costs, making it unsuitable for industrial production. Chemical synthesis, especially solid-phase synthesis, is currently the most commonly used method for peptide synthesis. For example, patent application number 202411431321.7 discloses a method for obtaining linear μ-conopod peptides using solid-phase synthesis, followed by oxidation to form cyclization, and finally purification using ultrafiltration combined with high-performance liquid chromatography to obtain refined μ-conopod peptides. In 2023, Shenzhen Viki Technology Co., Ltd. filed for registration of conopod peptides as a new cosmetic raw material. Its production process involves sequentially coupling protected amino acids to resin according to the amino acid sequence from C-terminus to N-terminus, cleaving the precursor with a trifluoroacetic acid-containing lysis buffer to obtain crude peptide precursors, oxidizing at room temperature, filtering, purifying with high-performance liquid chromatography, and lyophilizing to obtain refined conopod peptides. However, solid-phase peptide synthesis suffers from insurmountable drawbacks, including poor atom economy, the need for large amounts of excess reagents and solvents, high production costs, significant environmental impact, difficulty in monitoring reaction progress, and challenges in impurity control. In recent years, methods for biosynthesizing conopodeptides have emerged. These methods first involve genetically engineering microorganisms to express conopodeptide precursor proteins, followed by enzymatic treatment to generate conopodeptides of the target size. For example, patent application number 202211683244.5 discloses a conopodeptide fused with a leader peptide or thioredoxin, prepared through induced expression, ammonium sulfate precipitation, nickel column purification, enterokinase digestion, and nickel column purification. However, due to the small molecular weight and high disulfide bond content of conopodeptides, they are easily degraded or form unfolded, insoluble inclusion bodies during expression, resulting in complex purification processes, low recovery rates, and difficulties in industrialization.

[0004] With the deepening research on conospirin, market demand for conospirin in the cosmetics and pharmaceutical fields is constantly emerging. How to achieve efficient, low-cost, and environmentally friendly production of conospirin is a key factor in promoting its application, and it is also the technical problem that this invention aims to solve. Summary of the Invention

[0005] To address the problems mentioned in the background art, the present invention proposes a method for constructing engineered bacteria that produce conopeptides. By fusing conopeptides with ketosterol isomerase for expression, the method enables the efficient and soluble expression of conopeptides. High-purity conopeptides are obtained through one-step affinity chromatography using a Strep tag, thus solving the problems of difficult conopeptide expression and complex purification processes in the prior art.

[0006] The technical solution adopted by this invention to solve its technical problem is: a method for constructing an engineered bacterium that produces conopeptides, comprising the following steps:

[0007] (a) Design a gene fragment encoding a ketosteroid isomerase-Strep tag-conopeptide fusion protein, the nucleotide sequence of which is shown in SEQ ID NO: 2; the amino acid sequence of the ketosteroid isomerase-Strep tag-conopeptide fusion protein is shown in SEQ ID NO: 1;

[0008] (b) Insert the gene fragment into the multiple cloning site of the expression vector to obtain a recombinant expression vector;

[0009] (c) The recombinant expression vector was introduced into competent Escherichia coli cells by chemical transformation to obtain engineered bacteria that produce conopeptide.

[0010] Furthermore, in step (b), the expression vector is a pET series expression vector carrying the phage T7 promoter.

[0011] Furthermore, in step (b), the expression vector is pET-28a(+).

[0012] Further, in step (b), the nucleotide fragment is inserted between nucleotides 5070 and 5245 of the expression vector pET-28a(+).

[0013] Further, in step (c), the competent E. coli cells include BL21(DE3), BL21(DE3) / pLysS, BL21(DE3) / pLysE, Origami(DE3), OrigamiB(DE3), Origami(DE3), Rosetta(DE3), Rosetta-gami(DE3), or BL21 Star(DE3).

[0014] Further, in step (c), the chemical conversion method includes the following steps:

[0015] Ice bath: Mix the recombinant vector with E. coli BL21(DE3) competent cells and incubate on ice for 20-30 minutes;

[0016] Heat shock: Place the combined solution in a 42°C metal bath for heat shock for 60-90 seconds;

[0017] Cooling: Quickly transfer the mixture to an ice bath to cool for 2-3 minutes;

[0018] Resuscitation: Add non-resistant LB liquid medium to the mixture and incubate at 37°C and 100-150 rpm for 45-60 minutes with shaking.

[0019] Spreading: The bacterial culture after shaking culture was spread on an LB solid selection plate containing 50 μg / mL kanamycin sulfate. The correctly screened transformants were cultured and screened and then stored in glycerol to obtain engineered bacteria that produce conopeptides.

