Litopenaeus vannamei virus-resistant polypeptide litovirusin and application thereof

Abstract

The application discloses a white shrimp virus-resistant polypeptide Litovirusin and application thereof, and has the molecular formula of C 113 H 203 N 43 O 27 S2, and the amino acid sequence of S2 is shown as SEQ ID NO. 01. The application has good water solubility, is a cationic polypeptide with positive charge, has the characteristics of easy synthesis, good antiviral effect and stability, and has a good application prospect in the fields of drug research and aquaculture.

Description

TECHNICAL FIELD

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

[0002] Litopenaeus vannamei is one of the most important farmed crustaceans in the world, and is loved by people due to its high protein and low fat characteristics. However, due to the increase of farming density, the intensification of environmental pollution, and the influence of climate change and other factors, Litopenaeus vannamei farming is facing more and more serious disease threats, among which white spot syndrome virus (WSSV) causes white spot disease in shrimp, which is one of the most serious diseases. The disease has a fast onset and rapid spread, and can cause a large number of shrimp to die in a short time, and the mortality rate of infected shrimp is as high as 90-100% within 3-7 days.

[0003] Crustaceans lack acquired immunity and can only rely on innate immunity to resist the infection of pathogens. In recent years, research on antiviral polypeptides in Litopenaeus vannamei has gradually attracted widespread attention. Some studies have reported that there are some antiviral peptides with potential antiviral activity in Litopenaeus vannamei, such as Penaeidins, Crustins, etc. These antiviral polypeptides show certain inhibitory activity on shrimp viruses and have high application potential. The development of antiviral polypeptides can bring a new type of efficient disease prevention and control strategy to the aquaculture industry, and promote the sustainable development and improve the breeding efficiency of the aquaculture industry. SUMMARY

[0004] The application aims to provide a Litopenaeus vannamei antiviral polypeptide Litovirusin.

[0005] Another object of the application is to provide the application of the above-mentioned Litopenaeus vannamei antiviral polypeptide Litovirusin.

[0006] The technical solution of the application is as follows:

[0007] A Litopenaeus vannamei antiviral polypeptide Litovirusin, whose molecular formula is C 113 H 203 N 43 O 27 S2, and the amino acid sequence thereof is shown in SEQ ID NO. 01.

[0008] The application of the above-mentioned Litopenaeus vannamei antiviral polypeptide Litovirusin in preparing an antiviral composition.

[0009] In a preferred embodiment of the application, the antiviral composition has inhibitory and killing effects on white spot syndrome virus.

[0010] An antiviral composition, the effective component of which comprises the Litopenaeus vannamei antiviral polypeptide Litovirusin.

[0011] In a preferred embodiment of the present application, the effective component is the Litopenaeus vannamei antiviral polypeptide Litovirusin.

[0012] The application of the Litopenaeus vannamei antiviral polypeptide Litovirusin in the preparation of an aquatic feed additive.

[0013] In a preferred embodiment of the present application, the Litopenaeus vannamei antiviral polypeptide Litovirusin has inhibiting and killing effects on white spot syndrome virus.

[0014] An aquatic feed additive, the effective component of which comprises the Litopenaeus vannamei antiviral polypeptide Litovirusin.

[0015] In a preferred embodiment of the present application, the effective component is the Litopenaeus vannamei antiviral polypeptide Litovirusin.

[0016] The present application has the following advantages:

[0017] 1、 The present application is composed of 22 amino acids, and has a molecular formula of C 113 H 203 N 43 O 27 S2, a molecular weight of 2660.25 Dalton, 6 positively charged amino acid residues, an isoelectric point of 11.88 according to the charge of the amino acid residues, an average hydrophilic coefficient of 0.023, good water solubility, and is a positively charged cationic polypeptide with the characteristics of easy synthesis, good antiviral effect and stability, and has a good application prospect in the field of drug research and aquaculture.

[0018] 2、 The present application has good antiviral activity on foodborne pathogenic bacteria, and has no toxicity to hemopoietic tissue (Hpt) cells of Procambarus clarkii. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the toxicity experiment graph of the MTS-PMS method for detecting the Litopenaeus vannamei antiviral polypeptide Litovirusin in Example 2 of the present application, wherein the cells are hemopoietic tissue (Hpt) cells of Procambarus clarkii, the abscissa is the concentration of Litovirusin (μM), and the ordinate is the cell proliferation rate (%).

[0020] Figure 2Effect of Litovirusin on viral gene transcription in Litopenaeus vannamei, wherein the abscissa is different grouping, and the ordinate is the relative level of viral gene transcription.

[0021] Figure 3 Effect of Litovirusin on viral one-step growth curve in Litopenaeus vannamei, wherein the abscissa is infection time (h, hour), and the ordinate is viral copy number (copies / ng). DETAILED DESCRIPTION

[0022] The technical solutions of the present application are further described and explained in detail below by means of specific embodiments in conjunction with the accompanying drawings.

