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Ascidian antibacterial peptide, precursor peptide, coding gene and use thereof

A technology of antimicrobial peptides and sea squirts, applied in the field of bioengineering, can solve problems such as disrupting cell membrane permeability, cell energy status, and target cell death

Inactive Publication Date: 2008-08-27
SHENZHEN SUNSMILE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antimicrobial peptides have a special mechanism of action. The current popular view is: first, the positively charged antimicrobial peptide molecule contacts the negatively charged target cell membrane, and then the electromotive force generated by the target cell membrane will form a hydrophobic surface antimicrobial peptide dimer Injected into the cell membrane, finally multiple dimers or multimers together form a transmembrane ion channel, thereby disrupting the permeability of the cell membrane and the energy state of the cell, resulting in depolarization of the cell membrane, inhibition of respiration, and a severe decrease in cellular ATP content , resulting in target cell death

Method used

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  • Ascidian antibacterial peptide, precursor peptide, coding gene and use thereof
  • Ascidian antibacterial peptide, precursor peptide, coding gene and use thereof
  • Ascidian antibacterial peptide, precursor peptide, coding gene and use thereof

Examples

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Effect test

Embodiment 1

[0057] Example 1: Construction of cDNA library of pharyngeal tissue of Ascidian ascidian in South China Sea and EST sequence analysis

[0058] Extraction of total RNA and synthesis of cDNA: The pharyngeal tissue of Ascidian ascidian in South China Sea was isolated, and total RNA was extracted from the venom tube according to the instruction manual of TRIZOL LS reagent from GibcoBRL company. The library was constructed using the Smart cDNA Library Construction Kit kit from Clontech Company, and experiments were performed using the primers, vectors, and restriction endonucleases attached to the kit itself. Take 1 μg total RNA of sea squirt pharynx tissue and use SMART III oligonucleotide fragment (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII / 3'PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3') was reverse transcribed to synthesize the first strand to obtain 10 μl of cDNA first strand product.

[0059] Construction and identification of ascidian pharynge...

Embodiment 2

[0061] Example 2: Determination and Analysis of Ascidian Antibacterial Gene Clavanin H Sequence

[0062] The sequence of the antibacterial gene Clavanin H of Ascidian chinensis comes from 8 highly homologous EST sequences cloned from the cDNA library of the pharyngeal tissue of Ascidian spp. 4%, taking No. 5 clone D12 as an example to carry out bioinformatics analysis on it, the results showed that the length of its cDNA sequence was 523bp. Use the tool software SEQTOOL to analyze its base sequence, and obtain its largest open reading frame, with a length of 240bp (not including the stop codon), encoding a precursor peptide of 80 amino acid residues, and its nucleotide sequence is shown in the sequence table As described in SEQ ID NO: 1, the amino acid sequence of the precursor peptide of 80 amino acid residues is described in SEQ ID NO: 2 in the sequence listing. Further analysis showed that the 1-29 amino acid residues of the precursor peptide were its signal peptide region...

Embodiment 3

[0063] Example 3: Separation and purification of ascidian antimicrobial peptide Clavanin H

[0064] Take sea squirt pharyngeal tissue, cut it into pieces, extract with 50mmol / L Tris-Cl, pH8.6+100mmol / LNaCl, separate with G25Fine (i.d2.6*100cm), elute with extraction buffer, detect at 280nm, see four The first UV absorption peak is shown in Figure 2; the QSepharose fast flow separation on the second peak was collected, and NaCl gradient elution was used to obtain 5 elution peaks as shown in Figure 3. The first peak was collected and separated on a C18 reverse-phase column with acetonitrile gradient elution, and the peak with a retention time of 45.7 min was collected as shown in Figure 4. The isolated and purified ascidian antibacterial Clavanin H was analyzed by time-of-flight mass spectrometry, and the molecular weight was 2.71 kilodaltons, as shown in Figure 5. This is consistent with the theoretical molecular weight of 2.71 kilodaltons of the predicted expression sequence ...

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Abstract

The invention relates to a sea squirt antibacterial peptide, a relative precursor peptide, a relative encoding gene and an application, wherein the sea squirt antibacterial peptide is sea squirt antibacterial peptide Clavanin H, relative conservative variation polypeptide, relative active derivative or the polypeptide derived from the sea squirt antibacterial peptide Clavanin H with antibacterial function whose one or several amino acid residues are substituted, deleted or added. The precurose peptide of the sea squirt antibacterial peptide is provided with the amino acid sequence represented by SEQ ID NO: 2 in the sequence list. The inventive sea squirt antibacterial peptide can be used to prepare anti-virus product as forge additive, veterinary drug, preservative, pharmaceutical product as antibiotics.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an ascidian antibacterial peptide, its precursor peptide, coding gene and application of the ascidian antibacterial peptide. Background technique [0002] Antimicrobial peptides are a class of natural small molecular proteins with antibacterial activity. Since the Swedish scientist Boman et al. isolated the first antibacterial peptide cecropin (Cecropin) from silkworm chrysalis in 1975, people have been widely used in insects, amphibians, aquatic animals, mammals including humans, even plants and bacteria. At least 900 antimicrobial peptides have been found in the biological spectrum. This endogenous antimicrobial peptide is induced and synthesized, plays an important role in the body's resistance to pathogenic invasion, and is an important part of the body's non-specific immunity. [0003] Antimicrobial peptides have a broad spectrum of resistance. Most of them have a strong kil...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12A23K1/16A23L3/3526A61K38/17A61P31/00A23K20/147
Inventor 苏向东黄毓茂皮灿辉李永新
Owner SHENZHEN SUNSMILE BIOTECH
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