Transdermal analgesic peptide and preparation method and application thereof

Abstract

The application relates to a transdermal analgesic peptide and a preparation method and application thereof, and relates to the field of biological medicines. The transdermal analgesic peptide has an amino acid sequence of MRQIKIWFQNRRMKWKKWYKQSTGPSYYSVGRASGLLSGIRRSPDI. The preparation method of the transdermal analgesic peptide is that a neuropeptide B gene is combined with a transdermal peptide ANTP gene through a gene recombination technology, and the transdermal analgesic peptide is obtained by induction expression. The transdermal analgesic peptide obtained by creatively fusing a cell-penetrating peptide with the neuropeptide B has good cell-penetrating effect and analgesic effect, and overcomes defects such as strong addiction and large side effects of commercially available analgesics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a polypeptide. BACKGROUND

[0002] Inflammatory pain is caused by various stimulating factors to release inflammatory factors by mast cells, macrophages, neutrophils and nerve endings, causing local tissue inflammatory response. Because pain is often intertwined with autonomic nervous activity, psychological and emotional reactions, there is great variability, so it is more difficult to study than other senses.

[0003] Morphine as a representative of narcotic analgesics and aspirin as a representative of antipyretic analgesic anti-inflammatory drugs are commonly used analgesic drugs. Opioid analgesics have strong analgesic effect, but the problem of addiction greatly limits its clinical application, and the therapeutic effect on chronic pain is not ideal, and is usually used for patients with advanced cancer. Non-steroidal anti-inflammatory analgesic is the first choice for the treatment of inflammatory pain, although there is no addiction problem, but its analgesic effect is weak, and serious gastrointestinal reactions are prone to occur, and large doses or long-term use can cause liver damage, kidney damage, and affect blood clotting function. SUMMARY

[0004] The purpose of the present application is to provide a transdermal analgesic peptide to solve the above technical problems.

[0005] The purpose of the present application is also to provide a preparation method of transdermal analgesic peptide for preparing transdermal analgesic peptide.

[0006] The purpose of the present application is also to provide the application of transdermal analgesic peptide.

[0007] The technical problems solved by the present application can be solved by the following technical solutions:

[0008] The transdermal analgesic peptide is characterized in that the amino acid sequence is:

[0009] MRQIKIWFQNRRMKWKKWYKQSTGPSYYSVGRASGLLSGIRRSPDI.

[0010] The preparation method of transdermal analgesic peptide is characterized in that the neuropeptide B gene is combined with the transdermal peptide ANTP gene by gene recombination technology, and the transdermal analgesic peptide is obtained by inducing expression.

[0011] Specifically, the method comprises the following steps: step one, preparing BL21(DE3) / pET32a-TrxD-ANTP-BgNPB, wherein the BL21(DE3) / pET32a-TrxD-ANTP-BgNPB contains EcoR I and Hind III restriction endonuclease sites; step two, inducing expression of the fusion protein TrxD-ANTP-BgNPB in the BL21(DE3) / pET32a-TrxD-ANTP-BgNPB; step three, optimizing the fusion protein TrxD-ANTP-BgNPB; and step four, obtaining a mature fusion protein A NTP-BgNPB solution after separating and purifying the fusion protein TrxD-ANTP-BgNPB, wherein the mature fusion protein A NTP-BgNPB solution contains the transdermal analgesic peptide.

[0012] The application of the transdermal analgesic peptide is characterized in that the transdermal analgesic peptide is used for preparing an external analgesic.

[0013] The application of the transdermal analgesic peptide is characterized in that the transdermal analgesic peptide is used for preparing a cosmetic with an anti-inflammatory analgesic effect.

[0014] The transdermal analgesic peptide obtained by fusing the transmembrane peptide and the neuropeptide B has good transmembrane effect and analgesic effect, and overcomes the defects of strong addiction and large side effects of commercially available analgesics.

[0015] 1. High safety and small toxic side effects;

[0016] 2. Convenient transdermal administration and good patient compliance;

[0017] 3. Targeted treatment and strong specificity.

