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Optimized gene of zebrafish defensin defbl3 and preparation method of recombinant protein thereof

A technology of recombinant protein and defensin, which is applied in the field of genetic engineering, can solve the problems of troublesome post-processing of fermentation broth, no practical production application value, and high cost of culture medium, so as to facilitate later operation, save cost, and shorten the path of industrialization Effect

Inactive Publication Date: 2016-03-16
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is worth noting that the media used in the reports of Jin Junyan, Zhang Juan, and Wang Yuhua are all traditional laboratory media containing yeast extract and protein jelly, but the cost of this media is high, and the post-processing of the fermentation broth is troublesome. Generally, it is not used in production with fermentation tanks
Therefore, from the aspect of culture medium, the methods provided in these documents do not have practical production application value either.

Method used

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  • Optimized gene of zebrafish defensin defbl3 and preparation method of recombinant protein thereof
  • Optimized gene of zebrafish defensin defbl3 and preparation method of recombinant protein thereof
  • Optimized gene of zebrafish defensin defbl3 and preparation method of recombinant protein thereof

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Embodiment 1

[0039] Expression and purification of zebrafish recombinant defbl3 mature peptide in Pichia pastoris X-33

[0040] 1.1 Medium formula:

[0041] MGY (MinimalGlycerolMedium) medium: YNB13.4g / L, biotin 4×10 -4 g / L, glycerol 10mL / L; MM (MinimalMethanolMedium) medium: YNB13.4g / L, biotin 4×10 -4 g / L, methanol 10mL / L.

[0042] 1.2 Optimized synthesis of cDNA of mature peptide of zebrafish defbl3 and addition of enzyme cutting sites

[0043] Referring to the cDNA sequence of the zebrafish defensin defbl3 (GenBank: NM_001081555) published in GenBank, predict the cDNA sequence of its mature peptide (its nucleotide sequence is shown in SEQ ID NO: 1), according to the codon preference pair of Pichia pastoris Original ACA, GTG, CAG, ACA, TGC, GGA, CGA, GGA, CTA, TGC, AGG, TGC, TAT, GCA, CGG, GAG, TAT, ATT, TAT, CGT, GGC, TGC, CCT, CGC, AGG, TGC, CGA, TTT and other codons have been optimized, and the optimized corresponding codons are ACT, GTT, CAA, ACT, TGT, GGT, AGA, GGT, TTG, TGT, AG...

Embodiment 2

[0053] Antibacterial Activity of Recombinant Defensin defbl3 Mature Peptide by Agarose Diffusion Assay

[0054] The selected Gram-positive bacteria were Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus, and the Gram-negative bacteria were Escherichia coli BL21 (DE3), Escherichia coli Bacillus DH5α, Escherichia coli ATCC25922, Vibrio Parahaemolyticus, Salmonella, Pseudomonas aeruginosa. Cultivate the above-mentioned bacteria overnight in LB liquid medium, and then use fresh LB liquid medium to reduce the OD of the bacteria to 600 Adjust it to 0.4, add it to the nutrient agar that has not yet solidified according to the dilution ratio of 1:1000, shake it well and pour it on the plate. After the plate solidifies, use a puncher to make a 6mm hole, add 50 μL of the expression supernatant, and use the pPICZαA empty plasmid The expression supernatant of the above-mentioned supernatant was used as a control, and cultured in a 30°C incubator for 12 hours, and the inh...

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Abstract

The invention discloses an optimized gene of zebrafish defensin defbl3 and a preparation method of a recombinant protein of the optimized gene and further provides a recombinant expression vector and a pichia host cell including the optimized gene, application of the optimized gene in production of the zebrafish defensin defbl3 and application of the recombinant protein in preparation of antibacterial drugs. On the premise that an amino acid sequence of the zebrafish defensin defbl3 is not changed, a nucleotide sequence of the zebrafish defensin defbl3 is optimized according to a pichia preference codon, and the optimized sequence is shown as SEQ ID NO.2. The recombinant zebrafish defensin defbl3 is prepared by expressing the optimized gene in a pichia host cell. The zebrafish defensin defbl3 can be efficiently prepared by means of the preparation method, the adopted culture conditions are simple, the cost is lower, and the obtained defensin defbl3 is good in antibacterial effect.

Description

technical field [0001] The invention relates to a codon-optimized gene, in particular to an optimized gene transformed from the zebrafish defensin defbl3 gene according to the preferred codon of Pichia pastoris, and a method for preparing a recombinant protein coded by it, belonging to the field of genetic engineering. Background technique [0002] Looking at the domestic aquaculture industry, seawater and freshwater aquaculture areas are mostly distributed in the waters near coastal harbors and estuaries, and these waters are also the main receiving places for coastal land-based pollutants and marine sewage. According to statistics, the amount of waste water directly discharged into the sea in my country is as high as 8 billion tons every year. Agricultural sewage rich in nutrients, pathogenic microorganisms and organic pesticides also flows into coastal water bodies along with the surface, resulting in deterioration of water quality. Such aquaculture environmental pollutio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C12N1/19C07K14/46A61K38/17A61P31/04
CPCY02A50/30
Inventor 陶妍宋长丰李雯
Owner SHANGHAI OCEAN UNIV
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