Fused antibacterial peptide, and preparation method and application thereof

A technology of antimicrobial peptides and hyphae, which is applied in the fields of genetic engineering and biomedicine, and can solve problems such as difficulty in environmental release, long growth cycle, and difficulty in transgenics

Active Publication Date: 2015-10-21
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, animal and plant bioreactors still have problems such as difficulty in genetic modification, long growth cycle, difficulty in separation and purification, high cost, and difficulty in environmental release due to concerns about gene drift.

Method used

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  • Fused antibacterial peptide, and preparation method and application thereof
  • Fused antibacterial peptide, and preparation method and application thereof
  • Fused antibacterial peptide, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Construction of fungal-specific double T-DNA safe expression vector

[0024] Using the pCAMBIA1300 vector as the basic vector, first pass Hind III and EcoR I double enzyme digestion, remove the MCS region, after filling in with DNA polymerase Klenow enzyme, connect with T4 ligase to form the intermediate vector pCB130-1. Next, through gene synthesis technology in the intermediate vector Sph A sequence (SEQ ID NO.1) was introduced at the I site, including the left and right border sequences and multiple cloning sites, and the intermediate vector pCB130-2 was constructed. On the basis of this vector, through PCR technology and enzyme cutting and ligation methods, respectively in Pst Introduce a fungal specific promoter at the I restriction site GPD (SEQ ID NO.2), in Sac I and EcoR Introduced between I restriction sites nos Terminator (SEQ ID NO.3), constructed into a fungal-specific double T-DNA expression vector, named pCB130NG, the schemat...

Embodiment 2

[0025] Embodiment 2. The design of fusion antimicrobial peptide CB-TPI

[0026] In order to improve the antibacterial activity of antimicrobial peptides, cecropin B (CB) and tachyplesin I (TP I) were constructed in series, and the C-terminus of TP was amidated ; At the same time, in order to separate the two antimicrobial peptides in space after expression, a glycine-proline Linker is added between the two genes; for the convenience of detection after expression, a His tag is added to the 3' end of the TPI gene. The amino acid sequence of the obtained fusion antibacterial peptide CB-TP I is a C sequence.

Embodiment 3

[0027] Example 3. Construction of fusion antimicrobial peptide CB-TPI fungal expression vector

[0028] According to the codon preference of fungi, the nucleotide sequence of the fusion antimicrobial peptide was codon-optimized, and the sequence (SEQ ID NO.3) was inserted into the vector pUC57 by artificial synthesis, and enzymes were added at both ends of the sequence cutting site Xbal and Sac I.

[0029] The pUC57-CB-TP I vector and the fungal-specific expression double T-DNA safe expression vector pCB130NG ( figure 1 ), digested with Xbal and SacI respectively ( figure 2 ), after recovering the digested product, after ligation, transformation and identification ( image 3 ) to obtain the fusion antimicrobial peptide CB-TPI fungal expression vector pCB130NG-CB-TPI.

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Abstract

The invention discloses a fused antibacterial peptide, and a preparation method and an application thereof. Cecropin B (CB) and tachyplesin I (TP I) are serially connected to construct a fused antibacterial peptide CB-TP I, and the DNA sequence of the fused antibacterial peptide CB-TP I is synthesized through optimizing CB gene and TP I gene and is inserted to a fungal specific double T-DNA safe expression vector; and a fungus is transferred to produce a transgenic mycelium of the fused antibacterial peptide, and the extracted fused antibacterial peptide has the characteristics of greatly improved antibacterial ability, low dosage and no drug resistance. The produced fused antibacterial peptide mycelium is dried at a low temperature to directly prepare a feed additive, so the protein separation and purification process is omitted, the quality difference among all batches is small, the output is large, and the cost is low. The fused antibacterial peptide can increases the disease of livestock and poultry and reduce the use of antibiotics, and has wide social and economic benefits.

Description

technical field [0001] The present invention relates to a fusion antibacterial peptide, in particular to fusion of cecropin antibacterial peptide B and limulusin antibacterial peptide TP I; its preparation method adopts edible and medicinal fungus mycelium bioreactor to produce fusion antibacterial peptide CB-TP I, In particular, it relates to fungi prepared from Flammulina velutipes, Pleurotus ostreatus, Ganoderma lucidum and Cordyceps militaris, and belongs to the technical field of genetic engineering and biomedicine. Background technique [0002] Antimicrobial Peptides is a kind of cationic polypeptide with strong antibacterial effect produced by organisms. It is an important component of biological innate immune system. It has the characteristics of broad spectrum, high efficiency and stability, and it is not easy to produce drug resistance. sex. It not only has bactericidal effect, but also inhibits and kills fungi, parasites, viruses and tumor cells, and has less to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/80C12N1/15C07K1/36C07K1/34C07K1/22A23K1/17
Inventor 李海燕李晓薇李玉王法微张敏李校堃刘秀明姚娜董园园王南王琦王秀然卢天成王岩孙喆孙天旭
Owner JILIN AGRICULTURAL UNIV
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