Method for constitutive expression of target antibacterial peptide AgPlectasin in Pichia pastoris

An antimicrobial peptide and targeting technology, applied in the field of genetic engineering, can solve problems such as ApPlectasin that has not been seen yet, achieve broad application value and market prospects, and strong inhibitory effect

Active Publication Date: 2013-09-25
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
View PDF1 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] ApPlectasin is a plectasin derivative with ideal antibacterial activity, and there is

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for constitutive expression of target antibacterial peptide AgPlectasin in Pichia pastoris
  • Method for constitutive expression of target antibacterial peptide AgPlectasin in Pichia pastoris
  • Method for constitutive expression of target antibacterial peptide AgPlectasin in Pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0077] Example 1 Construction of Pichia pastoris constitutive expression vector pGAPAgPlectasin

[0078] (1) Design and synthesize the following gene fragments: According to the glyceraldehyde 3'-phosphate dehydrogenase promoter (GAP) sequence provided by Invitrogen, the enzyme cleavage site BglII was added at the 5' end of the sequence, and then added at the 3' end. α-factor secretion signal peptide sequence and XhoI restriction site, the designed gene sequence was sent to Shanghai Sangon Bioengineering Co., Ltd. for synthesis, the nucleotide sequence of the synthesized gene fragment is shown in SEQ ID No.4, GAP promoter The nucleotide sequence of the sequence is shown in SEQ IDNo.3;

[0079] (2) Double-enzyme digestion was performed on the synthesized gene fragment and the vector pUC57 with the restriction enzymes BglII and XhoI, respectively, and the pUC57 vector fragment and the GAP promoter + α-factor secretion signal peptide fragment were recovered and ligated to obtain ...

Example Embodiment

[0105] Example 2 Construction of recombinant yeast strain containing pGAPAgPlectasin

[0106] 2.1 Linearization of recombinant vector pGAPAgPlectasin

[0107] The constitutive recombinant expression vector pGAPAgPlectasin was digested with AvrII. The digestion system and reaction conditions were as follows:

[0108]

[0109] After the above enzyme digestion system was added, the reaction was carried out at 37°C for 3 hours, and detected by 2% agarose gel electrophoresis. Electrophoresis conditions: 120V, 30min. After electrophoresis is completed, the linearized recombinant expression vector pGAPAgPlectasin is recovered using a DNA recovery kit to detect the correct linearization of the recombinant expression vector.

[0110] The electrophoresis results showed that the linearization effect of the recombinant vector was good ( image 3 ), almost no uncleaved vector and star activity, the size is between 5000bp and 3000bp, which is consistent with the theoretical size (3224b...

Example Embodiment

[0129] Example 3 Screening of recombinant yeast strains expressing AgPlectasin constitutively at the shake flask level

[0130] 3.1 Constitutive expression at the shake flask level of transformants

[0131] Pick the positive transformants, inoculate them into YPD liquid medium, 30℃, 250rpm shaking culture for 18-20h; 0.5-1% inoculum is transferred to 50mL YPD liquid medium, 30℃, 250rpm shaking culture for 1 day, then 4 layers The sterilized gauze was replaced with cellophane sealing film to wrap the mouth of the shaker, and the flask was shaken at 30°C and cultured at 250rpm for 3-5 days until the fermentation was over.

[0132] 3.2 Detection of antibacterial activity of recombinant yeast fermentation broth

[0133] Zone of inhibition assay analysis (Zhang et al., Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus. Protein Expression and Purification, 2011, 78(2):189-196) was used to dete...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Lengthaaaaaaaaaa
The inside diameter ofaaaaaaaaaa
Login to view more

Abstract

The invention provides a method for constitutive expression of a target antibacterial peptide AgPlectasin in Pichia pastoris by use of a GAP promoter. According to the method, an AOX promoter in a vector pPICAgPlectasin is replaced by the GAP promoter to construct a constitutive expression vector pGAPAgpPICAgPlectasin and transform Pichia pastoris X-33; and the obtained recombinant yeast is subjected to fermentation culture and can secrete and generate AgPlectasin. The method provided by the invention realizes constitutive expression of the target antibacterial peptide AgPlectasin in Pichia pastoris for the first time, and after 120 hours of culture, the total protein concentration in the fermentation liquid reaches 532.4mg/L; the supernate of the fermentation liquid is subjected to G25desalination and SP ion-exchange chromatography, and a pure target product can be obtained with an output of 118.37mg/L; and the purity is 93.27% according to Genetools analysis. Bacteriostasis experiments indicate that AgPlectasin has a strong inhibition effect on the ATCC standard strain of staphylococcus aureus, and the MIC of AgPlectasin is 0.09-1.47muM. The AgPlectasin obtained by the method provided by the invention can be applied to the fields of antibacterial drugs, food additives, cosmetics, feed additives and the like, and has high application value and broad market prospect.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to the use of a constitutive promoter P GAP A method for highly expressing the targeting antimicrobial peptide AgPlectasin in recombinant Pichia pastoris. Background technique [0002] Plectasin is a highly effective anti-G + Bacterially active antimicrobial peptides. Plectasin gene encodes a polypeptide sequence containing 95 amino acid residues, of which 1-23 is a signal peptide sequence, 24-55 is a leader peptide sequence, and 56-95 is a mature peptide (Plectasin) sequence. The theoretical molecular weight of Plectasin is 4407.9Da, with six histidines (His) and five lysines (Lys). In different pH environments, due to the different dissociation states of histidine, the net charge of Plectasin ranges from +1 to +3 between (Mygind et al., Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature, 2005, 437(7061): 975-980). [0003] ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/81C12N1/19C12P21/02C07K14/39
Inventor 王建华毛若雨滕达王秀敏张勇
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap