Method for degrading BCL11B protein

A protein degradation and protein technology, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, microbial measurement/inspection, etc. It can solve the problems of KLF4 downstream pathway and apoptosis mechanism that have not been elucidated in detail

Active Publication Date: 2013-11-27
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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Problems solved by technology

Overexpression of KLF4 (Kruppel like factor4) gene in T-ALL cell lines can induce apoptosis, but the downstream pathway of KLF4 and the mechanism of apoptosis have not been elucidated in detail

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  • Method for degrading BCL11B protein
  • Method for degrading BCL11B protein
  • Method for degrading BCL11B protein

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Embodiment Construction

[0084] The content of the present invention will be further described below in conjunction with specific embodiments.

[0085] The method for degrading the specific protein of BCL11B gene of the present invention, the first step of the method is to infect Jurkat cells with lentivirus to obtain the Jurkat cell group that can induce the expression of KLF4 gene. For the DNA sequence of the KLF4 gene, see the DNA Sequence Listing.

[0086] The Jurkat cells (uninfected) are from the American ATCC cell bank, and are a kind of acute T-cell leukemia cell line.

[0087] The lentivirus needs to be packaged before infection. The lentivirus used in the present invention includes rtTA lentivirus and KLF4 lentivirus, and the mass ratio of the two in the lentivirus is 1:1. The method used when packaging these two kinds of lentiviruses respectively is a well-known technology, and the specific methods are as follows: 1. Prepare a large number of lentiviral packaging helper plasmids (pSPAX2 an...

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Abstract

The invention discloses a method for degrading BCL11B protein, which is characterized by comprising the following steps: (1) obtaining Jurkat cell population capable of realizing inducible expression of a KLF4 gene; (2) expressing the KLF4 gene by adding doxycycline to promote the Jurkat cell; (3) obtaining the Jurkat cell protein and RNA of the expressed KLF4 gene; (4) detecting the SUMO conjugation of BCL11B protein obtained in the step (3) in the Jurkat cell of the expressed KLF4 gene through Western Blot detection; (5) carrying out reverse transcription synthesis on the RNA obtained in the step (3) to obtain cDNA, detecting the expression condition of the BCL11B gene by adopting real-time quantitative PCR (polymerase chain reaction) and determining that the specific protein of the BCL11B gene is degraded. The method disclosed by the invention has the advantages that a new degradation way of specific BCL11B protein is found to partially explain the apoptosis reasons of T-ALL cells to provide theoretical guidance for leukemia treatment on one hand and provide a new train of thought for realizing human T cell transdifferentiation on the other hand.

Description

technical field [0001] The invention relates to the biotechnology field of protein degradation, in particular to a method for degrading the specific protein of the BCL11B gene. Background technique [0002] The currently known protein degradation pathway is protein SUMOylation: maintaining cellular metabolism requires maintaining protein self-renewal. In addition to synthesizing proteins according to the central dogma under the guidance of genes, there is also a set of protein degradation systems in cells to maintain the desired stability of the types and quantities of proteins in cells. The ubiquitin degradation pathway of proteins was discovered in the 1980s. In 2004, the inventors Aaron Ciechanover, Avram Hershko and Irwin Rose jointly shared the Nobel Prize in Chemistry. SUMO (Small Ubiquitin-like Modifier) ​​is a kind of ubiquitin, which can covalently modify proteins to be degraded like ubiquitin molecules. It is widely involved in various cellular events, including n...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12Q1/68G01N33/68
Inventor 李鹏李田忠蒋治武昊东海
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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