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Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as preparation method and application thereof

A technology of anti-lipopolysaccharide factor and pseudo-scylla, applied in antiviral agents, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of virus-free control methods, economic losses, etc., and achieve attractive applications Foreground, strong broad-spectrum antibacterial activity, effect of strong anti-WSSV activity

Active Publication Date: 2012-02-15
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the occurrence of WSSV disease in 1993, it has caused huge economic losses to the global aquaculture industry. In the past 20 years, domestic and foreign scholars have made some key progress in the research of WSSV infection and aquatic crustacean immunity against WSSV. Unfortunately, So far there is no effective control method against this virus

Method used

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  • Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as preparation method and application thereof
  • Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as preparation method and application thereof
  • Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of Eukaryotic Recombinant Expression Vector for Antiviral Anti-lipopolysaccharide Factor Sp-ALF2 of Scylla pseudocarpus

[0072] According to the multiple cloning site of the pPIC9k vector, a specific upstream primer F1 and a downstream primer R1 were designed to amplify the ORF of the gene coding for Scylla sclerophyllus Sp-ALF2 (cDNA). A SnaB I restriction site and a base encoding His-tag were added to the 5' end of the upstream primer F1; a stop codon and an Avr II restriction site were added to the 5' end of the downstream primer R1. The upstream primer F1: 5′-CGTACGTACACCATCATCATCATCATCAGTATGAAGCTCTGGTAGC-3′, the downstream primer R1: 5′-AACCTAGGTC ACCCCTTCAGCCAGGCGGCC-3′, amplify the coding region fragment of Sp-ALF2. The PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, repeating 30 cycles; extension at 72°C for 10 mi...

Embodiment 2

[0075] Example 2 Induced expression of pPIC9k / Sp-ALF2 recombinant plasmid in Pichia pastoris GS115

[0076] The sequenced correct plasmid pPIC9k / Sp-ALF2 was linearized by Sal I digestion (see figure 2 ), transformed into Pichia pastoris GS115 competent cells by electric shock method, and induced expression.

[0077] Pichia pastoris GS115 transformed with pPIC9k empty plasmid was used as the control, and Pichia pastoris GS115 transformed with pPIC9k / Sp-ALF2 recombinant plasmid was used as the experimental group. The expression of the target protein was detected by polyacrylamide gel electrophoresis (SDS-PAGE).

[0078] The results showed that Pichia pastoris GS115 transformed with the pPIC9k / Sp-ALF2 recombinant plasmid had significantly induced recombinant protein expression compared with before induction, and the protein band was around 14kDa (see image 3 ), which was similar to the calculated theoretical molecular weight, and was confirmed to be Sp-ALF2 after being identif...

Embodiment 3

[0079] Example 3 Purification, antiviral activity and antibacterial activity identification of pPIC9k / Sp-ALF2 recombinant plasmid induced expression product in Pichia pastoris GS115

[0080] 1. Purification of target protein by affinity chromatography

[0081] After a large amount of recombinant positive Pichia pastoris GS115 strain was induced and expressed, 1 L of the supernatant of the culture medium was collected by centrifugation to remove the bacterial cells, dialyzed twice with PBS (new dialysate was changed once every 12 hours), centrifuged at 12000 rpm for 35 minutes at 4°C, and the supernatant was obtained. Load the sample on the column. Then, the dialyzed protein was subjected to affinity chromatography using a metal chromatographic column. The elution peak of solution D was collected, and it was identified as Sp-ALF2 recombinant protein through SDS-PAGE electrophoresis analysis and mass spectrometry (see Figure 4 ).

[0082] 2. Anti-WSSV activity identification...

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Abstract

The invention provides a Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as a preparation method and application thereof and relates to an antiviral type lipopolysaccharide resistant factor. The invention provides a gene sequence and an amino acid sequence of a Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2, a preparation method of the Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2 and application of the Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2. The preparation method comprises the following steps: constructing a recombinant expression vector of the Scylla paramamosain antiviral type lipopolysaccharide resistant factor; leading the recombinant expression vector into a host cell, carrying out inducing expression on the host cell so as to obtain the expression product; separating and purifying the expression product so as to obtain recombinant protein, namely Sp-ALF2. The Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2 has obvious inhibition effect and killing effect on WSSV (white spot syndrome virus) and multiple pathogenic bacteria, and has wide application value in preparation of disease resistant new medicaments and the Scylla paramamosain antiviral type lipopolysaccharide resistant factor as an animal disease resistant additive also has wide application value.

Description

technical field [0001] The present invention relates to an anti-viral anti-lipopolysaccharide factor, in particular to an anti-viral anti-lipopolysaccharide factor of Scylla pseudomae and its preparation method and application. Background technique [0002] White spot syndrome virus (WSSV) is not only the main viral pathogen against shrimp, but also can infect many other crustacean decapods, including many important aquaculture species. Since the occurrence of WSSV disease in 1993, it has caused huge economic losses to the global aquaculture industry. In the past 20 years, domestic and foreign scholars have made some key progress in the research of WSSV infection and aquatic crustacean immunity against WSSV. Unfortunately, So far there is no effective control method against this virus. [0003] It is well known that compared with vertebrates, invertebrate crustaceans such as shrimps and crabs lack the acquired immune response system, and they only rely on non-specific immun...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C07K14/435A61K38/17A61P31/00A61P31/12A23K1/17A23K20/195
Inventor 刘海鹏陈荣元王克坚
Owner XIAMEN UNIV
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