Pro.AH8 Acetyl Hexapeptide-8 with the Function of Tightening and Anti-Wrinkle, Its Preparation Method and Application

Abstract

The present invention discloses a plasmid expression vector for hexapeptide-8, and the amino acid sequence expressed by the plasmid expression vector is as shown in SEQ ID No. 1 or 2. It also discloses its host bacteria, biosynthesis method, and a Pro.AH8 acetyl hexapeptide-8 with the efficacy of firming and anti-wrinkle and its application. The present invention provides a biosynthesis method for hexapeptide-8 and acetyl hexapeptide-8, which has stable quality and high safety. The acetyl hexapeptide-8 synthesized by the present invention has high biological activity, low immunogenicity, good solubility and safety. It can be expressed in a prokaryotic system, with a simple system, high yield and low cost.

Description

Technical Field

[0001] The present invention relates to a plasmid expression vector of hexa - peptide - 8 and its biosynthesis method, belonging to the technical field of protein expression. Background Art

[0002] The English name of acetyl hexapeptide - 8 is Argireline, and its sequence is Ac - Glu - Glu - Met - Gln - Arg - Arg - NH 2 , also known as botulinum - like toxin and argireline, is an oligopeptide that mimics the 6 amino acids at the N - terminus of the SNAP - 25 protein. Argireline is one of the commonly used raw materials in high - end cosmetics. Its main function is to reduce the wrinkles caused by the contraction of facial expression muscles, and it has an ideal effect on removing wrinkles on the forehead or around the eyes. Argireline is a safer, cheaper and milder alternative to botulinum toxin. Argireline participates in competing for the site of SNAP - 25 in the vesicle fusion complex, thus affecting the formation of the vesicle fusion complex, resulting in the vesicles being unable to effectively release neurotransmitters, weakening muscle contraction, and then relaxing the muscles, soothing fine lines to make the skin clear and flawless, and also promoting cell regeneration to make the skin restore soft elasticity. It is widely used in the preparation of cosmetics and health products.

[0003] At present, the synthesis methods of argireline mainly include solid - phase synthesis method and liquid - phase synthesis method. There are different defects in several currently disclosed synthesis methods of argireline. The preparation methods recorded in patent documents such as publication numbers CN103694316A, CN106632609A, CN102603869A, etc. use resin - supported solid - phase synthesis, with low substitution degree and high price, making it difficult to reduce costs and not suitable for large - scale production. The synthesis methods recorded in publication numbers CN103613642A and CN102199188A use liquid - phase methods for synthesis. Although the raw material cost can be effectively reduced, the production cycle is long and the purification of each intermediate is difficult, which is not suitable for large - scale production. The liquid - phase synthesis method recorded in publication number CN113845586A uses Pdf protection for two arginine side chains, Trt protection for the glutamine side chain, and tert - butyl ester protection for the glutamate side chain, resulting in an increase in raw material cost. Moreover, it uses palladium - carbon catalytic hydrogenation multiple times, and the dosage is 10%. Since palladium - carbon is expensive, this process is not competitive in terms of cost. Summary of the Invention

[0004] The purpose of the present invention is to provide a biosynthesis method of hexa - peptide - 8.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A plasmid expression vector of hexa - peptide - 8, wherein the amino acid sequence expressed by the plasmid expression vector is as shown in SEQ ID No.1 or 2.

[0007] A host bacterium containing the expression vector described in claim 1.

[0008] Preferably, the host bacterium is Escherichia coli.

[0009] The present invention also discloses a method for biosynthesizing hexa-peptide-8, the steps of which include:

[0010] (1) Construct the plasmid expression vector of the aforementioned hexa-peptide-8 and transform it into a host bacterium;

[0011] (2) Culture the host bacterium for expression and induce protein expression;

[0012] (3) Purify the protein, and digest the purified protein with enterokinase to obtain hexa-peptide-8.

[0013] Preferably, in step (3), the protein is purified by the method of Ni affinity chromatography column.

[0014] The present invention also discloses a method for biosynthesizing acetylated hexa-peptide-8, the steps of which include:

[0015] (1) Construct the plasmid expression vector of the aforementioned hexa-peptide-8 and transform it into a host bacterium;

[0016] (2) Culture the host bacterium for expression and induce protein expression;

[0017] (3) Purify the protein, and digest the purified protein with enterokinase to obtain hexa-peptide-8;

[0018] (4) Add glutamic acid acetyltransferase, acetyladenosine, and amidase to hexa-peptide-8 to carry out acetylation and amidation reactions;

[0019] (5) Purify with ion exchange resin to obtain acetylated hexa-peptide-8.

