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Trichosanthin protein derived peptide and use thereof

A technology of trichosanthin and derived peptides, which is applied in the direction of peptide/protein components, peptides, medical preparations containing active ingredients, etc., and can solve problems such as toxic reactions, destruction of the complete structure of epitopes, and constant cleavage points

Active Publication Date: 2009-07-01
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the technology of simply preparing and purifying Tk whole molecule can no longer meet the needs of application and treatment, because the whole molecule is too toxic as a drug
The second aspect is that the technology of using chemical drugs to crack Tk to obtain corresponding peptides is no longer applied. The main disadvantages are that the cleavage point of the drug is constant. If the cleavage point is located in the antigenic epitope, the complete structure of the epitope may be destroyed. , unable to show its biological function; second, the cracking conditions are difficult to master, and the purity is difficult to reach the standard

Method used

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  • Trichosanthin protein derived peptide and use thereof
  • Trichosanthin protein derived peptide and use thereof
  • Trichosanthin protein derived peptide and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of toxic peptide (p14), inhibitory peptide (p18), and inhibitory peptide p18 tetramer

[0031] (1) Materials

[0032] Solid phase extraction cartridge (Waters company); Fluorenemethoxycarbonyl (Fmoc) protected amino acid (Merck company); 1-Oxo-3-bisdimethylaminocarbonyl benzotriazole boron tetrafluoride salt (TBTU), 1-Hydroxybenzotriazole (HOBT) (ABI company); linear polylysine, DIEA, trifluoroacetic acid (TFA) (Sigma company); G-10, G-25 gel (Pharmacia company); Acetonitrile (Fisher Corporation).

[0033] (2) Method

[0034] 1. Synthesis of Linear Peptides

[0035]0.5 g of PAC-PEG-PS resin (degree of substitution 0.2 mmol / g) was soaked in N-N dimethylformamide (DMF) for 30 min for activation. The activated resin was linked to the first amino acid by the symmetrical anhydride method. Weigh 195 mg of the amino acid to be linked, add 1.5 ml of dichloromethane (DCM) and 3 drops of DMF to dissolve it, add 275 mL of 1 mol / L dicyclohexylcarbodiimide (DCC) / N-m...

Embodiment 2

[0049] Trypan blue exclusion test and in vitro apoptosis induction test (1) Trypan blue test

[0050] This experiment is a simple method to detect whether the cell membrane of the cell to be tested is ruptured, that is, whether the cell is dead. Peripheral blood mononuclear cells (PBMCs) were separated and washed with OptiPrep Lymphocyte Separation Medium (AXIS-SHIELD) from the blood, and then suspended in RPMI 1640 cell culture medium (Gibco), and the concentration was adjusted to 10 6 / ml, add 1-2 drops of trypan blue staining solution (The British DrugHouses.LTD), and count under the microscope after 5 minutes. Live cells are transparent, and dead cells become blue opaque cells due to membrane damage, and the dye enters the cytoplasm. The number of blue cells in 100 cells was counted, that is, the percentage of cell death.

[0051] (2) Apoptosis was detected by Annexin V / PI double staining.

[0052] Annexin-V / PI apoptosis kit (BD PharMingen) was used.

[0053] The exper...

Embodiment 3

[0061] Comparing the biological activities of Trichosanthes whole protein and related derived peptides in vitro and in vivo

[0062] (1) Materials

[0063] Ovalbumin OVA, Complete Freund's Adjuvant CFA (Sigma); Fetal Bovine Serum (Hyclone Lab, Inc), RPMI1640 Medium (Invitrogen Corp); 3 H-TdR (Shanghai Institute of Nuclear Research); crystalline pure trichosanthin (Shanghai Jinshan Pharmaceutical Factory), trichosanthin-derived peptide p18 tetramer and P18 peptide (prepared in Example 1), MOG 35-55 (MEVGWYRSPFSRVVHLYRNGK) (synthesized by Shanghai GL Biochem Company). The unrelated peptide is a tetrameric peptide with the same structure as the p18 tetramer, and its relative molecular weight is 13.7kDa, which is equivalent to that of the p18 tetramer. The following reagents were also used in the experiment: OptiPrep Lymphocyte Separation Solution (AXIS-SHIELD); Mouse CD4, CD8 Magnetic Bead Separation Reagent (Miltenyi Biotech); PE-Cy5-CD3, FITC-CD8, PE-CD28, APC-CTLA4 flow For...

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Abstract

The invention relates to a group of traditional Chinese medicine trichosanthin derived peptides and application thereof. The technical proposal is as follows: a trichosanthin derived peptide p18 has the amino acid sequence shown by SEQ ID NO: 1; a trichosanthin derived peptide p18 tetramer has the amino acid sequence shown by SEQ ID NO: 2; and a trichosanthin derived peptide p14 has the amino acid sequence shown by SEQ ID NO: 3. The trichosanthin derived peptides achieve effects in the experimental study of treating sthenic immune response autoimmune diseases. The invention has the advantage that the invention makes a breakthrough in a traditional method, and removes the part of a trichosanthin molecular structure with toxic and side effects while retaining the effective bioactivity so as to develop a low-toxicity high-efficiency new preparation truly capable of treating a plurality of clinical diseases.

Description

technical field [0001] The invention relates to a group of traditional Chinese medicine trichosanthin-derived peptides and their application. Background technique [0002] Trichosanthin (Tk) is a vegetable protein extracted from the tuberous root of Trichothansuskirilowii Max, a traditional Chinese medicine. Since the 1960s in my country, crystalline pure Tk preparations have been obtained and used in large quantities for mid-clinical induction of labor. It was later found that Tk also has a variety of biological functions, including anti-tumor, induction of apoptosis, anti-virus (including HIV) proliferation, and induction of immunosuppression. [0003] The separation and preparation technology of trichosanthin has mainly involved two aspects in the past. The first is to extract and purify the whole molecule of the plant protein from the root tuber. The technology was developed and provided by the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K38/10A61P37/06A61P37/08A61P37/02
Inventor 周光炎路丽明周芸
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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