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Preparation and use of recombinant gold needle mushroom immunomodulatory protein

An immunomodulatory protein, Flammulina velutipes technology, applied in the field of biopharmaceutical engineering, can solve the problems of imperfect advanced modification of target protein, low biological activity, time-consuming cost and the like

Inactive Publication Date: 2013-05-01
刘立侠
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the bacterial expression is simple and fast, the advanced modification of the target protein is not perfect, and the biological activity is lower than that of the natural protein; while the recombinant protein expressed in insect cells is close to the biological activity of the natural protein, but it is time-consuming and expensive

Method used

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  • Preparation and use of recombinant gold needle mushroom immunomodulatory protein
  • Preparation and use of recombinant gold needle mushroom immunomodulatory protein
  • Preparation and use of recombinant gold needle mushroom immunomodulatory protein

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Embodiment 1

[0034] The fruiting bodies of Flammulina velutipes were purchased from the market. Pichia pastoris GS115 and plasmid pPIC9 were purchased from Invitrogen Life Technologies, USA. Restriction endonucleases, plasmid extraction and DNA recovery kits and related biochemical reagents were purchased from Dalian Bao Biological Company, and primers were synthesized by Shanghai Sangong Company.

[0035] (1), the cloning of coding FIP-fve gene: get 0.5g dried mycelium and grind in liquid nitrogen, add 15ml DNA extraction liquid, then add K + , so as to effectively remove polysaccharides, and extract twice with phenol, chloroform and isoamyl alcohol at the same time, finally obtain and extract relatively pure genomic DNA, and detect the results by 0.8% agarose gel electrophoresis. The primers for cloning the gene encoding FIP-fve were designed as follows: upstream, 5`-ca gaattc atg tcc gcc acgtcg ctc acc-3`, downstream, 5`-ag gaattc ggatcc tca agt ctt ctt cca ctc agc-3`; 0.8% agarose ge...

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Abstract

The invention discloses a preparation method and application of a recombinant flammulina velutipes immunomodulatory protein that is expressed by Pasteur pichia pastoris, and the preparation method comprises following steps: (1) an encoding FIP-fve gene is cloned; (2) a recombinant Pasteur pichia pastoris expression carrier is constructed; (3) a recombinant Pasteur pichia pastoris transformant is obtained; and (4) the recombinant FIP-fve gene is induction expressed and purified. The preparation method clones an encoding flammulina velutipes immunomodulatory protein, constructs the Pasteur pichia pastoris expression carrier, and consequently obtains the recombinant flammulina velutipes immunomodulatory protein with high yield and high purity. The yield of the recombinant protein that is obtained by the 5-liter fermentation technique reaches 300mg / L to 350mg / L, the purity reaches over 90 percent, and the purified recombinant flammulina velutipes immunomodulatory protein is detected to have the same biologic features and physiological immune activity with natural flammulina velutipes immunomodulatory protein, has procoagulant activity and the promoting functions of mouse lymphocyte proliferation as well as interleukin and interferon secretion, and can induce tumor cell apoptosis.

Description

technical field [0001] The invention relates to a recombinant Flammulina velutipes immunoregulatory protein, in particular to a preparation method and application of the recombinant Flammulina velutipes immunomodulatory protein expressed by Pichia pastoris, belonging to the field of biopharmaceutical engineering. Background technique [0002] Fungal immunomodulatory protein (FIP) was first extracted from the fruiting bodies of Ganoderma lucidum as a small molecular protein (named LZ-8, Kino et al., 1989, J Biol Chem, 264: 472-478) , has the functions of stimulating the proliferation of peripheral blood lymphocytes, inducing the secretion of cytokines such as interleukins and interferons, promoting the synthesis of nucleic acids and proteins, inhibiting allergic reactions, and enhancing the body's immunity. This fungal immunomodulatory protein has been obtained from six kinds of edible and medicinal fungi, which are Ganoderma lucidum (LZ-8), Ganoderma lucidum (FIP-gts), and F...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12P21/02A61K38/16A61P37/02C12R1/84
Inventor 刘立侠孙非林景卫许守民张韧
Owner 刘立侠
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