An antibacterial and antiviral polypeptide Litomusin against Litopenaeus vannamei 81-100 and its application

Abstract

The application discloses an antibacterial and antiviral polypeptide Litomusin for white shrimp 81‑100 and application thereof, the molecular formula of the polypeptide is C 106 H 183 N 35 O 25 S1, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 01. The polypeptide has good water solubility, has significant antibacterial activity on various bacteria and fungi, has good antibacterial effect and high efficiency, has a wide antibacterial spectrum, has strong activity on white spot syndrome virus, has no toxicity on hematopoietic tissue (Hpt) cells of Procambarus clarkii, and has a good application prospect in drug research and development.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of marine molecular biology, and particularly relates to a Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 and application thereof. BACKGROUND

[0002] Since the 1950s, shrimp culture has begun to rise, and in the 1980s, the shrimp culture industry has entered a rapid development stage. Asia is not only the birthplace of shrimp culture, but also the main production area, and the culture yield accounts for more than 85% of the total yield in the world. The culture yield of shrimp in China has ranked first in the world after decades of continuous development. Although the shrimp industry is booming and presents a prosperous scene, there are still many problems that cannot be ignored in the culture process, among which the disease problem is particularly challenging. Biological diseases are usually caused by organisms around the host, and these organisms mainly refer to pathogenic organisms, including bacteria, viruses and parasites.

[0003] Antibacterial peptides are a class of small molecule polypeptides naturally existing in organisms, which are well-known for their few enzyme action sites and good thermal stability, and have significant drug properties. These polypeptides have broad-spectrum antimicrobial activity, including antiviral effects. Compared with traditional antibiotics, their antimicrobial mechanism is not easy to cause microbial drug resistance, and they are not easy to accumulate in organisms, and are a new type of efficient and environmentally friendly antimicrobial drug. At present, a variety of antibacterial peptides derived from shrimps have been reported. For example, ALF is identified in many crustaceans and has strong antibacterial activity, which can significantly inhibit the transcription and replication of white spot syndrome virus (WSSV). Penaeidin5 in Chinese white shrimp has antibacterial activity, and inhibition of its expression will lead to increased susceptibility of shrimps to WSSV infection. In addition, serum proteins, as the main protein components of hemolymph, have the effect of resisting bacterial and fungal invasion, and can inhibit the proliferation of WSSV in shrimps.

[0004] The research and development of new antibacterial peptides of Litopenaeus vannamei provide a new idea and method for the prevention and treatment of aquatic culture diseases and the screening of antibacterial and antiviral drugs. SUMMARY

[0005] The application aims to provide a Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 .

[0006] Another object of the application is to provide the application of the above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 .

[0007] The technical solution of the application is as follows:

[0008] A Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 , the amino acid sequence of which is shown as SEQ ID NO. 01.

[0009] The above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 Application in preparing an antibacterial composition.

[0010] In a preferred embodiment of the present application, the antibacterial composition has inhibitory and killing effects on Acinetobacter baumannii, Vibrio fluvialis and Staphylococcus aureus.

[0011] An antibacterial composition, the effective component of which comprises the above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 .

[0012] The above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 Application in preparing an antifungal composition.

[0013] In a preferred embodiment of the present application, the antifungal composition has inhibitory and killing effects on Cryptococcus neoformans, Fusarium oxysporum, Fusarium solani and Aspergillus niger.

[0014] An antifungal composition, the effective component of which comprises the above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 .

[0015] The above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 Application in preparing an antiviral composition, the antiviral composition having inhibitory and killing effects on White Spot Syndrome Virus.

[0016] An antiviral composition, the effective component of which comprises the above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 .

[0017] The above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 Application in preparing an aquatic feed additive.

[0018] An aquatic feed additive, the effective component of which comprises the above-mentioned Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 .

[0019] The beneficial effects of the present application are:

[0020] 1、The present application is composed of 20 amino acids, and the molecular formula is C 106 H 183 N 35 O 25S1, the molecular weight is 2379.90 Dalton, wherein 6 positively charged amino acid residues are contained, the isoelectric point is 12.48 according to the amino acid residue charge prediction, the average coefficient of hydrophilicity is-0.120, has good water solubility, has good water solubility.

[0021] 2、The present application has significant antibacterial activity to various bacteria and fungi, has good antibacterial effect and high efficiency, has a wide antibacterial spectrum, in addition, has strong anti-white spot syndrome virus activity, and has no toxicity to the hemopoietic tissue (Hpt) cells of the red claw crayfish, and has a good application prospect in drug research and development. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Litomusin for the antibacterial and antiviral polypeptide of Litopenaeus vannamei in embodiment 3 of the present application 81-100 The antibacterial activity stability diagram of Staphylococcus aureus is shown in the figure, wherein figure A is a thermal stability diagram, and figure B is a salt ion stability diagram. The abscissa is time (h, hour), and the ordinate is OD value.