[0018] 4. Strong transdermal analgesic activity. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a protein surface conformation diagram;

[0020] Figure 2 is an active site diagram of the transdermal analgesic peptide;

[0021] Figure 3 is a pain threshold improvement rate comparison diagram. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application is further described below in combination with specific diagrams.

[0023] Transdermal analgesic peptide

[0024] The amino acid sequence of the transdermal analgesic peptide is:

[0025] MRQIKIWFQNRRMKWKKWYKQSTGPSYYSVGRASGLLSGIRRSPDI.

[0026] Transdermal analgesic peptides are preferably in lyophilized powder form, which makes them convenient to use and facilitates absorption.

[0027] Preparation method of transdermal analgesic peptide

[0028] The method for preparing transdermal analgesic peptides involves combining the neuropeptide B gene with the transdermal peptide ANTP gene using gene recombination technology, and inducing expression to obtain transdermal analgesic peptides. Specific Implementation

[0030] Step 1: Preparation of BL21(DE3) / pET32a-TrxD-ANTP-BgNPB. BL21(DE3) / pET32a-TrxD-ANTP-BgNPB contains EcoR I and Hind III restriction endonuclease sites. The specific operation is as follows:

[0031] 1.1 ANTP-BgNPB gene amplification

[0032] 1) Amplify the target gene using PCR.

[0033] The whole-gene synthesis method with codon optimization was used to obtain the fragment. The synthesized whole-gene fragment was inserted into the EcoRI and Hind III restriction endonuclease sites of the pET28a(+) plasmid. The target gene was amplified from the pET28a-ANTP-BgNPB plasmid using PCR to obtain the ANTP-BgNPB fragment containing EcoRI and Hind III restriction endonuclease sites.

[0034] The table below shows the PCR amplification system (50 μL).

[0035]

[0036] The table below shows the PCR amplification conditions.

[0037]

[0038]

[0039] 2) The ANTP-BgNPB fragment was obtained by agarose gel electrophoresis and gel recovery.

[0040] Agarose gel electrophoresis: Add an appropriate amount of nucleic acid dye to the uniformly melted 1% agarose gel, mix well, pour into the assembled gel plate, and let stand until the gel solidifies; place the gel and gel plate into the electrophoresis tank, so that the 1x TAE electrophoresis buffer covers the gel surface by about 2 mm; mix the DNA sample and 10x loading buffer in proportion, add an appropriate amount of sample and marker to the gel wells; electrophoresis at 130V for 30 min; observe and photograph the gel under UV light using a gel imaging system, and cut out the agarose gel containing the target DNA and store it in a 1.5mL EP tube.

[0041] Gel recovery: Calculate the gel weight (as one gel volume); add 3 gel volumes of Buffer DE-A and heat at 65°C, mixing intermittently until the gel block is completely melted; add 1.5 gel volumes of Bufftr DE-B, mix thoroughly until the solution turns yellow (if the isolated DNA fragment is less than 400 bp, add another gel volume of isopropanol); transfer the mixture to the assembled preparation tube, centrifuge at 12,000 x g for 1 min, and discard the filtrate; add 500 μL of Buffer W1, centrifuge at 12,000 x g for 30 s, and discard the filtrate; add 700 μL of Buffer W2, centrifuge at 12,000 x g for 30 s, and discard the filtrate (repeat twice); centrifuge an empty column at 12,000 x g for 1 min; place the preparation tube in a clean 1.5 mL centrifuge tube, open the cap and let stand for 8 min (to evaporate ethanol), and add 25-30 μL of [unclear text - possibly a specific ingredient or solution] to the center of the membrane. Use 65℃ deionized water, let stand for 2 min, centrifuge at 12,000xg for 1 min to elute DNA (the eluted liquid can be repeated once); finally, use an ultra-micro spectrophotometer to determine the concentration of DNA fragments.

[0042] 1.2 Construction of vector pET32a-TrxD-ANTP-BgNPB

[0043] 1) Obtain the target ANTP-BgNPB fragment with sticky ends and the vector pET32a fragment by double digestion with EcoR I / Hind III restriction endonucleases.

[0044] The table below shows the double enzyme digestion reaction system (20 μL).

