A multifunctional hymenoptera skin peptide tsin4 and its application

Abstract

The present invention discloses a multifunctional skin peptide tsin4 of the common salamander and its application. The skin peptide tsin4 of the common salamander is obtained by collecting and isolating the skin secretions of the common salamander Qinba, and its amino acid sequence is Val Ala Phe Ile Gly His Cys Lys Gly Lys Thr Leu Arg Pro Lys Cys Phe. Tsin4 has good antibacterial activity and can inhibit the tested Gram-positive and Gram-negative bacteria with a low MIC value. At the same time, tsin4 has good inflammation-regulating activity and can activate mast cells to release histamine and β-aminoglycosidase. Experimental results also show that tsin4 has no toxicity to mammalian cells and has good safety. For exogenous pathogens, tsin4 can not only directly exert an antibacterial effect, but also can activate the host immune system by regulating the inflammatory response, thereby achieving the purpose of anti-infection, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and in particular relates to a multifunctional skin peptide tsin4 of Hystrix davidianus and an application thereof. Background Art

[0002] For 2 billion years, before organisms evolved an acquired immune system, the primitive innate immune system constituted the most basic defense system of organisms. Compared with acquired immunity, innate immunity is characterized by rapid and timely immune response, non-specific immune targets, and diverse immune responses. Antimicrobial peptides (AMPs), as the host's primary defense factor against invasion by external pathogenic microorganisms, are an important component of the innate immune system of living organisms. AMPs are encoded by specific genes in organisms and are widely present in nature in prokaryotes and eukaryotes such as plants, insects, amphibians, and mammals.

[0003] Antimicrobial peptides were first discovered due to their antibacterial properties, hence their name. In recent years, the overuse of antibiotics has exerted significant selective pressure on pathogens and environmental microorganisms, inducing the emergence of microbial strains with multi-antibiotic resistance, posing a serious threat to human health. Antimicrobial peptides, with their small molecular weight, good water solubility, low antigenicity, and high stability, are particularly resistant to developing drug resistance. Therefore, they hold promise as promising treatments for diseases caused by drug-resistant pathogens.

[0004] The transition of amphibians from aquatic to terrestrial life was a significant event in the history of biological evolution, ushering in the era of biological evolution beyond the constraints of water. In animal taxonomy, the class Amphibians is divided into the orders Urodela, Anura, and Gymnophiona. Among these, the family Hymenoptera is considered to be a group of tailed amphibians endemic to Asia. The Qinba giant salamander (Ranodon tsinpaensis) was first discovered in Zhouzhi, Shaanxi Province. Further research has led to its current name, the Qinba giant salamander (Liua tsinpaensis), and is reportedly found in Shaanxi, Sichuan, and Henan Provinces of my country. Due to their unique amphibious habitats, amphibians are highly susceptible to microbial invasion and invasion. Consequently, over the course of their long evolutionary history, they have developed a well-developed innate immune defense system, of which antimicrobial peptides are a key component. Previous studies have reported that frogs and giant salamanders can produce antimicrobial peptides with broad-spectrum antibacterial activity, but there are few reports on antimicrobial peptides from amphibians of the family Hynobiidae.

