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Multi-copy high expressed recombined plectasin by pichia pastoris

A technique of lectin and expression cassette, which is applied in the field of the preparation of a recombinant lectomycin gene multi-copy expression vector and its recombinant yeast, can solve the problem of limited expression amount, large workload of screening multi-copy, low multi-copy gene, etc. question

Active Publication Date: 2012-04-11
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Application Information

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Problems solved by technology

[0004] The invention patent application with the Chinese patent application number 201010115149.6 discloses a method for preparing recombinant plectasiacin by yeast engineering bacteria, but the method has the following disadvantages: 1) it is mainly single-copy integration, and the expression amount is limited; 2) multiple occurrences The probability of spontaneous exchange to produce multiple copies of genes is extremely low, 3) the workload of screening multiple copies is large

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  • Multi-copy high expressed recombined plectasin by pichia pastoris
  • Multi-copy high expressed recombined plectasin by pichia pastoris
  • Multi-copy high expressed recombined plectasin by pichia pastoris

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Embodiment 1

[0035] The construction of embodiment 1 plectasin genetically engineered bacteria

[0036] 1.1 Design of Plectasin gene based on yeast preferred codons

[0037] The Plectasin gene was artificially designed according to the codon usage preference of Pichia pastoris (http: / / www.kazusa.or.jp / codon / ), and the obtained sequence is shown in SEQ ID NO.1.

[0038] 1.2 Construction of single-copy expression vector of recombinant plectasin

[0039] At the 5'-end of the Plectasin gene, a restriction endonuclease XhoI cleavage site, which is not available in the Plectasin gene but on the multiple cloning site of the vector, is designed, and the yeast Kex2 cleavage site after the XhoI cleavage site is retained (KR) coding sequence, in order to cleave the signal peptide after secretion and expression, to obtain recombinant Plectasin, design the TAATAA terminator sequence and XbaI restriction site at the 3'-end of the gene, in order to terminate the expression of the polypeptide and constru...

Embodiment 2

[0071] Induced expression of embodiment 2 recombinant plectasin

[0072] 1% of the inoculum was inoculated into 10 mL of BMGY medium with two copies, four copies, and eight copies of the correctly identified plectasin, shaken to OD at 30°C and 250 rpm 600nm 4.0 (logarithmic growth phase, about 16-18h); centrifuge at 2500g for 5min at room temperature. Discard the supernatant and resuspend the cells to OD with 50mL BMMY medium 600nm 1.0; add the above culture into a 250mL Erlenmeyer flask, cover with 4 layers of sterilized gauze, and put it in a shaker to continue to grow; every 24 hours, add methanol to a final concentration of 0.5% to continue induction; at the following time points, 0h , 24h, 48h, 72h, 96h, 120h Take 1mL of medium to a 1.5mL centrifuge tube and measure the OD value at this time point, centrifuge at 12000rpm at room temperature for 2-3min, and the induction time is 120h. Dilute the bacterial suspension to the same OD value, centrifuge to collect the superna...

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Abstract

The invention discloses a preparation method of multi-copy high expressed recombined plectasin by pichia pastoris. The method comprises the following steps: a plectasin expressing gene sequence is designed according to preference performance to codon translated by pichia pastoris; the optimized plectasin gene is fused on an alpha-factor signal peptide C terminus of an expression vector pPICZalphaA to construct a single-copy expression vector, the vector comprises a plectasin expression cassette containing a start signal element alcohol oxygen dehydrogenase strong promoter (AOX), alpha-factor signal peptide gene and a plectasin gene fused in C terminus, a stop signal element AOX (TT) and the like. A complementation principle of restriction endonuclease Bg1II and BamHI cohesive end is used to obtain plectasin gene-containing recombinant plasmid of different copy cascade expression cassettes, pichia pastoris is electrotransformed and secreted and expressed plectasin with high efficiency under the methanol induction. The expression level and plectasin gene copy number exist a linear relation. The constructed multi-copy high expressed yeast cells can be used for raising the output and reducing the cost, and is adapted to large scale production of plectasin.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multi-copy expression vector of a recombinant plectasin gene and a method for preparing the recombinant yeast. Background technique [0002] Plectasin is the first defensin isolated from a fungus (the saprophytic ascomycete Pseudoplecticania nigrella) by Mygind et al. (Mygind et al., Nature, 2005, 437: 975-980). Its higher order structure contains an α-β model consisting of an α-helix and two antiparallel β-sheets, stabilized by three disulfide bonds (Cys4-Cys30, Cys15-Cys37, Cys19-Cys39). Plectasin has high resistance to Gram-positive bacteria, no cytotoxicity, no hemolysis, and good permeability to cerebrospinal fluid. It has considerable potential in the treatment of Gram-positive mycosis, and is a new peptide antibiotic with therapeutic potential. [0003] Using genetic engineering means to construct plectasin genetically engineered bacteria has the advantages of s...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/69C12R1/865C12R1/84C12R1/78
CPCC12N15/81C07K14/37C07K2319/00
Inventor 王建华杨雅麟张军滕达王少然
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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