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Mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38

A technology of Mycobacterium tuberculosis, secreted protein, applied in the field of cell biology

Active Publication Date: 2014-01-22
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the research on the influence of PtpA protein on cell signaling pathways is still a blank

Method used

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  • Mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38
  • Mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38
  • Mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Mycobacterium tuberculosis secretory protein PtpA

[0025] The amino acid sequence of the Mycobacterium tuberculosis secretory protein PtpA that can dephosphorylate host p-JNK and p-P38 proteins is shown in SEQ ID NO:1. PtpA and its structural analogues can be used in the development of JNK and P38 activity inhibitors.

Embodiment 2

[0026] Example 2 Ubiquitin molecules improve the phosphatase activity of PtpA

[0027] The protein molecule Ubiquitin (Ubiquitin), which can effectively improve the activity of the Mycobacterium tuberculosis secreted protein PtpA phosphatase, has an amino acid sequence as shown in SEQ ID NO:2. Ubiquitin and its structural analogues can be used in the development of PtpA phosphatase activity activators.

[0028] The ubiquitin protein preparation method used in this experiment is to construct the recombinant plasmid pET30a-Ubiquitin containing the Ubiquitin gene, and transform the plasmid into Escherichia coli BL21, induce protein expression with IPTG; ultrasonically break the bacteria, and collect the supernatant after high-speed centrifugation; Slowly flow the supernatant through the packed His-NTA nickel column, then add column washing buffer to fully wash the column, and finally add 2-3ml eluent to elute the protein; add the collected eluate to a 10KD Centrifuge and concent...

Embodiment 3

[0029] Example 3 PtpA and ubiquitin are applied to the dephosphorylation reaction of p-JNK and p-P38 protein

[0030] In vitro experimental system:

[0031] (A) Preparation of phosphatase PtpA protein

[0032] 1) Construct the recombinant plasmid pGEX-6P-1-PtpA containing the gene encoding PtpA, and transform the plasmid into Escherichia coli BL21;

[0033]2) Escherichia coli BL21 carrying the plasmid was added IPTG at 30°C to induce protein expression, and the induction time was 4 hours;

[0034] 3) Sonicate the bacteria, and collect the supernatant after high-speed centrifugation;

[0035] 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;

[0036] 5) Add the collected eluate into a 30KD protein concentrator tube (millipore), and concentrate by centrifugation at 3500rpm for 20 minutes;

[0037] 6) Add 2 ...

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Abstract

The invention discloses a mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38, and belongs to the cytobiology field. The invention ascertains the mycobacterium tuberculosis secretory protein PtpA capable of inhibiting two signal pathways of p-JNK and p-P38, and provides a protein, namely ubiquitin, capable of increasing PtpA activity for the first time. The secretory protein PtpA and the ubiquitin can provide novel tools and ideas for treatment of a plurality of clinical diseases, particularly have a broad prospect in aspects of development and clinical applications of a plurality of medicines such as antineoplastic medicines, and other aspects, and can be directly used for the scientific research field or for guiding development of JNK inhibitors and P38 inhibitors. In addition, the secretory protein PtpA and the ubiquitin have important theory meaning for searching new medicine targets and screening new medicines.

Description

technical field [0001] The invention relates to a mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38, and belongs to the field of cell biology. Background technique [0002] Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis. It, together with AIDS and malaria, is known as the three major infectious diseases threatening human health in the world today. Studies have shown that the Mycobacterium tuberculosis genome encodes 11 serine / threonine protein kinases (Serine / threonine protein kinase, STPK) and two eukaryotic-like tyrosine phosphatases (PtpA and PtpB), which in Mycobacterium tuberculosis During the process of infecting host macrophages, they can be secreted into the cytoplasm of host cells. These proteins interact with different protein molecules in host cells, and interfere with the true effect by regulating the phosphorylation level of each target protein in host cells. The purpose of signaling pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/70C07K14/35
CPCC07K14/35C12N9/16C12N15/70C12Y301/03048
Inventor 刘翠华汪静
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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