[0023] Figure 2 The toxicity experiment diagram of Litomusin for the antibacterial and antiviral polypeptide of Litopenaeus vannamei detected by MTS-PMS method in embodiment 4 of the present application 81-100 , wherein the abscissa is Litomusin concentration (μM), and the ordinate is cell proliferation rate (%). 81-100

[0024] Figure 3 The antiviral activity diagram of Litomusin for the antibacterial and antiviral polypeptide of Litopenaeus vannamei 81-100 , wherein the abscissa is different groups, and the ordinate is the relative level of viral gene transcription. DETAILED DESCRIPTION

[0025] The technical solutions of the present application are further described and explained by specific embodiments in combination with the drawings.

[0026] Preparation of Litomusin for the antibacterial and antiviral polypeptide of Litopenaeus vannamei in embodiment 1 81-100

[0027] The amino acid sequence of the antibacterial and antiviral polypeptide Litomusin 81-100 is as follows:

[0028] Ala-Lys-Arg-Gln-Arg-Ile-Thr-Val-Ser-Ala-Met-Arg-Arg-Leu-Phe-Ala-Lys-Phe-Ser-Leu (SEQ ID NO. 01, AKRQRITVSAMRRLFAKFSL). ​​

[0029] The antibacterial and antiviral polypeptide Litomusin with purity of more than 95% can be obtained by using the existing solid-phase chemical synthesis method 81-100 The antibacterial and antiviral polypeptide Litomusin in this embodiment 81-100 Nanjing Kingsrui Biological Technology Co., Ltd. was commissioned to obtain the antibacterial and antiviral polypeptide Litomusin by using the solid-phase chemical synthesis method, and provide the polypeptide molecular weight and HPLC detection information.

[0030] The antibacterial and antiviral polypeptide Litomusin 81-100 The physicochemical parameters are shown in Table 1.

[0031] Table 1 The antibacterial and antiviral polypeptide Litomusin 81-100 Physicochemical parameters

[0032] Physico-chemical parameter class Litomusin 81-100 ]]> Amino acid residues 20 Molecular weight 2379.90 Da Molecular formula C 106 H 183 N 35 O 25 S1]]> Isoelectric point 12.48 Net charge +6 Hydrophobicity 30.5% Total average hydrophilicity -0.120

[0033] As can be seen from Table 1, the antibacterial and antiviral polypeptide Litomusin has the following characteristics: 81-100 The molecular weight is small, has good water solubility, and is a cationic polypeptide with positive charge.

[0034] Example 2 Antibacterial and antiviral polypeptide Litomusin of Litopenaeus vannamei 81-100 Determination of minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC)

[0035] The strains involved in this embodiment include Acinetobacter baumannii, Vibrio fluvialis, Staphylococcus aureus, Cryptococcus neoformans, Fusarium oxysporum, Fusarium solani, and Aspergillus niger. All strains are from the Culture Collection of Microorganisms of the Chinese Academy of Sciences and are preserved and stored by the laboratory.

[0036] The specific method is as follows:

[0037] (1) Spread the preserved bacteria on nutrient broth plates, and incubate at the appropriate temperature for 18-24 h; spread the Vibrio on 2216 plates, and incubate at 28°C for 18-24 h; spread the yeast on YPD plates, and incubate at 28°C for 2-3 d; spread the filamentous fungal spores on potato glucose plates, and incubate at 28°C for 3-7 d.

[0038] (2) Set the final concentration of bacteria at 5x10 5 CFU / mL, and adjust the spore concentration so that the final concentration of mold spores is 5x10 4 CFU / mL.

[0039] (3) Dissolve the synthesized Litomusin 81-100 powder in sterilized ddH2O, filter through a 0.22 μm filter, and dilute the polypeptide concentration by a factor of 2 to 3 μM, 6 μM, 12 μM, 24 μM, 48 μM, 96 μM, 192 μM, and store on ice.

[0040] (4) On a 96-well cell culture plate, set up a blank control group, a negative control group, and a test group for each test bacterium, and set up three parallels for each group:

[0041] a Blank control group: 50 μL of the test polypeptide sample and 50 μL of the culture medium

[0042] b Negative control group: 50 μL of sterilized ddH2O and 50 μL of the bacterial suspension

[0043] c Test group: 50 μL of the test polypeptide sample and 50 μL of the bacterial suspension

[0044] (5) Place the 96-well cell culture plate in a 28°C incubator, and incubate for 1-2 d, observe the MIC results in the test group; mix the test group by blowing, take an appropriate amount of bacterial solution, and drop it on the corresponding solid culture medium plate, and incubate at the appropriate temperature for 1-2 d, and observe the MBC results. The antibacterial results are shown in Table 2:

[0045] Table 2 Antibacterial activity of Litomusin 81-100 antibacterial and antiviral polypeptide for white-spotted seabream (Pagrosomus nitidus)

[0046] Strain Latin name MIC MBC Gram-negative bacteria (G - )]]> Acinetobacter baumannii A. baumannii 3-6 6-12 Vibrio fluvialis S. aureus 1.5-3 3-6 gram-positive bacteria (G + )]]> Fungi Cryptococcus neoformans 6-12 12-24 Fusarium oxysporum Fusarium solani Aspergillus niger 3-6 6-12 Figure 1 Figure 1 3-6 6-12 Figure 2 Figure 3 3-6 6-12 ​ ​ 6-12 12-24

[0047] Note: MIC: minimum inhibitory concentration (μM), represented by a-b. a: the highest polypeptide concentration at which bacterial growth is visible to the naked eye; b: the lowest polypeptide concentration at which bacterial growth is not visible to the naked eye;

[0048] MBC: Minimum bactericidal concentration (μM), denoted as ab. a: The highest peptide concentration at which colonies are visible on the plate; b: The lowest peptide concentration at which no colonies are visible on the plate.

