Protein related to cell stress, and coding gene and application thereof

A technology that encodes genes and proteins, which can be used in applications, gene therapy, genetic engineering, etc., and can solve problems such as unclear biological functions of OCT4B protein

Inactive Publication Date: 2011-02-09
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, OCT4A is currently known to maintain the self-renewal and pluripotency of embryonic stem cells (ES), but the biological function of OCT4B protein is still unclear

Method used

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  • Protein related to cell stress, and coding gene and application thereof
  • Protein related to cell stress, and coding gene and application thereof
  • Protein related to cell stress, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Acquisition of the protein OCT4B-190 related to cell stress and its coding gene

[0033] Utilize the TRIZOL kit (purchased from Invitrogen Company) to extract the total RNA of PA-1 cells (purchased from ATCC), and use M-MLV reverse transcriptase (purchased from Promega Company) to reverse transcribe the total RNA of the above-mentioned PA-1 cells as cDNA, using the cDNA as a template, respectively design the upstream primer 5'-GTAGTCCTTTGTTACATG-3' and the downstream primer 5'-TTACTGTGTCCCAGGCTT-3', using Pfx DNA polymerase (purchased from Invitrogen) was used for PCR reaction.

[0034] The above PCR reaction product was recovered and detected by 1.0% agarose gel electrophoresis. As a result, a band of 1150 bp was obtained, which was recovered and ligated into pMD18-T cloning vector for sequence determination. The sequencing results showed that a single band of 1150bp was obtained, and the 1150bp nucleotide fragment was OCT4B-190, its nucleotide sequence wa...

Embodiment 2

[0035] Example 2, the subcellular localization of OCT4B-190 protein

[0036] The OCT4B-190 gene obtained in the above example 1 was double digested with AgeI and PacI, and the digested product was inserted into the vector pQCXIN (purchased from Clontech Company) through the same digestion; at the same time, the pEGFP-1 vector (purchased from Clontech Company) as a template, the GFP gene was amplified by PCR using the upstream primer 5'-ATTGGATCCATGGTGAGCAAGGGCGA-3' and the downstream primer 5'-ACTGAATTCTTACTTGTACAGCTCGT-3'.

[0037] The GFP gene obtained above was double digested with BamHI and EcoRI, and the digested product was inserted between the BamHI and EcoRI restriction sites downstream of the OCT4B-190 gene in the above pQCXIN vector, and the obtained recombinant expression vector was named pQCXIN -OCT4B-190-GFP. At the same time, the above GFP gene was inserted between the BamHI and EcoRI restriction sites of the pQCXIN vector not containing the OCT4B-190 gene, and ...

Embodiment 3

[0039] Example 3. Expression of OCT4B-190 protein under stress conditions and its inhibitory effect on apoptosis

[0040] 1. The expression of OCT4B-190 protein after heat shock treatment or oxidation treatment

[0041] Heat shock treatment: HepG2 cells, PA-1 cells, MCF-7 cells, Hela cells and human ES cells (purchased from ATCC) were respectively cultured in cell culture plates, and when the above-mentioned cells grew to 90% confluence, The cell culture plate was placed in a 45°C electric constant temperature water-proof incubator and cultured for 0.5h, 1.0h, 1.5h and 2.0h respectively.

[0042] Oxidative stress treatment: HepG2 cells, PA-1 cells, MCF-7 cells, Hela cells and human ES cells were cultured in cell culture plates respectively. Hydrogen peroxide (H 2 o 2 ), so that the final concentration in the cell culture medium was 800 μM, PA-1 cells were treated with hydrogen peroxide for 1h, 3h, 6h and 9h, and HepG2 cells were treated with hydrogen peroxide for 3h, 6h, 12...

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Abstract

The invention discloses a protein related to cell stress, and a coding gene and application thereof. The protein is 1) a protein which has an amino acid sequence shown as a sequence 2 in a sequence table, or 2) a protein which is related to the cell stress and derived from the protein 1) with the amino acid sequence shown as the sequence 2 in the sequence table by substituting and/or losing and/or adding one or more amino acid residues. Cells which overexpress an OCT4B-190 protein are constructed; results prove that the apoptosis of the cells which overexpress the OCT4B-190 protein under the stress condition is obviously reduced, namely the OCT4B-190 is the protein related to the cell stress; and the OCT4B-190 protein and the coding gene thereof can be used for inhibiting cell apoptosis under the stress condition.

Description

technical field [0001] The invention relates to a protein related to cell stress, its coding gene and application. Background technique [0002] OCT4 is one of the most important transcription factors involved in regulating the self-renewal of embryonic stem (ES) cells and maintaining their pluripotency, and is also a key gene for establishing induced pluripotent stem (iPS) cells in vitro. The pre-mRNA of human OCT4 can generate two mRNA isoforms through alternative splicing: OCT4A and OCT4B. Among them, OCT4A is currently known to maintain the self-renewal and pluripotency of embryonic stem cells (ES), but the biological function of OCT4B protein is still unclear. Contents of the invention [0003] The purpose of the present invention is to provide a protein related to cell stress and its coding gene. [0004] The protein related to cell stress provided by the present invention, named OCT4B-190, is the following 1) or 2) protein: [0005] 1) A protein consisting of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/63C07K14/435A61K35/48A61K35/00A61K48/00C12N1/00A61K38/17C12N15/11C12N5/10A61K35/74A61P43/00C12N15/12
Inventor 王霞戴建武
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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