[0045] Reagent Amount Plasmid DNA / Fragment DNA 1 μg / 500 ng 10x H buffer 2 μL EcoR I endonuclease 1 μL Hind III endonuclease 1 μL Sterile water Make up to 20 μL

[0046] Double enzyme digestion conditions: 37℃ constant temperature metal bath for 8 hours.

[0047] 2) Purify the ANTP-BgNPB and pET32a fragments using a PCR / enzyme digestion product purification kit.

[0048] Add 3 sample volumes of Buffer PCR-A to the enzyme digestion reaction solution (if Buffer PCR-A is less than 100 μL, add to 100 μL), mix well, and transfer to the assembled preparation tube. Centrifuge at 12,000 x g for 1 min and discard the filtrate. Add 700 μL of Buffer W2, centrifuge at 12,000 x g for 1 min, and discard the filtrate. Add 400 μL of Buffer W2, centrifuge at 12,000 x g for 1 min, and centrifuge the tube empty at 12,000 x g for 1 min. Place the preparation tube in a clean 1.5 mL centrifuge tube, open the cap, and let it stand for 8 min. Add 25-30 μL of 65℃ deionized water to the center of the membrane in the preparation tube, let it stand at room temperature for 1-2 min, and centrifuge at 12,000 x g for 1 min to elute the DNA (the elution liquid can be repeated once). Finally, use a micro spectrophotometer to determine the concentrations of the ANTP-BgNPB fragment and the pET32a fragment.

[0049] 3) Construct the vector pET32a-TrxD-ANTP-BgNPB using T4 ligase.

[0050] The table below shows the Ligase reaction system (20 μL).

[0051] Reagent Amount Plasmid DNA 6 μL Fragment DNA 10 μL T4 buffer 2 μL T4 Ligase 2 μL

[0052] T4 ligase reaction conditions: incubate overnight in a constant temperature metal bath at 16°C.

[0053] 1.3 Recombinant Clonal Strains and Expression Strains

[0054] 1) Thermal shock transformation of plasmids

[0055] Remove 50 μL of competent cells from the -80℃ freezer and thaw them on ice (about 5 min). Add 2 μL of target DNA (2 μL of plasmid or 10 μL of ligation product) to the EP tube in a clean bench and gently mix by tapping the bottom of the tube. Let it stand on ice for 25 min. Heat shock it in a 42℃ water bath for 45 s, then quickly return it to ice and let it stand for 2 min. Add 350 μL of antibiotic-free sterile LB medium to the EP tube in a clean bench, mix well, and incubate at 37℃ and 200 rpm for 60 min. Centrifuge at 5000 rpm for 1 min to collect the cells, collect about 100 μL of supernatant, resuspend the cells, and spread them onto LB (LB / Amp) solid plates containing 0.1 mg / mL Amp. Invert the plates and incubate overnight at 37℃.

[0056] 2) Colony PCR verification

[0057] Inside the clean bench, select 6 single colonies of varying sizes and shapes from each LB / Amp plate and incubate them overnight (at least 6 hours) in an EP centrifuge tube containing 1 mL of LB / Amp on a shaker at 37°C and 200 rpm / min.

[0058] The table below shows the colony PCR amplification system (20 μL).

[0059] Reagent Amount Upstream primer T7 (10 uM) 0.8 μL Downstream primer T7t (10 uM) 0.8 μL 2x MasterMix (with dye) 10 μL Bacterial solution 1 μL Sterile water Make up to 20 μL

[0060] The table below shows the colony PCR reaction procedure.

[0061]

[0062]

[0063] 3) Agarose gel electrophoresis

[0064] The steps are the same as above. Compare the markers to determine if the band sizes are correct. Send the bacterial culture with the correct PCR band size to a sequencing company for sequencing and identification.

[0065] 4) Agarose gel electrophoresis

[0066] After successful sequencing, the bacterial culture was mixed with 50% sterile glycerol at a 1:1 ratio, labeled, and stored at -80°C.

