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Group of NZ2114 histidine mutants, and preparation method thereof

An amino acid, a series of technologies, applied in the field of protein engineering and genetic engineering, can solve the problems such as histidine, mutation, etc. that have not yet been seen

Inactive Publication Date: 2017-01-18
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

At present, there is no report on the mutation of histidine in the NZ2114 sequence

Method used

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  • Group of NZ2114 histidine mutants, and preparation method thereof
  • Group of NZ2114 histidine mutants, and preparation method thereof
  • Group of NZ2114 histidine mutants, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 NZ2114 histidine mutant molecular design and gene synthesis

[0040] Based on the presence of two histidines at the 16th and 18th positions in the NZ2114 sequence, arginine and lysine were used to mutate them. A total of 5 sequences (SEQ ID No. 1-SEQ ID No. 5) were designed for single-site mutants and double-site mutants.

[0041] The mutant gene sequences (SEQ ID No.6-SEQ ID No.10) 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

[0042] Example 2 Construction of Pichia pastoris inducible expression vector pPICH1~pPICH5

[0043] The synthetic gene fragment and vector pPICZaA were double-digested with restriction endonucleases XhoI and XbaI respectively, and the pPICZaA vector fragment and mutant gene fragment were recovered and ligated to obtain vectors pPICH1-pPICH5;

[0044] Detailed construction process of vectors pPICH1~pPICH5: use restriction endonucleases XhoI and XbaI to carry out double enzyme digestion on the synthesized gene fragment and pPICZaA respectively. The enzyme digestion system and conditions are as follows:

[0045]

[0046]

[0047] 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 ultraviolet light, and use the Tia...

Embodiment 3

[0063] Example 3 Construction of recombinant yeast strain containing pPICH1~pPICH5

[0064] 3.1 Linearization of recombinant vector pPICH1~pPICH5

[0065] Use PmeI to digest the constitutive recombinant expression vectors pPICH1~pPICH5. The enzyme digestion system and reaction conditions are as follows:

[0066]

[0067] 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.

[0068] 3.2 Pichia pastoris electrotransformation and identification of linearized vector

[0069] 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;

[0070] 2) Inoculate 50μL overnight culture so...

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Abstract

The invention provides a group of NZ2114 histidine mutants, and a preparation method thereof. Two histidine sites existing in an NZ2114 sequence are selectively mutated by using a protein directional transformation. The histidine mutants with the amino acid sequences represented by SEQ ID NO:1 to SEQ ID NO:5 are designed, and recombinant expression of the histidine mutants in Pichia pastoris is realized. Design of an NZ2114 histidine mutantderivative is realized for the first time, and a determination result shows that the mutants (especially the 16th histidine mutant) has substantial bacteriostatic activity on Staphylococcus aureus ATCC 25923, ATCC 43300 and ATCC 6538 and Streptococcus suis CVCC 3928 and CVCC 3309, and the MIC value is 0.0625-2 [mu]g / ml. The NZ2114 histidine mutants obtained through the method can be applied to the fields of antibacterial drugs, food additives, cosmetics and feed additives, and has wide application values 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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IPC IPC(8): C07K14/37C12N15/31C12N15/81C12N1/19C12R1/84
CPCC07K14/37
Inventor 王建华陈惠娴毛若雨滕达王秀敏郝娅
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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