[0005] Currently, research on peptides derived from frogs is relatively in-depth. Frogs not only express antimicrobial peptides; different frog species produce a variety of peptides with diverse biological activities, including antiviral, antifungal, antiparasitic, insecticidal, antioxidant, enzyme inhibitory, immunomodulatory, and antitumor properties. Research on active peptides from tailed amphibians has largely focused on the giant salamander, with the majority of reports focusing on antimicrobial peptides, but some peptides also possess other biological activities. For example, the antimicrobial peptides Cathelicidin-ME1 and Cathelicidin-ME2 (invention patent applications CN107759679A and CN107663234A) not only exhibit antimicrobial activity but also increase blood albumin and antibody levels in piglets, as well as blood T lymphocyte transformation rate and IL-2 levels. However, the antimicrobial activity of Cathelicidin-ME1 and Cathelicidin-ME2 is relatively weak, with minimum inhibitory concentrations (MICs) ≥125 μg / mL. Invention patent CN106047968B reported two antioxidant peptides that can increase the IgG level, macrophage and T cell counts in mouse serum, but there is no report that these two peptides have antibacterial activity. Similarly, invention patent application CN111363773A improved the processing method to obtain a giant salamander active peptide with a high F value, which is actually a mixture. This active peptide increased the IgG level in mouse serum and also had antioxidant activity, but no antibacterial activity was reported. Currently, there are even fewer studies on the antibacterial peptides of small salamanders. Invention patent application CN102764273A reported an active peptide derived from the Xinjiang giant salamander of the Hynobiidae family, but only reported that it had anti-tumor activity.

[0006] Mast cells are granulocytes, a type of white blood cell. Their cytoplasm is filled with uniformly sized, bluish-purple granules, primarily composed of substances such as histamine, heparin, and serotonin. Because they mediate inflammatory responses, they are also known as inflammatory mediators. Mast cells are primarily located in areas of the body that connect to the external environment, such as the skin, airways, and digestive tract, around blood vessels. These areas are frequently exposed to environmental foreign bodies such as pathogens, making mast cells one of the first cell groups in the hematopoietic immune system to interact with invading pathogens and other foreign substances. Upon contact with stimulation, mast cells degranulate (i.e., release intracellular inflammatory mediators), causing the surrounding blood vessels to dilate and recruiting more immune cells and factors to the infected or damaged area, resulting in an inflammatory response and the removal of foreign substances to resist infection. Summary of the Invention

[0007] The purpose of the present invention is to provide a multifunctional skin peptide of Hymenoptera siliqua, named as tsin4, and to provide a new application for tsin4.

[0008] For the above purpose, the present invention provides a multifunctional skin peptide tsin4 having an amino acid sequence of Val AlaPhe Ile Gly His Cys Lys Gly Lys Thr Leu Arg Pro Lys Cys Phe.

[0009] The above-mentioned method for extracting the skin peptide tsin4 of Hymenoptera diversifolia comprises the following steps:

[0010] (1) Scrape the back of the Babirusa qinbaensis with a sterile plastic scraper. Rinse the skin with a 0.1 mol / L NaCl solution containing 0.01 mol / L EDTA to collect mucus. Mix the mucus with a 5% acetic acid solution at a volume ratio of 1:3 to 1:5, shake overnight at 4°C, and centrifuge to obtain the supernatant. Adjust the pH of the supernatant to 7.0-7.2 with phosphate buffer and centrifuge again to obtain the supernatant. Use an ultrafiltration tube to retain the fraction with a molecular weight less than 10,000 MW, filter through a 0.45 μm filter membrane, and finally freeze-dry.

[0011] (2) The freeze-dried sample obtained in step (1) was dissolved in 0.1 mol / L Na2HPO4-NaH2PO4 buffer solution with a pH of 6.0, and then loaded onto a Sephadex G-50 gel filtration chromatography column. The column was eluted with the same buffer solution to obtain three elution peak solutions.

[0012] (3) Streak Staphylococcus aureus and Escherichia coli plates, culture at 37°C overnight, pick single colonies and transfer them to liquid culture medium, culture at 37°C, 250 rpm, shaking for 4-6 hours, and then dilute the culture to 10 6 cfu / mL, and obtain the bacterial solution. After coating the plate with the bacterial solution, immerse the filter paper pieces in the elution peak solution of step (2), stick the filter paper pieces on the plate, and observe whether a transparent inhibition zone is formed after incubation at 37°C overnight.

[0013] (4) The elution peak solution producing the transparent inhibition zone in step (2) was freeze-dried and loaded onto a Sephadex G-25 gel filtration chromatography column. Elution was performed in the same manner as in step (2) to obtain three elution peak solutions. The antibacterial activity of the three elution peak solutions obtained was detected according to the method of step (3).