[0067] Step 2: Induce expression of the fusion protein TrxD-ANTP-BgNPB in BL21(DE3) / pET32a-TrxD-ANTP-BgNPB. The specific procedures are as follows:

[0068] 2.1 Expression of fusion protein

[0069] 1) Activation of microbial strains

[0070] Remove the glycerol bacterium BL21(DE3) / pET32a-TrxD-ANTP-BgNPB from the -80℃ freezer and thaw it on ice; take 10 μL of bacterial culture and streak it on LB / Amp solid medium for activation, and incubate it in a constant temperature incubator at 37℃ for 10-14 h; pick a single colony and add it to an EP tube containing 1 mL of LB / Amp, and incubate it overnight (10-14 h) at 37℃ and 200 rpm on a shaker to make a seed culture.

[0071] 2) Prokaryotic induced expression

[0072] At a 2% inoculation rate, add 500 μL of bacterial suspension to a conical flask containing 25 mL LB / Amp and incubate at 37℃ and 200 rpm for 3-4 h. Measure the absorbance (OD) of the bacterial suspension at 600 nm using a UV-9100 spectrophotometer. When the bacterial concentration reaches 0.6-1.0, add 5 μL of 1M IPTG (final concentration 0-5.0 mM) to the bacterial suspension and incubate at 16℃-37℃ and 200 rpm for 1-12 h.

[0073] 3) Collect bacterial cells

[0074] Transfer the bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 4 °C and 8000 rpm / min for 10 min, and discard the supernatant. Resuspend the bacterial cells in an appropriate amount of PBS, centrifuge at 4 °C and 8000 rpm / min for 10 min, and discard the supernatant. Repeat this process 3 times. Then, resuspend the bacterial cells in 1 mL of PBS. Dilute the bacterial culture 100 or 200 times, measure the OD600 using a spectrophotometer, and calculate the dilution or concentration based on the principle that 1 mL of bacterial culture with OD=2 should be concentrated to 100 μL (OD=20). Add a certain amount of protease inhibitor (mixed protease inhibitor, PMSF, EDTA, etc.) and finally store the bacterial culture at 4 °C for short-term storage or at -80 °C for long-term storage.

[0075] 4) Obtain soluble and insoluble protein solutions

[0076] Remove the pre-treated bacterial culture from -80℃ and thaw it in a 37℃ water bath (approximately 30 minutes). Pre-cool it in a 4℃ freezer for 30 minutes, then freeze it again at -80℃ (approximately 30 minutes). Repeat this process 5 times. Add lysozyme solution to the bacterial culture to a final concentration of 0.25 mg / mL, mix thoroughly by inverting, and incubate on ice for 30 minutes. Then incubate at 37℃ for 5 minutes (inverting and mixing every 30 seconds during this period), and finally incubate on ice for another 30 minutes.

[0077] Add an appropriate amount of sterile glass beads with a diameter of 1 mm to the bacterial culture and sonicate them. Place the culture on ice throughout the process. The sonication conditions are: power 100W, working time 5s, interval 10s, sonication time 10-45min. Finally, centrifuge at 4℃ and 12000rpm / min for 20min, collect the supernatant, adjust the pH to 7.4 to obtain a soluble protein solution. Resuspend the precipitate in an equal volume of PBS and mix well. Remove the glass beads and centrifuge at 4℃ and 12000rpm / min for 10min. Discard the supernatant and dissolve the protein in 8M urea. Finally, centrifuge at 4℃ and 12000rpm / min for 10min. The supernatant is the treated insoluble protein solution (containing inclusion bodies).

[0078] 2.2 SDS-PAGE identification of recombinant protein expression

[0079] The expression of recombinant proteins in Escherichia coli was identified using a 15% separating gel and a 5% stacking gel.

[0080] Step 3: Optimize the conditions for the fusion protein TrxD-ANTP-BgNPB. The specific steps are as follows:

[0081] 3.1 Optimization of IPTG Concentration as an Inducer

[0082] Glyceryl bryophyte BL21(DE3) / pET32a and BL21(DE3) / pET32a-TrxD-ANTP-BgNPB plates were activated and cultured overnight to obtain seed culture. The seed culture was inoculated into 25 mL / 250 mL LB / Amp medium shake flasks at a 2% inoculation rate and cultured at 37°C and 200 rpm. When BL21(DE3) / pET32a cells reached mid-logarithmic growth (OD600 = 1.0), 0.3 mM IPTG solution was added for induction. When BL21(DE3) / pET32a-TrxD-ANTP-BgNPB cells reached mid-logarithmic growth, different amounts of 1 M IPTG solution were added for induction, resulting in final IPTG concentrations of 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 1.0 mM, 2.5 mM, and 5.0 mM. Soluble proteins were collected from the bacterial cells after culturing at 30℃ for 5 h, and the expression level of soluble recombinant proteins was identified using SDS-PAGE.

