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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
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  • 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 no report on the constitutive expression of ApPlectasin using the Pichia pastoris GAP promoter

Method used

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  • 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

Embodiment 1

[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 restriction site BglII was added to the 5' end of the sequence, and the 3' end was sequentially added α-factor secretion signal peptide sequence and XhoI restriction site, and 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 ID No.3;

[0079] (2) The synthetic gene fragment and the vector pUC57 were double-digested with restriction endonucleases 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 the vector p...

Embodiment 2

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

[0106] 2.1 Linearization of recombinant vector pGAPAgPlectasin

[0107] Use AvrII to digest the constitutive recombinant expression vector pGAPAgPlectasin. The enzyme digestion system and reaction conditions are as follows:

[0108]

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

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

Embodiment 3

[0129] Example 3 Shake Flask Level Constitutive Expression AgPlectasin Recombinant Yeast Strain Screening

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

[0131] Pick positive transformants, inoculate into YPD liquid medium, 30°C, 250rpm shaking culture for 18-20h; Sterilized gauze was replaced with cellophane sealing film to wrap the mouth of the shaker flask, and cultured with shaking at 30°C and 250rpm for 3-5 days until the end of fermentation.

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

[0133] The inhibition zone assay (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 detect recombinant yeast Anti-Staphylococcus aureus activity of fermentation broth. S.aureus ATCC25923 was used as the test bacteria. Pick a single colony of...

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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

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Application Information

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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
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