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An anti-WSSV peptide lvhcl48 derived from hemocyanin of Litopenaeus vannamei and its application

A technology of hemocyanin and lvhcl48, which is applied in the field of short peptides and their coding genes for inhibiting white spot syndrome virus

Active Publication Date: 2021-09-10
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, by looking for active substances that can inhibit the proliferation of WSSV, it is a feasible method to add them to the feed to enhance the physique of prawns and improve disease resistance.

Method used

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  • An anti-WSSV peptide lvhcl48 derived from hemocyanin of Litopenaeus vannamei and its application
  • An anti-WSSV peptide lvhcl48 derived from hemocyanin of Litopenaeus vannamei and its application
  • An anti-WSSV peptide lvhcl48 derived from hemocyanin of Litopenaeus vannamei and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Isolation and identification of shrimp hemocyanin degradation peptides related to inhibition of WSSV

[0029] (1) Dilute WSSV to 1.4×10 2 copy / μl, each shrimp was injected with 100 μl, that is, 10 4 copy / shrimp, the hemolymph of the prawns was extracted 2 hours after the injection. Use 75% alcohol cotton balls to wipe the rear edge of the prawn head breastplate, draw hemolymph from the heart of the prawns with a disposable syringe and mix it with anticoagulant (1:1). Centrifuge at 800g for 10min at 4°C to remove blood cells and take the supernatant. Ultracentrifuge at 32,000 rpm for 10 h at 4°C to remove most of the hemocyanin, and take the supernatant. Pre-cool acetone to precipitate, remove insoluble protein, dissolve with loading buffer, and store at -20°C for later use.

[0030] (2) The protein concentration was determined by BCA method. Add 20 μl of BSA Protein Standard of different concentrations, samples to be tested and blank control to each well...

Embodiment 2

[0035] Example 2: Obtaining the molecular characteristics of the degradation peptide of shrimp hemocyanin related to the inhibition of WSSV

[0036] (1) According to the mass spectrometry results, the protein spots that were identified as hemocyanin and had significant differences were recovered by gel, using the Shanghai Sangon PAGE Protein Micro-Glue Recovery Kit.

[0037] (2) Send the gel-recovered protein to Shanghai Zhongke New Life for Edman N-terminal sequencing.

[0038] (3) Send the protein sample to Shenzhen Micronafe Biotechnology Co., Ltd. for C-terminal sequencing.

[0039] (5) According to the results of Edman N-terminal sequencing and C-terminal sequencing, deduce the amino acid sequence of the hemocyanin degradation peptide. Obtain the amino acid sequence composition of the degraded peptide.

[0040]The amino acid composition of LvHcL48 is: Arg-Phe-Leu-Ile-Pro-Lys-Gly-Asn-Glu-Gln-Gly-Leu-Glu-Phe-Asp-Leu-Val-Val-Ala-Val-Thr-Asp- Gly-Ala-Ala-Asp-Ala-Ala-Val-As...

Embodiment 3

[0041] Example 3: Gene cloning and prokaryotic expression of the shrimp hemocyanin degradation peptide related to the inhibition of WSSV

[0042] (1)根据多肽的N端和C端序列,定位至血蓝蛋白氨基酸序列中,结合核苷酸序列:CGATTCCTCATCCCCAAGGGTAATGAACAGGGTCTGGAGTTCGACCTTGTTGTTGCCGTGACTGATGGCGCAGCCGACGCAGCAGTGGATGGCCTCCACGAAAACACCGAATTCAATCATTATGGTTCCCATGGCAAGTACCCTGACAATCGCCCACATGGCTACCCTCTGGATCGCAGGGTTCCCGATGAACGTGTATTCGAAGATCTTCCCAACTTCGGCCACATCCGAGTTAAG,设计PCR引物,用肝胰腺的cDNA为模板进行PCR扩增。 The target band was recovered and purified using an agarose gel DNA recovery kit (Shanghai Sangong). For the method, refer to the kit instruction manual, and the concentration was measured for the next experiment.

[0043] (2) The purified PCR product and pGEX-6p-1 were digested with restriction endonucleases, the concentration was measured after recovery from the gel, and ligated with T4 ligase overnight at 16°C. The ligation product was transformed into Escherichia coli strain Rosetta, and cultured upside down on LB solid medium con...

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Abstract

The invention relates to an anti-WSSV peptide LvHcL48 derived from Litopenaeus vannamei hemocyanin, the amino acid sequence is shown in SEQ ID NO:1, and the coding gene is shown in SEQ ID NO:2. The short peptide LvHcL48 of the present invention can significantly inhibit the transcription of the very early gene wsv069 and the late gene wsv421 of WSSV no matter at the animal level or at the cell level in vitro, and can also significantly inhibit the proliferation of WSSV in vivo; The interaction between outer membrane protein VP28 may be the mechanism of its function. The anti-WSSV peptide LvHcL48 of the present invention can be used as a preparation for inhibiting WSSV, and can be added to feed as an additive in order to improve the anti-WSSV ability of prawns.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a short peptide derived from hemocyanin of Litopenaeus vannamei for inhibiting White Spot Syndrome Virus (WSSV), its coding gene and its application. Background technique [0002] White spot syndrome virus (WSSV) is the most harmful virus in the shrimp farming industry. The virus is highly contagious and has a high lethal rate. After prawns are infected with WSSV, the mortality rate can reach 100% within 7-10 days . It has not been fully controlled so far and has become one of the main obstacles to the development of the shrimp farming industry. [0003] At present, methods such as cultivating species with strong disease resistance and cutting off the transmission route of WSSV are adopted in the production of prawns to prevent the spread of WSSV disease. At the same time, many scholars at home and abroad have tried to control shrimp WSSV by enhancing the shrimp's physique and impr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/795C12N15/12A61K38/41A61P31/20A23K20/147
CPCA61K38/00A23K20/147A61P31/20C07K14/795
Inventor 章跃陵詹世雄陶梦圆姚德福魏杰
Owner SHANTOU UNIV
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