Antimicrobial peptide gene of portunus trituberculatus and clone method

A technology of swimming crabs three warts and antibacterial peptides, which can be applied in the directions of genetic engineering, plant genetic improvement, botanical equipment and methods, etc., and can solve problems such as the loss of swimming crab aquaculture.

Inactive Publication Date: 2009-08-19
ZHEJIANG OCEAN UNIV
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Problems solved by technology

However, with the development of the portunus aquaculture industry, the diseases of the portunus crab, like other cultured species, show an increasing trend year by year, such as vibriosis, gill...

Method used

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  • Antimicrobial peptide gene of portunus trituberculatus and clone method
  • Antimicrobial peptide gene of portunus trituberculatus and clone method
  • Antimicrobial peptide gene of portunus trituberculatus and clone method

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Embodiment Construction

[0096] 1. Extraction of total RNA from blood lymphocytes of Portunus trituberculatus

[0097] Select healthy Portunus trituberculatus limbs to collect 10ml of blood, add 10ml of anticoagulant (NaCl 8.2g, glucose 19.8g, citric acid 6.3g, sodium citrate 7.6g, EDTA 3.7g, pH7.4, add distilled water to make up to 1L , sterilized at 121°C for 15min), centrifuged at 3000rpm for 5min, discarded the supernatant, and washed the precipitate once with marine saline (NaCl 33.9g, CaCl 2 2.95g, KCl 0.90g, Na 2 HPO 4 0.2g, Tris-base 6.05g, pH7.4, add water to 1L, sterilize at 121°C for 15 minutes), add 0.5ml Trizol, mix well with pipette tip, use 1ml syringe, 6 # Aspirate the homogenate twice with the needle to cut the genomic DNA, then transfer it into a 1.5ml Eppendoff tube (treated with DEPC), add 100ul chloroform / isoamyl alcohol (24:1), shake vigorously for 30sec, and centrifuge at 12,000rpm for 5min at room temperature. Transfer the supernatant (about 450ul) to a 1.5ml centrifuge tu...

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Abstract

The invention provides a Portunus trituperculatus antibacterial peptide gene and a cloning method thereof. The Portunus trituperculatus crustin antibacterial peptide gene cDNA is 565nt except poly A, and contains 5' non-coding region (5' UTR) of 75nt, a reading frame of 330nt, and 3' non-coding region (3' UTR) of 160nt, wherein the sequence number of GenBank is FJ467931. The method successfully clones complete sequence of Portunus trituperculatus crustin cDNA, can construct pronucleus or eucaryon gene engineering strain through a gene engineering mode, efficiently express recombinant protein with physiological activity, and can be produced in scale. As a feed immunity additive, the gene can replace certain antibiotics in the traditional feed for seaculture industry, thereby realizing green culture, reducing and even eliminating accumulation of the antibiotics in aquatic products, and improving quality of the aquatic products; the gene can also be used as an effective substitute of certain drug resistance antibiotics for the medicine of preventing and treating drug resistance bacteria in seaculture.

Description

technical field [0001] The invention relates to a type I Crustin antimicrobial peptide gene cloned from Portunus trituberculatus and belongs to the field of biotechnology. Background technique [0002] China is the largest aquaculture country in the world. The total output of aquatic products (fishing + aquaculture) accounts for 35% of the world, of which the output of aquaculture accounts for about 70% of the world. In 2005, the output of aquatic products reached 51.81 million tons. Among them, 28.54 million tons of marine products, accounting for 55% of the total output, about 10.22 million tons of fishing, about 18.88 million tons of artificial breeding products. With the global consumption of aquatic products increasing year by year, domestic consumption is growing rapidly. On the other hand, global marine fishing resources are declining, and the supply is gradually shifting to farmed aquatic products. China's aquaculture industry is becoming more and more important in g...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C07K14/435
Inventor 申望王日昕石戈廖智王建鑫
Owner ZHEJIANG OCEAN UNIV
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