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Method for preparing recombinant glossy ganoderma immunomodulatory protein

A technology of immunoregulatory protein and Ganoderma lucidum, which is applied in the field of fermentation engineering, can solve the problems of small production scale and low yield, and achieve the effects of reduced electrical conductivity, rich nutrition, and increased yield

Inactive Publication Date: 2008-06-25
张喜田
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Purpose of the invention: to provide an engineering bacterium for efficiently expressing recombinant Ganoderma lucidum immunoregulatory protein, to provide a fermentation process and purification technology for preparing Ganoderma lucidum immunomodulatory protein on a scale of 100 liters, to improve the yield, and to overcome the small production scale and the production capacity of the prior art. low rate

Method used

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  • Method for preparing recombinant glossy ganoderma immunomodulatory protein
  • Method for preparing recombinant glossy ganoderma immunomodulatory protein

Examples

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Embodiment 1

[0014] Example 1: Construction of engineering bacteria

[0015] 1. Experimental materials

[0016] Pichia pastoris host strain GS115 and plasmid pPIC9K were purchased from Invitrogen, and yeast integrated expression vector p819 was constructed by the Chinese Center for Disease Control and Prevention.

[0017] Yeast Nitrogen Base (without amino acid), D-Biotin and Casein (Acid hydrolysates) are imported reagents from Sigma, and G418 is a product of GIBCOBRL.

[0018] 2. Experimental methods and results

[0019] 2.1 Construction of α-rLZ-8 fusion gene

[0020] 1) According to the preference of the genetic code of Pichia pastoris, redesign the base sequence encoding LZ-8, and commission Shanghai Shenggong Biotechnology Service Co., Ltd. to synthesize the full sequence of Ganoderma lucidum immunomodulatory protein gene. The synthesized recombinant plasmid is named pUC57-rLZ -8. The redesigned rLZ-8 gene is as follows:

[0021] CTCGAG AAAAGAATGTCTGATACTGCTTTGATCTTCAGATTGGCTTGGGATGTTA...

Embodiment 2

[0045] Example 2: Fermentation process under the scale of 100L fermentor

[0046] Fermentation pilot test result: the output of rLZ-8 reached 800mg·L -1 , About 10 times the expression in the shake flask, the wet weight of the bacteria is 420g·L -1 . The specific process is as follows:

[0047] 1. Equipment and equipment

[0048] Digital display peristaltic pump (7523-47), fermentation tank (BioFlo-5000), low temperature centrifuge (Microfuge R), low temperature centrifuge (Allegra 6R), constant temperature shaker (Scientific Allegra 6), biological safety cabinet (Scientific Model 1205A) ), gel analysis system (Biotech), ultra-low temperature refrigerator, UV spectrophotometer (DU7400), incubator (Scientific Model 3111), chromatography UV detection system (REC 112), 3K hollow fiber column, cation exchange resin (SP Sepharose XL), three-foot sedimentation centrifuge (SSC-600-NG), tubular separator (GQLB-105), vertical electrophoresis apparatus (OYC-40B), transfer electrophoresis ap...

Embodiment 3

[0121] Example 3: Purification process

[0122] The purification process is designed according to the characteristics of rLZ-8, the main process is as follows:

[0123] Fermentation broth separator centrifugation (3000rpm)→Using the supernatant with a tubular separator (16000rpm)→Ultrafiltration (30000-5000Da)→Anion exchange purification column (Q Sepharose Fast Flow)→Hydrophobic interaction purification column (PhenylSepharose 6 Fast Flow) )→Ultrafiltration to remove salt→High performance liquid phase (gel chromatography) to prepare the target protein.

[0124] 1. Ion Exchange Chromatography

[0125] Use the chromatographic column Q sepharose HP (1ml) to explore the chromatographic conditions and find the best pH value for purification. Mobile phase A: A1 liquid is 0.07M Bis-Tris and 0.05M Tris, A2 liquid is 0.1M HCl. The effective buffer range of the buffer is pH=6.0-9.0. Mobile phase B phase: B1 is pure water, B2 is 2M NaCl solution. The instrument automatically adjusts the pH ...

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Abstract

The invention provides a preparing method of reconstructing immunoregulatory protein of a ganoderma lucidum. The method includes the following steps: construction of engineering bacteria, wherein, LZ-8 protein gene is designed and coded, and is connected with a leading peptide coding sequence of saccharomyces cervisiae alpha-factor into fusion gene, which is transferred to a Pichia Pastoris expression system GS115 for reconstructing expression, high expressed rLZ-8 protein bacterial strain is acquired; the fermentation under the scale of 100L fermentation cylinder, wherein, improved seed culture medium and fermentation medium are used, additions of glycerol and methanol are regulated, 0.5M(NH4)2SO4 is added at the late stage of the fermentation; the purification on a column via centrifugal separation, wherein, salt is hyperfiltrated, powder is prepared after being frozen and dried. The method has large scale of fermentation, optimized technological condition and high production rate.

Description

Technical field [0001] The invention belongs to the field of fermentation engineering, and specifically relates to a method for preparing recombinant ganoderma immunomodulatory protein. Background technique [0002] Immunoregulatory Protein of Ganoderma lucidium (Immunoregulatory Protein of Ganoderma lucidium), a small molecule protein isolated and purified from Ganoderma lucidum mycelium extract by Kino et al., Japan in 1989 (Kohsuke Kino et al., J. Boil. Chem. 1989, 1:472-478), named LZ-8, and determined its amino acid sequence and immunophysiological activity. Protein sequencing showed that LZ-8 is composed of 110 amino acid residues, the amino terminal is acetylated, the molecular weight is 12.4KD, and the isoelectric point is 4.4. The main function of Ganoderma lucidum immunomodulatory protein is that it can promote the proliferation of peripheral lymphocytes and spleen cells, induce animal and human macrophages to secrete a variety of cytokines (such as interleukin, tumor n...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N1/19C12N15/09C12N15/29A61K9/19A61P37/02
Inventor 孙非刘立侠许守民梁重阳
Owner 张喜田
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