[0020] Further, the screening method was colony PCR verification; the verification was performed using primers T7-F and T7-R, the sequence of primer T7-F is shown in SEQ ID No. 3, and the sequence of primer T7-R is shown in SEQ ID No. 4; the size of the colony PCR product transformed with empty vector pET-28a(+) was 361 bp, and the size of the colony PCR product of the positive transformant containing the correct recombinant vector was 658 bp.

[0021] An application of a method for constructing engineered bacteria that produce conopeptides in the preparation of engineered bacteria that produce conopeptides.

[0022] The application of conopeptide-producing engineered bacteria prepared using a method for constructing conopeptide-producing engineered bacteria in the production of anti-wrinkle drugs.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] (1) The method for constructing engineered bacteria that produce conospirin proposed in this invention achieves efficient and soluble expression of conospirin by fusing conospirin with a ketosterone isomerase. High-purity conospirin is then obtained through one-step affinity chromatography using a Strep tag, solving the problems of difficult conospirin expression and complex purification processes in existing technologies. The engineered bacteria constructed in this invention synthesize conospirin with advantages such as simple production process and high production efficiency, significantly reducing the demand for chemical reagents and organic solvents, greatly alleviating environmental pressure, and effectively reducing production costs.

[0025] (2) This invention ultimately realizes the efficient, low-cost and environmentally friendly production of conotoxin, with high product purity and significant anti-wrinkle bioactivity, meeting the needs of large-scale production and market application. Attached Figure Description

[0026] Figure 1 This is a plasmid map of the expression vector pET-28a(+)-KSI-Strep-CTX in Example 1 of this application;

[0027] Figure 2 This is an electrophoresis image of colony PCR verification in Example 1 of this application;

[0028] Figure 3 This is an SDS-PAGE electrophoresis image of Example 2 of this application;

[0029] Figure 4 This is an SDS-PAGE electrophoresis image of Example 3 of this application;

[0030] Figure 5 This is an SDS-PAGE electrophoresis image of Example 4 of this application;

[0031] Figure 6 This is a plasmid map of the expression vector pET-28a(+)-KSI-RBS-Strep-CTX of Comparative Example 1 of this application;

[0032] Figure 7 This is a comparative example 1 of this application, an SDS-PAGE electrophoresis image. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Prepare the culture medium and buffer solution. Culture medium and buffer solution formulations:

[0035] (1) LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar 15 g / L;

[0036] (2) LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L;

[0037] (3) Fermentation medium: yeast extract 20~40g / L, glycerol 10~20 g / L, dipotassium hydrogen phosphate trihydrate 4~6g / L, sodium chloride 3~6g / L, ammonium sulfate 2~4g / L, citric acid monohydrate 2~4g / L, magnesium sulfate heptahydrate 1~2g / L, ferric chloride hexahydrate 200~500mg / L;

[0038] (4) Feeding medium: glycerol 600~800g / L, yeast extract 50~80g / L, magnesium sulfate heptahydrate 1~3g / L;

[0039] (5) Sample loading buffer: 20mM Na2HPO4·12H2O, 280mM NaCl, 6mM KCl, pH=7.4

[0040] (6) Elution buffer: 20 mM Na2HPO4·12H2O, 280 mM NaCl, 6 mM KCl, 2.5 mM d-dethiobiotin, pH=7.4

[0041] The materials used in the preparation of the culture medium and buffer solution in this invention are all commercially available analytical grade reagents, and other reagents are from conventional commercial sources.

[0042] Example 1: Construction of engineered bacteria producing conopeptides.

[0043] The fusion protein sequence of ketosteroid isomerase-Strep tag-conopeptide (SEQ ID No. 1) was designed, and its corresponding DNA sequence (SEQ ID No. 2) was delivered to a gene synthesis company for gene synthesis. The gene fragment was then inserted between positions 5070 and 5245 of the vector pET-28a(+) to obtain the recombinant vector pET-28a(+)-KSI-Strep-CTX. The plasmid map is shown below. Figure 1 As shown.

