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Chicken lysozyme and chicken beta-defensin 7 fused gene and preparation method thereof

A technology of fusion gene and lysozyme, applied in the field of genetic engineering

Inactive Publication Date: 2017-02-15
易道生
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Feng Xuan constructed the expression vector of antibacterial peptide B-human lysozyme fusion protein; Ouyang Ping expressed the fusion expression of human lysozyme-antibacterial peptide, and used it in the treatment test of mouse mastitis, and achieved good antibacterial effect. However, there is no report on the fusion expression of chicken lysozyme-antibacterial peptide

Method used

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  • Chicken lysozyme and chicken beta-defensin 7 fused gene and preparation method thereof
  • Chicken lysozyme and chicken beta-defensin 7 fused gene and preparation method thereof
  • Chicken lysozyme and chicken beta-defensin 7 fused gene and preparation method thereof

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Embodiment 1

[0026] Embodiment 1, PCR primer design

[0027] On the basis of retaining the functional regions of the chicken lysozyme gene and the chicken β-defensin gene, PCR primers are designed; the present invention is a fusion protein transitionally connected by a connecting peptide, so the connecting peptide coding sequence (Linker) is introduced into the connecting region of the two genes .

[0028] 1. Chicken lysozyme gene primer:

[0029] Extract chicken oviduct total RNA, reverse transcribe into cDNA, use it as a template, according to the sequence of chicken lysozyme gene published by Genbank, use primer design software (primer5.0), design upstream and downstream primers, P1 and P2, for amplification Chicken lysozyme gene with addition and deletion of stop codon. The BamH I restriction site sequence is introduced into the upstream primer, and the linker sequence is added to the downstream primer.

[0030] The upstream primer sequence is as follows, and the primer name is P1: ...

Embodiment 2

[0044] Embodiment 2, PCR amplification

[0045] 1. Amplify chicken lysozyme gene fragment:

[0046] This is PCR1, and its reaction system is: containing Lyz gene plasmid 0.5μl, upstream primer (P1) 0.5μl, downstream primer (P2) 0.5μl, 2×HiFi-PCR Master 12.5μl, add ddH2O to a total volume of 25μl; amplify The conditions are: denaturation at 95°C for 5 minutes and cycle, denaturation at 95°C for 30s, annealing at 57°C for 30s, extension at 72°C for 30s, and extension at 72°C for 10 minutes after 30 cycles.

[0047] 2. Amplify chicken β-defensin 7 gene fragment:

[0048]This is PCR2, and its reaction system is: 0.5 μl of β-Gal7gene plasmid, 0.5 μl of upstream primer (P1), 0.5 μl of downstream primer (P2), 12.5 μl of 2×HiFi-PCR Master, add ddH2O to a total volume of 25 μl; The growth conditions are as follows: denaturation at 95°C for 5 minutes, followed by cycling, denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 30 seconds, and exten...

Embodiment 3

[0051] Embodiment 3, the construction of fusion gene Lyz-beta-Gal7 (overlap extension PCR method)

[0052] First, carry out PCR1 reaction to amplify chicken lysozyme gene Lyz gene and PCR2 reaction to amplify chicken antimicrobial peptide gene β-Gal7gene respectively, take 25ul of PCR products respectively, carry out 1.5% agarose gel electrophoresis respectively, and cut out with a clean scalpel. Gel the target fragment between 100bp and 250bp, between 250bp and 500bp, and use the DNA gel recovery kit to purify and recover the DNA fragment in the gel, take 5-10ul recovery solution and measure the OD with a spectrophotometer, and calculate the genes in it Molecular concentration, respectively take appropriate volumes of Lyz gene recovery solution and β-Gal7gene recovery solution to mix the two genes in equal amounts, and perform PCR3 reaction construction and amplification of Lyz-β-Gal7gene. Here, the reaction system and reaction conditions of the PCR3 reaction are shown in the ...

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Abstract

The invention discloses a chicken lysozyme and chicken beta-defensin 7 fused gene. The nucleotide sequence of the chicken lysozyme and chicken beta-defensin 7 fused gene is shown as SEQ ID NO:1. Fused protein encoded by the fused gene has the amino acid sequence shown as SEQ ID NO:2. The invention further discloses a construction method of an expression vector of the fused gene, the fused protein prepared by adopting the expression vector has the obvious bacteriostasis activity for staphylococcus aureus, and the minimal inhibitory concentration is lower than 4mg / ml.

Description

technical field [0001] The invention relates to a fusion gene of chicken lysozyme and chicken β-defensin 7 and a preparation method thereof, belonging to the technical field of genetic engineering. Background technique [0002] At present, due to the overuse of antibiotics, public safety issues such as the rapid increase of drug-resistant strains and food safety crises are emerging one after another. Finding efficient, residue-free, green and environmentally friendly alternatives to antibiotics has become a research hotspot. [0003] Lysozyme mainly exists in the tissue fluid of animals and plants and some microorganisms, such as nasal mucus, tears, saliva, egg protein, Bacillus subtilis culture and some vegetables. This enzyme can hydrolyze the β-1,4-glucosidic bond between N-acetylglucosamine and N-acetylmuramic acid in the cell wall of bacteria, so it is also called muramidase, that is, N-acetylmuramic acid glycan hydrolase. Among them, the research on egg white lysozym...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/10C12N15/70C12N1/21C12R1/19
CPCC12N9/2462C07K14/4723C07K2319/00C12N15/70C12N2800/101C12Y302/01017
Inventor 易道生
Owner 易道生
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