[0014] (5) The elution peak solution with antibacterial activity obtained in step (4) was freeze-dried and then dissolved in a 0.1% trifluoroacetic acid aqueous solution. The sample was loaded onto a Hypersil BDS C18 reverse phase semi-preparative chromatography column. The mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and the mobile phase B was acetonitrile. The elution gradient was: 100% solution A for 0-3 min, 100%-20% solution A for 3-35 min, and 20% solution A for 35-40 min. The column flow rate was 4 mL / min. The recovered main peak sample was freeze-dried.

[0015] (6) The main peak sample freeze-dried in step (5) was dissolved in a trifluoroacetic acid aqueous solution with a mass concentration of 0.1%, and the sample was loaded onto a liquid chromatography-tandem quadrupole time-of-flight mass spectrometer. After mass spectrometry detection, data analysis was performed to obtain a polypeptide with a molecular weight of 1905.35 Da, named tsin4, with a total of 17 amino acids, and its amino acid sequence was Val Ala Phe Ile Gly His Cys Lys Gly Lys Thr Leu Arg Pro Lys Cys Phe.

[0016] The multifunctional skin peptide tsin4 of the present invention has good antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, and can be used to prepare antibacterial infection drugs, veterinary antibacterial drugs, feed additives, preservatives, agricultural antibacterial agents, etc.

[0017] The multifunctional skin peptide tsin4 of the present invention has good inflammation regulating activity, can activate mast cells to release histamine and beta-aminoglycosidase, and can be used to prepare antibacterial and anti-inflammatory drugs.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention collects skin secretions from the Qinbaba salamander and separates and purifies a small molecule polypeptide, named tsin4. The full sequence of tsin4 was searched and compared with the NCBI protein database, and no identical polypeptides were found. Tsin4 has good antibacterial activity and can inhibit the tested Gram-positive and Gram-negative bacteria, and the MIC value of the polypeptide is low; at the same time, tsin4 has good inflammation-regulating activity and can activate mast cells to release histamine and β-aminoglycosidase. The experimental results also show that tsin4 has no toxicity to mammalian cells and has good safety. For exogenous pathogens, tsin4 can not only directly exert an antibacterial effect, but also activate the host immune system by regulating the inflammatory response to achieve the purpose of anti-infection, and has good application prospects. The Qinbaba salamander is an amphibian unique to my country, and its bioactive polypeptide resources have broad application prospects in clinical, food, agriculture, feed industry and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The gel filtration chromatograms of skin secretions of Babirusa qinba using Sephadex G-50 (A) and Sephadex G-25 (B) are shown.

[0021] Figure 2 This is the analytical RP-HPLC chromatogram of the skin secretions of Babirusa qinba. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples.

[0023] Example 1

[0024] 1. Extraction of tsin4 peptide from Hymenoptera skin

[0025] (1) Use a sterile plastic scraper to vigorously scrape the back of the Qinbaba Salamander. Repeat this process several times and rinse the skin with a 0.1 mol / L NaCl aqueous solution containing 0.01 mol / L EDTA to collect mucus. The mucus was mixed evenly with a 5% acetic acid aqueous solution at a volume ratio of 1:4, shaken overnight at 4°C and 120 rpm, and then centrifuged at 12,000 rpm for 30 min to obtain the supernatant. The pH of the supernatant was adjusted to 7.0-7.2 with phosphate buffer, and the supernatant was centrifuged at 12,000 rpm for 30 min to obtain the supernatant. The fraction with a molecular weight less than 10,000 MW was cut off using an ultrafiltration tube, and then filtered through a 0.45 μm filter membrane and finally freeze-dried.