[0044] SEQ ID No.1

[0045] MHTPEHITAVVQRFVAALNAGDLDGIVALFADDATVEDPVGSEPRSGTAAIREFYANSLKLPLAVELTQEVRAVANEAAFTVSFEYQGRKTVVAPIDHFRFNGAGKVVSIRALFGEKNIHACQWSHPQFEKQGCCNGPKGCSSKWCRDHARCC

[0046] SEQ ID No.2

[0047] ATGCATACCCCAGAACACATCACCGCCGTGGTACAGCGCTTTGTGGCTGCGCTCAATGCCGGCGATCTGGACGGCATCGTCGCCTGTTGCCGATGACGCCACGGTGGAAGACCCCGTGGGTTCCGAGCCCAGGTCCGGTACGGCTGCGATTCGTGAGTTTTACGCCAACTCGCTCAAACTGCCTTTGGCGGTGGAGCTGACGCAGGAGGTACGCGCGGTCGCCAACGAAGCG GCCTTCGCTTTCACCGTCAGCTTCGAGTATCAGGGCCGCAAGACCGTAGTTGCGCCCATCGATCACTTTCGCTTCAATGGCGCCGGCAAGGTGGTGAGCATCCGCGCCTTGTTTGGCGAAGAATATTCACGCATGCCAGTGGAGCCACCCGCAGTTCGAAAAACAGGGTTGCTGCAACGGCCCGAAAGGCTGTTCTTCTAAATGGTGCCGTGACCACGCGCGCTGCTGTTAA

[0048] 2 μL of the recombinant vector pET-28a(+)-KSI-Strep-CTX was added to 200 μL of commercially available *E. coli* BL21(DE3) competent cells, gently mixed, and incubated on ice for 30 min. The competent cells were then heat-shocked in a 42°C metal bath for 90 s, quickly returned to the ice bath, and incubated for 3 min. 800 μL of LB broth was then added, and the cells were incubated at 37°C with shaking at 100 rpm for 1 h. 100 μL of the bacterial culture was spread onto LB agar plates containing 50 μg / mL kanamycin sulfate and incubated overnight at 37°C. Colony PCR was performed using primers T7-F and T7-R to verify the negative control (transformed *E. coli* BL21(DE3) with the empty vector pET-28a(+)) and the obtained positive transformants, respectively (the correct band size for the negative control was 361 bp, and the correct band size for the positive transformant was 658 bp). The correctly verified transformants were used to prepare glycerol-containing bacteria for preservation, resulting in the engineered strain CTX. The electrophoresis results of colony PCR are as follows Figure 2 As shown.

[0049] Table 1 lists the primer names and sequences used in Example 1.

[0050] Primer name Serial Number Sequence (5'-3') T7-F SEQ ID No. 3 TAATACGACTCACTATAGGGG T7-R SEQ ID No. 4 CAAAAAACCCCTCAAGACCCG

[0051] Example 2: Shake-flask culture expression of engineered bacteria CTX.

[0052] The engineered bacteria CTX obtained in Example 1 were streaked and activated on LB solid medium supplemented with 50 μg / mL kanamycin sulfate, and cultured at 37°C for 16 h. Then, 10 mL of LB liquid medium supplemented with 50 μg / mL kanamycin sulfate was inoculated, and the culture was incubated at 37°C with shaking at 220 rpm for 12 h. Finally, 50 mL of fermentation medium supplemented with 50 μg / mL kanamycin sulfate was inoculated at a 4% inoculation rate, and the culture was incubated at 37°C with shaking at 220 rpm until OD600 = 0.6. Induction was then initiated with 0.2 mM IPTG, and the culture was continued at 28°C with shaking at 220 rpm for 24 h. The bacterial cells were collected by centrifugation at 4°C and 8000–10000 rpm for 10–15 min, and the supernatant was discarded. The bacterial cells were resuspended in loading buffer at a ratio of culture medium to loading buffer of 4–5:1, and the cells were sonicated in an ice-water bath (150 W, 15 min). The lysate was centrifuged at 4℃ and 8000-10000 rpm for 20-30 min to remove insoluble cell debris and impurities. Using *E. coli* BL21(DE3) transformed with empty vector pET-28a(+) as a negative control, the expression of conopeptide (CTX) in the engineered bacteria was detected by SDS-PAGE. The SDS-PAGE results are shown below. Figure 3As shown, compared with the negative control, the engineered bacteria CTX successfully expressed soluble conotoxin with a size of 16.9 kDa.

[0053] Example 3: Expression of engineered bacteria CTX in a 5L tank.

