Preparation and application of recombinant plectasin

A technology of plectasin and yeast, applied in the field of treatment or prevention of Gram-positive bacteria, especially streptococci, preparation of recombinant plectasiacin by yeast genetic engineering bacteria, the field of preparation of recombinant plectasiacin, Achieve good pH stability

Active Publication Date: 2014-01-15
SHENZHEN SUNSMILE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the expression of Plectasin is limited to heterologous expression in Aspergillus niger, so far there is no report on its expression in yeast

Method used

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  • Preparation and application of recombinant plectasin
  • Preparation and application of recombinant plectasin
  • Preparation and application of recombinant plectasin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1 plectasin genetically engineered bacteria

[0045] 1.1 Design of Plectasin gene based on yeast preferred codons

[0046] The Plectasin gene was artificially designed according to the codon usage preference of Pichia pastoris (http: / / www.kazusa.or.jp / codon / ), and the obtained sequence is shown in SEQ ID NO.1.

[0047] 1.2 Construction of recombinant plectasin expression vector

[0048] At the 5'-end of the Plectasin gene, a restriction endonuclease XhoI cleavage site, which is not available in the Plectasin gene but on the multiple cloning site of the vector, is designed, and the yeast Kex2 cleavage site after the XhoI cleavage site is retained (KR) coding sequence, in order to cleave the signal peptide after secretion and expression, to obtain recombinant Plectasin, design the TAATAA terminator sequence and XbaI restriction site at the 3'-end of the gene, in order to terminate the expression of the polypeptide and construct the vector d...

Embodiment 2

[0055] Induced expression of embodiment 2 recombinant plectasin

[0056] 2.1 Induced expression at shake flask level

[0057] The recombinant Pichia pastoris with high expression level obtained by screening was cultured in BMGY medium with glycerol as carbon source at 29°C with shaking at 250rpm to OD 600 2-6 hours; centrifuge at 5000rpm for 5min, collect the cells, and resuspend the cells with methanol-containing BMMY medium to OD 600 1.0, 250rpm at 29°C for induction culture; methanol was added every 24h to a final concentration of 0.5%, and the fermentation broth was centrifuged at 120h of induction, and the supernatant was collected and freeze-dried for later use.

[0058] Such as figure 1 As shown, after induced expression, Tricine-SDS-PAGE electrophoresis showed that the secreted protein contained a 4.4kDa peptide.

[0059] 2.2 Induced expression at the level of 5L fermenter

[0060] Sartorius Bplus 5L fermenter (Sartorius, Germany) was used for high-density fermenta...

Embodiment 3

[0063] The purification of embodiment 3 recombinant plectasin

[0064] 3.1 Gel Filtration Chromatographic Separation of Recombinant Plectasin

[0065] The chromatographic column material is dextran gel Sephadex G-25 (purchased from Huamei Bioengineering Company), the separation range is 1000-5000Da, the purification conditions: the column bed volume is 40mL, the diameter-to-height ratio is 1:10, and deionized water is used for elution , the flow rate is 0.5mL / min, the detection wavelength: 280nm, after purification, it is tested by antibacterial test, and the components with the highest antibacterial activity are collected for further reversed-phase high performance liquid chromatography.

[0066] 3.2 Reversed-phase high-performance liquid chromatography of recombinant plectasin

[0067] Columns are XBridge TM BEH300 C18 (5μm, 4.6mm×250mm), using TFA-containing water and acetonitrile as the mobile phase, separated by gradient elution mode, dissolved the sample in ultrapure ...

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Abstract

The invention discloses preparation and application of recombinant plectasin. The method comprises designing plectasin gene according to preferred codons of Pichia pastoris, wherein possible nucleotide sequences of the plectasin gene are expressed in SEQ ID NO. 1, constructing recombinant expression vectors pPICPlectasin and recombinant genetic engineering bacteria Pichia pastoris X33pPICPlectasin (CGMCC NO. 3564), carrying out a high density fermentation process on the recombinant genetic engineering bacteria Pichia pastoris having a high expression level, wherein a total protein concentration of supernate from the high density fermentation process is 729 microgrammes per milliliter, dialyzing and freeze-drying the supernate, and orderly carrying out a gel filtration chromatography treatment and a reversed phase high performance liquid chromatogram treatment on the freeze-dried supernate to obtain high purity recombinant plectasin. The high purity recombinant plectasin is not hemolytic, has favorable PH stability, heat stability and anti-pepsin activity, and can inhibit effectively the growth of gram-positive pathogen Streptococcus pneumonia, staphylococcus aureus and staphylococcus epidermidis. Therefore, the high purity recombinant plectasin can be utilized for treating and preventing gram-positive bacterium and especially streptococcus and has potential antimicrobial drug development values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing recombinant plectasiacin and its application, in particular to the preparation of recombinant plectasiacin by yeast genetically engineered bacteria and its application in the treatment or prevention of Gram-positive bacteria, especially streptococci Applications. Background technique [0002] Plectasin is the first defensin isolated from a fungus (the saprophytic ascomycete Pseudolectania nigrella) by Mygind et al. (Mygind et al., Nature, 2005, 437: 975-980). The open reading frame of the plectasin gene encodes a 95-residue long peptide consisting of a signal peptide sequence (residues 1-23), a pro-fragment (residues 23-55) and a 40-residue C-terminal Region (residues 56-95) with 50-55% sequence similarity to several invertebrate defensins. No sequence similarity to mammalian α- or β-defensins. Plectasin, has six cysteines and five lysines, and a differentiate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/81C12N1/19C07K14/37C12P21/02A61K38/16A61P31/04C12R1/84
Inventor 王建华杨雅麟张军滕达王少然田子罡
Owner SHENZHEN SUNSMILE BIOTECH
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