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Blunt snout bream beta-defensin gene and protein encoded by the same

A technology of β-defensins and bream, applied in the field of genetic engineering, can solve the problems of limited sources of natural defensins, high cost of chemical synthesis, difficulties in mass production, etc., and achieve shortened preparation time, obvious bacteriostatic effect, and good bacteriostasis active effect

Inactive Publication Date: 2013-07-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sources of natural defensins are limited, and chemical synthesis also has high costs and difficulties in mass production; the antibacterial activity of defensins is closely related to the spatial structure of peptide molecules. Although the primary structure of defensins synthesized in vitro is consistent with natural defensins, the spatial structure is different are different, resulting in less active

Method used

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  • Blunt snout bream beta-defensin gene and protein encoded by the same
  • Blunt snout bream beta-defensin gene and protein encoded by the same
  • Blunt snout bream beta-defensin gene and protein encoded by the same

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Extraction of total RNA and synthesis of cDNA from bream

[0026] 1) Extraction of total RNA from bream: The glassware used in the experiment was treated with 0.1% DEPC water and baked at 150°C for 4 hours. Plastic containers were soaked in 0.1% DEPC water overnight, and sterilized under high temperature and high pressure at 121°C for 20 minutes. Metal utensils were soaked in 1mol / L NaOH for 2h, rinsed thoroughly with 0.01% DEPC water, and dried at 37°C. Each reagent was prepared with 0.01% DEPC water without ribonuclease (Ribonuclease, RNase). The bream was anesthetized with anesthetic MS222, dissected, and the liver was removed, and 0.1 g of the liver was cut to extract RNA. Total RNA was extracted according to the instructions of Trizol reagent. The extracted RNA was subjected to 1.5% agarose gel electrophoresis, observed and identified using a gel imager, and three clear bands ( figure 1 ). Total RNA samples were frozen at -80°C for later use.

[00...

Embodiment 2

[0038] Example 2: Cloning and sequence analysis of the bream beta defensin gene

[0039] 1) Primer design

[0040] According to the fish β-defensin sequence information registered in NCBI / GenBank, according to the conservation of the sequence, a pair of primers (F1, R1) were designed in the upstream and downstream regions of the cDNA sequence open reading frame (ORF) with Primer 5.0 software; F1 : 5'-GCCATCATCCGAAGAAAC-3'; R1: 5'- TTCCAAATCAAAGGCATG-3'.

[0041] 2) PCR amplification: PCR amplification was performed using the reverse transcription product (cDNA) as a template, and the reaction system was 50 μL, namely: Premix Taq 2.025 μL; upstream primer F1 (100 mM) 0.5 μL; downstream primer R1 (100 mM) 0.5 μL ; Reverse transcription product (cDNA) 2 μL; sterilized deionized water 22 μL, the total volume is 50 μL, mix well. The PCR amplification reaction conditions are: pre-denaturation at 94°C for 3 minutes; then 30 cycle reactions, the temperature cycle conditions are dena...

Embodiment 3

[0051] Example 3: Construction of recombinant eukaryotic expression vector and induced expression in Pichia pastoris

[0052] 1) Design primers for expression of bream β-defensin:

[0053] Expression primers (F2, R2) were designed according to the ORF sequence of bream β defensin. The upstream primer F2 removed the 5' signal peptide sequence and added EcoR I restriction site, and the downstream primer added a stop codon (TGA, Note: Complementary codon sub-TCA) and Xba I restriction site, as follows:

[0054] F2: 5' CG GAATTC ATACTTGTCTTGCTTGTCC 3'

[0055] EcoRI

[0056] R2: 5' GC TCTAGATCA ATGTGATACACAGCATAAG 3'

[0057] XbaⅠ

[0058] 2) Construction of recombinant expression plasmids

[0059] PCR amplification of the recombinant plasmid with expression primers (F2, R2) (see image 3 ), the target fragment and the expression vector pPICZαA were digested step by step with restriction endonucleases EcoR I and XbaⅠ, ligated overnight at 16°C with T4 ...

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Abstract

The invention discloses blunt snout bream beta-defensin gene, which has a nucleotide sequence represented by SEQ ID NO:1. The invention further discloses recombinant protein encoded by the blunt snout bream beta-defensin gene, and a preparation method thereof, wherein the recombinant protein has an amino acid sequence represented by SEQ ID NO:2. The blunt snout bream beta-defensin recombinant protein provides good bacterial inhibition activities for staphylococcus aureus, streptococcus, ampicillin resistance type escherichia coli, aeromonas hydrophila and the like, and provides a class of new drugs with good therapeutic effects for anti-infective drugs and drug-resistant bacteria. The preparation method for the blunt snout bream beta-defensin recombinant protein has the following advantages that: high purity products can be easily obtained; and compared to the existing chemical synthesis method, the method of the present invention has characteristics of good activity, cost reduction, preparation time shortening, and easy industrial production achievement.

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 bream beta defensin gene and its encoded protein and application. Background technique [0002] Bacterial resistance to traditional antibiotics has become a major public health issue of global concern. WHO describes antibiotic resistance as a "global public health emergency affecting all countries", and the proportion of global health problems caused by this problem is gradually increasing. Therefore, the development of new antibiotics to overcome drug resistance is becoming more and more important. The antimicrobial peptides that have emerged in recent years are a new type of antibacterial drugs with great development potential. They not only have a broad-spectrum killing effect on bacteria and fungi, but also have good antiviral and antitumor effects. In addition, they are comparable to traditional antibiotics. C...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/81C07K14/46A61K38/17A61P31/04A23K1/17A23L3/3544A23K20/195
CPCY02A50/30
Inventor 袁改玲陈思思张涓黄金海熊文静刘小玲王卫民
Owner HUAZHONG AGRI UNIV
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