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Rat's IL-17RE span film region and cell inner region poly peptide and coding sequence, fusion protein and clone

A fusion protein, transmembrane region technology, applied in interleukins, animal/human proteins, cytokines/lymphokines/interferons, etc.

Inactive Publication Date: 2008-06-04
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the understanding of IL-17 ligand-receptor interaction is still in the early stage, and many issues still need to be further studied

Method used

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  • Rat's IL-17RE span film region and cell inner region poly peptide and coding sequence, fusion protein and clone
  • Rat's IL-17RE span film region and cell inner region poly peptide and coding sequence, fusion protein and clone
  • Rat's IL-17RE span film region and cell inner region poly peptide and coding sequence, fusion protein and clone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1, Cloning of Rat IL-17RE Transmembrane Region and Intracellular Region (rIL-17Reid) Coding Sequence

[0042] According to the DNA sequence of SEQ ID №: 2, primers were designed for RT-PCR to amplify the coding sequence of rIL-17RE transmembrane region and intracellular region polypeptide (named rIL-17Reid), and the primer sequences are as follows:

[0043] P1 (upstream primer): 5'-tatagaattcagacacctcgggctcttgatc-3';

[0044] P2 (downstream primer): 5′-tatatctagacagacaactgaagccacaagg-3′

[0045] Total RNA of rat liver tissue was extracted with Trizol kit (purchased from Invitrogen). Using 2 g of rat liver total RNA as a template, under the guidance of primers P1 and P2, a one-step RT-PCR amplification kit (purchased from Takara) was used to amplify rIL-17RE transmembrane by RT-PCR according to the kit instructions The coding sequence of the domain and the intracellular domain polypeptide. The RT-PCR amplification system is: 50ng of template DNA, 100pmol of pr...

Embodiment 2

[0046] Example 2, bioinformatics analysis of rIL-17Reid

[0047] Bioinformatics analysis was carried out on the amino acid residue sequence of rIL-17Reid, rIL-17Reid has the amino acid residue sequence of SEQ ID №: 1 in the sequence table, consisting of 225 amino acid residues, wherein the amino acid residue from the amino terminal (N terminal) The 124th to 126th amino acid residues are the phosphorylation site of protein kinase C, the 166th to 187th amino acid residues from the N-terminal are the conserved leucine zipper, and the 195th to 198th amino acid residues from the N-terminal are casein Kinase phosphorylation site, the 1st to 15th amino acid residues from the N-terminal is the Erk1 kinase binding site.

Embodiment 3

[0048] Example 3, Verification experiment of the activation effect of rIL-17Reid on the RAS / MAPK signaling pathway

[0049] EPOR / rIL-17RE was overexpressed in 293 cells to detect the effect of EPOR / rIL-17RE on the phosphorylation level of intracellular ERK. First, the activating effect of EPOR / rIL-17RE on the RAS / MAPK signaling pathway was detected with a luciferase reporter system, and pCDNA3.1 empty vector was used as a control. The transcription factor Elk1 is a target gene activated by RAS / MAPK signaling. If IL-17RE can activate the RAS / MAPK signaling pathway, the expression level of Elk1-Luciferase will be up-regulated. If EPOR / rIL-17RE can activate the RAS / MAPK signaling pathway, the expression level of Elk1-Luciferase will be up-regulated. The specific method is: each hole 4 × 10 5 293 cells were plated in a 6-well plate to grow to 80% density. Divided into two groups, transfected 3.5 μg pcDNA3.1 empty vector and pcDNA3.1 (EPOR / rIL-17RE) into 293 cells in different ...

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Abstract

The invention discloses white rat IL-17RE membrane crossing area and inside cell area polypeptide and the coding sequence, fusion albumen and cell line. The polypeptide is one sequence of the following aminoacid residue: SEQ ID No:2, the albumen relative to cell transferring of SEQ ID No:1 that has been replaced and / or lacking and / or adding one to ten aminoacid residue and passing RAS / MAPK signal channel path to take mitosis. Interfusing albumen of the polypeptide and the out area of the rat resource erythropoietin has one sequence of the following aminoacid residue: SEQ ID No:2, the albumen relative to cell transferring of SEQ ID No:1 that has been replaced and / or lacking and / or adding one to ten aminoacid residue and passing RAS / MAPK signal channel path to take mitosis. It has great actual value and wide application prospect.

Description

technical field [0001] The invention relates to a polypeptide composed of a rat interleukin 17 receptor (IL-17RE) transmembrane region and an intracellular region, its coding sequence, fusion protein and a transgenic cell line. Background technique [0002] The interleukin 17 (IL-17) and its receptor (IL-17R) family is a unique immune molecule-receptor family that plays an important role in normal body immunity, inflammatory response and tumorigenesis (J.K.Kolls, A . Linden, Interleukin-17 family members and inflammation. Immunity 21(4) (2004) 467; T.A. Moseley, D.R. Haudenschild, L. Rose, A.H. Reddi, Interleukin-17 family and IL-17 receptors. Cytokine Growth Factor Rev 14(2 ) (2003) 155). Currently known IL-17 ligands have six members, and each ligand acts on a different immune process. For example, the earliest discovered IL-17A can stimulate a variety of tissue cells to release chemokines to attract neutrophils to the inflammatory response area (M.Laan, Z.H.Cui, H.Hoshi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K14/54C12N15/12C12N15/24C12N15/63C07K19/00C12N15/62C12N5/10
Inventor 刘力李铁石李雪妮傅新元常智杰张淑平
Owner TSINGHUA UNIV
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