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Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof

A technology of trichosanthin and immunosuppression, applied in the direction of medical preparations containing active ingredients, peptides, peptide sources, etc., can solve problems such as constant cleavage point, inability to display biological functions, and difficulty in mastering cleavage conditions

Active Publication Date: 2014-04-16
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 effective epitope peptide fragment with immunosuppression effect and application thereof
  • Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof
  • Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, preparation PA, PAQ peptide

[0035] (1) Materials

[0036] Solid-phase extraction cartridge (Waters); amino acid protected by fluorenylmethoxycarbonyl (Fmoc) (Merck); 1-oxo-3-bisdimethylaminocarbonylbenzotriazole boron tetrafluoride (TBTU), 1-Hydroxybenzotriazole (HOBT) (ABI); linear polylysine, DIEA, trifluoroacetic acid (TFA) (Sigma); G-10, G-25 gel (Pharmacia); Acetonitrile (Fisher).

[0037] (2) Method

[0038] 1. Synthesis of Linear Peptides

[0039] Put 0.8g of 2-CL-TRT resin (substitution degree 0.25mmol / g) into the chromatography column, soak it in dichloromethane for 30min to swell. Weigh 240mg of the amino acid Fmoc-Asn (T to RT)-OH to be connected, add 5ml of dichloromethane (DCM) and 3 drops of DMF to dissolve it, add 1mol / LDIEA, stir with nitrogen bubbling, react at room temperature for 60min, and the reaction is complete , filter the reaction solution and rinse the resin with DCM and DMF. Add 20% piperidine / DMF to remove the Fmoc prote...

Embodiment 2

[0047] Example 2. Lactate dehydrogenase (LDH) experiment and in vitro apoptosis induction experiment

[0048] (1) Lactate dehydrogenase (LDH) experiment

[0049] This test is a general method to detect whether the cell membrane of the cell to be tested is broken, that is, whether the cell is dead. OptiPrep Lymphocyte Separation Medium (AXIS-SHIELD) was used to separate mouse spleen mononuclear cells of different backgrounds (C57 and BALB / C), washed and suspended, and adjusted to 10 with RPMI 1640 cell culture medium (Gibco). 6 / ml, add 96-well round-bottom plate, add 100μl to each well, add different concentrations of Tk, PA and PAQ to the experimental group, and set up positive control group and negative control group, set up 4 duplicate wells for each group, 37℃, 5% Culture in CO2 environment for 6 days, take 50 μl of cell culture supernatant to 96-well flat bottom plate every day, add 50 μl of LDH substrate complex, keep away from light for 30 minutes, add 50 μl of stop so...

Embodiment 3

[0059] Embodiment 3, compare the biological activity experiment that trichosanthin and PA, PAQ play in vivo and in vitro

[0060] (1) Materials

[0061] Peripheral blood from different individuals, human AB serum, C57BL / 6J mice, BALB / C mice, fetal bovine serum (Hyclone Lab, Inc), RPMI 1640 culture medium (Invitrogen Corp); 3 H-TdR (Shanghai Nuclear Research Institute); crystalline pure trichosanthin (Shanghai Jinshan Pharmaceutical Factory), effective epitope peptide PA and PAQ peptide of trichosanthin (prepared in Example 1). The following reagents were used in the experiment at the same time: OptiPrep lymphocyte separation solution (AXIS-SHIELD); Real-time PCR primers (Shanghai Sangon Bioengineering Technology Service Co., Ltd.).

[0062] (2) Method

[0063] 1. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

[0064] Take peripheral blood from different individuals, dilute blood samples with PBS, separate mononuclear cells by Ficoll density gradient centrif...

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Abstract

The invention relates to a trichosanthin effective epitope peptide fragment with an immunosuppression effect and application of the trichosanthin effective epitope peptide fragment, and discloses a trichosanthin effective epitope peptide PA and a trichosanthin effective epitope peptide PAQ, wherein an amino acid sequence of the trichosanthin effective epitope peptide PA is as shown in SEQ ID NO: 1; an amino acid sequence of the trichosanthin effective epitope peptide PAQ is as shown in SEQ ID NO: 4. The trichosanthin effective epitope peptide fragment PA and PAQ obtain effects in the experiment research on treatment of allogeneic mixed lymphocyte proliferation of immune response hyperfunction and obtains effects in the research on treatment on latent autoimmune diabetes in adults. According to the invention, the conventional method is broken through; parts with toxic or side effects are removed from a trichosanthin molecular structure and effective biological activity parts of the trichosanthin molecular structure are kept; a novel low-toxicity high-efficiency preparation which really can be used for treating various clinical diseases is developed.

Description

technical field [0001] The invention relates to a group of effective epitope peptides of trichosanthin of traditional Chinese medicine and its inhibitory effect. Background technique [0002] Trichosanthin (Tk) is a plant protein extracted from the root of the Chinese medicine Trichothansus kirilowii 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] Regarding the separation and preparation technology of trichosanthin protein, in the past, the full-length molecule of the plant protein was extracted and purified from Trichosanthes tuber. Relevant technologies were developed and provided by Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and were used for batch pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K7/08A61K39/36A61P37/08A61P37/06
CPCA61K39/00C07K14/415
Inventor 路丽明周光炎李青周琳
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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