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Multi-copy expression vector, pichia pastoris expressing plectasin and preparation method thereof

A technique for plectasiamycin and expression vectors, applied in the field of Pichia pastoris expressing myceliamycin and its preparation, and multi-copy expression vectors, which can solve the problems of low expression, heavy screening workload, and unstable strains, etc. Achieve the effect of improving stability and reducing gene copy number

Pending Publication Date: 2019-05-31
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a multi-copy expression vector, Pichia pastoris expressing mycelia and its preparation method, aiming to solve the problem of using Pichia strains to construct mycelia in the prior art. The process of mymycin expression strain is cumbersome, the copy number of mycelia in the constructed expression strain is low, the expression level is small, the screening workload is large, and the strain is unstable

Method used

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  • Multi-copy expression vector, pichia pastoris expressing plectasin and preparation method thereof
  • Multi-copy expression vector, pichia pastoris expressing plectasin and preparation method thereof
  • Multi-copy expression vector, pichia pastoris expressing plectasin and preparation method thereof

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Embodiment 1

[0033] Single copy expression vector construction

[0034] Insert the Plectasin NZ2114 gene sequence into the C-terminus of the α-factor signal peptide of the pPICzαA expression vector to construct a single-copy expression vector, which contains the methanol dehydrogenase AOX1 promoter, α-factor signal peptide, mycelium Plectasin expression cassettes such as mymycin NZ2114 gene and AOX1 (TT) terminator. The specific construction method is as follows:

[0035] Add Xho at the 5' end of Plectasin NZ2114 gene Enzyme cleavage site and Kex2 cleavage site (CTCGAGAAAAGA) ensure that the secreted plectasin can be correctly cleaved signal peptide. Add Xba at the 3' end of the NZ2114 gene Restriction site (TCTAGA). xho and Xba The plectasin gene fragment treated with restriction endonuclease double digestion was connected to the same Xho and Xba Restriction endonuclease double-digested pPICzαA vector to construct a single-copy expression vector pPICzαA-NZ2114, such as figu...

Embodiment 2

[0039] Multi-copy integration vector construction

[0040] Use the yeast genome extraction kit to extract the genome of Pichia pastoris X33 strain, use the X33 genome as a template, and use amplification primers 5'-CCCGGATCCCCTAAGATTCGAAAA-3' (SEQ ID No.2) and 5'-CCCAGATCTAACGCCTCTAAGTCA-3' (SEQ ID No. .3), amplifying the rDNA non-transcribed spacer gene fragment (NTS fragment), as shown in the sequence SEQ ID No.1. Add BamH at the 5' end of the NTS fragment by amplification primers Restriction site (GGATCC), add Bgl at the 3' end Restriction site (AGATCT). Bgl The single-copy expression vector pPICzαA-NZ2114 was digested with a single restriction enzyme, and the digested product was dephosphorylated by calf intestinal alkaline phosphatase (CIP), and the linearized single-copy expression vector obtained was combined with BamH and Bgl The NTS fragments treated with restriction endonuclease double digestion were connected to construct a multi-copy integration vector pP...

Embodiment 3

[0044] Preparation of Pichia pastoris cells expressing plectasin

[0045] Extract the multi-copy integration vector, Spe Restriction endonuclease single digestion treatment, electrophoresis to recover the digested product, electroporation (1.5KV) to X33 competent cells, after recovery, spread on YPDZ plate (0.25-4mg / mL bleomycin concentration), culture at 30°C Until a single colony grows, screen positive multi-copy transformants, which are Pichia pastoris cells expressing plectasin.

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Abstract

The invention discloses a multi-copy expression vector, pichia pastoris expressing plectasin and a preparation method thereof. The method includes utilizing a gene fragment of a non-transcribed spacerregion of rDNA as an integration site to integrate a plectasin NZ2114 expression cassette into a pichia pastoris genome to obtain a pichia pastoris strain with high plectasin expression level and good stability, and the problem in the prior art that the process is cumbersome for constructing the plectasin expression strain by using the pichia pastoris strain, the constructed expression strain haslow plectasin copy number, less expression and high screening workload, and the strain is unstable is solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multi-copy expression vector, Pichia pastoris expressing plectasin and a preparation method thereof. Background technique [0002] Plectasin has obvious killing effect on Gram-positive bacteria, especially the bactericidal effect on Streptococcus pneumoniae is equivalent to that of vancomycin and penicillin, and has good salt ion tolerance, pH stability and thermal stability. stability. At the same time, Plectasin has no cytotoxicity, no hemolysis and good permeability of cerebrospinal fluid, so it is a potential drug for treating diseases caused by Gram-positive bacteria. As one of the most commonly used protein expression systems, the Pichia pastoris expression system has the advantages of clear genetic background, simple gene operation, mature fermentation process, high protein expression level, and post-translational modification, and is suitable for large-scale production of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N1/19
Inventor 梁伟凡周玉岩逯佩凤丁小云
Owner GUANGDONG HINAPHARM PHARMA CO LTD
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