Genetic engineering protein TAT-VP28-GH capable of resisting white spot syndrome virus (WSSV) as well as preparation and application thereof

A fusion protein, shrimp technology, applied in antiviral agents, peptide/protein components, biochemical equipment and methods, etc., can solve problems such as economic losses in shrimp farming industry

Inactive Publication Date: 2011-11-23
广西众达生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Huge economic losses t...

Method used

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  • Genetic engineering protein TAT-VP28-GH capable of resisting white spot syndrome virus (WSSV) as well as preparation and application thereof
  • Genetic engineering protein TAT-VP28-GH capable of resisting white spot syndrome virus (WSSV) as well as preparation and application thereof
  • Genetic engineering protein TAT-VP28-GH capable of resisting white spot syndrome virus (WSSV) as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Preparation of tat-vp28 gene.

[0046] (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.

[0047] (2) PCR amplification of VP28 gene: PCR primers were designed according to the sequence of WSSV (GeneBank: EU414753) published in GeneBank, and the above supernatant was used as template DNA to amplify the target gene VP28 by PCR, and the PCR product was detected by DNA gel electrophoresis , recover the PCR product and clone it into the pGEM-T vector (purchased from Promega), pick a single colony and use alkaline lysis to extract a small amount of plasmid for enzyme digestion identification, and the positive clone obtained by sequencing is the one containing VP28 coli, named pGEM-T-vp28, for th...

Embodiment 2

[0049] Embodiment 2: Preparation of carp growth hormone mature peptide gene (GH):

[0050] (1) Extraction of carp growth hormone RNA:

[0051] Take carp pituitary gland, according to RNeasy R The total RNA was extracted according to the operating instructions of the Kit, and the extracted total RNA was stored at -80°C for later use.

[0052] (2) Synthesize the first strand of GH cDNA:

[0053] According to the instructions of the Reverse Transcription System kit, with Oligo(dT) 20 Synthesize cDNA first strand for primers. The reaction system is as follows, and the following reagents are sequentially added to the PCR tubes treated with DEPC water and sterilized:

[0054]

[0055]

[0056] The reaction parameters are:

[0057] 42℃ 1h

[0058] 95℃ 5min

[0059] 3°C 5min

[0060] The reacted product was placed at -20°C as a template for cloning the target gene.

[0061] (3) Preparation of carp growth hormone GH:

[0062] Based on the cDNA sequence of carp growth h...

Embodiment 3

[0073] Example 3: Recovery, purification and subcloning of PCR products.

[0074] The TAT-VP28 obtained in Example 1 and 2, the PCR product of the carp growth hormone mature peptide gene GH, was electrophoresed on an agarose gel, and the band to be recovered was cut out rapidly under an ultraviolet light, and the TIANGEN agarose gel was used to DNA recovery kit for purification, put a single target DNA band into a clean Eppendorf tube, and weigh it. Add three times the volume of sol solution PN 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 temperature (the same below 20-25°C) for 2 minutes, centrifuge at 12000rpm for 1 minu...

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Abstract

The invention discloses a genetic engineering protein TAT-VP28-GH capable of resisting white spot syndrome virus (WSSV) as well as preparation and application thereof. The sequences of the genetic engineering protein are a nucleotide sequence shown in SEQIDNO:1 and an amino acid sequence shown in SEQIDNO:2; and the strain of the genetic engineering protein is a recombinant genetic engineering strain EscherichiacoliBL21pEt-32A-TAT-VP28-GH. The preparation method comprises the following steps: extracting WSSV from prawn, and obtaining VP28 gene by a polymerase chain reaction (PCR); amplifying aTAT-VP28 sequence through the PCR; extracting total mRNA (messenger RNA) from hypophysis cerebri of carp, and obtaining total cDNA through RT-PCR (reverse transcription-PCR); obtaining a mature peptide gene of carp growth hormone through PCR, inserting acid hydrolysis site aspartate and proline (Asp-Pro-Asp) in the upstream of the mature peptide gene; then constructing a recombinant plasmid pEt-32A-TAT-VP28-GH; and transforming the recombinant plasmid pEt-32A-TAT-VP28-GH to Escherichia coil BL21, and efficiently expressing fusion protein TAT-VP28-GH in a dissoluble form under the induction of isopropyl beta-D-1-thiogalatopyranoside (IPTG). The fusion protein can effectively prevent the prawn from suffering WSSV and promoting growth development of the prawn.

Description

technical field [0001] The invention relates to the technical field of genetic bioengineering. More specifically, it relates to a recombinant fusion protein TAT-VP28-GH subunit vaccine, and also relates to a method for preparing a genetically engineered E. PTD), the genetically engineered strain of the fusion protein of shrimp white spot syndrome virus envelope protein VP28 and carp growth hormone (carp mature growth hormone) mature peptide (abbreviated as GH); also relates to the fusion protein expressed by the above strains against shrimp white spot syndrome virus And the use of promoting the growth of prawns. Background technique [0002] Shrimp farming is the pillar industry of mariculture in my country's coastal areas. In the early 1990s, the national shrimp farming area reached 160,000 hectares, and the highest annual output reached 220,000 tons, accounting for about 30% of the world's total shrimp farming production. From 1988 to 1992, the output of my country's shr...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N1/21C12N15/70A61K38/16A61P31/20A01K61/00C12R1/19
CPCY02A40/81
Inventor 孟小林徐进平王健张毅
Owner 广西众达生物工程有限公司
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