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Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris

A technology of NZ2114 and Pichia pastoris, which is applied in the field of genetic engineering, can solve problems such as no gene cloning and expression research reports, achieve broad application value and market prospects, and overcome the effect of low yield

Active Publication Date: 2013-04-17
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, the NZ2114 used in the research on the antimicrobial peptide NZ2114 is chemically synthesized (Andes, Craig et al., 2009; Brinch, Tulkens et al., 2010; Xiong, Hady et al., 2011), and there is no research on its gene cloning and expression to report

Method used

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  • Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris
  • Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris
  • Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The acquisition of embodiment 1 antimicrobial peptide NZ2114 gene fragment

[0062] 1.1 Optimization of gene expression sequence of antibacterial peptide NZ2114

[0063] According to the yeast codon table Pichia pastoris[gbpln]:137CDS's(81301codons):

[0064]

[0065]

[0066] According to the AA sequence of antimicrobial peptide NZ2114: GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY (SEQ ID No.1), the gene encoding antimicrobial peptide NZ2114 was designed, and the DNA sequence (rNZ2114) after codon optimization was as follows:

[0067] GGT TTT GGT TGT AAC GGT CCA TGG AAC GAA GAT GATTTG AGA TGT CAT AAC CAT TGT AAG TCT ATT AAG GGT TACAAG GGT GGT TAC TGT GCT AAG GGT GGT TTT GTT TGT AAGTGT TAC (SEQ ID No. 2).

[0068] Sequence feature: 149bp; type: nucleic acid; chain type: double-stranded; topology: linear; GC%: 40.9%. Molecule Type: Double-stranded DNA.

[0069] 1.2 Design of gene expression cassette

[0070] In order to effectively terminate the translation express...

Embodiment 2

[0097] Example 2 Construction of Yeast Recombinant Expression Vector

[0098] 1.1 The NZ2114 gene fragment that embodiment 1 obtains reclaims transformation fragment after XhoI and XbaI endonuclease double digestion. At the same time, the pPICZαA vector (purchased from Invitrogen) was double digested with XhoI and XbaI.

[0099] The double enzyme digestion system is as follows:

[0100]

[0101] Digestion conditions: 37°C water bath for 4h.

[0102] The digested product was recovered with a DNA product recovery kit (purchased from Tiangen Biological Co., Ltd.), and stored at -20°C for future use. After the NZ2114 gene and the pPICZαA vector were digested with XbaI and XhoI, the NZ2114 gene was ligated with the linearized pPICZαA vector with T4 DNA ligase. The connection system is as follows:

[0103]

[0104] Connection conditions: 25°C, 1h.

[0105] 1.2 Transform the obtained recombinant vector into Escherichia coli DH5α: Take out the competent cells of Escherichia...

Embodiment 3

[0126] Example 3 Preparation of recombinant yeast strain X-33 containing NZ2114 gene

[0127] 1.1 Linearization of recombinant pPICZαA-NZ2114 vector

[0128] The recombinant pPICZαA-NZ2114 vector obtained in Example 2 was linearized with BglII endonuclease and used for yeast transformation.

[0129] The linearization system is as follows:

[0130]

[0131] Reaction conditions: 37°C, 4h.

[0132] Electrophoresis results ( image 3 ) shows that the pPICZαA-NZ2114 recombinant vector is completely linearized.

[0133] 1.2 Preparation of competent Pichia pastoris X-33

[0134] Pick a single colony of Pichia pastoris X-33 (purchased from Invitrogen) and inoculate it into 10ml of YPD medium, shake it at 250rpm at 30°C overnight, and inoculate the overnight culture of Pichia pastoris X-33 into 50ml of YPD with 1% inoculum Medium, 250rpm, 30°C shaking culture to OD 600nm =1.3-1.5, 4°C 4000rpm, centrifuge for 5min, remove the supernatant, resuspend the bacteria in 50ml ice-cold...

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Abstract

The invention provides a method for efficiently expressing an antibacterial peptide NZ2114 in recombinant pichia pastoris. An expression vector comprising a DNA (Deoxyribose Nucleic Acid) sequence of the antibacterial peptide NZ2114 which is subjected to optimization coding by yeast preferred codons is constructed, pichia pastoris is transformed, the obtained recombinant pichia pastoris secretes to generate the antibacterial peptide NZ2114 by fermentation cultivation. According to the invention, by optimizing the genic sequence of the antibacterial peptide NZ2114 and constructing the special expression vector, efficient expression of the antibacterial peptide NZ2114 in the pichia pastoris is realized for the first time; yield breaks through the gram-grade level; the problem of excessively low yield or excessively high chemical synthesis cost in the large-scale production is solved; a perfect purification system is established; scale production can be realized; the method can be applied to the fields of development of antibacterial agents, development of feed additives and the like and has high application values and broad market prospects.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for highly expressing antimicrobial peptide NZ2114 in recombinant Pichia pastoris. Background technique [0002] Antimicrobial peptides (AMPs) are small molecule polypeptides with biological activity in organisms. The main characteristics are as follows: high antibacterial activity, wide antibacterial spectrum, and many types, and they have killing or inhibiting effects on fungi, protozoa, even viruses and tumor cells (Brandenburg, Merres et al. 2012). Due to its outstanding biological characteristics, it has the most potential as a new type of safe and harmless antibiotic substitute, and thus has attracted the attention of the scientific community (Eckert 2011; Brandenburg, Merres et al., 2012). [0003] Plectasin is an antimicrobial peptide with antibacterial function isolated from the northern European pine forest surface saprophytic ascomycete (Pseudolectania nigr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P21/02C12R1/84
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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