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Polypeptide combined with T cell surface co-stimulation molecule CD137 and its use

A costimulatory molecule, cell surface technology, applied in the direction of peptides, etc., can solve the problem of unclear key sequences

Inactive Publication Date: 2006-03-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, the key sequence of the specific binding site between CD137 and CD137L has not been clarified

Method used

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  • Polypeptide combined with T cell surface co-stimulation molecule CD137 and its use
  • Polypeptide combined with T cell surface co-stimulation molecule CD137 and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Screening of CD137-binding peptides from a phage random heptapeptide library

[0059] materials, reagents

[0060] Random heptapeptide phage display library (Ph.D.7 TM Phage Display Peptide Library Kit) was purchased from New England Biolabs, USA, and the titer of the phage was 2.0×10 13 pfu / ml with a complexity of 2.8 × 10 9 The transformant, the host bacterium is E.coli ER2738.

[0061] Recombinant human CD137 was purchased from R&D Company.

[0062] IPTG and Xgal were purchased from Takara Company.

[0063] PEG8000 was purchased from Promega.

[0064] The sequencing primer 5'-CCC TCA TAG TTA GCG TAA CG-3' was synthesized by Shanghai Boya Company.

[0065] Coating solution: 0.1M NaHCO3 pH 8.6

[0066] Blocking solution: 0.5% BSA-NaHCO3

[0067] Eluent: 0.2M Glycine-HCl (pH 2.2), 1mg / ml BSA

[0068] Neutralizing solution: 1M Tris-HCl (pH 9.1)

[0069] Wash solution: 0.1% TBST, 0.5% TBST

[0070] experiment

[0071] 1.1 Phage random heptapeptide li...

Embodiment 2

[0086] Embodiment 2: ELISA experiment detects the affinity of peptide and CD137

[0087] materials, reagents

[0088] Horseradish peroxidase (HRP)-labeled anti-M13 phage monoclonal antibody (HRP / anti-M13) is a product of Pharmacia.

[0089] experiment

[0090] 2.1 Sandwich ELISA to detect the binding of panned phage-displayed polypeptides to target molecules

[0091] When amplifying the above-mentioned phage plaques for DNA sequencing, the remaining supernatant containing phage plaques was stored at 4°C. For each plaque clone to be identified, inoculate a tube of host bacteria ER2738 in 20ml LB medium, and culture at 37°C until slightly turbid. Add 5 μl of phage supernatant to each tube of ER2738 culture medium, and incubate at 37°C for 4.5hrs. The culture was transferred to a centrifuge tube and centrifuged for 10 min. Transfer the supernatant to a fresh centrifuge tube and centrifuge again. Take 80% of the supernatant in a fresh centrifuge tube and add 1 / 6 volume of PE...

Embodiment 3

[0099] Example 3: Phage Displayed Polypeptide Inhibits Anti-CD137 Antibody-Induced T Cell Proliferation in Vitro

[0100] materials, reagents

[0101] PHA was purchased from Kerui Biotechnology Company

[0102] 3 H-TdR was purchased from China Institute of Atomic Energy

[0103] Anti-CD137Ab was purchased from R&D Company.

[0104] experiment

[0105] For the screened phage clones containing consensus sequences, 1×10 12 Phages were added to a 96-well plate and irradiated with ultraviolet light for three hours to inactivate the phages infectivity and viability, but still maintain the displayed polypeptides. The effect of each phage-displayed polypeptide on the proliferation response of human peripheral blood lymphocytes stimulated by PHA and anti-CD137Ab was detected in vitro. At the same time, an irrelevant phage VCSM13 was used as a control.

[0106] Routine separation of human peripheral blood lymphocytes, adjust the cell concentration to 1×10 6 / ml, add 200ul per we...

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Abstract

The present invention discloses one group of polypeptide combined with T cell surface co-stimulating molecule CD137 with the amino acid sequence shown in SEQ ID No. 1 to SEQ ID No. 4; and the application of the polypeptides combined with T cell surface co-stimulating molecule CD137 in preparing medicine for treating autoimmune disease, tumor graft rejection, preparing medicine for activating or blocking CD137-CD137L passage, and developing and preparing CD137-CD137L agonist peptide and peptoid.

Description

technical field [0001] The present invention relates to a key peptide for regulating the immune function of T lymphocytes, in particular, the present invention relates to a group of polypeptides combined with co-stimulatory molecule CD137 on the surface of T cells and application thereof. Background technique [0002] Activation of T cells during an immune response requires dual-signal stimulation. The first signal is provided by the specific binding of the antigen receptor (TCR) on the T cell to the antigenic peptide-major histocompatibility complex (MHC) on the surface of the antigen-presenting cell (APC); the second signal (also known as Co-stimulatory signal) comes from the interaction between T cells and co-stimulatory molecules on the surface of APC. Enhancing co-stimulatory signals required for T lymphocyte activation is an important method to enhance anti-tumor immunity. Blocking the co-stimulatory signal required for T cell activation is an effective method for in...

Claims

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Application Information

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IPC IPC(8): C07K7/06
Inventor 张利宁李娜王群
Owner SHANDONG UNIV
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