[0020] Preferably, in step (3), the protein is purified by the method of Ni affinity chromatography column.

[0021] The present invention also discloses a Pro.AH8 acetylated hexa-peptide-8 with the efficacy of firming and anti-wrinkle, which is prepared by the aforementioned biosynthetic method.

[0022] Application of the aforementioned Pro.AH8 acetylated hexa-peptide-8 in the preparation of anti-wrinkle products.

[0023] Advantages of the present invention:

[0024] The present invention provides a method for biosynthesizing hexa - peptide - 8 and acetyl - hexa - peptide - 8, which has stable quality, high safety and low cost. The acetyl - hexa - peptide - 8 synthesized by the present invention has high biological activity, low immunogenicity, good solubility and safety. It can be expressed in a prokaryotic system, with a simple system, high yield and low cost. It has an anti - wrinkle effect and can be used to prepare anti - wrinkle products. Brief Description of the Drawings

[0025] Figure 1 Plasmid map of the expression vector pET30a - hexa - peptide plasmid of the present invention.

[0026] Figure 2 SDS - PAGE gel diagram of the induced expression of hexa - peptide - 8 of the present invention: Lane 1 is the broken precipitate of the induced group of DE3 - pET30a - hexa - peptide - 8 - 1 engineering bacteria, and lane 2 is the broken supernatant of the induced group of DE3 - pET30a - hexa - peptide - 8 - 1 engineering bacteria; Lane 3 is the broken precipitate of the induced group of DE3 - pET30a - hexa - peptide - 8 - 2 engineering bacteria, and lane 4 is the broken supernatant of the induced group of DE3 - pET30a - hexa - peptide - 8 - 2 engineering bacteria.

[0027] Figure 3 HPLC detection chromatogram of acetyl - hexa - peptide - 8 of the present invention

[0028] Figure 4 Cytotoxicity detection results of acetyl - hexa - peptide - 8 of the present invention.

[0029] Figure 5 Cell scratch detection results of acetyl - hexa - peptide - 8 of the present invention. Detailed Embodiments

[0030] The present invention is further illustrated by the following examples, but it is not intended to limit the present invention. The specific materials and their sources used in the embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials with the same or similar types, models, qualities, properties or functions as the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0031] Example 1: Preparation of the hexa - peptide expression vector

[0032] (1) Gene design and synthesis

[0033] In the present invention, an expression vector for hexa-peptide-8 was constructed. The amino acid sequences designed for the expression of hexa-peptide-8 are shown in SEQ ID No.1 and SEQ ID No.2. The amino acid sequence shown in SEQ ID No.1 includes a 6*His sequence, an enterokinase recognition site, and hexa-peptide-8 connected in sequence. SEQ ID No.2 is based on SEQ ID No.1 with an additional amino acid sequence that promotes protein expression to increase the protein expression level. The corresponding coding genes are shown in SEQ ID No.3 and SEQ ID No.4. SEQ ID No.3 and SEQ ID No.4 were designed into primers, and the primer synthesis was entrusted to Sangon Biotech (Shanghai) Co., Ltd.

[0034] Primer design:

[0035]

[0036] (2) Construction of the expression vector

[0037] Using the above primers, PCR amplification was performed. The amplified product was subjected to gel extraction, and the recovered product was subjected to blunt-end T4 ligation to obtain the corresponding plasmid.

[0038] PCR amplification system:

[0039] System components Component volume Primer F 2.5 μL Primer R 2.5 μL 2xSuperNova PCR Mix (Dye) 25 μL pET30a plasmid 1 μL <![CDATA[ddH 2 O]]> To 50 μL

[0040] After preparing the PCR system, mix well and centrifuge. The PCR amplification conditions were as follows: the first stage was pre-denaturation at 98°C for 30 s; the second stage was denaturation at 98°C for 10 s, annealing at 50 - 72°C for 30 s, extension at 72°C for 30 s / kb, with 33 cycles; the third stage was final extension at 72°C for 2 min. The above PCR product was recovered using a universal DNA purification kit (Tiangen Biochemical Technology Co., Ltd.) according to the operating steps in the product manual.

[0041] T4 ligation system:

[0042]

[0043]

[0044] T4 ligation procedure:

[0045] The first stage The second stage The third stage 37 °C, 120 min 65 °C, 30 min 4℃，∞

[0046] The ligation product obtained above was stored on ice or at -20°C for subsequent competent cell transformation.