[0049] Example 3: Litomusin, an antibacterial and antiviral polypeptide from Litopenaeus vannamei. 81-100 stability

[0050] In this embodiment, Staphylococcus aureus was selected as the test bacterium, and the antibacterial and antiviral polypeptide Litomusin was tested against Litopenaeus vannamei. 81-100 The stability was determined. The specific method is as follows:

[0051] Thermal stability: First, the antibacterial and antiviral peptide Litomusin 81-100 Heat in a boiling water bath for 0, 10, and 30 minutes. After cooling, mix the Staphylococcus aureus solution with the heated Litomusin. 81-100 The bacteria were added to the wells of a 96-well plate and incubated at the optimal growth temperature. After different incubation times, the bacterial growth status was monitored at 600 nm using a microplate reader. Results are as follows: ​ As shown in A, Litomusin 81-100 It has good thermal stability.

[0052] Salt stability: Staphylococcus aureus culture, antibacterial and antiviral peptide Litomusin 81-100 Different concentrations of NaCl were added to the wells of a 96-well plate and incubated at the optimal growth temperature. After different incubation times, the bacterial growth was monitored at 600 nm using a microplate reader. The results are as follows: ​ As shown in B, Litomusin 81-100 It exhibits good salt ion stability.

[0053] Example 4: Litomusin, an antibacterial and antiviral polypeptide from Litopenaeus vannamei. 81-100 Cytotoxicity assay

[0054] This embodiment describes the activity of the antibacterial and antiviral polypeptide Litomusin in Litopenaeus vannamei in the hematopoietic tissue (Hpt) cells of redclaw crayfish. 81-100 Cytotoxicity was measured.

[0055] (1) Collect hematopoietic tissue (Hpt) cells from shrimp in good growth condition and adjust the cell concentration to 5×10⁻⁶. 5 Cells / mL: Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate at a suitable temperature.

[0056] (2) Carefully aspirate the culture medium and add different concentrations of Litomusin. 81-100The culture medium was placed in an incubator at a suitable temperature and incubated for 24 hours.

[0057] (3) After adding 20 μL of MTS-PMS solution and incubating in the dark for 2 h, the OD was measured using a microplate reader. 492 Value, evaluation of Litomusin 81-100 Cytotoxicity.

[0058] The results are as follows ​ As shown, at the experimental concentration, Litomusin 81-100 It has no cytotoxic effect on the hematopoietic tissue (Hpt) cells of red claw crayfish.

[0059] Example 5: Litomusin, an antibacterial and antiviral polypeptide from Litopenaeus vannamei. 81-100 Antiviral activity assay

[0060] This embodiment measures the proliferation of vitiligo syndrome virus and evaluates Litomusin. 81-100 Inhibitory effect on viral transcription.

[0061] Different concentrations of antibacterial and antiviral peptides were mixed with vitiligo syndrome virus and added to the corresponding cells. A negative control group and a test group were set up, with three replicates in each group. Cells were collected at 6 h, RNA was extracted, and reverse transcribed into cDNA. Viral transcription was detected by qPCR.

[0062] The results are as follows ​ As shown in A and B, Litomusin 81-100 The inhibition rate of transcription of the vitiligo syndrome virus genes ie1 and vp28 can reach more than 50%.

[0063] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin 81-100 characterized in that: The amino acid sequence thereof is shown as SEQ ID NO.

01.

2. The Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin according to claim 1 81-100 Use in the manufacture of an antibacterial composition, characterized in that: The antibacterial composition has inhibitory and killing effects on Acinetobacter baumannii, Vibrio fluvialis and Staphylococcus aureus.

3. An antibacterial composition characterized in that: The effective component includes the Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin in claim 1 81-100 .

4. The Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin for use according to claim 1. 81-100 Use in the manufacture of an antifungal composition, characterized in that: The antifungal composition has inhibitory and killing effects on Cryptococcus neoformans, Fusarium oxysporum, Fusarium solani and Aspergillus niger.

5. An antifungal composition characterized in that: The effective component includes the Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin in claim 1 81-100 .

6. The Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin of claim 1 81-100 Use in the preparation of an antiviral composition, characterized in that: The antiviral composition has inhibitory and killing effects on white spot syndrome virus.

7. An antiviral composition, characterized by: The effective component includes the Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin in claim 1 81-100 .

8. The Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin of claim 1 81-100 In the preparation of aquaculture feed additives.

9. An aquafeed additive, characterized by: The effective component includes the Litopenaeus vannamei antibacterial and antiviral polypeptide Litomusin in claim 1 81-100 .

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