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Method for producing trametes versicolor immunomodulatory protein by virtue of pichia pastoris expression system

A technology of immunoregulatory protein and Pichia pastoris, which is applied in the field of biomedicine to achieve the effect of short cycle, high purity and low cost

Inactive Publication Date: 2015-11-18
HENAN ACAD OF SCI INST OF BIOLOGY LIABILITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there has been no report on the successful expression and mass production of Versicolor versicolor immunoregulatory protein FIP-tvc in Pichia pastoris

Method used

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  • Method for producing trametes versicolor immunomodulatory protein by virtue of pichia pastoris expression system
  • Method for producing trametes versicolor immunomodulatory protein by virtue of pichia pastoris expression system

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Embodiment Construction

[0019] The specific implementation of the present invention will be described in detail below in conjunction with the accompanying drawings and specific conditions.

[0020] The present invention in concrete implementation, comprises the following steps:

[0021] (1) Synthesize the nucleotide sequence SEQ ID NO.1 of the Yunzhi immunoregulatory protein FIP-tvc gene according to the codon preference of Pichia pastoris (published in "Lietal.2012, ProExpPurif, 82:339-344");

[0022] The Pichia pastoris is Pichia pastoris cell GS115 (commercially available products, such as strains preserved by ACCC China Agricultural Microorganism Culture Collection Management Center, ISF Soil and Fertilizer Research Institute of Chinese Academy of Agricultural Sciences or CGMCC General Microbiology Culture Collection Management Center) ;

[0023] (2) Using restriction endonucleases to digest the Yunzhi immunoregulatory protein FIP-tvc synthesized in step (1) and the expression plasmid vector X t...

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Abstract

The invention relates to a method for producing a trametes versicolor immunomodulatory protein by virtue of a pichia pastoris expression system. Problems about the preparation of trametes versicolor immunomodulatory proteins are effectively solved. The method comprises the following steps of synthesizing a nucleotide sequence SEQ ID NO.1 of a trametes versicolor immunomodulatory protein FIP-tvc according to a codon usage bias of pichia pastoris; constructing a recombinant expression plasmid X-FIP-tvc by virtue of the trametes versicolor immunomodulatory protein FIP-tvc synthesized by restriction enzyme digestion and an expression plasmid vector X, linearizing the recombinant expression plasmid X-FIP-tvc by virtue of a restriction enzyme, then transforming a competent cell of pichia pastoris GS115, screening a constitutive positive transformant or inducible positive transformant by virtue of an antibiotic-containing plate and PCR (Polymerase Chain Reaction) method, selecting an inducible high-level rFIP-tvc secretion expression pichia pastoris recombinant strain, performing fermental cultivation or induction expression for 3 to 7 days, precipitating fermentation supernatant with ammonium sulfate, performing dialysis on precipitates for 24 to 48 hours, performing affinity exchange chromatography with a chromatographic column, and performing drying to obtain solid powder of the trametes versicolor immunomodulatory protein FIP-tvc. The method is low in cost, short in cycle, easy to implement technically, suitable for industrial production and high in purity.

Description

technical field [0001] The invention relates to biomedicine, in particular to a method for producing versicolor immunoregulatory protein using a Pichia pastoris expression system. Background technique [0002] Fungal immunomodulatory proteins (fungalimmunomodulatoryproteins, FIPs) are a new type of biologically active small molecule proteins discovered from edible and medicinal fungal cells. The discovery of this protein broke the traditional view that "fungal polysaccharides are the main active ingredients of edible and medicinal fungi", and stimulated people's interest in finding new active ingredients from edible and medicinal fungi. Eight fungal immunomodulatory proteins have been obtained from edible and medicinal fungi: LZ-8, FIP-fve, FIP-vvo, FIP-gts, FIP-gmi, FIP-gja, FIP-gsi and FIP-tvc, respectively from Ganoderma lucidum Gandermalucidum , Ganoderma lucidum G. tsugae , Microspore Ganoderma lucidum G. microsporum , Ganoderma lucidum G.japnoicum , Zizhi G. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N15/81C12N1/19C12R1/84
Inventor 周伏忠李锋解复红刁文涛王雪妍陈晓飞冯菲
Owner HENAN ACAD OF SCI INST OF BIOLOGY LIABILITY
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