[0026] (2) Prepare a Sephadex G-50 gel filtration chromatography column (length 500 mm, diameter 26 mm), equilibrate the column with 0.1 mol / L Na2HPO4-NaH2PO4 buffer at pH 6.0, dissolve 2 g of the freeze-dried sample obtained in step (1) in 0.1 mol / L Na2HPO4-NaH2PO4 buffer at pH 6.0, load the sample, and elute with 0.1 mol / L Na2HPO4-NaH2PO4 buffer at pH 6.0 at a flow rate of 0.3 mL / min. Draw an elution curve at a wavelength of A280 nm, and three elution peaks, 1 to 3, can be observed (see Figure 1 A). The elution peak solution is collected by an automatic collector.

[0027] (3) The elution peak solution collected in step (2) was tested for antibacterial activity using Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli as test bacteria. The above-mentioned strains were streaked on NA plates and cultured overnight at 37°C. Single colonies were picked from the plates and inoculated into NA liquid culture medium. The culture was shaken at 37°C and 250 rpm for 6 hours, and then the culture was diluted to 10 6 cfu / mL to obtain bacterial solution. Take 0.1mL of bacterial solution and inject it into NA plate, spread it evenly, and let it stand at room temperature for 20 minutes to allow the bacterial solution to be fully absorbed. Divide the NA plate into 6 areas, set up parallel groups diagonally, and use sterile water as a negative control and ampicillin (100U) as a positive control. Immerse the filter paper (diameter 0.6cm) in step (2) to collect the elution peak solution, and make 2 filter paper pieces for each sample. Stick the filter paper on the NA plate, let it stand at room temperature for 2h, then invert the plate, incubate it at 37℃ overnight, observe whether a transparent inhibition zone is produced, and measure the diameter of the inhibition zone with a ruler. The results showed that among the elution peak solutions No. 1-3 obtained after filtration through Sephadex G-50 gel filtration chromatography column, the No. 2 elution peak solution had the main antibacterial activity.

[0028] (4) After freeze-drying the elution peak solution No. 2, the solution was separated using a Sephadex G-25 gel filtration chromatography column (length 600 mm, diameter 16 mm) in the same manner as in step (2). A total of three elution peaks No. ac were observed (see Figure 1 B) The elution peak solutions were collected using an automated collector and subsequently assayed for antibacterial activity. The results showed that, of the peak solutions A to C obtained after filtering the product from peak 2 through a Sephadex G-25 gel filtration column, peak C exhibited the primary antibacterial activity. Finally, peak C was freeze-dried and stored at 4°C.

[0029] (5) Take the freeze-dried elution peak product c from step (4), dissolve it in 200 μL of a 0.1% trifluoroacetic acid aqueous solution, centrifuge it at 12000 r / min for 2 min, take the supernatant, filter it through a 0.22 μm filter membrane, and separate and purify it by reverse-phase high performance liquid chromatography. First, use an analytical column to determine the optimal elution conditions (see Figure 2), then collect the elution peaks with a semi-preparative column, and finally detect the purity of each elution peak with an analytical column. The analytical column is a Hypersil BDS C18 reverse phase chromatography column (length 250mm, diameter 4.6mm, filler 5μm), and the semi-preparative column is also a Hypersil BDS C18 reverse phase chromatography column (length 250mm, diameter 20mm, filler 12μm). Mobile phase A is a trifluoroacetic acid aqueous solution with a mass concentration of 0.05%, and mobile phase B is acetonitrile. Gradient elution is adopted, and the elution gradient is: 100% A solution 0-3min, 100%-20% A solution 3-35min, 20% A solution 35-40min; the flow rate of the analytical column is 1mL / min, and the flow rate of the semi-preparative column is 4mL / min. The recovered main peak sample is freeze-dried.

