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A group of trypsin-resistant antimicrobial peptides and preparation method thereof

An antibacterial peptide, Pichia pastoris technology, applied in the fields of protein engineering and genetic engineering, can solve the problems of loss of activity, affecting the stability of NZ2114 trypsin, restricting the effect of oral therapy and other issues

Active Publication Date: 2020-03-31
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

However, it is very sensitive to trypsin and almost loses its activity after incubation with trypsin
Analysis showed that there were 6 trypsin cleavage sites in the NZ2114 sequence, which were R14, K20, K23, K26, K32, and K38. Effect
At present, there is no relevant report on the stability modification of NZ2114 trypsin

Method used

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  • A group of trypsin-resistant antimicrobial peptides and preparation method thereof
  • A group of trypsin-resistant antimicrobial peptides and preparation method thereof
  • A group of trypsin-resistant antimicrobial peptides and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Molecular design and gene synthesis of trypsin-resistant antimicrobial peptides

[0035] According to the presence of 6 trypsin cleavage sites (R14, K20, K23, K26, K32, K38) in the NZ2114 sequence, it binds to its active site and key structural sites. A total of 33 sequences (SEQ ID No. 1-SEQ ID No. 33) were designed for single-site mutants, double-site mutants and triple-site mutants.

[0036] The mutant gene sequences (SEQ ID No. 34-SEQ ID No. 66) were designed according to the codon preference of Pichia pastoris. In order to ensure the integrity of the sequence during expression, an XhoI restriction site and a Kex2 restriction site were added to the 5' end of the mutant gene sequence, and a TAA, TAG terminator sequence and XbaI restriction site were added to the 3' end. The above sequence was completed by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 2

[0037] Example 2 Construction of Pichia pastoris constitutive expression vector pGAPNZTYR1-pGAPNZTYR33

[0038] The synthetic gene fragment and vector pGAPZaA were double-digested with restriction endonucleases XhoI and XbaI respectively, and the pGAPZaA vector fragment and mutant gene fragment were recovered and ligated to obtain vectors pGAPNZTYR1-pGAPNZTYR33;

[0039] The detailed construction process of the vector pGAPNZTYR1~pGAPNZTYR33: use restriction endonucleases XhoI and XbaI to carry out double enzyme digestion on the synthesized gene fragment and pGAPZaA respectively, the enzyme digestion system and conditions are as follows:

[0040]

[0041] After adding the sample to the above enzyme digestion system, react at 37°C for 3 hours, and detect by 2% agarose gel electrophoresis, electrophoresis conditions: 120V, 30min. After electrophoresis, use a scalpel to cut the electrophoretic bands corresponding to the carrier fragment and the gene fragment under the ultraviol...

Embodiment 3

[0058] Example 3 Construction of recombinant yeast strains containing pGAPNZTYR1~pGAPNZTYR33

[0059] 3.1 Linearization of recombinant vector pGAPNZTYR1~pGAPNZTYR33

[0060] Use AvrII to digest the constitutive recombinant expression vector pGAPNZTYR1~pGAPNZTYR33. The enzyme digestion system and reaction conditions are as follows:

[0061]

[0062] After adding the sample to 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 is completed, it is detected that the recombinant expression vector is correctly linearized, and then the linearized recombinant expression vector is recovered using a DNA recovery kit.

[0063] 3.2 Pichia pastoris electrotransformation and identification of linearized vector

[0064] 1) Pick a single colony of X-33 on the YPD plate, inoculate it into 10mL YPD liquid medium, culture at 30°C, 250rpm overnight;

[0065] 2) Inocula...

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Abstract

The invention provides a group of trypsin resistant antimicrobial peptides and a preparation method thereof. Six trypsin active sites in a NZ2114 sequence are subjected to selective mutation with a protein directional transformation technology. Trypsin resistant mutants with amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:33 are designed, and recombinant expression of the trypsin resistant mutants in pichia pastoris is realized. NZ2114 trypsin resistant derivatives are designed for the first time, and measurement shows that the trypsin degradation rate of part of the mutants (particularly double mutants and all triple mutants) is increased remarkably by 16.67%-25%. The trypsin resistant antimicrobial peptides show significant antibacterial activity against staphylococcus aureus ATCC25923, ATCC43300 and ATCC6538, and MIC (minimal inhibitory concentration) is 0.25-16 mu g / ml. The trypsin resistant antimicrobial peptides obtained with the method can be applied to fields such as antimicrobial drugs, food additives, cosmetics and feed additives and other fields, and have a broad application value and market prospect.

Description

technical field [0001] The invention relates to the fields of protein engineering and genetic engineering, in particular to a method for directional modification and genetic engineering production of a series of antimicrobial peptides. 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 are signal peptide sequences, 24-55 are leader peptide sequences, and 56-95 are mature peptide (Plectasin) sequences. 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 is between +1 +3 between (Mygind et al., Plectasin is a peptide antibiotic with therapeutic potential from asaprophytic fungus. Nature, 2005, 437(7061): 975-980). [0003] Plectasin has a strong killin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/81C12N1/19A61P31/04C12R1/84
CPCC07K14/37
Inventor 王建华毛若雨滕达王秀敏郝娅
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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