Recombinant ganoderma lucidum immunoregulatory protein mutant and application thereof

An immunomodulatory protein and mutant technology, which can be used in peptide/protein components, drug combinations, medical preparations containing active ingredients, etc., and can solve problems such as unclear mechanisms

Active Publication Date: 2017-09-01
张喜田 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This confirms the above speculation to a certain extent, but the mechanism that promotes the macropinocytosis of EGFR into cells is not clear, and this way of cell entry is accompanied by a large amount of degradation of receptors

Method used

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  • Recombinant ganoderma lucidum immunoregulatory protein mutant and application thereof
  • Recombinant ganoderma lucidum immunoregulatory protein mutant and application thereof
  • Recombinant ganoderma lucidum immunoregulatory protein mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Prokaryotic and eukaryotic expression of recombinant Ganoderma lucidum immunomodulatory protein and mutants thereof

[0065] In this embodiment, "target protein" represents "rLZ-8 and its mutants", and "target gene" represents "rLZ-8 and its mutant genes".

[0066] Construction and expression of prokaryotic expression strains of recombinant Ganoderma lucidum immunomodulatory protein and its mutants

[0067] In this example, the representative Escherichia coli BL21 (DE3) strain in the prokaryotic expression system was used to express the target protein. The target protein gene is codon-optimized, and according to the direction of the T7 promoter of the pET-28a vector, an Xba I restriction site and a ribosome binding site DNA sequence are added at the 5' end of the target gene, and a stop codon and Xho are added at the 3' end I restriction site for whole gene synthesis. The recombinant expression plasmid was constructed by double digestion with Xba I and XhoI ...

Embodiment 2

[0072] Example 2 Preparation of rLZ-8 and its mutants using fermentation engineering technology

[0073] Ganoderma lucidum immunomodulatory protein and its mutants were constructed and screened using the same expression vector and strain. In this example, Pichia pastoris was used for genome recombination construction. The obtained strains were cultured in shake flasks in the early stage and screened for engineering strains that can be used for fermenter-scale production. In the following descriptions, "target protein" is used to represent rLZ-8 and its mutants.

[0074] The specific process of the fermentation process

[0075] Recover working seeds: take out constitutive working seeds from -80°C refrigerator, dissolve slowly at room temperature, take out 10 μL and put them into 100mL shake flasks with 10mL YPD liquid medium, shake and culture at 28.5°C and 225rpm for 24h.

[0076] Constitutive seed liquid culture: the OD value of the resuscitated working seed bacterial liqui...

Embodiment 3

[0091] Example 3 Separation and purification process of ganoderma lucidum immunomodulatory protein and its mutants

[0092] 1. Experimental method

[0093] After experiments, rLZ-8 and its mutants were separated and purified in the order of microfiltration, ultrafiltration, cation exchange chromatography and hydrophobic interaction chromatography. Since the difference in physical and chemical properties between rLZ-8 and its mutants is small, the purification process is similar, and the purification process using rLZ-8 can successfully prepare mutants, which is not the best separation and purification process for each mutant. In this example The purification process for either mutant was not optimized. The following text is a detailed description of the details of the purification process of rLZ-8.

[0094] ① Fermentation broth microfiltration treatment process

[0095] Wash the well-preserved 0.7m with 3L water for injection 2 Hollow fiber microfiltration column, wash and...

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Abstract

The invention discloses a recombinant ganoderma lucidum immunoregulatory protein mutant and application thereof. The invention finds that an anti-EGFR structural domain exists in an rLZ-8 protein structure, and particularly an EGFR abnormal expression tumor killing effect is induced through the positive potential characteristic of the structural region. On the basis of the scientific discovery, the rLZ-8 mutant protein with a stronger anti-tumor effect is obtained by a computer simulation technology.

Description

technical field [0001] The technical field of the present invention includes the optimization and transformation of protein mutations by computer simulation technology. Background technique [0002] Human epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) is a transmembrane receptor with a molecular weight of 170kDa, which is encoded by a proto-oncogene and exhibits intrinsic tyrosine kinase activity. Epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin, heparin-binding EGF and β animal cellulose are all ligands that bind to EGFR. After EGFR is activated by ligand, it regulates many cellular processes through tyrosine kinase-mediated signal transduction pathways, including but not limited to activation control of cell proliferation, differentiation, cell survival, programmed cell death, etc. [0003] A large number of studies have shown that although EGFR is expressed in normal tissue cells, the expression level is very lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375A61K38/16A61P35/00
CPCC07K14/375A61K38/00A61P35/00A61P35/04A61K38/1754
Inventor 孙非梁重阳
Owner 张喜田
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