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Pelteobagrus fulvidraco beta defensin gene, and beta defensin antibacterial peptide and application thereof

A technology of β-defensins and antimicrobial peptides, applied in the field of molecular biology, can solve the problems of recombinant protein antibacterial experiments, different antibacterial effects of recombinant proteins, and in-depth research, and achieve good broad-spectrum antibacterial activity in vitro Effect

Active Publication Date: 2017-08-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on other few fish β-defensins is not in-depth, and the lack of sufficient and reliable recombinant protein antibacterial experiments and antibacterial effect statistics is currently affecting the evaluation of fish β-defensins antibacterial effect and its application in fish disease control. The problem with practice
Due to the differences in the β-defensin sequences of different fishes, the antibacterial effects of their recombinant proteins must be different

Method used

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  • Pelteobagrus fulvidraco beta defensin gene, and beta defensin antibacterial peptide and application thereof
  • Pelteobagrus fulvidraco beta defensin gene, and beta defensin antibacterial peptide and application thereof
  • Pelteobagrus fulvidraco beta defensin gene, and beta defensin antibacterial peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Cloning of the open reading frame (ORF) fragment of the β-defensin gene of yellow catfish.

[0052] 1. Cloning of the open reading frame (ORF) fragment of the β-defensin gene of yellow catfish

[0053] According to the partial transcriptome data of the yellow catfish in our laboratory, the predicted sequence of the yellow catfish β-defensin gene was retrieved, and Primer 5.0 was used to design the primer Pf_ΒD-F1 / R1 for amplifying the ORF region of the β-defensin:

[0054] Pf_ΒD-F1: ATGAAGTATCAAGGGATGACCAT

[0055] Pf_ΒD-R1: TCAGAGAATAACGTGAGACACAC

[0056] The PCR reaction system is as shown in Table 1-1:

[0057] Table 1-1 PCR reaction system

[0058]

[0059] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, a total of 35 cycles; extension at 72°C for 10 min; 16°C for 5 min. After the reaction, 20 μL of the PCR product was added to 4 μL of 6×loading buff...

Embodiment 2

[0089] Example 2 The cloning of the core coding region of the yellow catfish β-defensin gene (with the signal peptide removed from the ORF) and the construction of the prokaryotic recombinant expression plasmid.

[0090] According to the characteristics of the restriction site on the prokaryotic expression plasmid PET-32a, a pair of specific primers Pf_BD-F2 / R2 with added BamHI and EcoR I restriction sites were specially designed:

[0091] Pf_ΒD-F2: CGC GGATCC GCTAAAGGAAATGCAATGGC

[0092] Pf_ΒD-R2: CCG GAATTC TCAGAGAATAACGTGAGACACAC

[0093]The underlined parts of the primer sequences are the restriction sites of BamH I and EcoR I, respectively, and the bold parts (CGC and CCG) at the 5' end of the restriction sites are protective bases. Using PCR amplification technology, the ORF fragment cloned in Experiment 1 was used as the cDNA template, and the reaction program was (first, pre-denaturation at 95°C for 5 minutes; then, denaturation at 95°C for 30 seconds, annealing a...

Embodiment 3

[0094] Example 3: Induction and expression of prokaryotic recombinant expression vectors in Escherichia coli.

[0095] The constructed recombinant expression vector PET-32a-Pf_BD was introduced into Escherichia coli BL21 (DE3) competent cells, and the positive clones were picked on the plate and inoculated in 10 mL of LB liquid medium containing ampicillin, cultivated overnight at 37 ° C, 200 rpm, and added An equal volume of 30% glycerol was stored at -35°C for later use.

[0096] Exploration of the best induction expression conditions:

[0097] (1) Induction of expression at different times: Take 1 mL of spare bacteria and add it to 9 mL of LB liquid medium, cultivate to OD 600 0.6 to 0.8, then take 1mL of the bacterial solution and divide it into 2mL preparation tubes, a total of 7 tubes, at the same time take 1mL of the PET-32a empty plasmid bacterial solution under the same conditions as a control, and add 8 tubes of bacterial solution with a final concentration of 1mM ...

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Abstract

The invention discloses a pelteobagrus fulvidraco beta defensin gene, and beta defensin antibacterial peptide and application thereof. An in vitro recombinant expression technology is used for successfully expressing a pelteobagrus fulvidraco beta defensin recombinant protein PET-32a-Pf_BD, and antibacterial activity identification results show that the pelteobagrus fulvidraco beta defensin gene has a certain bacteriostasis for staphylococcus aureus in gram-positive bacterium, escherichia coli in gram-negative bacterium, aeromonas hydrophila, edwardsiella ictaluri and flavobacterium columnare. Therefore, a foundation is laid for application of the defensin recombinant protein, used as a broad-spectrum antibacterial drug, in aquaculture.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a yellow catfish β-defensin gene, a β-defensin antibacterial peptide and applications thereof. Background technique [0002] Yellow catfish is an important small and medium-sized freshwater economic fish in my country. It is favored by consumers because of its absence of intermuscular spines, delicious meat and rich nutrition. In recent years, it has become an important famous and high-quality farmed fish in my country's aquaculture industry. In 2015, the total output of yellow catfish in my country reached 355,725 tons, ranking the thirteenth among all freshwater fish output in my country. With the continuous increase of market demand, the current farming scale of yellow catfish is expanding year by year. However, large-scale and high-density farming will inevitably lead to the deterioration of the breeding water environment, and various bacterial diseases frequently o...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/46C12N15/70C12N1/21C12P21/02C07K1/34A61K38/17A61P31/04
CPCA61K38/00C07K14/461C12N15/70
Inventor 魏开建陈俭勇张桂蓉姬伟杨瑞斌樊启学
Owner HUAZHONG AGRI UNIV
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