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Acipenser sinensis antibacterial peptide and preparation method and application thereof

A Chinese sturgeon and antimicrobial peptide technology, applied in the field of genetic engineering, can solve the problem of not finding proteins and peptides

Inactive Publication Date: 2012-07-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no protein peptides similar to natural antimicrobial peptides of Chinese sturgeon have been found in the protein database

Method used

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  • Acipenser sinensis antibacterial peptide and preparation method and application thereof
  • Acipenser sinensis antibacterial peptide and preparation method and application thereof
  • Acipenser sinensis antibacterial peptide and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Cloning of Chinese sturgeon antimicrobial peptide gene

[0049] (1) Primer design

[0050] According to the fish hepcidin sequence information registered in NCBI / GenBank, according to the conservation of the sequence, a pair of degenerate primers (F1, R1) were designed in the upstream and downstream regions of the cDNA sequence ORF with Primer 5.0 software; F1: 5'-ACAYCAGAACWAACAYTC- 3'

[0051] R1: 5'-CYACTTTTAYAAGGCWTA-3'.

[0052] (2) Extraction of blood total RNA by Trizol method

[0053] Draw blood from the caudal artery of Chinese sturgeon with a sterile disposable syringe. After standing at room temperature for a period of time, centrifuge at 1000 rpm for 30 minutes at room temperature, discard the supernatant, and precipitate to become blood cells.

[0054] The glassware used in the experiment was treated with 0.1% DEPC water and baked at 150°C for 4h. Plastic containers were soaked in 0.1% DEPC water overnight, and then sterilized under high temp...

Embodiment 2

[0076] Example 2: Expression and antibacterial activity analysis of recombinant eukaryotic expression vector in Pichia pastoris

[0077] (1) Linearization of recombinant plasmids

[0078] Recombinant expression plasmid pPICZαA-pen-epcidin and pPICZαA empty vector were cut with restriction endonuclease BstX I respectively, the system is as follows:

[0079]

[0080] After reacting at 45°C for 5h, the digested product was purified with a DNA cleaning kit, eluted with 20 μL ddH2O, and stored at -20°C.

[0081] (2) Electrotransformation of yeast competent cells with linearized recombinant plasmids

[0082] Mix 80 μL of Pichia pastoris competent cells (Pichia pastoris) with BstX I linearized pPICZαA-antimicrobial peptide plasmid (20 μL), transfer to a pre-cooled 0.2 cm electroporation cup (Bio-Red), and place 5min on ice, 2kV, 25μF, 400Ω, after electric shock for 8 milliseconds, immediately add 1mL of pre-cooled 1mol / L sorbitol, take 200μL and spread them on YPDS containing Ze...

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Abstract

The invention discloses an acipenser sinensis antibacterial peptide and a preparation method and an application thereof, and particularly discloses a novel acipenser sinensis natural antibacterial peptide pen-hepcidin, an amino acid sequence for encoding a gene and an application of a protein to preparation of a medicament for treating infectious diseases and a feed additive. An antibacterial peptide gene pen-hepcidin is cloned from acipenser sinensis blood with an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method, and the DNA (Deoxyribose Nucleic Acid) sequence of the antibacterial peptide gene pen-hepcidin is shown as a sequence SEQ ID:1 in a sequence table. The amino acid sequence of a protein (pen-hepcidin) encoded by using the gene is shown as SEQ ID:3 in a sequence table. According to amino acid sequence analysis, a protein polypeptide which is similar to the antibacterial peptide is not found in a protein database, and belongs to a novel antibacterial peptide. The antibacterial peptide disclosed by the invention has antibacterial activity on a part of Gram-positive bacteria and Gram-negative bacteria, has a remarkable effect on drug-resistant strains, and can be used for preparing a medicament for treating infectious diseases or applied as antibacterial feed additives and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a new natural antibacterial peptide, in particular to a Chinese sturgeon antibacterial peptide and its preparation method and application. Background technique [0002] Antimicrobial peptides are a class of small molecule polypeptides, generally consisting of less than 100 amino acid residues, most of which have a certain amount of positive charge. Antimicrobial peptides have activities such as inhibiting bacteria, fungi, protists (Nicolas et al., 1995), antiviral (Mohan et al., 2010), antitumor (Papo et al., 2005), etc., and are a class of broad-spectrum antibacterial peptides , and is nontoxic to eukaryotic cells. At present, antimicrobial peptides have become a new research hotspot in recent years. The research contents include: isolation and purification of antimicrobial peptides, analysis of amino acid sequences, expression levels of antimicrobial peptides in vario...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12C12N15/10A61K38/17A61P31/04A23K1/17A23K20/195
Inventor 袁改玲高宇陈思思
Owner HUAZHONG AGRI UNIV
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