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Trichosanthin protein derived peptide and use thereof

A technology of trichosanthin and derived peptides, which can be applied in the fields of peptide/protein components, peptides, and medical preparations containing active ingredients, etc., and can solve the problems of inability to display biological functions, high toxicity, and difficult to meet the purity standards.

Active Publication Date: 2011-09-07
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

[0031] Preparation of toxic peptide (p14), inhibitory peptide (p18), inhibitory peptide p18 tetramer

[0032] (1) Materials

[0033] Solid-phase extraction cartridge (Waters Company); fluorenylmethoxycarbonyl (Fmoc) protected amino acid (Merck Company); 1-oxo-3-bisdimethylaminocarbonylbenzotriazole boron tetrafluoride (TBTU), 1-Hydroxybenzotriazole (HOBT) (ABI company); Linear polylysine, DIEA, trifluoroacetic acid (TFA) (Sigma company); G-10, G-25 gel (Pharmacia company); Acetonitrile (Fisher).

[0034] (2) Method

[0035] 1. Synthesis of Linear Peptides

[0036]Soak 0.5g of PAC-PEG-PS resin (degree of substitution 0.2mmol / g) in N-N dimethylformamide (DMF) for 30min to activate. The activated resin was connected with 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 1 mol / L dicyclohexylcarbodiimide (DCC) / N-methylpyrrolidone (NMP) 275 mL, Stir m...

Embodiment 2

[0050] Trypane blue rejection test and in vitro apoptosis induction test

[0051] (1) Trypan blue test

[0052] This test is a simple method to detect whether the cell membrane of the cell to be tested is broken, that is, whether the cell is dead. Peripheral blood mononuclear cells (PBMCs) were fractionated and washed with OptiPrep lymphocyte separation medium (AXIS-SHIELD) from the blood, 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 Drug Houses.LTD), and count under the microscope after 5 minutes. Viable cells are transparent, and dead cells become blue opaque cells due to membrane damage and the dye enters the cytoplasm. Count the number of blue cells out of 100 cells, ie the percentage of cell death.

[0053] (2) AnnexinV / PI double staining method to detect cell apoptosis.

[0054] Annexin-V / PI cell apoptosis kit (BD PharMingen) was used.

[0055] The expe...

Embodiment 3

[0063] Comparing the biological activity of trichosanthin whole protein and related derivative peptides in vivo and in vitro

[0064] (1) Materials

[0065] Ovalbumin OVA, complete Freund's adjuvant CFA (Sigma); fetal bovine serum (Hyclone Lab, Inc), RPMI 1640 culture medium (Invitrogen Corp); 3 H-TdR (Shanghai Institute of Nuclear Research); crystallized 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 irrelevant peptide is a tetrameric peptide segment 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 used in the experiment at the same time: OptiPrep lymphocyte separation medium (AXIS-SHIELD); mouse CD4, CD8 magnetic bead sorting reagent (Miltenyi Biotech); PE-Cy5-CD3, FITC-CD8, P...

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PUM

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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 asto 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 applications thereof. Background technique [0002] Trichosanthin (Tk) is a plant protein extracted from the root of the traditional Chinese medicine Trichothansuskirilowii Max. Since the 1960s in my country, crystalline pure Tk preparations have been obtained and widely used in mid-term clinical labor induction. 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] In the past, the separation and preparation technology of trichosanthin mainly involved two aspects. One is to extract and purify the whole plant protein molecule from Kuolou tuber. Relevant technologies were developed and provided by Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and were used for batch preparation and...

Claims

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Application Information

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Patent Type & Authority Patents(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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