Method for implementing constitutive expression of plectasin derivative MP1102 in Pichia pastoris

A technology of MP1102 and plectasin, which is applied in the field of genetic engineering, can solve the problems of MP1102 that have not been seen yet, and achieve the effect of broad application value and market prospect

Active Publication Date: 2013-10-02
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] MP1102 is a plectasin derivative with ideal antibacterial activity, and there is no report on the constitutive expression of MP1102 using the Pichia pastoris GAP promoter

Method used

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  • Method for implementing constitutive expression of plectasin derivative MP1102 in Pichia pastoris
  • Method for implementing constitutive expression of plectasin derivative MP1102 in Pichia pastoris
  • Method for implementing constitutive expression of plectasin derivative MP1102 in Pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Construction of Pichia pastoris constitutive expression vector pGAPMP1102

[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 pUCG...

Embodiment 2

[0104] Example 2 Construction of recombinant yeast strain containing pGAPMP1102

[0105] 2.1 Linearization of recombinant vector pGAPMP1102

[0106] Use AvrII to digest the constitutive recombinant expression vector pGAPMP1102. The enzyme digestion system and reaction conditions are as follows:

[0107]

[0108] After adding the above enzyme digestion system, react at 37°C for 3 hours, and detect by 2% agarose gel electrophoresis, electrophoresis conditions: 120 V, 30 min. After the electrophoresis was completed, the linearized recombinant expression vector pGAPMP1102 was recovered using a DNA recovery kit to detect the correct linearization of the recombinant expression vector.

[0109] 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 5000 bp and 3000 bp, which is consistent with the theoretical size (3186 bp).

[0110] 2.2 Pi...

Embodiment 3

[0128] The results showed that the positive clone rate of transformants was high, and the recombinant yeast PCR band size was 700-900bp ( Figure 4 ), which are consistent with the theoretical size (879bp), indicating that the target gene has been integrated into the yeast genome, and the recombinant yeast Pichia pastorisX-33 / GAPMP02 was obtained. Example 3 Shake Flask Level Constitutive Expression MP1102 Recombinant Yeast Strain Screening

[0129] 3.1 Constitutive expression of transformants at shake flask level

[0130] Pick positive transformants and inoculate them into YPD liquid medium, 30°C, 250rpm shaking culture for 18-20h, transfer 0.5-1% of the inoculum to 50mL YPD liquid medium, 30°C, 250rpm shaking culture for 1 day, then in four layers 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.

[0131] 3.2 Detection of antibacterial act...

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Abstract

The invention provides a method for implementing constitutive expression of plectasin derivative MP1102 in Pichia pastoris by using a GAP promoter. According to the invention, the GAP promoter is substituted for an AOX promoter in a vector pPICMP1102 to construct a constitutive recombinant expression vector pGAPMP1102 and convert Pichia pastoris X-33, and the obtained recombinant yeast is fermented and cultured to secrete MP1102. The invention implements constitutive expression of the plectasin derivative MP1102 in Pichia pastoris for the first tine, and after culturing for 12 hours, the total protein concentration in the fermentation liquid reaches 387.6 mg / L; the supernatant of the fermentation liquid is subjected to G25 desalting and SP ion-exchange chromatography to obtain the pure target product, wherein the yield is 105.84 mg / L; the Genetools analysis indicates that the purity is 95.13%. The antibacterial experiment indicates that the MP1102 has strong inhibiting action on Staphylococcus aureus ATCC standard strain, and the MIC of MP1102 is 0.0028-0.11 mu M. The MP1102 obtained by the method can be used in the fields of antibacterial drugs, food additives, cosmetics, feed additives and the like, and has wide application value and market prospects.

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 plectasin derivative MP1102 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 (Mygind et al., Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature, 2005, 437(7061): 975-980). [0003] Plectasin has a strong killi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P21/02C07K14/39
Inventor 王建华张勇滕达王秀敏毛若雨
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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