Construction and application of universal carrier for performing heterologous protein expression on pichia pastoris and hansenula polymorpha

A technology of Hansenula and Pichia pastoris, applied in the direction of using vectors to introduce foreign genetic material, chemical instruments and methods, and peptide peptides

Inactive Publication Date: 2015-04-01
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The expression of green fluorescent protein in Hansenula was successfully directed using the phytase yeast translation elongation fact...

Method used

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  • Construction and application of universal carrier for performing heterologous protein expression on pichia pastoris and hansenula polymorpha
  • Construction and application of universal carrier for performing heterologous protein expression on pichia pastoris and hansenula polymorpha
  • Construction and application of universal carrier for performing heterologous protein expression on pichia pastoris and hansenula polymorpha

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Embodiment

[0090] Example: Application method of universal vector for heterologous protein expression in methanolotrophic Pichia pastoris and Hansenula

[0091] 1) Analysis of the effect of Hansenula ribosomal DNA and autonomously replicating sequence elements on the transformation efficiency of plasmids in Pichia pastoris and Hansenula cells

[0092] ① Construction of recombinant plasmids pHanMOX-rDNA and pHanMOX-HARS

[0093] The plasmid pHanMOX-GFP was single-digested with restriction endonuclease Sal Ⅰ, and the digested product was recovered by gel and ligated with T4 ligase overnight at 16°C, transformed into Escherichia coli DH5α competent cells, and coated with 0.1mg / mL Kana The LB plate of the prime, the extracted plasmid was digested and identified, and the correct plasmid was identified as pHanMOX-rDNA. The enzyme digestion reaction system is: 15 microliters of plasmid pHanMOX-GFP 0.6 μg / μl, 1 microliter of Sal Ⅰ, 4 microliters of 10×H buffer, and 20 microliters of double dist...

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Abstract

The invention relates to universal carriers constructed based on methylotrophic hansenula polymorpha and pichia pastoris, in partituclar to a construction method of a universal carrier for performing heterologous protein expression on pichia pastoris and hansenula polymorpha. The construction method comprises the following steps that (1) a plasmid pHanMOX-GFP is digested through restriction enzymes Bgl II and BamH I, so that a pHan carrier is obtained; (2) a carrier plasmid pPinkAOX1-HPV16L1 is digested through restriction enzymes Bgl II and BamH I, a fragment PAOX1-HPV16L1-CYC1TT is recovered and then connected with the pHan carrier obtained in the step (1) through a T4 ligase at 16 DEG C overnight, an LB flat plate containing 0.1 mg/mL kanamycin is converted and coated, and plasmid enzyme digestion is extracted for identification, so that a restructured hansenula polymorpha plasmid pHanAOX1-HPV16L1 is obtained. The universal carrier is used for guiding expression of human papillomavirus type 16 major capsid protein L1 in pichia pastoris and hansenula polymorpha.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the construction of a universal vector based on methanolotrophic Hansenula and Pichia pastoris, by investigating the ribosomal DNA of Hansenula yeast and the autonomously replicating sequence and the Pichia alcohol oxidase type 1 promoter, The role of the translation elongation factor 1-α promoter and the Hansenula methanol oxidase promoter in the shuttle vector, and the construction of a shuttle vector based on the Hansenula ribosomal DNA and the Pichia promoter in Hansenula and Pichia pastoris A method for the universal vector of shuttle expression. Background technique [0002] The methanolotrophic yeasts Pichia and Hansenula are two host systems for heterologous protein expression. They not only have the characteristics of eukaryotic expression system with high secretion efficiency and post-translational protein modification, but also have the advantag...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/81C07K14/025
Inventor 马雁冰金晓媚刘存宝姚宇峰杨旭黄惟巍孙文佳龙琼李杨褚晓杰
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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