[0023] Example 1 Preparation of Litovirusin in Litopenaeus vannamei

[0024] The amino acid sequence of the Litovirusin in Litopenaeus vannamei is as follows:

[0025] Leu-Phe-Cys-Val-Leu-Cys-Val-Val-Leu-Arg-Leu-Arg-Ala-Thr-Arg-Gly-Arg-Glu-Arg-Ser-Arg-Arg (SEQ ID NO. 01, LFCVLCVVLRLRATRGRERSRR).

[0026] Litovirusin with a purity of more than 95% can be obtained by using the existing solid-phase chemical synthesis method. In this embodiment, the Litovirusin is obtained by solid-phase chemical synthesis method by Nanjing Kingsrui Biological Technology Co., Ltd., and the polypeptide molecular weight, HPLC and other detection information are provided.

[0027] The physicochemical parameters of the Litovirusin are shown in Table 1.

[0028] Table 1 Physicochemical parameters of the Litovirusin

[0029]

[0030] As can be seen from Table 1, Litovirusin has a small molecular weight, good water solubility, and is a cationic polypeptide with positive charge.

[0031] Example 2 Cytotoxicity determination of Litovirusin in Litopenaeus vannamei

[0032] The cytotoxicity of Litovirusin in Litopenaeus vannamei was determined in the hemopoietic tissue (Hpt) cells of the red claw crayfish, including the following steps:

[0033] (1) Collect hematopoietic tissue (Hpt) cells from crayfish in good growth condition and adjust the cell concentration to 1×10 5 ~5×10 5 The cells were evenly dispersed by blowing, and 100 μL of cell suspension was added to each well of a 96-well cell culture plate, and the plate was placed in an incubator at a suitable temperature for culture.

[0034] (2) Carefully aspirate the culture medium and add culture medium containing different concentrations of litovirusin (0 μM, 1 μM, 5 μM, 10 μM, 15 μM), and place in an incubator at a suitable temperature for 24 h.

[0035] (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 The cytotoxicity of Litovirusin was evaluated by RT-PCR.

[0036] The results are as follows Figure 1 As shown in the results, litovirusin had no toxic effect on red claw crayfish hematopoietic tissue (Hpt) cells at the experimental concentrations.

[0037] Example 3 Effect of Litovirusin, an antiviral peptide from Litopenaeus vannamei, on viral gene transcription

[0038] In this example, the proliferation of white spot syndrome virus was measured to evaluate the inhibitory effect of Litovirusin on viral transcription.

[0039] Different concentrations of litovirusin were mixed with white spot syndrome virus and then added to Hpt cells. A negative control group and a test group were set up, with three replicates per group. Cells were harvested at 6 hours, RNA was extracted, reverse transcribed into cDNA, and viral transcription was detected by qPCR.

[0040] The results are as follows Figure 2 As shown in AB, the inhibition rate of Litovirusin on the transcription of white spot syndrome virus genes ie1 and vp28 can reach more than 50%.

[0041] Example 4 Effect of Litovirusin, an antiviral peptide from Litopenaeus vannamei, on the one-step growth curve of the virus

[0042] In this example, the growth of white spot syndrome virus was measured to evaluate the inhibitory effect of Litovirusin on viral replication.

[0043] Different concentrations of Litovirusin were mixed with white spot syndrome virus and added to Hpt cells, and a negative control group and a test group were set up, with three parallel groups in each group. At 6h, 12h, 24h, different cells were collected, total DNA was extracted using a blood / cell / tissue genomic DNA extraction kit, and used as a template to detect the viral load using the established qPCR method.

[0044] The results are shown in Figure 3 Litovirusin can inhibit the replication of white spot syndrome virus by more than 50% at 24h.

[0045] The above is only a preferred embodiment of the present application, and therefore cannot limit the scope of the present application, i.e. equivalent changes and modifications made in accordance with the scope of the present patent and the content of the specification should still be within the scope of the present application.

Claims

1. A Litovirusin antiviral polypeptide of Penaeus vannamei, characterized by: Its molecular formula C 113 H 203 N 43 O 27 S2, the amino acid sequence of which is shown in SEQ ID NO.

01.

2. The use of the antiviral peptide Litovirusin of Litopenaeus vannamei according to claim 1 in the preparation of an antiviral composition, characterized in that: The antiviral composition has an inhibitory effect on white spot syndrome virus.

3. An antiviral composition, characterized in that: The effective component comprises the antiviral polypeptide Litovirusin of Penaeus vannamei according to claim 1.

4. The antiviral composition according to claim 3, wherein: The effective component is the antiviral polypeptide Litovirusin of Penaeus vannamei.

5. The use of the antiviral peptide Litovirusin of Litopenaeus vannamei according to claim 1 in the preparation of an aquatic feed additive, characterized in that: The vannamei antiviral polypeptide Litovirusin has an inhibitory effect on white spot syndrome virus.

6. An aquatic feed additive, characterized in that: The effective component comprises the antiviral polypeptide Litovirusin of Penaeus vannamei according to claim 1.

7. The aquatic feed additive according to claim 6, wherein: The effective component is the antiviral polypeptide Litovirusin of Penaeus vannamei.

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