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Polypeptide with resistance to gram-positive bacteria

A technology for gram-positive bacteria and antibacterial activity, applied in the field of antimicrobial peptide screening, can solve the problems of reduced growth rate of farmed animals

Active Publication Date: 2018-01-19
NINGBO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, exogenous antimicrobial peptides often cause stress reactions in fed animals due to bioincompatibility; cause problems such as reduced growth rates in farmed animals

Method used

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  • Polypeptide with resistance to gram-positive bacteria
  • Polypeptide with resistance to gram-positive bacteria
  • Polypeptide with resistance to gram-positive bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Screening and detection of antimicrobial peptides

[0022] 1. Strain activation

[0023] 1) Nocardia seriolae was provided by Professor Wang Guoliang of Ningbo University. Nocardia seriolae was inoculated into the culture medium (glucose 0.02g / ml, yeast powder 0.015g / ml, potassium dihydrogen phosphate 0.75x10 -3 g / ml, calcium chloride 0.2x10 -3 g / ml, pH value is 6.5); then cultured at 25°C for 24h, then centrifuged at 3000rpm to obtain bacterial precipitate, diluted with sterile normal saline until the concentration of Nocardia ichthyosa was 1×10 5 The cfu / mL Nocardia spp. solution is ready for use;

[0024] 2) The Streptococcus agalactiae obtained from the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences was activated on a blood agar plate, and then inoculated in the brain-heart infusion medium (BHI) for expansion; cultured at 30°C for 24h, then 3000rpm After centrifugation, the bacterial pellet was obtained and dilu...

Embodiment 2

[0049] Example 2: PA-G + Recombinant expression of -1 polypeptide

[0050] 1. In PA-G + The nucleotide sequence of the -1 polypeptide (SEQ ID NO: 1) was linked into the expression vector of Pichia pastoris. Both the vector containing the antimicrobial peptide gene and the yeast expression vector were digested with XhoI and XbaI, and the digested products were recovered and ligated for PCR identification and sequencing.

[0051] 2. After the positive plasmid was linearized by SacI single enzyme digestion, it was added to the competent cell suspension of Pichia pastoris. After electroporation, spread evenly on YPDS selection plate containing 100 μg / mL Zeocin, and incubate at 30°C for 3-5 days. When the positive transformants on the YPDS plate grow larger, each transformant is inoculated onto the YPDS selection plate containing Zeocin 200 μg / mL, 500 μg / mL, and 1000 μg / mL in turn, and the colonies that grow normally on the high-concentration Zeocin plate are Possibly high copy...

Embodiment 3

[0055] Example 3: PA-G + Antibacterial detection of -1 polypeptide

[0056] Detect PA-G + Antibacterial properties of -1 polypeptide against Streptococcus agalactiae, Nocardia, Vibrio parahaemolyticus and Edwardsiella lentus; meanwhile, the pa1-1 polypeptide with amino acid sequence SQGVKPSINVGSYSLATIIQDLL (SEQ ID NO: 3) was used as a control.

[0057] Specific steps are as follows:

[0058] 1. Activation treatment of bacterial strains: Obtain purified single colonies of Streptococcus agalactiae, Nocardia, Vibrio parahaemolyticus and Edwardsiella tarda by streak culture on solid medium. Select and expand the culture in 25ml liquid LB medium respectively, and dilute the cultured bacteria solution to a concentration of 5×10 5 CFU / mL, take 60 μl in turn and add to each well of a 96-well plate to prepare for the experiment.

[0059] 2. The recombinantly expressed PA-G + After the -1 polypeptide and pa1-1 polypeptide are quantified, they are sequentially diluted in liquid medi...

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Abstract

The invention provides polypeptide with resistance to gram-positive bacteria, and a gene for coding the polypeptide. The sequence of the polypeptide is as show in SEQ ID NO:1, and the nucleotide sequence of the gene is as shown in SEQ ID NO:2. Pomfret is infected by gram positive microorganisms frequently used in the field of aquatic products, the polypeptide with resistance to gram-positive bacteria is obtained by using a subtractive hybridization method, and the polypeptide can be used as a feed additive for the pomfret.

Description

technical field [0001] The invention belongs to the technical field of antimicrobial peptide screening, and in particular relates to a polypeptide resistant to Gram-positive bacteria. Background technique [0002] Silver Pomfret (Pampus argenteus) belongs to Perciformes (Perciformes), Stromateoidei (Stromateoidei) Pomfret family (Stromateidae), Pomfret (Pampus). It spends the winter in the open sea where the warm current passes, and has the habit of migrating. Its meat is delicious, tender, thick and soft, and is deeply loved by consumers. It is a fish with high commercial value but has not yet been successfully cultured on a large scale. [0003] Since the 1990s, due to overfishing and deterioration of the marine environment, silver pomfret resources have declined rapidly. After entering this century, domestic scholars have done a lot of research on the resource distribution and changes of silver pomfret. The results show that the current silver pomfret resources have dec...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P31/04A23K50/80A23K20/147
Inventor 郑俊勇王亚军胡佳宝余娜匡思雯张曼曹小欢李渊博陶顺顺徐芳君
Owner NINGBO UNIV
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