Erk signal pathway inhibitor

A technology of signaling pathway and inhibitor, applied in the field of cell biology

Active Publication Date: 2015-02-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, the research on Mce3E protein's role in inflamm

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Mycobacterium tuberculosis secretory protein Mce3E

[0022] The amino acid sequence of Mycobacterium tuberculosis secreted protein Mce3E, which can prevent host Erk1 / 2 protein phosphorylation, is shown in SEQ ID NO:1. Mce3E and its structural analogs can be used in the development of inhibitors of Erk1 / 2 activity. The sequence of the Erk2 gene is shown in GeneID:5594, and the sequence of the MEK1 gene is shown in GeneID:5604.

Embodiment 2

[0023] Example 2 Mce3E can inhibit the activation of the Erk1 / 2 pathway and can prevent the phosphorylation of the Erk1 / 2 protein

[0024] (A) The effect of Mce3E on the activation of Erk1 / 2 pathway was detected by dual fluorescent reporter gene technology

[0025] 1) Construct the recombinant plasmid p3XFlag-CMV14-Mce3E containing the gene encoding Mce3E;

[0026] 2) Construct the recombinant plasmid pCS2HA-MEK1-ED containing the gene encoding MEK1-ED (SEQ ID NO.2), and use the overlapping extension PCR method to mutate the 218th serine and 222th serine in the MEK1 protein into glutamic acid and glutamic acid respectively. Obtain the MEK1-ED gene fragment after aspartic acid;

[0027] 3) The recombinant plasmid p3XFlag-CMV14-Mce3E and the plasmids pGal4-Elk, pGal4-luc, and pRL-TK (purchased from Promega) for detecting the activation of the Erk1 / 2 pathway were composed by calcium phosphate transfection method, and the Erk1 / 2 pathway was expressed The plasmid pCS2HA-MEK1-ED o...

Embodiment 3

[0033] Example 3 Mce3E protein can directly interact with Erk protein

[0034] (A) Preparation of Mce3E protein

[0035] 1) Construct the recombinant plasmid pGEX-6P-1-Mce3E containing the gene encoding Mce3E, and transform the plasmid into Escherichia coli BL21;

[0036] 2) Escherichia coli BL21 carrying the plasmid was added IPTG at 30°C to induce protein expression, and the induction time was 4 hours;

[0037] 3) Ultrasonic to destroy bacteria, collect the supernatant after high-speed centrifugation;

[0038] 4) Slowly flow the supernatant through the filled Glutathione-Sepharose beads (GE Company, GST column material), then add column washing buffer to fully wash the column, and finally add 2-3ml eluent to elute the protein;

[0039] 5) Add the collected eluate into a 10kD protein concentration tube (millipore), and concentrate by centrifugation at 3500rpm for 20min;

[0040] 6) Add 2ml of phosphate buffered saline (PBS) to the protein concentrator tube and mix well, th...

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Abstract

The present invention discloses an Erk signal pathway inhibitor, and belongs to the field of cell biology. According to the present invention, the Mycobacterium tuberculosis secretory protein Mce3E capable of inhibiting the Erk1/2 signal pathway is firstly determined, and the Mce3E achieves the inhibition on the Erk1/2 signal pathway through spatial regulation on the Erk1/2 protein; the results of the present invention can provide new tools and ideas for the clinical treatment of various diseases, especially provide wide prospects in the fields of development, clinical application and the like of anti-tumor drugs and other drugs, and can further be directly used in the scientific research field or be used for guide the development of Erk1/2 inhibitor; and the result further provides the important theoretical significance for finding of the new drug target and screening of the new drug.

Description

technical field [0001] The invention relates to an Erk signaling pathway inhibitor, which is a mycobacterium tuberculosis secreted protein capable of inhibiting the Erk signaling pathway, and belongs to the field of cell biology. Background technique [0002] Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (Mtb), and it is still one of the infectious diseases that seriously threaten human health around the world. [0003] Through comparative genomics research, the researchers found that the genome of the vaccine Bacilli Calmette Guerin (BCG) used to prevent tuberculosis has 16 deletions compared to the human Mycobacterium tuberculosis Mtb, and these corresponding regions are named in the Mtb genome RD1-RD16, and studies have shown that the proteins encoded by these RD regions play a crucial role in the pathogenicity of Mtb. Among these RD regions, RD15 contains six genes Mce3A-F. There are four mce operons (mce1-4) in the Mtb genome, and each o...

Claims

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Application Information

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IPC IPC(8): C07K14/35C12N15/70A61K38/16A61P31/06
CPCA61K38/00C07K14/35
Inventor 刘翠华李杰汪静
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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