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Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof

A mannosyltransferase and Pichia pastoris technology is applied in the field of Pichia pastoris strains and their construction, and can solve the problems of strain instability, restriction, reverse mutation and the like

Active Publication Date: 2008-06-11
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after the target gene is inserted and inactivated by single crossover, the strain is unstable during the passage process and prone to reverse mutations, requiring continuous screening pressure, which limits its use in subsequent research and production applications.

Method used

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  • Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof
  • Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof
  • Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] 1. Construction of knockout plasmid

[0038]The genome of Pichia pastoris GS 115 (purchased from Invitrogen life technologies, carlsbad, california 92008, USA) was extracted by glass bead preparation method (A. Adams et al., "Experimental Guide to Yeast Genetics Methods", Science Press, 2000), and The genome was used as a template to amplify the homology arms on both sides of α-1,6-mannosyltransferase (OCH1), URA3 gene and ADE gene (pyrobest enzyme used in PCR reaction was purchased from Bao Biological Engineering Co., Ltd., Dalian; primers were obtained from Synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., Beijing), the homology arms on both sides of OCH1 are 900bp, and the coding gene of 1000bp is missing in the middle, and the primer used to amplify the homology arm at the 15' end of OCH is p1105 (SEQ ID No. 5) and p1106 (SEQ ID No.6), the primer sequences are respectively: 5'-acggatccccggaaaaccgagagaactct-3' and 5'-acgcggccgcagagcgatagagaa...

Embodiment 2

[0046] Example 2. Expression of fusion protein HSA / GM-CSF in OCH1 knockout bacteria

[0047] HSA / GM-CSF is a fusion protein of human serum albumin (HSA) and granulocyte-macrophage colony-stimulating factor (GM-CSF), with two N-glycosylation sites. The fusion protein can stimulate the proliferation of granulocytes and macrophages, and has important clinical application value, but the fusion protein expressed by Pichia vulgaris is excessively glycosylated, which seriously affects its clinical application. This example discloses a method for expressing non-hyperglycosylated modified HSA / GM-CSF using an α-1,6-mannosyltransferase knockout strain.

[0048]1. Expression of HSA / GM-GM-CSF

[0049] The hGM-CSF cDNA was obtained from the human liver fetal cDNA library (purchased from Clontech Laboratories Inc. 1290 Terra Bella Ave. Mountain View, CA94043, USA) by PCR, and the primers used were GMCSF1: 5'ATGGATCC GCACCC GCC CGC TCG CCC AGC 3 ' (SEQ ID No. 15) and GMCSF2: 5' ATGAATTC TTA...

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Abstract

The invention discloses Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent and the establishing method, which belongs to the biological engineering field. The collection number of the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent is CGMCC No.1853. The invention has the advantages that the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent built by the invention can prevent the generation of excessive manna saccharify, when expressing extrinsic glucoprotein, reduce the immunogenicity of the pressed protein, and therefore, the invention has important application value in the biomedical field, and other fields; simultaneously, the strain can also be applied to the further gene knockout or the metabolic engineering reconstruction. The invention also builds a method for knocking out the alpha-1, 6-mannosyl transferase gene through secondary homologous recombination, mutant strain can be relatively easily obtained through the method, therefore the sieving workload is greatly reduced, and the success ratio is improved.

Description

technical field [0001] The invention relates to a pichia yeast strain lacking alpha-1,6-mannosyltransferase and a construction method thereof, belonging to the field of bioengineering. Background technique [0002] Gene knockout refers to the design of experiments on a gene whose sequence is known at the molecular level to remove the gene or replace it with other genes. In addition to suspending the expression of a certain gene, gene knockout also includes the introduction of new genes and site-directed mutations, either by knocking out the corresponding normal gene with a mutant gene or other genes, or knocking out the corresponding mutant gene with a normal gene . [0003] Gene knockout utilizes the principle of gene homologous recombination. Homologous recombination can be divided into single crossover homologous recombination and double crossover homologous recombination in principle. When single crossover homologous recombination occurs, the exogenous gene is inserte...

Claims

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Application Information

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IPC IPC(8): C12N1/19C07K19/00C12N15/09C12R1/84
Inventor 吴军巩新唱韶红马清钧
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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