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Novel purpose of OCT4B protein isomer

A technology of OCT4B-265, 1.OCT4B-265, which is applied in the new application field of OCT4B protein isomers, and can solve problems such as unclear biological functions

Active Publication Date: 2014-11-26
BEIJING ZKZKTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

OCT4B can produce multiple isoforms, among which OCT4B-190 has been proved to have anti-apoptotic function under cell stress conditions, while the biological functions of other isoforms (such as OCT4-265, OCT4-164) are mostly still unclear

Method used

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  • Novel purpose of OCT4B protein isomer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Expression of OCT4B-265 protein and its subcellular localization in different cell lines under cell stress conditions

[0036] OCT4B-265 protein is a protein isoform with 265 amino acids translated from OCT4B mRNA with ATG as the start codon. Its amino acid sequence is sequence 1 in the sequence listing, and its full-length cDNA sequence is sequence listing Sequence 2, the entire open reading frame has a total length of 798 bases.

[0037] 1. Expression of OCT4B-265 protein in different cell lines under cell stress conditions

[0038] NTERA-2、MCF-7、293T、PA-1、HepG2、RT-4细胞细胞系购自北京协和细胞中心;资源编号依次分别为3111C0001CCC000019、3111C0001CCC000013、3111C0001CCC000091、3111C0001CCC000021、3111C0001CCC000035、3131C0001001200015;

[0039] The human embryonic stem cell line (hES) H9 was purchased from the stem cell technology platform of the Institute of Biochemical Cells, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences.

[0040] The medium used for the above-me...

Embodiment 2

[0079] Example 2, OCT4B-265 protein promotes apoptosis

[0080] 1. Adenovirus transfection

[0081] The adenovirus containing the gene encoding the OCT4B-265 (m) protein was purchased from Beijing Nuosai Gene Company (the nucleotide sequence of the gene encoding the OCT4B-265 (m) protein is sequence 4 in the sequence listing) and the negative control virus; the virus The titers were all 1×10 10 pfu / ml.

[0082] According to the standard procedure of the manufacturer, the adenovirus containing the gene encoding OCT4B-265 (m) protein and the negative control virus (MOI=35) diluted to different titers (MOI=70 and MOI=35) were transfected into MCF-7 cells, Transgenic cells V-265 transfected with different titers (MOI=70 and MOI=35) and control V-null cells were obtained respectively.

[0083] Transgenic cells V-265 transfected with different titers (MOI=70 and MOI=35) and control V-null cells at 36 hours of transfection above were treated with mitomycin C (GR-311, Enzo Life Sci...

Embodiment 3

[0090] Example 3. Genes related to OCT4B-265 protein expression

[0091] 1. The effect of small interfering RNA transfection on the apoptosis rate of MCF-7 cells overexpressing OCT4B-265 protein

[0092] The nucleotide of the human p53 tumor suppressor gene is sequence 7 in the sequence list.

[0093] Human p53 small interfering RNA was purchased from Shanghai Gemma Company, and its forward sequence was: 5'CUACUUCCUGAAAACACGdTdT3' (sequence 5); the reverse sequence was: 5'CGUUGUUUUCAGGAAGUAGdTdT3' (sequence 6).

[0094] Follow the standard steps in the instructions, using Lipofectamine TM RNAiMAX (Invitrogen) transfection reagent, human p53 small interfering RNA was transfected into the transgenic cell V-265 obtained in 1 of Example 2 (identified as positive, MOI=35), and the transgenic cell p53i-V- 265 (MCF-7);

[0095] Control p53i-V-null cells (MCF-7): Human p53 small interfering RNA was transfected into the control group V-null cells obtained in 1 of Example 2 to obtain...

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Abstract

The invention discloses a novel purpose of an OCT4B protein isomer. The invention provides an application of OCT4B-265 protein in preparing a product promoting cell apoptosis. The OCT4B-265 protein is (1) or (2) of the following: (1) a protein composed of an amino acid sequence represented by sequence 1 in the sequence list; (2) a protein derived from the sequence 1 and has a same function with the sequence 1, wherein the protein (2) is obtained by that the amino acid sequence represented by the sequence (1) in the sequence list is subjected to substitution and / or deletion and / or addition of one or more amino acid residues. As a result of experiments, the invention identifies specific expression of the OCT4B-265 protein in stem cells for a first time, and discloses the apoptosis-promoting function of the OCT4B-265 protein in cell genotoxic stress response for a first time, wherein the stress response is regulated buy a cancer suppressor gene p53.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a new application of an OCT4B protein isoform. Background technique [0002] OCT4 is a type of homeodomain transcription factor encoded by the POU5F1 gene. Typical OCT4 is mainly expressed in embryonic stem cells, germ stem cells and teratoma cells with differentiation potential, and is a key transcription factor for maintaining the self-renewal ability and pluripotency of embryonic stem cells. factor. Loss or overexpression of OCT4 can cause the differentiation of embryonic stem cells. At the same time, OCT4 is also the most critical factor in in vitro induced totipotent stem cell technology. [0003] The human OCT4 gene can form three mRNA isoforms, OCT4A, OCT4B1 and OCT4B, through alternative splicing. Among them, OCT4A can generate a typical 360 amino acid OCT4A protein, which plays a key role in maintaining the self-renewal and pluripotency of ES cells. OCT4B1 can be cleaved...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K48/00A61P35/00C12N15/861
Inventor 肖志峰陈冰王霞高原魏建树韩津戴建武
Owner BEIJING ZKZKTECH CO LTD
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