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Recombinant-plectasin-expressing Pichia pastoris

A technology of mycelia and Pichia pastoris, which is applied in the fields of biotechnology and genetic engineering, can solve the problems of reduced gene copy number of bacterial strains, heavy screening workload, unstable strains, etc., and achieves the goal of improving stability and reducing copy number Lost, increased dispersion effects

Pending Publication Date: 2018-02-16
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a Pichia pastoris of recombinant Pichia, which aims to solve the problem of low expression of Pichia expressing Pichia in the prior art, and screening The workload is heavy, and the gene copy number of the strain gradually decreases during the passaging process, and the strain is unstable

Method used

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  • Recombinant-plectasin-expressing Pichia pastoris
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  • Recombinant-plectasin-expressing Pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Construction of Plectasin Expression Vector

[0068] 1.1 Design of plectasin NZ2114 gene based on the codon preference of Pichia pastoris

[0069] According to Pichia pastoris codon preference

[0070] Codon Frequency ‰ Quantity

[0071] UUU 24.1 1963

[0072] UCU 24.4 1983

[0073] UAU 16.0 1300

[0074] UGU 7.7 626

[0075] UUC 20.6 1675

[0076] UCC 16.5 1344

[0077] UAC 18.1 1473

[0078] UGC 4.4 356

[0079] UUA 15.6 1265

[0080] UCA 15.2 1234

[0081] UAA 0.8 69

[0082] UGA 0.3 27

[0083] UUG 31.5 2562

[0084] UCG 7.4 598

[0085] UAG 0.5 40

[0086] UGG 10.3 834

[0087] CUU 15.9 1289

[0088] CCU 15.8 1282

[0089] CAU 11.8 960

[0090] CGU 6.9 564

[0091] CUC 7.6 620

[0092] CCC 6.8 553

[0093] CAC 9.1 737

[0094] CGC 2.2 175

[0095] CUA 10.7 873

[0096] CCA 18.9 1540

[0097] CAA 25.4 2069

[0098] CGA 4.2 340

[0099] CUG 14.9 1215

[0100] CCG 3.9 320

[0101] CAG 16.3 1323

[0102] CGG 1.9 158

[0103] AU...

Embodiment 2

[0166] Embodiment 2 Construction of Plectasin Engineering Bacteria

[0167] see Figure 6 , SpeI restriction endonuclease single-digestion treatment of pTRP-NZ2114 expression vector, cutting gel to recover linear expression vector, electroporation (1.5-2.5kv) to X33 Pichia competent cells or other Pichia such as GS115, SMD1168, etc. , spread the bacterial solution on a YPD plate (containing 0.25-4mg / mL bleomycin), and incubate at 30°C for 2-3 days until a single colony grows. Pick a single colony, cultivate and evaluate the expression of plectasin, and measure the size of the inhibition zone after the fermentation supernatant is diluted by a certain number of times. The optimal strain PLE-1 is selected for the next experiment.

[0168] Such as Figure 7As shown, the pPIC9K-NZ2114 expression vector was treated with SalI restriction endonuclease single enzyme digestion, the linearized expression vector was recovered by cutting the gel, electroporated (1.5-2.5kv) to PLE-1 compe...

Embodiment 3

[0170] Example 3 Induced Expression of Recombinant Plectasin

[0171] The Pichia cells expressing Pichia pastoris prepared in Example 2 were picked, inoculated in 25 mL of BMGY medium, and cultured at 30° C. and 220 rpm for 24 hours to prepare a primary seed solution.

[0172] Inoculate 20 mL of primary seed liquid into 200 mL of BMGY medium to prepare secondary seed liquid, and culture at 30°C and 220 rpm for 24 hours.

[0173] All the secondary seed liquids were inoculated in a 5L fermenter (2L BSM medium), the temperature was controlled at 30°C±0.5°C, the dissolved oxygen was 20%±5%, and the pH=5.0±0.5.

[0174] After the basic glycerol was exhausted, 10% glycerol was added continuously. After the glycerol was exhausted, until the dissolved oxygen rose to 90%, starvation was performed for half an hour, and methanol was added to induce the expression of Plectasin. The total induction was 72 hours, and centrifuged (6000×g, 5min) to take The fermentation supernatant was teste...

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Abstract

The invention discloses recombinant-plectasin-expressing Pichia pastoris. The Pichia pastoris is successively transformed by three different plectasin expression vectors, i.e., pTRP-NZ2114, pPIC9K-NZ2114 and pPIC6alphaA-NZ2114, and strains obtained after each transformation are subjected to high-copy screening with different antibiotics so as to obtain an engineered Pichia pastoris strain with a high plectasin expression quantity, wherein the highest expression quantity of plectasin in the engineered Pichia pastoris strain is up to 6.0 g / L, much higher than plectasin expression quantities in the prior art. The three plectasin expression vectors utilize three different integration sites, so the dispersibility of plectasin expression cassettes in the genomes of multi-copy strains is improved, and loss of the copy number of the plectasin expression cassettes is reduced to a certain extent; and thus, the stability of the engineered Pichia pastoris strain is improved.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and genetic engineering, in particular to a Pichia pastoris expressing recombinant plectasin. Background technique [0002] Plectasin (Plectasin) was obtained from the saprophytic ascomycete Pseudomonas nigella ( Pseudoselectania nigrella ) isolated from the first case of fungal defensin (patent WO 2003044049 A1). The complete open reading frame of plectasin gene encodes a 95-amino acid polypeptide consisting of 3 parts. Signal peptide sequence (1-23 amino acids), propeptide (24-55 amino acids) and C-terminal mature peptide (56-95 amino acids). Mature plectasin consists of 40 amino acid residues, and its secondary structure belongs to the α-β model, consisting of an α-helix and two antiparallel β-sheets, which contain three disulfide bonds (Cys4- Cys30, Cys15-Cys37, Cys19-Cys39), the net charge number is +1~+3. The NZ2114 mutant is a variant containing 3 amino acid mutations obtained by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P21/00C12R1/84
CPCC07K14/37C12N1/165C12R2001/84
Inventor 梁伟凡周玉岩李洪金丁小云
Owner GUANGDONG HINAPHARM PHARMA CO LTD
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