[0083] 3.2 Optimization of Induction Temperature

[0084] When the bacterial cells BL21(DE3) / pET32a-TrxD-ANTP-BgNPB reached the mid-logarithmic growth phase (OD600 = 1.0), 5 μL of 1M IPTG (final concentration 0.2 mM) was added for induction. The cells were cultured at 16℃, 20℃, 25℃, 30℃, and 37℃ for 5 h, respectively. The soluble protein in the bacterial cells was collected, and the expression level of the soluble recombinant protein was identified by SDS-PAGE.

[0085] 3.3 Optimization of Induction Time

[0086] When the bacterial cells BL21(DE3) / pET32a-TrxD-ANTP-BgNPB reached the mid-logarithmic growth phase (OD600 = 1.0), 5 μL of 1M IPTG (final concentration 0.2mM) was added for induction. The cells were cultured at 30℃ for 1h, 2h, 3h, 4h, 5h, 6h, 7h, and 8h, respectively, and the soluble protein in the cells was collected. The expression level of the soluble recombinant protein was identified by SDS-PAGE.

[0087] 4. Western Blot identification of recombinant protein expression

[0088] 4.1 Induced expression and electrophoresis

[0089] Glyceryl bacillus BL21(DE3) / pET32a and glyceryl bacillus BL21(DE3) / pET32a-TrxD-ANTP-BgNPB plates were activated and cultured overnight to obtain seed culture. The seed culture was inoculated into 25 mL LB / Amp medium shake flasks at a 2% inoculation rate for expansion. When the cells reached mid-logarithmic growth (OD600 = 1.0), both bacteria were induced under optimized conditions. A separate batch of BL21(DE3) / pET32a-TrxD-ANTP-BgNPB was prepared without an inducer. Soluble proteins from the cells were collected and electrophoresed using a 15% SDS-PAGE gel.

[0090] 4.2 Transfer of film

[0091] After removing the gel, cut off a piece of gel between 15 kDa and 35 kDa (the fusion protein TrxD-ANTP-BgNPB is approximately 25 kDa), place it in a dish with a small amount of electrophoresis buffer, and store at 4°C for later use. Cut a PVDF membrane the same size as the gel and two pieces of thick filter paper slightly smaller than the gel. Always wear clean gloves during the cutting process, otherwise protein on your hands will contaminate the membrane. Make a notch in the upper left corner of the smooth side of the PVDF membrane as a mark. Soak the membrane in methanol for 10 minutes beforehand, then soak it, the thick filter paper, clean tweezers, and a 50 mL centrifuge tube in electrophoresis buffer for at least 30 minutes.

[0092] 4.3 Enclosure

[0093] After the transfer was completed, the PVDF membrane was immersed in the blocking solution (5% skim milk powder in TBST solution) and blocked on a decolorizing shaker at room temperature for 1-3 hours.

[0094] 4.4 Primary Antibody Incubation

[0095] After blocking, the PVDF membrane was thoroughly washed with TBST on a decolorizing shaker at room temperature, at least 6 times, 5 min each time. The primary antibody was thawed at 4°C and diluted to an appropriate concentration with blocking buffer, then the PVDF membrane was immersed and incubated overnight at 4°C or at room temperature for 1-2 h.

[0096] 4.5 Secondary Antibody Incubation

[0097] After the primary antibody incubation is complete, wash the PVDF membrane thoroughly with TBST, washing 6 times on a decolorizing shaker for 5 minutes each time. Similarly, dilute the secondary antibody according to the instructions, immerse the PVDF membrane in the secondary antibody dilution solution, and incubate at room temperature for 1-2 hours.

