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Engineered protein against shrimp white spot syndrome virus, preparation and use thereof

A fusion protein and prawn technology, which is applied in antiviral agents, viral antigen components, peptide/protein components, etc., can solve the problems of high cost of antibacterial peptides, inability to obtain expression products, harmful hosts, etc., to improve immunity and improve resistance The ability to infect WSSV and the effect of improving disease resistance

Inactive Publication Date: 2008-10-08
GUANGXI TIANCHI HALOBIOS PHARMA
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  • Abstract
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AI Technical Summary

Problems solved by technology

2000, 45(23): 2539-2544) transferred the green fluorescent protein gene as a reporter gene to the plasmid pGDNA-RZI of the recombinant anti-WSSV ribozyme gene of prawns by gene gun method, and successfully obtained the transgenic green fluorescent protein gene of Chinese shrimp individual, but its technical requirements are high and its efficiency is low
[0017] However, due to the small size of antimicrobial peptide molecules, there are certain difficulties in separation and purification, so natural resources are limited
Chemical synthesis and genetic engineering are the main means to obtain antimicrobial peptides, but the cost of chemical synthesis of antimicrobial peptides is high, and direct expression of antimicrobial peptide genes in microorganisms through genetic engineering may be harmful to the host and the expression product cannot be obtained

Method used

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  • Engineered protein against shrimp white spot syndrome virus, preparation and use thereof
  • Engineered protein against shrimp white spot syndrome virus, preparation and use thereof
  • Engineered protein against shrimp white spot syndrome virus, preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The preparation of the VP28 gene of embodiment 1WSSV

[0056] (1) Preparation of WSSV virus genome: take gills, stomach and heart of diseased prawns on ice, homogenate in ice bath, then add proteinase K (100ug / ml), boil in boiling water for 15 minutes, and immediately ice bath for 5 minutes , centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken and stored at 4°C.

[0057] (2) Preparation of the vp28 gene of WSSV

[0058] Primers were designed according to the sequence of vp28 published in GenBank, the upstream primer primer1 of vp28: GAATTC GATCTTTTCTTTCACTCT (the underline is the EcoR I site, and the bold is the initiation codon), the downstream primer primer2 of vp28: AAGCTT CTCGGTCTCAGTGCCAGA (the underline is the HindIII site). Use the above supernatant as a template for PCR, PCR reaction system: 10×Taq Buffer with KCl 2.5uL, MgCl2 (25mM) 1.5uL, dNTP Mixture (2.5mM) 1uL, upstream and downstream primers 1uL (25pmol), template 1uL, Taq DNA po...

Embodiment 2

[0059] Example 2 Preparation of silkworm antimicrobial peptide mature peptide gene (cd)

[0060] (1) Extraction of total RNA from silkworm fat body

[0061]Get 5-10 silkworms induced by Escherichia coli JM109 (purchased at INVETROGEN company) for 24h, and clean them with diethyl pyrocarbonate (DEPC, purchased at Shanghai Sangong) water (aqueous solution of 0.1% diethyl pyrocarbonate) Finally, the abdominal cavity was cut open, the fat was squeezed out and collected, and ground into powder in liquid nitrogen, according to RNeasy R The operating instructions of the Kit were used to extract total RNA from the fat body, and the extracted total RNA was stored at -80°C for later use.

[0062] (2) Synthesis of the first strand of Cecropin D cDNA

[0063] According to the instructions of the Reverse Transcription System kit (purchased from TIANGEN Company), with Oligo(dT) 20 Synthesize cDNA first strand for primers. The reaction system is as follows, and the following reagents ar...

Embodiment 3

[0109] The recovery, purification and subcloning of embodiment 3PCR product

[0110] The PCR product of vp28 obtained in Example 1 was electrophoresed on an agarose gel, and when the color of the gel loading buffer showed that the target band to be recovered was completely separated from other bands, the electrophoresis was stopped. Cut off the band to be recovered quickly under the ultraviolet light, purify it with TIANGEN Agarose Gel DNA Recovery Kit, put the single target DNA band into a clean Eppendorf tube, and weigh it. Add 3 times the volume of the sol solution to the gel block (the weight of the gel is 0.1g, its volume can be regarded as 100uL, and so on). Water bath at 50°C for 10 minutes, during which time the Eppendorf tube was gently turned up and down every 2 minutes to ensure that the gel was fully dissolved. Take 750uL of the resulting solution and add it to an adsorption column (the adsorption column is placed in a collection tube), place it at room temperatur...

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Abstract

The invention discloses a White Spot Syndrome Virus(WSSV) resisting engineering protein VP28-CD, the preparation and application thereof. The engineering protein VP28-CD is an isolated protein with sequence of ribonucleotide of SEQ ID NO:1 plus the amino acid sequence of SEQ ID NO:2; the bacterial strain is recombinant gene engineering scherichia coli BL21 (pET-32a-VP28-CD), CCTCC No:M208032; White Spot Syndrome Virus(WSSV) is isolated from prawn and gene VP28 is obtained through PCR; silkworm is induced with colon bacillus JM109 to obtain the total mRNA of fat body of the silkworm and the total cDNA is obtained through RT-PCR; mature gene of silkworm antibacterial peptide Cecropin D is obtained through PCR, with the upper reach inserted with aspartic acid-proline at the acid hydrolysis locus; after that recombinant plasmid Pet-32a(+)-VP28-CD is constructed; the recombinant plasmid Pet-32a(+)-VP28-CD is transformed into colon bacillus BL21(DE3); under IPTG induction, the fused protein VP28-CD achieves soluable and high-level expression. The fused protein is applicable in the preparation of the drugs for curing and preventing White Spot Syndrome Virus(WSSV).

Description

technical field [0001] The invention relates to the technical field of genetic bioengineering. More specifically, it relates to a recombinant fusion protein VP28-CD subunit vaccine, and also relates to a preparation method of Escherichia coli genetically engineered strains, specifically relating to the construction and high-efficiency expression of soluble prawn white spot syndrome virus (WSSV) envelope protein VP28 and A genetically engineered strain of the silkworm antimicrobial peptide Cecropin D mature peptide (CD) fusion protein; involving the insertion of the acid hydrolysis site aspartic acid-proline (Asp-Pro); also involving a bifunctional fusion expressed by the above strains The application of the protein in resisting WSSV and inhibiting and killing other pathogenic microorganisms in prawns. Background technique [0002] Since the 1980s, the shrimp aquaculture industry in the coastal countries of the world has developed vigorously. With the commercial production o...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N1/21C12N15/70A61K39/12A61P31/20A61K38/17C12R1/19
Inventor 孟小林徐进平王健鲁伟孟海洋王岚
Owner GUANGXI TIANCHI HALOBIOS PHARMA
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