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Mdm2 and p53 protein interaction detection system

A technology of dcas9-mdm2 and protein, which is applied in the field of Mdm2 and p53 protein interaction detection system, can solve the problems of weak fluorescent signal, loss of transcriptional activity, and difficult detection of interaction, so as to improve detection sensitivity and strong operability , the effect of simple process

Pending Publication Date: 2020-10-16
UNIV OF SCI & TECH OF CHINA
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Problems solved by technology

But at the same time, Y2H also has defects: for some protein pair interactions that exist in non-yeast cells, false negative results may be generated in yeast cells due to stimulation different from the normal physiological state, and the experiment cannot It completely simulates the cell type, cell space, and different stages of growth and development of the target protein, so it cannot be used to study protein interactions that occur under specific cell conditions
There are some problems with this detection method: only when the fluorescent protein is assembled in the correct way can the fluorescent signal be generated, so the fluorescent signal is weak, and it is difficult to detect some weak interactions
[0021] In cancer cells, Mdm2 protein is usually overexpressed, and excessive Mdm2 may lead to p53 gene silencing, making it lose transcriptional activity and fail to activate the expression of downstream apoptosis genes, resulting in uncontrolled proliferation of cancer cells

Method used

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  • Mdm2 and p53 protein interaction detection system
  • Mdm2 and p53 protein interaction detection system
  • Mdm2 and p53 protein interaction detection system

Examples

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Embodiment 1

[0051] Example 1: Establishment of p53-Mdm2 protein interaction detection system

[0052] 1. Construction of two fusion protein expression vectors of dCas9-Mdm2 and p53-VPR

[0053] Firstly, the p53 (SEQ ID NO.2) and Mdm2 (SEQ ID NO.1) gene fragments were obtained by PCR reaction with Prime STAR max mix (Takara, Japan) premix. Then use primers VPR F (SEQ ID NO.6) and VPR R (SEQ ID NO.7) to PCR amplify the VPR vector (purchased from addgene) to add homologous recombination sequences at both ends of the VPR vector fragment to linearize it. Next, the dCas9 vector (purchased from addgene) was digested with Bsu36I and EcoRV to obtain linear fragment 1 (2837bp fragment comprising dCas9 gene) and linear fragment 2 (1267bp fragment comprising dCas9 gene), and then primer Bsu36I F (SEQ ID NO. 8) and EcoRV R (SEQ ID NO.9) PCR amplification linear fragment 1, use primer EcoRV F (SEQ ID NO.10) and Bsu36I R (SEQ ID NO.11) PCR amplification linear fragment 2, in two Homologous recombinati...

Embodiment 2

[0060] Example 2: Detection of the effect of the small molecule inhibitor nutlin-3a on p53-Mdm2 protein interaction

[0061] The Mdm2 described in the above Example 1 can not only interact with p53, but also be the binding protein of the imidazoline small molecule inhibitor nutlin-3a. Nutlin-3a is a specific Mdm2 inhibitor, which can effectively bind to the hydrophobic cleft of the N-terminus of Mdm2 protein, thereby preventing its binding to p53. 293T cells were transfected with the above system and treated with nutlin-3a to detect the effect of nutlin-3a on the interaction between p53 and Mdm2.

[0062] The specific method is that after the cells described in Example 1 were transfected for 4 hours, the transfection mixture was sucked off, and replaced with 1 ml of DMEM+10% FBS containing 0, 10, 20, 30, 40 μM small molecule inhibitor nutlin-3a for culture Medium culture for 48h.

[0063] After 48 hours of cell transfection, the expression of red fluorescent protein dtomato ...

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Abstract

The invention relates to an Mdm2 protein function detection composition or kit. The composition or kit is characterized by comprising a coding gene of dCas9-Mdm2 fusion protein, a coding gene of P53-transcriptional activator (such as P53-VP64-P65-Rta (VPR)) fusion protein, a transcription gene of sgRNA and a reporter gene, wherein the sgRNA is used for guiding the dCas9-Mdm2 protein to target thereporter gene, and optionally, the gene is constructed on one or more (such as 1, 2, 3 and 4) expression vectors. The composition or kit can detect functions of Mdm2, particularly in mammalian cells,and regulates and controls Mdm2 and P53 protein interaction by changing concentration of nutlin-3a.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection system for the interaction between Mdm2 and p53 proteins. Background technique [0002] 1. Gene transcription regulation [0003] Gene regulation refers to the regulation of the process from gene to protein expression, and the ways to achieve gene regulation can be divided into three aspects: regulation at the transcriptional level, regulation at the post-transcriptional level, and regulation at the translational level. Among them, transcriptional regulation is an important part of gene expression regulation. [0004] Transcription in eukaryotes is achieved through the joint action of RNA polymerase, related transcription factors and some regulatory elements. And only when the chromatin is in a certain state (that is, accessible), it can be bound by transcription factors to regulate transcription. Because the accessibility of chromatin is affected by DNA modifications ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/62C12Q1/6897
CPCC07K14/4746C07K14/82C07K2319/00C12N9/22C12N15/85C12N15/907C12N2800/107C12Q1/6897
Inventor 付瑜何苗梁好均
Owner UNIV OF SCI & TECH OF CHINA
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