[0047] The ligation product was transformed into the host bacterium E. coli - DH5α by heat shock method, spread on an LB culture resistance plate, incubated overnight at 37°C. Positive clones were randomly picked and cultured in LB liquid medium overnight at 37°C with 220 rpm shaking. Plasmids were extracted using a plasmid rapid extraction kit. The successfully constructed plasmids pET30a - hexapeptide - 8 - 1 and pET30a - hexapeptide - 8 - 2, and their maps are shown in Figure 1 .

[0048] pET30a is the most commonly used plasmid vector. It can be expected that other commonly used plasmid vectors, such as pET28a and pET32a, can also be used to construct the expression of the amino acids shown in SEQ ID No.1 or SEQ ID No.2.

[0049] (3) Construction of engineered bacteria

[0050] The above - obtained recombinant expression plasmid was transferred into Escherichia coli competent cell BL21(DE3) by heat shock method, and positive Escherichia coli genetically engineered bacteria were screened. The specific process was as follows: ① Take 5 μL of the recombinant expression plasmid and add it to 100 μL of Escherichia coli competent cell BL21(DE3), and let it stand on ice for 30 min; ② Heat - shock the mixture in a 42°C water bath for 90 s, and then quickly place it on ice for 2 min; ③ Add 500 μL of non - resistant LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) to the mixture and culture it at 37°C with 220 rpm for 0.5 h; ④ Take 200 μL of the bacterial solution and evenly spread it on an LB solid medium plate containing kanamycin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 50 μg / mL kanamycin); ⑤ Invert the plate and culture it in a 37°C incubator for about 16 h until clear colonies grow, and the corresponding DE3 - pET30a - hexapeptide - 8 - 1 and DE3 - pET30a - hexapeptide 8 - 2 engineered bacteria were obtained.

[0051] Example 2: Induced expression of engineered bacteria

[0052] The single colonies on the above plate were placed in LB liquid medium containing kanamycin antibiotic and cultured at 37°C with 220 rpm for 10 hours. This was the primary seed liquid. It was inoculated into a new LB medium at an inoculation amount of 1%, cultured overnight at 37°C. This was the secondary seed liquid. Then it was inoculated into a new LB medium at an inoculation amount of 5% and cultured at 37°C for 2 h. IPTG with a final concentration of 0.5 mM was added and induced expression was carried out at 18°C for 20 hours. The cells were collected by centrifugation at 4000 g for 20 min at 4°C.

[0053] The bacterial cells were resuspended in lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 500 mM NaCl, pH 8.5), 100X protease inhibitor PMSF was added, and then the cells were disrupted by sonication for 30 min (sonication for 3 s with a 6 s interval) using a 360 W ultrasonic instrument. After that, 1 mL of the disrupted solution was centrifuged at 4 °C and 10,000 g for 10 min. 80 μL of the supernatant was taken, the precipitate was resuspended in 1000 μL of lysis buffer, 80 μL of the resuspended solution was taken, and 20 μL of 5X protein loading buffer was added to both the supernatant and the precipitate. After mixing, they were heated in a boiling water bath for 10 min for SDS-PAGE electrophoresis. The results were as Figure 2 shown. Under the induction condition, both the constructed engineering bacteria DE3-pET30a-hexapeptide-8-1 and DE3-pET30a-hexapeptide-8-2 had protein expression, and the protein expression level of DE3-pET30a-hexapeptide-8-2 was significantly higher than that of DE3-pET30a-hexapeptide-8-1. According to the gray scale analysis of the SDS-PAGE gel, the expression level of DE3-pET30a-hexapeptide-8-1 was 0.2494 mg / mL; the expression level of DE3-pET30a-hexapeptide-8-2 was 0.6317 mg / mL. The following experiments were all carried out using DE3-pET30a-hexapeptide-8-2 for expression, purification, and bioactivity detection experiments.

[0054] Example 3: Purification of the expression product

[0055] The disrupted solution obtained in Example 2 was centrifuged at 10,000 g and 4 °C for 30 min, and the supernatant was collected. The Ni affinity column material was washed with distilled water and equilibrated with buffer 1 (25 mM Tris, 200 mM NaCl, pH 8.0). The sample was loaded, and the miscellaneous proteins were rinsed with a washing buffer containing 20 mM imidazole (20 mM imidazole, 25 mM Tris, 200 mM NaCl, pH 8.0), and the target protein was eluted with a solution containing 250 mM imidazole (250 mM imidazole, 25 mM Tris, 200 mM NaCl, pH 8.0); the column material was washed with a solution containing 1 M imidazole, then with distilled water, and finally filled with 20% ethanol.

