Mytilus coruscus antibacterial peptide and preparation method and application thereof

A thick-shelled mussel, antibacterial peptide technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibacterial drugs, etc. Effect

Inactive Publication Date: 2011-04-20
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Mytilus coruscus is one of the cultured mussels with important economic v

Method used

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  • Mytilus coruscus antibacterial peptide and preparation method and application thereof
  • Mytilus coruscus antibacterial peptide and preparation method and application thereof
  • Mytilus coruscus antibacterial peptide and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Embodiment one: cDNA gene cloning of the antimicrobial peptide myticin of thick shell mussel

[0053] Using TRIZOL Reagent to extract the total RNA of mussel blood cells, and identify it by 1.2% agarose gel electrophoresis, and measure the OD at the same time 260 and OD 280 The concentration and purity of the extracted RNA were analyzed. According to Invitrogen FastTrack 2.0Kit kit operation manual, through poly(T) cellulose column for affinity chromatography, to obtain poly A from total RNA of blood cells of mussel thick shell + mRNA. Use MMLV to reverse transcribe into the first strand of cDNA;

[0054] The upstream primer (myticin2F: 5'-CAAACGTACAACATGAAGG-3') was designed according to the conserved region in the gene sequence of Mediterranean mussel (M. galloprovincialis) myticin (GenBank accession numbers: AF162335 and AF162334) searched from NCBI, and the downstream primer was Carrier-specific primers (T7: 5'-TAATACGACTCACTATAGGG-3'), using the first strand ...

Embodiment 2

[0057] Example 2: Construction of eukaryotic expression vector of mussel antimicrobial peptide myticin

[0058] Forward and reverse primers were designed according to the two end sequences of the mature peptide sequences of cDNA genes of mytilin-1, 2, 3 and myticin-3, 5 (102 bp for mytilin and 120 bp for myticin). The forward primer adds two protective bases CG and restriction site TCTAGA (XbaI) at the 5' end respectively; a T is added thereafter to prevent ORF shifting; then the codon AAGAGA is added, which encodes two amino acids of KR, so that The precursor processing enzyme Kex2 secreted by yeast itself can cleave the mature peptide and secrete the expression product into the medium; the reverse primer adds three protective bases CCG and an enzyme cleavage site AAGCTT (HindⅢ ); pVT-1 and pVT-2 are sequencing primers. All primers were synthesized by Shanghai Meiji Biological Co., Ltd., and the relevant primers are as follows:

[0059] Myticin-F CGTCTAGATAAGAGACATTCGCATGCT...

Embodiment 3

[0065] Example Three: Transformation of Expression Vectors and Fermentation of Recombinant Saccharomyces cerevisiae

[0066] The expression plasmid DNA was transformed into yeast competent cells S78 by LiAC transformation method, and the cell suspension was coated with YSD (YNB 6.7g / L, Glucose 20g / L, Leucine 200mg / L, Adenine 50mg / L, Inositol 200mg / L and Agar 20g / L, sterilized at 121°C for 15min) plate, cultured at 30°C for 4-6 days; picked the plaques on the YSD plate and expanded them in YSD solution (50mL) for 24h; finally transferred to 1LYPD (Peptone 20g / L, Yeast extract 10g / L, Glucose 20g / L, sterilized at 121°C for 15min) solution, 30°C, 250 rpm for 3-4 days.

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Abstract

The invention discloses a mytilus coruscus antibacterial peptide. The name of the antibacterial peptide is myticin, and the open reading frame of cDNA gene coded by the antibacterial peptide has a special nucleotide sequence. The invention also discloses a preparation method and a eukaryotic expression vector of the mytilus coruscus antibacterial peptide and application thereof in antibiotic preparation. The antibacterial peptide provided by the invention can be used as a feed additive to substitute some antibiotics in the traditional feed and used in the aquaculture industry, thereby realizing green culture, reducing or even eliminating the accumulation of antibiotic in aquatic products, and improving the quality of the aquatic products; and the antibacterial peptide also can be used as novel biological antibiotic and used for treatment or prevention of bacterial disease.

Description

technical field [0001] The invention relates to a mussel antimicrobial peptide, a method for preparing the mussel antimicrobial peptide, and a use of the mussel antimicrobial peptide. 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 im...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/81C12N1/19A61K38/17A61P31/04A23K1/17C12R1/865A23K20/195
Inventor 廖智武梅王健鑫范美华石戈王日昕
Owner ZHEJIANG OCEAN UNIV
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