[0054] The engineered strain CTX obtained in Example 1 was activated by streaking on LB solid medium supplemented with 50 μg / mL kanamycin sulfate and cultured at 37°C for 16 h. Then, 80 mL of LB liquid medium supplemented with 50 μg / mL kanamycin sulfate was inoculated and cultured at 37°C with shaking at 220 rpm for 12 h to obtain the seed culture. 2 L of fermentation medium supplemented with 50 μg / mL kanamycin sulfate was inoculated at a 4% inoculum size. The culture temperature was 37°C, the initial rotation speed was 200 rpm, the aeration rate was 2 L / min (1 vvm), and the pH was controlled at 7.0 ± 0.05 using 25-28% ammonia. After inoculation, dissolved oxygen was controlled at 30% using a dissolved oxygen-stirring correlation method, and OD600 was measured every 2 hours. When dissolved oxygen recovered, fed-batch culture medium was started at a flow rate of 20 mL / h. When OD600 reached 50, the temperature was lowered to 28°C, and IPTG was added to a final concentration of 0.2 mM for induction. Fermentation was completed after 30 h. The culture medium of the engineered bacteria was centrifuged at 4℃ and 8000-10000 rpm for 10-15 min to collect the bacterial cells, and the supernatant was discarded. The bacterial cells were resuspended in loading buffer at a ratio of culture medium to loading buffer of 4-5:1, and the cells were sonicated in an ice-water bath (150W, 15 min). The lysate was centrifuged at 4℃ and 8000-10000 rpm for 20-30 min to remove insoluble cell debris and impurities. Using *E. coli* BL21(DE3) transformed with the empty vector pET-28a(+) as a negative control, the expression of conopeptide (CTX) in the engineered bacteria was detected by SDS-PAGE. The SDS-PAGE results are shown below. Figure 4 As shown, compared with the negative control, the engineered bacteria CTX successfully expressed soluble conotoxin with a size of 16.9 kDa.

[0055] Example 4: Purification of conopeptide.

[0056] The culture medium of the engineered bacteria obtained in Example 3 was centrifuged at 4°C and 8000 rpm for 15 min to collect the bacterial cells, and the supernatant was discarded. The bacterial cells were resuspended in loading buffer at a ratio of culture medium to loading buffer of 5:1, and the cells were sonicated in an ice-water bath (150W, 15 min). The lysate was centrifuged at 4°C and 8000 rpm for 30 min to remove insoluble cell debris and impurities, and the supernatant was filtered through a 0.22 μm aqueous microporous membrane. Conopeptide was purified using a StrepTrap HP affinity chromatography column. First, 5 column volumes were equilibrated with buffer, and the sample was loaded after baseline equilibration. After loading, 20 column volumes were equilibrated with buffer until the breakthrough peak returned to baseline. Finally, 10 column volumes were eluted with elution buffer, and the elution peak was collected. The purification effect of conopeptide was detected by SDS-PAGE, and the results are as follows: Figure 5 As shown, purified conospirin was successfully obtained.

[0057] Example 5: Detection of the efficacy of conopeptide.

[0058] The anti-wrinkle efficacy of conopeptide was assessed using an elastase activity inhibition assay. The concentration of conopeptide obtained in Example 4 was determined by the Coomassie Brilliant Blue method. Then, the conopeptide concentration was diluted to 1 ppm, 5 ppm, and 10 ppm using 50 mM Tris-HCl (pH 8.0) buffer to prepare sample solutions, which were then added to 96-well microplates.

[0059] Add 25 μL of sample solution, 25 μL of elastase solution, and 150 μL of AAAPAN solution to the sample wells. Add 25 μL of sample solution, 25 μL of 50 mM Tris-HCl (pH 8.0) buffer, and 150 μL of AAAPAN solution to the sample background wells. Add 25 μL of 50 mM Tris-HCl (pH 8.0) buffer, 25 μL of elastase solution, and 150 μL of AAAPAN solution to the enzyme reaction wells. Add 50 μL of 50 mM Tris-HCl (pH 8.0) buffer and 150 μL of AAAPAN solution to the solvent background wells. Add 25 μL of 2 mg / mL EGCG solution, 25 μL of elastase solution, and 150 μL of AAAPAN solution to the positive control wells. Add 25 μL of 2 mg / mL EGCG solution, 25 μL of 50 mM Tris-HCl (pH 8.0) buffer, and 150 μL of AAAPAN solution to the positive control background wells. The ELISA plate was incubated at 25°C for 15 min, and the absorbance was measured at 410 nm using an ELISA reader. The inhibition rate of the sample on elastase activity was then calculated.

[0060]

[0061] In the formula, C is the absorbance of the enzyme reaction well, C0 is the absorbance of the solvent background well, T is the absorbance of the sample well, T0 is the absorbance of the sample background well, P is the absorbance of the positive control well, and P0 is the absorbance of the positive control background well.

[0062] Table 2 shows the elastase activity inhibition rate in Example 5.

[0063]

[0064] The results are shown in Table 2. It can be seen that when the concentration of conopeptide is 1 ppm, 5 ppm and 10 ppm, it has a significant inhibitory effect on elastase, with inhibition rates of 6.06±1.76*, 9.32±0.41* and 11.36±1.62*, respectively, and has anti-wrinkle effect.

