Antimicrobial peptide SETIT, encoding DNA and expression system

Abstract

The present invention discloses an antimicrobial peptide SETIT, which is derived from Paspalum vaginatum and has an amino acid sequence as shown in SEQ ID NO: 1. Also disclosed are its encoding DNA and expression system. The antimicrobial peptide SETIT of the present invention has an obvious inhibitory effect on Gram-positive, Gram-negative bacteria and fungi, has high antibacterial efficiency and good broad-spectrum. By expressing the antimicrobial peptide SETIT using an edible Kluyveromyces marxianus cell-free expression system, an edible millet antimicrobial peptide can be obtained.

Description

Technical Field

[0001] The invention relates to an antimicrobial peptide SETIT, encoding DNA and an expression system, and relates to the technical field of biological products. Background Art

[0002] Since the advent of antibiotics, antimicrobial drugs have been used in all aspects of our lives and health, such as human infections, poultry, livestock, pets, aquaculture, food and crops, especially in the treatment of human infections. However, due to long-term and high-frequency use, existing antibiotics have developed widespread drug resistance in the human body. Among them, super bacteria such as vancomycin-resistant Enterococci that have been discovered are almost incurable by antibiotics. Therefore, the development of new antimicrobial drugs is urgent. At present, most antimicrobial drug development focuses on the exploration of new antimicrobial natural products. These natural products often have complex chemical structures and are synthesized or biosynthesized on a large scale. In addition, these chemicals have a single antibacterial mechanism and are prone to drug resistance with frequent use. In addition, these complex compound drugs are often accompanied by kidney burden and multiple side effects, so the development of new antimicrobial drugs is the current focus of research and development.

[0003] Antimicrobial peptides are protein molecules that have bactericidal and antiviral properties. Antimicrobial peptides can be produced naturally in animals and plants, or they can be synthesized. Antimicrobial peptides can be used to make feed additives, antimicrobial agents, drugs and health products, etc., and have a wide range of applications. The size of antimicrobial peptides can vary, depending on its source and type. Summary of the invention

[0004] The invention discloses an antimicrobial peptide SETIT, which is derived from Paspalum vaginatum and has an amino acid sequence as shown in SEQ ID NO:1.

[0005] Also disclosed is a DNA encoding the antimicrobial peptide SETIT, the sequence of which is shown as SEQ ID NO:2.

[0006] Also disclosed is an expression system of the antimicrobial peptide SETIT.

[0007] Preferably, the expression system is an edible Kluyveromyces marxianus cell-free expression system, and the antimicrobial peptide obtained by using the Kluyveromyces marxianus cell-free expression system is an edible antimicrobial peptide. Other conventional cell expression systems may also be used for expression.

[0008] Preferably, the expression system comprises: 30-70% V / V Kluyveromyces marxianus cell extract, 1-3 mM ribonucleotide mixture, 0.05-0.2 mM amino acid mixture, salt ion solution, glucose, and an expression DNA template for the antimicrobial peptide SETIT, wherein the expression DNA template for the antimicrobial peptide SETIT is a promoter added upstream of the coding DNA for the antimicrobial peptide SETIT.

[0009] Preferably, the DNA sequence of the promoter is shown in SEQ ID NO:3.

[0010] Preferably, the salt ion solution includes 10-100 mM potassium acetate, 10-100 mM sodium chloride and 0.2-1 mM magnesium chloride; and the concentration of glucose is 50 mM.

[0011] Preferably, the concentration of the expression DNA template of the antimicrobial peptide SETIT is 0.1-1 mg / L.

[0012] The invention discloses an antimicrobial peptide SETIT derived from millet Paspalum vaginatum, which has obvious inhibitory effects on Gram-positive, Gram-negative bacteria and fungi, has high antibacterial efficiency and good broad-spectrum. The antimicrobial peptide SETIT is expressed by using an edible Kluyveromyces marxianus cell-free expression system to obtain an edible millet antimicrobial peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the test results of the antibacterial activity of the antimicrobial peptide SETIT mixture against Staphylococcus aureus.

[0014] Figure 2 These are the test results of the antibacterial activity of the antimicrobial peptide SETIT mixture against Bacillus thuringiensis.

[0015] Figure 3 These are the test results of the antibacterial activity of the antimicrobial peptide SETIT mixture against Pseudomonas putida.

[0016] Figure 4 These are the test results of the antibacterial activity of the antimicrobial peptide SETIT mixture against Klebsiella oxytoca.

[0017] Figure 5 These are the test results of the antibacterial activity of the antimicrobial peptide SETIT mixture against Candida tropicalis. DETAILED DESCRIPTION

[0018] The present invention is further illustrated by examples below in conjunction with the accompanying drawings, but is not intended to limit the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, and materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0019] Example 1

[0020] A single clone of Kluyveromyces marxianus was inoculated into 50 ml of YD medium (2% yeast extract, 4% glucose) and cultured at 30°C and 220 rpm for 48 hours. The cells were collected by centrifugation, suspended with physiological saline, crushed under high pressure, and centrifuged to prepare the yeast extract. Cell-free synthesis system: 50% Kluyveromyces marxianus cell extract, 2mM ribonucleotide mixture (NTP mix), 0.1mM amino acid mixture, salt ion solution (20mM potassium acetate, 0.5mM magnesium chloride), 50mM glucose.

[0021] 10 ug of antimicrobial peptide SETIT expression DNA template was added to a 5 ml system, wherein the sequence of the antimicrobial peptide SETIT expression DNA template was the promoter sequence shown in SEQ ID No. 3 added to the upstream of the coding sequence shown in SEQ ID No. 2. After reacting at 37° C. overnight, an edible antimicrobial peptide SETIT mixed solution was obtained.

[0022] Example 2

[0023] To test the antibacterial activity of the antimicrobial peptide SETIT, the following bacteria were selected: Gram-positive bacteria: Staphylococcus aureus (CCTCC: AB 94004), Bacillus thuringiensis (CCTCC: AB93100); Gram-negative bacteria: Pseudomonas putida (CCTCC: PP), Klebsiella oxytoca (CCTCC: AB2010143); fungi: Candida tropicalis. The above Gram-positive bacteria, Gram-negative bacteria and fungi were purchased from China Center for Type Culture Collection.

[0024] The above Gram-positive bacteria, Gram-negative bacteria, fungi and multi-drug-resistant bacteria were cultured in a 96-well plate containing 100uL of the response culture medium, and the OD600 was cultured to 0.2. 0, 0.01%, 0.1%, 1% and 10% V / V of the antimicrobial peptide SETIT mixed solution obtained in Example 1 and 10ug / L Nisin were added, respectively, and cultured at 37°C for 12h. The OD600 values ​​of the samples in the 96-well plate were detected by a microplate reader. The results are as follows: Figure 1-5 As shown, it is shown that the antimicrobial peptide SETIT fermentation broth produced by the present invention has good antibacterial ability.

Claims

1. The use of the antimicrobial peptide SETIT in the preparation of an antimicrobial agent, characterized in that: The amino acid sequence of the antimicrobial peptide SETIT is shown in SEQ ID NO:

1. The antimicrobial agent is used to inhibit Staphylococcus aureus. Staphylococcus aureus , Bacillus thuringiensis Bacillus thuringiensis , Pseudomonas putida Pseudomonas putida , Klebsiella oxytoca Klebsiella oxytoca or Candida tropicalis Candida tropicalis .

2. The use according to claim 1, characterized in that The coding DNA sequence of the antimicrobial peptide SETIT is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that The expression system of the antimicrobial peptide SETIT is a cell-free expression system of Kluyveromyces marxianus.

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