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Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain

A technology of defective and genetically engineered bacteria, applied in the field of construction and utilization of Pichia pastoris X-33 strain, can solve the problems of reduced activity, shortened half-life, enhanced immunogenicity of glycoproteins, etc.

Inactive Publication Date: 2011-07-13
JIANGNAN UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the glycosylation modification of proteins by Pichia pastoris produces high-mannose sugar chains, which are different from the complex sugar chains of mammalian cells, which leads to enhanced immunogenicity, shortened half-life, and reduced activity of glycoproteins. A series of adverse effects, which is the main reason why Pichia pastoris cannot be used in the preparation of most glycoprotein drugs

Method used

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  • Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain
  • Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain
  • Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain

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Embodiment 1

[0030] Knockout and identification of OCH1 gene: the knockout plasmid pYXZ-OCH1( figure 1 ) was sexualized with MluI and then electroporated into X-33 (Δura3) competent cells to undergo double exchange homologous recombination with the target. The knockout plasmid contains the URA3 marker gene, so the strains that can grow on MD medium are homologous recombination strains; at the same time, the och1 knockout bacteria grow well at 25°C but cannot grow at 37°C. Select the strains that meet the above two traits, Genomic PCR identification was performed. Since the sequence of about 1000bp in the reading frame of the OCH1 gene was replaced by a 2.8kb selection marker sequence, the external primers OCH5F and OCH3R were used to identify, and the PCR amplification product of X-33 bacteria was 2.8kb, and that of X-33 (Δoch1) bacteria was 4.4kb ( figure 2 ); (in)3R in the internal primers (in)5F and (in)3R is located in the replaced reading frame sequence, so the X-33 strain can amp...

Embodiment 2

[0032] Construction and expression of GM-CSF expression vector

[0033] 1) Construction of the expression vector: the plasmid pUC19 / GM-CSF stored in our laboratory was digested with EcoRI and NotI, and the GM-CSF gene fragment was recovered (6xHis tag sequence was introduced at the end), and cloned into Pichia On the yeast expression vector pPICZaA, the expression vector pPICZaA / GM-CSF( Figure 4 ), positive clones were screened, identified by enzyme digestion and sequenced.

[0034] 2) Expression of GM-CSF in P. pastoris X-33 and X-33 (Δoch1): the expression vector pPICZaA / GM-CSF was linearized with the restriction endonuclease SacI and transferred into X-33 and X-33 respectively In -33 (Δoch1) competent cells, the bacterial solution after electric shock was applied to YPDZ (containing 100ug / mL, 300ug / mL, and 500ug / mL Zeocin respectively) medium, and X-33 transformants were cultured at 30°C for 3 -5d, X-33 (Δoch1) transformants were cultured at 25°C for about a week, and th...

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Abstract

The invention discloses preparation and application of an alpha-1,6 mannose transferase gene (och1) flaw type P. pastoris X-33 bacterial strain, belonging to the technical field of biological engineering. The engineering bacterial strain P. pastoris X-33 (delta och1) is characterized in that a target gene knockout method by double-exchange homologous recombination is adopted, and a URA3 gene serves as a selective marker to knock out the alpha-1,6 mannose transferase gene (och1) of the P. pastoris X-33 (delta och1) to obtain the P. pastoris X-33 (delta och1) bacterial strain with OCH1 gene knockout. The bacterial strain provides a P. pastoris expression system which modifies protein by virtue of low N-glycosylation modification and provides a favourable basis for further glycosylation modification. The bacterial strain has the advantage of great potential application prospect.

Description

technical field [0001] The invention discloses the construction and utilization of a Pichia pastoris X-33 (Δoch1) strain, belonging to the technical field of bioengineering. Background technique [0002] Glycoprotein drugs (such as antibodies, cytokines, vaccines, etc.) have become one of the fastest-growing drugs in recent years, and their demand is growing rapidly. At present, mammalian cells are mainly used for the production of these glycoproteins. However, there are many disadvantages in the production of pharmaceutical proteins by mammalian cells, such as complicated cell culture, low protein expression, and high production costs. [0003] The Pichia pastoris expression system has the characteristics of fast growth, convenient gene manipulation, low cost, large-scale culture and high-density fermentation, etc., and has most of the post-translational processing and modification functions of eukaryotic cells (such as glycosylation process, etc.), has been widely used in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/09C12P21/02C12R1/84
Inventor 金坚朱瑞宇张大成许永利雷楗勇陈蕴
Owner JIANGNAN UNIV
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