[0098] 4.6 Chemiluminescence Developing

[0099] After the secondary antibody incubation, wash the PVDF membrane with TBST at least 6 times, 5 minutes each time. Mix the ECL developer and stabilizer in a 1:1 ratio. Add an appropriate amount of the ECL mixture and evenly cover the smooth surface of the PVDF membrane, then incubate in the dark for 5 minutes. After incubation, expose the membrane to light using a chemiluminescence analyzer and take photographs to record the results.

[0100] Step 4: After separating and purifying the fusion protein TrxD-ANTP-BgNPB, a mature fusion protein ANTP-BgNPB solution is obtained. The mature fusion protein ANTP-BgNPB solution contains transdermal analgesic peptides. The specific operation is as follows:

[0101] 5.1 Treatment of Fermentation Broth

[0102] After activating the glycerol bacterium BL21(DE3) / pET32a-TrxD-ANTP-BgNPB, the fermentation broth was induced under optimized conditions. The total fermentation broth volume was 500 mL. The broth was then crushed and concentrated to obtain 10 mL of soluble protein solution.

[0103] 5.2 Affinity chromatography purification

[0104] Protein purification was performed using NI-IDA-agarose gel FF. The gel was equilibrated with 5 column volumes (1 mL) of equilibration buffer W0. 10 mL of concentrated soluble protein solution was added and passed through the NI-IDA-agarose gel FF, and flow-through was collected (F1). The gel was then washed with 5 column volumes of equilibration buffer W0, and flow-through was collected (F2). Elution was performed using 5 column volumes of elution buffers W1-W5 containing different concentrations of imidazole (10 mM, 20 mM, 30 mM, 50 mM, 100 mM), in a gradient from low to high concentration, and flow-through was collected for each imidazole concentration (F3-F7). Finally, the protein on the NI-IDA-agarose gel FF was eluted with 2 column volumes of elution buffer W6 containing 500 mM imidazole, and flow-through was collected (F8). After using the NI-IDA-agarose gel FF, wash it with 2 column volumes of elution buffer W7, then equilibrate it 3 times with equilibration buffer W0, and finally fill it with stock solution W8 and store it at 4°C.

[0105] Replace the imidazole solution in flow-through solutions F3-F8 with equilibration buffer W0 using a 3kDa ultrafiltration tube. Perform SDS-PAGE and Western blot analysis on all flow-through solutions F1-F8 to verify whether the fusion protein TrxD-ANTP-BgNPB was eluted and separated.

[0106] 5.3 Enterokinase cleavage to isolate fusion protein

[0107] The concentration of the fusion protein TrxD-ANTP-BgNPB in the flow-through solution was determined using a BCA kit. Following the Solarbiok enterokinase instructions, the fusion protein concentration was 0.1-1 mg / mL (total protein 0.5-1.0 mg) in a 50 mM Tris-HCl pH 80 system, using 1-2 μL, and digested at 25°C in a metal bath for 12-16 h. After the double digestion reaction, the fusion protein ANTP-BgNPB (approximately 6 kDa) in the reaction solution was separated from other components using 3 kDa and 10 kDa ultrafiltration tubes (12000 rpm / min, 10 min each time). The obtained mature fusion protein ANTP-BgNPB solution was freeze-dried into powder and stored at -80°C.

[0108] Assay of the anti-inflammatory and transmembrane activity of transdermal analgesic peptides

[0109] 1.1 Hot Plate Method

[0110] 1) Two to three days before the experiment, the backs of 30 eight-week-old male ICR mice were shaved using a razor followed by depilatory cream, resulting in a 4 cm² hairless area on the backs of the mice. They were randomly divided into four groups: a blank control group (NS, physiological saline), a positive control group (DDE, diclofenac diethylamine cream), a low-dose ANTP-BgNPB group (L-AB, 5 mg / kg), and a high-dose ANTP-BgNPB group (H-AB, 50 mg / kg), with seven mice in each group.

[0111] 2) Adjust the constant temperature water bath to 55℃±0.2℃ and place a 1L beaker inside. Place mice from each group into the beaker sequentially, immediately start a timer, and observe and record the time required from placement to the start of licking the hind paw. This time is used as the pain threshold. Predict the pain threshold twice, with a 10-minute interval between each prediction. Mice with an average pain threshold between 5 and 30 seconds are considered acceptable. A pain threshold less than 5 seconds indicates hypersensitivity, while a threshold greater than 30 seconds indicates sluggishness. Mice with either hypersensitivity or sluggishness should not be used.