[0030] (6) The main peak sample freeze-dried in step (5) was dissolved in a trifluoroacetic acid aqueous solution with a mass concentration of 0.1% for mass spectrometry detection and bioinformatics analysis. The mass spectrometer was a liquid chromatography-tandem quadrupole time-of-flight mass spectrometer (AB Sciex, Triple TOF 5600plus). The mass spectrometry conditions were as follows: ion mode was ESI+, capillary voltage was 3.5 kV; ionization temperature was 100 °C; desolvation temperature was 400 °C; detection mass range was 50-2000 Da. First, the ion to be sequenced was selected by the primary mass spectrometer, and then the parent ion of the ion to be sequenced was impact-fragmented into an ordered series of ions by adjusting the collision gas energy, obtaining a multi-charged original secondary mass spectrum. Finally, the peptide was analyzed by MaxEnt3 and PepSeq software to obtain a peptide named tsin4. The physicochemical properties of tsin4 were analyzed by online ProtParam software. The peptide had a molecular weight of 1905.35 Da and a theoretical pI value of 9.85. The amino acid sequence of tsin4 is Val Ala Phe Ile Gly His Cys Lys Gly Lys Thr Leu ArgPro Lys Cys Phe, which contains 17 amino acids. It carries four positive charges, an instability index of 22.08, and an average hydrophilicity (GRAVY) of 0.141. The secondary structure of tsin4 was analyzed using online PSIPRED software, which revealed that the peptide's primary secondary structure is a random coil.

[0031] 2. Biosynthesis and antibacterial activity detection of Tsin4

[0032] Nanjing GenScript was commissioned to synthesize tsin4 with a purity of 95% to 97%. The synthesized tsin4 was dissolved in deionized water to prepare a 2 mg / mL stock solution. The test bacteria species included: Staphylococcus aureus, Rhodococcus rhodochrous, Enterococcus faecium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. The above bacteria were streaked on NA plates and cultured overnight at 37°C; a single colony was picked from the plate and inoculated into MH liquid medium. After shaking culture at 37°C and 250 rpm for 6 hours, the culture was diluted to 1×10 6 cfu / mL. tsin4 was also diluted in MH medium, and 0.1 mL of each dilution was added to a 96-well plate, with 3 wells in parallel for each concentration. Then 0.1 mL of 1×10 6 cfu / mL of bacterial solution, mix well, and incubate at 37℃ for 20h. The negative control is 0.1mL 1×10 6 cfu / mL of bacterial solution was mixed with 0.1mL MH medium and plated in 3 parallel wells; the positive control was 0.1mL 1×10 6 cfu / mL of bacterial solution, mixed with 0.1mL of ampicillin solution prepared in MH medium, with the final antibiotic concentration of 100U, in parallel 3 wells. After the incubation, the OD 600 After analyzing the data, the results showed that tsin4 had a certain antibacterial effect on most tested bacterial species and had no selectivity for Gram-positive bacteria and Gram-negative bacteria (see Table 1).

[0033] Table 1 MIC values of Tsin4

[0034]

[0035]

[0036] 3. Detection of hemolytic activity of Tsin4

[0037] Take healthy Mianyang blood, wash the red blood cells with normal saline until the supernatant is colorless, centrifuge at 3000r / min for 1-2 minutes, discard the supernatant, and prepare a 2% red blood cell suspension with normal saline. Dilute the antimicrobial peptide solution with normal saline to 200, 100, 50, 25, and 12.5μg / mL solutions. Mix equal volumes of 2% red blood cell suspension and equal volumes of antimicrobial peptide solutions with different dilutions. The positive control is 0.2% TritonX-100, and the negative control is normal saline. Set up 3 parallels for each sample and incubate at 37°C for 1.5h. After incubation, centrifuge at 3000r / min and take the supernatant at OD 540 The absorbance values of each sample were measured. The results showed that tsin4 had no hemolytic effect on erythrocytes at concentrations of 12.5 to 50 μg / mL. However, as the concentration increased, it exhibited hemolytic activity, causing 8.8% and 20.6% erythrocyte lysis at concentrations of 100 μg / mL and 200 μg / mL, respectively. This suggests that tsin4 has a weak hemolytic activity and exhibits little hemolytic activity at concentrations that have an inhibitory effect on bacteria.