[0056] The protein solution purified by the Ni affinity column was desalted by a desalting column to remove the high-concentration imidazole. Then, enterokinase was added to the protein solution, and the reaction was carried out overnight at 25 °C to remove the His tag. Glutamate acetyltransferase, acetyladenosine, and amidase were added to the protein solution, and the acetylation and amidation reactions were carried out in a 37 °C water bath for 30 min. Then, it was centrifuged at 4 °C and 10,000 g for 30 min, and the supernatant was collected for enrichment and purification by positive ion exchange resin to obtain acetylated hexapeptide-8.

[0057] The collected acetyl hexapeptide-8 was subjected to liquid phase detection. Instrument: Agilent 1260 high performance liquid chromatograph, chromatographic column: Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm), column oven at 35 °C; mobile phase A: ultrapure water, mobile phase B: acetonitrile; flow rate 1.0 mL / min, injection volume 20 μL, detection wavelength 215 nm, gradient elution was used, elution program: 0 min, A: 95%, B: 5%; 2.5 min, A: 95%, B: 5%; 10 min, A: 40%, B: 60%; 12.5 min, A: 40%, B: 60%; 12.6 min, A: 95%, B: 5%; 15 min, A: 95%, B: 5%. The liquid chromatogram is shown in Figure 3 , with only a single significant peak and a purity greater than 99%.

[0058] Example 4: Biological activity detection

[0059] The acetyl hexapeptide-8 obtained in Example 3 was subjected to cytotoxicity and cell scratch experiments

[0060] (1) Preliminary experiment (cytotoxicity experiment)

[0061] Cytotoxicity detection. The sample was dissolved in ultrapure water. The results of the cytotoxicity experiment are as Figure 4 shown. It can be seen that the prepared acetyl hexapeptide-8 has no cytotoxicity.

[0062] (2) Formal experiment (change in acetylcholine content of sample on neuron cells)

[0063] Cell seeding: Neuron cells were seeded in 6-well plates, 6×10 5 cells per well, cultured at 37 °C, 5% CO 2 for 24 h. A normal control group (without sample) and a sample group (aqueous solution of 0.1% acetyl hexapeptide-8) were set up respectively.

[0064] Drug administration: The corresponding concentration of medium containing the sample was added to the sample group, the normal control group was replaced with fresh medium, and the blank group was added with blank medium, incubated at 37 °C, 5% CO 2 for 24 h.

[0065] Sample collection and detection: After incubation, the cell culture supernatant was collected from each well, and the acetylcholine content was detected by ELISA method.

[0066] The results are as Figure 5 shown. Compared with the control group, the acetylcholine content in the sample group was significantly down-regulated, indicating that the sample has anti-wrinkle efficacy.

Claims

1. A plasmid expression vector of hexapeptide-8, characterized in that: The amino acid sequence expressed by the plasmid expression vector is shown as SEQ ID No.

2.

2. A host bacterium containing the expression vector according to claim 1.

3. The host bacteria according to claim 2, characterized in that The host bacteria is Escherichia coli.

4. A biosynthetic method of hexapeptide-8, characterized in that: The steps include: (1) Constructing the plasmid expression vector of hexapeptide-8 as described in claim 1, and transforming it into a host bacterium; (2) Cultivate the expression host bacteria and induce protein expression; (3) Purify the protein and digest the purified protein with enterokinase to obtain hexapeptide-8.

5. The biosynthesis method according to claim 4, characterized in that In step (3), the protein is purified using a Ni affinity chromatography column.

6. A biosynthetic method of acetyl hexapeptide-8, characterized in that: The steps include: (1) Constructing the plasmid expression vector of hexapeptide-8 as described in claim 1, and transforming it into a host bacterium; (2) Cultivate the expression host bacteria and induce protein expression; (3) Purify the protein, and digest the purified protein with enterokinase to obtain hexapeptide-8; (4) Add glutamate acetyltransferase, acetyladenosine and amidating enzyme to hexapeptide-8 to carry out acetylation and amidation reactions; (5) Purification by ion exchange resin to obtain acetyl hexapeptide-8.

7. The biosynthesis method according to claim 6, characterized in that In step (3), the protein is purified using a Ni affinity chromatography column.

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