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Polypeptide related to RTN4B, and preparation and application thereof

A technology for linear polypeptides and uses, applied in the field of RTN4B-related polypeptides and their preparation and application, to achieve the effect of inhibiting the adhesion of vascular endothelial cells

Active Publication Date: 2013-11-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, how RTN4B regulates tumor growth and how to use RNT4B as a molecular target to achieve tumor growth inhibition is still an urgent scientific problem to be solved.

Method used

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  • Polypeptide related to RTN4B, and preparation and application thereof
  • Polypeptide related to RTN4B, and preparation and application thereof
  • Polypeptide related to RTN4B, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 The solid-phase synthesis of polypeptide of the present invention

[0065] Synthetic raw materials and related reagents:

[0066] (1) Protected amino acid raw materials: Fmoc-L-Cys(Trt)-OH, etc. (fluorenylmethoxycarbonyl-S-trityl-L-cysteine) (Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.);

[0067] (2) Condensation reagent and fluorescence, biotin raw material: HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) (Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.) , HOBT (1-hydroxybenzotriazole) (Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.), DIEA (N,N-diisopropylethylamine) (Sinopharm Shanghai Chemical Reagent Company), FITC (isosulfur Fluorescein cyanate) (SIGMA), D-BIOTIN (D-biotin) (SIGMA);

[0068] (3) Solvent: DMF (dimethylformamide) (Dikma), DCM (dichloromethane) (Dikma), acetonitrile (Fisher);

[0069] (4) Resin: 2-chlorotrityl chloride resin (2-chlorotrityl chloride resin) (Tianjin Nankai S...

Embodiment 2

[0106] Example 2 Application of polypeptide of the present invention in in vitro cell adhesion

[0107] Raw materials and related reagents: phosphate buffer, trypsin (Invitrogen), M200 medium and LSGS serum (cascade biologicals), bovine serum albumin (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), MTS / PMS kit ( Promega), human HUVEC cells (ScienCell).

[0108] Instruments and equipment: cell culture box (Heraeus Company), inverted microscope (Leica Company), horizontal centrifuge (Eppendorf centrifuge), microplate reader (Bio-Rad Company).

[0109] experiment procedure:

[0110] (1) Well plate preparation: Dilute the RTN4B-P4 protein in PBS (part of the extracellular segment of the RTN4B protein disclosed in ZL00111791.2, which has the activity of promoting cell adhesion and can be synthesized by polypeptide), and coat a 96-well plate, each well 100 μL. overnight at 4°C. After washing once with PBS the next day, 1% BSA was added for blocking, 150 μL per well...

Embodiment 3

[0119] Example 3 Application of polypeptide of the present invention in cell growth in vivo

[0120] Raw materials and related reagents: phosphate buffer, trypsin (Invitrogen Company), DMEM medium and fetal bovine serum (Gibco Company), 3-4 week-old nude mice (Shanghai Slack Company), tumor cell line SMMC-7721, etc. .

[0121] Instruments and equipment: cell culture box (Heraeus Company), inverted microscope (Leica Company), horizontal centrifuge (Eppendorf centrifuge), SPF animal room.

[0122] experiment procedure:

[0123] 1. The cyclic peptide was diluted with PBS to prepare a 5 mg / mL stock solution, and the pH value was adjusted to 7.4.

[0124] 2. Breed nude mice (3-4 weeks old).

[0125] 3. Culture SMMC7721 liver cancer cells, collect cells, and resuspend to 2*10 in serum-free medium 7 / mL.

[0126] 4. The cell suspension was inoculated subcutaneously into nude mice, 200uL per mouse, 40 mice in total.

[0127] 5. Four days after cell injection, 40 mice were e...

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Abstract

The present invention relates to an RTN4B-related polypeptide, and, preparation and applications thereof. The RTN4B-related polypeptide of the present invention contains the amino acid sequence as expressed by SEQ ID NO. 1. Further disclosed are a chemical synthesis-based preparation method for the RTN4B-related polypeptide and a use of the RTN4B-related polypeptide. The RTN4B-related polypeptide of the present invention is capable of in vitro inhibition of the biological activity of RTN4B and of in vivo inhibition of tumor cell growth.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RTN4B-related polypeptide and its preparation and application. Background technique [0002] RTN4B belongs to the reticolon family (RTNs), named for its specific endoplasmic reticulum location, and has a conserved RHD structural box (reticulon-homology domain). This family contains four genes, RTN1, RTN2, RTN3 and RTN4, among which there are many research reports on RTN4. The RTN4 (also known as Nogo / ASY / RTN-X) gene has multiple alternative splices, mainly encoding RTN4A (GenBank accession number AF148537), RTN4B (GenBank accession number AF148538) and RTN4C (GenBank accession number AF087901) three proteins, Some other splices are mostly expressed specifically in the testis [Oertle T, Schwab ME. Nogo and its paRTNers. Trends Cell Biol. 2003Apr;13(4):187-94.]. [0003] RTN4B is widely expressed in all normal tissues except the liver, and previous research work suggests that RTN4...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/64C12N15/12A61K38/08A61P35/00A61P1/16A61P7/00
CPCC07K7/06A61K38/00C07K7/00C12P21/00A61P1/16A61P7/00A61P35/00C07K14/47
Inventor 余龙吴燕华季国庆韩丁丁唐丽莎
Owner FUDAN UNIV
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