[0038] 4. Detection of aminoglycosidase release activity of Tsin4

[0039] The rat basophil leukemia cell line RBL-2H3 was isolated from Wistar rats in 1978. Its biological activity is similar to that of mast cells and can be used as a substitute for mast cells in related experiments. The cells were cultured in DMEM medium. The cells were diluted to a concentration of 5×10 5 Cells / mL cell suspension was added to each well of a 96-well cell culture plate, 100 μL was added, and cultured in a 5% CO2 constant temperature incubator at 37°C for 16 hours. After the cells adhered, the culture medium was discarded, the cells were washed 2 to 3 times with Tyroder solution, and 100 μL of Tyroder solution was added; then 50 μL of 150 μg / mL tsin4 solution was added to each well. After incubation at 37°C for 15 minutes, centrifugation was performed at 2000 rpm for 5 minutes, and 50 μL of supernatant was transferred to a new 96-well plate. 50 μL of substrate solution was added to each well and mixed. After incubation at 37°C for 6 hours, the reaction was terminated with 150 μL of Na2CO3 stop solution, and the OD was measured. 405 (A) The experiment was repeated three times, and the activation activity of tsin4 on RBL-2H3 cells was expressed by aminoglycosidase release rate, as follows: aminoglycosidase release rate (%) = (A 实验组 -A 阴性组 ) / (A 阳性组 -A 阴性组) × 100%. The experimental group consisted of 100 μL of cells treated with 50 μL of tsin4 solution (final concentration 50 μg / mL); the positive group consisted of 100 μL of cells treated with 50 μL of Triton X-100 (final concentration 1%); and the negative group consisted of 100 μL of cells treated with 50 μL of Tyrode solution. A system control group was also established, consisting of 100 μL of cells treated with 50 μL of C48 / 80 (final concentration 50 μg / mL). C48 / 80 is a synthetic polyamine that can induce mast cell degranulation and is often used as a release agent for mast cell mediators. Statistical results showed that treatment with C48 / 80 at a final concentration of 50 μg / mL resulted in a β-aminoglycosidase release rate of 12.12 ± 3.10%; treatment with tsin4 at a final concentration of 50 μg / mL resulted in a β-aminoglycosidase release rate of 68.54 ± 5.30%. The experimental results showed that tsin4 has a certain ability to induce mast cells to release β-aminoglycosidase, indicating that tsin4 can regulate inflammatory responses and has immunomodulatory activity.

