Ribosome inactivated protein attenuated modification method for blocking receptor binding

A technology of ribosome inactivation and receptor binding, applied in the field of bioengineering

Active Publication Date: 2020-08-07
成都富岱生物医药有限公司
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Problems solved by technology

Therefore, the above two attenuating transformations still cannot solve the toxicity problem well.

Method used

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  • Ribosome inactivated protein attenuated modification method for blocking receptor binding
  • Ribosome inactivated protein attenuated modification method for blocking receptor binding
  • Ribosome inactivated protein attenuated modification method for blocking receptor binding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0047] 1. Protein-protein docking between α-MMC and the CR56 and CR17 binding subunits of the LRP-1 receptor to predict the receptor binding site and binding site

[0048] Apply Zdock3.0.2 software package, select α-MMC protein (PDB: 1AHC) and LRP CR56 and LRP CR17 (such as figure 1 ) for protein-protein docking, four modes are set for the protein docking interface, namely Residue 1-14 (Site1), Residue71-136 (Site2) and Residue 195-222 (Site3) and random binding without special setting of the binding interface (Non -site), after the docking is completed, select the binding modes with the top 10 scores (top10) for conformational analysis.

[0049] The experimental process of molecular docking is roughly as follows: (1) Prepare the spatial structure (pdb file) of the two proteins of ligand and receptor, and manually delete all water molecules, hydrogen atoms and other heteroatoms in the PDB file; (2) use the command mark_sur processes the pdb file of the receptor, and generates...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses a ribosome inactivated protein attenuated transformation method for blocking receptor binding, which comprises the following steps of: A, predicting a binding site, namely predicting a part where ribosome inactivated protein is bound with a receptor and the binding site by using molecular docking software; b, performing mutation transformation on the amino acid residues of the binding sites according to the prediction result in the step A; c, performing codon optimization according to the mutant gene obtained in the step B; d, synthesizing the whole gene of the target mutant optimized in the step C, and constructing an expression vector; and E, detection: detecting the target mutant gene protein constructed in the step D, detecting whether the target mutant gene protein enters cells or not through a toxicity test and a fluorescence test, and detecting whether the target mutant gene protein is expressed or not. The structural modification mode of the mutation binding site amino acid residues created by the scheme can significantly block alpha-MMC from entering cells and causing cytotoxicity thereof, thereby achieving a significant attenuation effect.

Description

Technical field: [0001] The application of the present invention belongs to the technical field of bioengineering, and more specifically relates to a method for attenuating and transforming a ribosome inactivating protein that blocks receptor binding. Background technique: [0002] α-Momorcharin (α-MMC) is a type I ribosome-inactivating protein extracted from bitter gourd seeds, which can treat lung cancer, colon cancer, liver cancer, epithelial cell carcinoma, melanoma, choriocarcinoma, Both breast cancer and nasopharyngeal carcinoma have a strong growth inhibitory effect. Studies have found that its growth inhibitory effect on breast cancer is better than that of clinical first-line anti-breast cancer drugs (such as epirubicin), and it has great potential as a clinical anti-tumor drug. medicine. However, when α-MMC is used in vivo, it has obvious tissue cytotoxicity, causing cell damage such as liver cells, brain cells, and monocytes / macrophages. obstacle. At present, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/70G01N21/64
CPCC12N15/66C12N15/70G01N21/6428Y02A50/30
Inventor 刘梦铃
Owner 成都富岱生物医药有限公司
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