[0065] Comparative Example 1: Construction and shake-flask culture expression of engineered bacteria producing conopeptides.

[0066] Following the method described in Example 1, the gene sequences of ketosterol isomerase, Strep tag, and conopod peptide were inserted between positions 5070 and 5245 of the vector pET-28a(+). The difference was that the ketosterol isomerase gene sequence and the Strep tag were not directly linked; instead, a sequence RBS (SEQ ID No. 5: TAAAAGGAGATATACC) was added, resulting in the recombinant vector pET-28a(+)-KSI-RBS-Strep-CTX, in which ketosterol isomerase, Strep tag, and conopod peptide were not fused. The plasmid map is shown below. Figure 6 As shown in Example 1, the recombinant vector pET-28a(+)-KSI-RBS-Strep-CTX was then transformed into *E. coli* BL21(DE3) competent cells to obtain engineered CTX(unfused). Following the method described in Example 2, the engineered CTX(unfused) was cultured in shake flasks for expression, and the expression of conostopeptides in the engineered CTX(unfused) was detected by SDS-PAGE. The SDS-PAGE results are shown below. Figure 7 As shown, compared with the negative control, the engineered bacteria CTX(unfused) only expressed ketosteroid isomerase (correct size 13.5kDa), while conostin was not expressed normally (correct size 3.4kDa). This indicates that the fusion of ketosteroid isomerase with the Strep tag and conostin is a necessary factor to achieve soluble expression of conostin in this invention.

[0067] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an engineered bacterium that produces conopeptides, characterized in that, Includes the following steps: (a) Design a gene fragment encoding a ketosteroid isomerase-Strep tag-conopeptide fusion protein, the nucleotide sequence of which is shown in SEQ ID NO: 2; the amino acid sequence of the ketosteroid isomerase-Strep tag-conopeptide fusion protein is shown in SEQ ID NO: 1; (b) Insert the gene fragment into the multiple cloning site of the expression vector to obtain a recombinant expression vector; (c) The recombinant expression vector was introduced into competent Escherichia coli cells by chemical transformation to obtain engineered bacteria that produce conopeptide.

2. The method for constructing an engineered bacterium producing conopeptide according to claim 1, characterized in that, In step (b), the expression vector includes the pET series expression vectors with the phage T7 promoter.

3. The method for constructing an engineered bacterium producing conopeptide according to claim 2, characterized in that, In step (b), the expression vector is pET-28a(+).

4. The method for constructing an engineered bacterium producing conopeptide according to claim 3, characterized in that, In step (b), the nucleotide fragment is inserted between nucleotides 5070 and 5245 of the expression vector pET-28a(+).

5. The method for constructing an engineered bacterium producing conopeptide according to claim 3, characterized in that, In step (c), the competent E. coli cells include BL21(DE3), BL21(DE3) / pLysS, BL21(DE3) / pLysE, Origami(DE3), OrigamiB(DE3), Origami(DE3), Rosetta(DE3), Rosetta-gami(DE3), or BL21 Star(DE3).

6. The method for constructing an engineered bacterium producing conopeptide according to claim 1, characterized in that, In step (c), the chemical conversion method includes the following steps: Ice bath: Mix the recombinant vector with E. coli BL21(DE3) competent cells and incubate on ice for 20-30 minutes; Heat shock: Place the combined solution in a 42°C metal bath for heat shock for 60-90 seconds; Cooling: Quickly transfer the mixture to an ice bath to cool for 2-3 minutes; Resuscitation: Add non-resistant LB liquid medium to the mixture and incubate at 37°C and 100-150 rpm for 45-60 minutes with shaking. Spreading: The bacterial culture after shaking culture was spread on an LB solid selection plate containing 50 μg / mL kanamycin sulfate. The correctly screened transformants were cultured and screened and then stored in glycerol to obtain engineered bacteria that produce conopeptides.

7. The method for constructing an engineered bacterium producing conopeptide according to claim 6, characterized in that, The screening method was colony PCR verification; the verification was performed using primers T7-F and T7-R, the sequence of primer T7-F is shown in SEQ ID No. 3, and the sequence of primer T7-R is shown in SEQ ID No. 4; the size of the colony PCR product transformed with empty vector pET-28a(+) was 361 bp, and the size of the colony PCR product of the positive transformant containing the correct recombinant vector was 658 bp.

8. The application of a method for constructing engineered conopeptide-producing bacteria as described in any one of claims 1-7 in the preparation of engineered conopeptide-producing bacteria.

9. The use of conostin-producing engineered bacteria prepared using the method for constructing conostin-producing engineered bacteria as described in any one of claims 1-7 in the production of anti-wrinkle drugs.

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