[0040] 5. Detection of histamine release activity of Tsin4

[0041] Rat basophil leukemia cell line RBL-2H3 was cultured in DMEM medium. The cells were diluted to a concentration of 5×10 5A cell suspension of 100 μL of Tyrode's solution (100 μL / well) was added to each well of a 96-well cell culture plate and incubated at 37°C in a 5% CO2 incubator for 16 hours. After the cells adhered, the medium was discarded and the cells were washed two to three times with Tyrode's solution. 100 μL of Tyrode's solution was added, followed by 50 μL of a 150 μg / mL tsin4 solution per well. After incubation at 37°C for 15 minutes, the reaction was terminated by adding 100 μL of pre-chilled Tyrode's solution. Centrifuge at 2000 rpm for 5 minutes, transfer the entire supernatant to a small test tube, and refrigerate until further use. The histamine content of this sample is the amount of histamine in the supernatant. 250 μL of Tyrode's solution was added to the pellet in each well of the original 96-well plate. The pellet was repeatedly frozen and thawed several times at -80°C and 40°C to completely lyse the cell pellet. Centrifuge at 2000 rpm for 5 minutes, and again transfer the supernatant to a small test tube. The histamine content of this sample is the amount of histamine in the pellet. Add 100 μL of 0.4 mol / L NaOH aqueous solution to a small test tube, mix well to make the solution alkaline, immediately add 20 μL of 0.1% OPT solution and mix well, react at 21-22°C for 10 minutes to generate fluorescent substances, and finally add 100 μL of 0.1 mol / L HCl aqueous solution to acidify and terminate the reaction. After the reaction is completed, draw 100 μL of the reaction solution from each small test tube into a black 96-well plate, with 3 parallels for each sample, and use a multifunctional microplate to detect the fluorescence value F (excitation wavelength 342 nm, emission wavelength 430 nm). The readings of the histamine content in the supernatant and the precipitate are added together to obtain the total histamine content in each well of the initial 96-well plate. The above experiment was repeated 3 times, and the histamine release activity of tsin4 was expressed as the histamine release rate, as follows: Histamine release rate (%) = (F 实验组 -F 阴性组 ) / (F 实验组 +F 沉淀组胺量 )×100%. The experimental group consisted of 100 μL of cells added with 50 μL of tsin4 solution (final concentration 50 μg / mL), and the negative group consisted of 100 μL of cells added with 50 μL of Tyrode's solution. A system control group was also established, consisting of 100 μL of cells added with 50 μL of C48 / 80 (final concentration 50 μg / mL). The statistical results of the experimental data showed that after the cells were treated with C48 / 80 at a final concentration of 50 μg / mL, the histamine release rate reached 42.68±3.23%; after the cells were treated with tsin4 at a final concentration of 50 μg / mL, the histamine release rate reached 77.34±2.50%. The experimental results show that tsin4 has a certain ability to induce histamine release from mast cells, indicating that tsin4 can regulate inflammatory responses and has immunomodulatory activity.

Claims

1. A multifunctional skin peptide tsin4 from Hymenoptera siliqua, characterized in that: Its amino acid sequence is Val Ala Phe Ile Gly His Cys Lys Gly Lys Thr Leu Arg Pro Lys Cys Phe.

2. The multifunctional skin peptide tsin4 of claim 1 is used in the preparation of anti-Staphylococcus aureus ( Staphylococcμs aμreμs ), Rhodococcus rhodochrous ( Rhodococcus rhodochrous ), Enterococcus faecium ( Enterococcus faecium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Escherichia coli ( Escherichia coli ) and Acinetobacter baumannii ( Acinetobacter Baumannii ) Application of infection drugs.

3. The multifunctional skin peptide tsin4 of claim 1 is used in the preparation of a drug for inhibiting Staphylococcus aureus ( Staphylococcμs aμreμs ), Rhodococcus rhodochrous ( Rhodococcus rhodochrous ), Enterococcus faecium ( Enterococcus faecium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Escherichia coli ( Escherichia coli ) and Acinetobacter baumannii ( Acinetobacter Baumannii ) in the application of preservatives.

4. The multifunctional skin peptide tsin4 of claim 1 is used in the preparation of veterinary anti-Staphylococcus aureus ( Staphylococcμs aμreμs ), Rhodococcus rhodochrous ( Rhodococcus rhodochrous ), Enterococcus faecium ( Enterococcus faecium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Escherichia coli ( Escherichia coli ) and Acinetobacter baumannii ( Acinetobacter Baumannii ) Application of infection drugs.

5. The multifunctional skin peptide tsin4 of Hymenoptera minor according to claim 1 is used in the preparation of agricultural anti-Staphylococcus aureus ( Staphylococcμs aμreμs ), Rhodococcus rhodochrous ( Rhodococcus rhodochrous ), Enterococcus faecium ( Enterococcus faecium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Escherichia coli ( Escherichia coli ) and Acinetobacter baumannii ( Acinetobacter Baumannii ) Application in infectious preparations.

6. The multifunctional skin peptide tsin4 of claim 1 is used in the preparation of a drug for inhibiting Staphylococcus aureus ( Staphylococcμs aμreμs ), Rhodococcus rhodochrous ( Rhodococcus rhodochrous ), Enterococcus faecium ( Enterococcus faecium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Escherichia coli ( Escherichia coli ) and Acinetobacter baumannii ( Acinetobacter Baumannii ) in feed additives.

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