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Application of human polypeptide in preparation of immune regulator

An immunomodulator, human technology, applied in peptide/protein components, medical preparations containing active ingredients, allergic diseases, etc.

Inactive Publication Date: 2013-12-25
赵海潞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports at home and abroad that hIAPP can induce Treg cells and have immunosuppressive effects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of hIAPP

[0025] (1) The amino acid sequence of human amylin (islet amyloid polypeptide, hIAPP / Amylin):

[0026] KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY

[0027] (2) Synthesis and purification of hIAPP:

[0028] full-length peptide hIAPP 1-37 Provided by the Keck Biotechnology Center, Yale University, New Haven, CT, the center uses t-boc chemistry to synthesize hIAPP, and uses reversed-phase high-pressure liquid chromatography to purify, with a purity of more than 99.95%.

[0029] (3) Preparation of hIAPP solution

[0030] The lyophilized peptide hIAPP was pretreated with a 1:1 mixture of trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP). Transfer the dissolved hIAPP to a new tube and let the HFIP / TFA evaporate under a gentle flow of nitrogen for 5-10 minutes. The lyophilized peptide was dissolved in 0.5% acetic acid solution to prepare a 50 mmol / L stock solution. Before use, prepare fresh hIAPP solution by diluting the stock soluti...

Embodiment 2

[0031] Example 2: Effect of hIAPP on mouse splenocytes and human peripheral blood mononuclear cells

[0032] 2.1 Primary mouse spleen cell culture

[0033] Six clean-grade C57BL / 6 mice aged 5-6 months were provided by the Animal Experiment Center of the school. The mice were anesthetized and sacrificed. The spleen was removed under aseptic conditions, ground to prepare a fresh single-cell suspension, and then buffered with ammonium acid hemolysis. solution (BD company kit) to treat splenocytes, remove red blood cells, wash 3 times, and adjust the cell concentration to 5×10 4 cells / ml. Primary splenocytes were cultured using RPMI medium 1640 from Invitrogen Gibco, USA. Add 1, 5, 10, 50, 100, 500, 1000 or 10000 nmol / L hIAPP freshly prepared solutions to splenocytes (100ml) on a 96-well plate, respectively, and control group without hIAPP, in CO 2 The incubators were incubated for 24, 48, or 72 hours, respectively. Finally, Olympus phase contrast microscope CKX41-32PH was use...

Embodiment 3

[0039] Example 3: hIAPP induces human PBMCs to transform into CD4+CD25+Foxp3+ regulatory T cells

[0040] 3.1 Isolation of human PBMC

[0041] 300 ml of peripheral blood (900 ml in total) were collected from 3 healthy blood donors, anticoagulated with ethylenediaminetetraacetic acid (EDTA), and PBMCs were separated using the density gradient method. After red blood cell lysis and 3 washes, PBMCs were counted up to 5′10 6 cells / ml;

[0042] 3.2 hIAPP induced PBMC

[0043] At 37°C, hIAPP solution (10 nmol / L) was used to incubate for 24 hours, and the control group was the same except that hIAPP was not added;

[0044] 3.3 Detection of CD4+CD25+Foxp3+ regulatory T cells by flow cytometry

[0045] Treated human PBMCs were reacted with T cell surface antibodies CD4 and CD25 at 37°C for 30 minutes, and then washed with PBS buffer. Next, add BD Foxp3 buffer at 50:1 and fix for 10 minutes. The fixed PBMCs were permeable with BD Perm buffer for 30 minutes, and then washed twice....

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PUM

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Abstract

The invention discloses an application of a human polypeptide in preparation of an immune regulator. The invention is characterized in that a human polypeptide of which the amino acid sequence is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY is prepared into an immune regulator for inducing CD4+CD25+Foxp3+regulatory T cells. The polypeptide preprocesses human peripheral blood mononuclear cells (PBMC) in vitro, thereby obviously increasing the quantity of human CD4+CD25+Foxp3+regulatory T cells; and the polypeptide immunizes non-obese diabetic (NOD) mice in vivo, thereby effectively preventing and treating autoimmune diabetes, improving immune pathological reaction and preventing and treating autoimmune diseases. The human polypeptide has wide application prospects in the aspects of autoimmune diseases, tissue and organ transplantation, inflammatory pathology and the like.

Description

technical field [0001] The present invention relates to a human polypeptide, in particular to the application of a human polypeptide in the preparation of immunomodulators. Background technique [0002] The immune system consists of immune organs (such as thymus, spleen), immune tissues (skin, mucosal tissue), immune cells (such as T, B lymphocytes), immune factors (such as interleukin IL, interferon IFN, tumor necrosis factor TNF-a, TGF-b) and other components maintain the balance of the body's response to infectious antigens and self-tolerance. Under normal circumstances, the body recognizes self-antigens and foreign antigens through immune regulation mechanisms and appropriate immune responses, prevents the invasion of pathogenic microorganisms, monitors and eliminates cancerous cells in the body, and maintains a stable internal environment. [Sakaguchi,2005;Sakaguchi et al,2008] [0003] Regulatory T cells (Treg) are a specific subset of T cells with unique regulatory f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61P37/06
Inventor 赵海潞张均智牟学晶
Owner 赵海潞
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