Hypha mycin gene and karyogamy gene-engineering bacteria thereof

A kind of pectin and gene technology, applied in genetic engineering, plant genetic improvement, bacteria and other directions, can solve the problems of no E. Effect

Inactive Publication Date: 2011-09-21
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no research report on the expression of Plectasin in Escherichia coli

Method used

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  • Hypha mycin gene and karyogamy gene-engineering bacteria thereof
  • Hypha mycin gene and karyogamy gene-engineering bacteria thereof
  • Hypha mycin gene and karyogamy gene-engineering bacteria thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The construction of embodiment 1 plectasin prokaryotic fusion genetic engineering bacteria

[0033] 1.1 Design of Plectasin gene based on Escherichia coli preferred codons

[0034] According to the preference of Escherichia coli (Escherichia coli) codon usage (http: / / www.kazusa.or.jp / codon / ) optimization design, the obtained plectasin gene has as shown in the sequence table SEQ ID NO.1 Nucleotide sequence.

[0035] 1.2 Construction of plectasin prokaryotic fusion expression vector

[0036] Design the recognition cleavage site (IEGR) coding sequence of factor Xa at the 5′-end of the Plectasin gene, which is used to cut the fusion protein to obtain recombinant Plectasin, and design the multiple cloning sites of the vector that are not available in the Plectasin gene at both ends of the gene The restriction endonuclease BamHI and XhoI restriction sites on the site were directly synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the plectasin ...

Embodiment 2

[0039] Induced expression of embodiment 2 fusion protein and purification of recombinant plectasin

[0040] 2.1 Induced expression of fusion protein

[0041] Inoculate the transformed colonies in 10 mL of LB medium containing 100 μg / mL Amp, culture overnight in a shaker at 37°C, transfer 1% of the inoculum to 100 μg / mL Amp in TB medium, and wait for the bacteria to grow to logarithmic In the growth phase, when OD600 is about 1.0, add 100mmol / LIPTG to make the final concentration 0.4mmol / L. After induction at 30°C for different times, centrifuge at 5000rpm for 5min, wash once with PBS buffer (pH 7.4), collect the cells by centrifugation, and SDS- PAGE electrophoresis showed ( figure 1), the molecular weight of the protein expressed in the bacterial protein was close to the expected fusion protein molecular weight (22.4 kDa), and the expression level of the fusion protein Trx-plectasin reached the highest level at 4 h of induction, accounting for 53.6% of the total cell protein...

Embodiment 3

[0044] The activity of embodiment 3 recombinant Plectasin

[0045] 3.1 Hemolytic property of recombinant plectasin

[0046] Use a 10mL vacuum blood collection tube (containing heparin sodium as an anticoagulant) to collect blood from the ear vein of Japanese long-eared white rabbits, wash the cells three times with 10mM phosphate buffer (PBS, pH 7.3), and then centrifuge at 4°C and 1500rpm 10min until the supernatant is colorless and transparent. Take 180 μL of 10 mM PBS suspension of red blood cells (concentration of red blood cells is about 2%), mix with 20 μL of purified recombinant plectasiacin diluted in different ratios, and the mixed system is incubated at 37° C. for 30 minutes, and then centrifuged at 1500 rpm for 5 minutes. The supernatant was transferred to a 96-well plate, and the absorbance was measured at 540 nm using a microplate reader. The values ​​of hemolysis percentage 0% and 100% were determined simultaneously under the same conditions using 10 mM PBS and...

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Abstract

The invention discloses a preparation method and an application of recombinant hypha mycin. The hypha mycin gene designed by using Escherichia coli codon-bias has a nucleotide sequence represented by SEQ ID No.1, a recombinant expression pETPlectasin and a recombinant Escherichia coli BL21(DE3)pETPlectasin(CGMCC No.3563) are constructed, induced by IPTG, the expression of a fusion protein reaches 53.6% of the total cell protein. The recombinant hypha mycin prepared by utilizing Xa factor for enzyme cutting is nonhemolytic and can effectively inhibit the growth of Gram-positive bacterial pathogen Streptococcus pneumoniae and staphylococcus aureus, and is a novel anti infectious agent with the potential to prevent and cure streptococcosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a gene engineering bacterium fused with a plectasin gene and Escherichia coli. Background technique [0002] Plectasin is the first fungus isolated by Mygind and other research groups in 2005 by screening the cDNA library of the secreted protein of the saprophytic ascomycete Pseudoplectania nigrella, finding out some fragments highly similar to the defensin sequences of invertebrates Defensins (the research results were published in the journal "Nature" (Nature, 2005, 437: 975-980)). The open reading frame of the Plectasin gene encodes a 95-residue peptide consisting of a signal peptide sequence (residues 1-23), a pro-fragment (residues 23-55) and a 40-residue C-terminal region mature peptide ( residues 56-95). Plectasin has high resistance to Gram-positive bacteria, no cytotoxicity, no hemolysis, and good cerebrospinal fluid permeability. It has considerable potential in the treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N1/21C12P21/02C07K14/37C07K1/36A61K38/16A61P31/04C12R1/19
Inventor 杨雅麟王建华滕达张军田子罡
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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