[0112] 3) At least 5 mice with acceptable pain thresholds should be selected for each group. The drug should be applied topically to the hairless area, with each mouse receiving 0.2 mL of the drug. The pain thresholds of the four groups of mice should be measured 60 min and 70 min after administration, using the same method. If a mouse shows no pain response within 60 seconds after administration, it should be removed immediately to avoid burns. The pain threshold is calculated based on a 60-second timeframe.

[0113] The table below shows the hot plate test results for mice.

[0114]

[0115] The percentage increase in pain threshold at different time points after medication was calculated using the following formula: Pain threshold increase rate (%) = (Pain threshold after medication - Pain threshold before medication) / Pain threshold before medication × 100%.

[0116] 1.2 Statistical Analysis

[0117] Data are expressed as mean ± standard deviation. Data analysis was performed using SPSS 26.0 statistical software (IBM, Armonk, USA). The Kolmogorov-Smirnov test was used to determine the normality and homogeneity of variance of all data. One-way ANOVA or chi-square test was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant. For details on the improvement rate of pain relief using the hot plate test in the blank control group NS (normal saline NS), the positive control group DDE (diclofenac diethylamine cream DDE), the low-dose ANTP-BgNPB group L-AB (5mg / kg), and the high-dose ANTP-BgNPB group H-AB (50mg / kg), please refer to [link to relevant documentation]. Figure 1 Note: Results are the mean ± standard deviation of each group; *P<0.05, **P<0.01 compared with physiological saline (60 min later), #P<0.05, ##P<0.01 compared with physiological saline (70 min later). Experimental results are as follows. Figure 1 As shown, both the low-concentration (5 mg / kg) and high-concentration (50 mg / kg) transdermal analgesic peptides resulted in a positive increase in the pain threshold rate in mice using the hot plate test, indicating good transdermal analgesic activity. Furthermore, the high-concentration (50 mg / kg) transdermal analgesic peptide exhibited stronger transdermal analgesic activity than the commercially available diclofenac diethylamine cream (DDE).

[0118] Transdermal analgesic peptides can be used to prepare topical analgesics and cosmetics with anti-inflammatory and analgesic effects. Neuropeptide B is widely distributed in the central and peripheral nervous systems, functioning as a neurotransmitter or neuromodulator and participating in biological functions such as pain transmission, neuroinflammatory reactions, and vasodilation. Transdermal peptides can promote transdermal drug delivery, helping proteins or drugs enter the body through the skin. Transdermal drug delivery is a novel route of administration. It offers numerous advantages and has fewer side effects. (Appendix) Figure 2 This is a protein surface conformation diagram of the transdermal analgesic peptide binding to G protein-coupled receptor 7 (GPR7). The transdermal analgesic peptide appears in red and blue, while GPR7 appears in gray. Figure 3This diagram shows the active sites of transdermal analgesic peptides. As can be seen, the active sites are WYK at the N-terminus and GRAAGLLSGI at the C-terminus. Peptides are a class of compounds whose molecular structure lies between amino acids and proteins, enabling proteins to possess certain physiological functions. In human life activities, peptides are digested and absorbed more readily than free amino acids and have a unique in vivo transport system. Peptide drugs offer advantages such as lower dosage, higher selectivity, better specificity, better efficacy, fewer side effects, ease of synthesis, modification, and optimized combination, allowing for rapid determination of their medicinal value.

[0119] The foregoing has shown and described 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 to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A transdermal analgesic peptide, characterized in that, Its amino acid sequence is MRQIKIWFQNRRMKWKKWYKQSTGPSYYSVGRASGLLSGIRRSPDI.

2. The method for preparing the transdermal analgesic peptide according to claim 1, characterized in that, Transdermal analgesic peptides were obtained by combining the neuropeptide B gene with the transdermal peptide ANTP gene using gene recombination technology and inducing their expression.

3. The application of the transdermal analgesic peptide according to claim 1, characterized in that, Used in the preparation of topical analgesics.

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