A target of anti-tuberculosis mycobacterium and its application

A technology of Mycobacterium tuberculosis and target, applied in the field of cell biology, can solve the problems of unclear specific molecular mechanism and decreased survival ability, and achieve the effect of inhibiting interaction and inhibiting intracellular survival.

Active Publication Date: 2021-05-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with wild-type Mtb, the survival ability of PknG-knockout Mtb in macrophages is significantly reduced, but the specific molecular mechanism of PknG regulating host innate immunity is still unclear

Method used

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  • A target of anti-tuberculosis mycobacterium and its application
  • A target of anti-tuberculosis mycobacterium and its application
  • A target of anti-tuberculosis mycobacterium and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 TPR domain of Mycobacterium tuberculosis secretory protein PknG

[0022] Through bioinformatics analysis, it was found that the conserved TPR domain (amino acid sequence shown in SEQ ID NO: 1) that exists in eukaryotic proteins also exists at position 506 of the amino acid sequence of Mycobacterium tuberculosis effector protein PknG (GI: 15607551) -568, but its function is not clear. Studies have found that PknG can inhibit cell autophagy and promote the intracellular survival process of Mycobacterium tuberculosis by interacting with host proteins Rab14 (Gene ID: 612673) and Akt (Gene ID: 207) through the TPR domain. wxya The sequence of the gene is shown in GeneID: 886397.

Embodiment 2

[0023] Example 2 PknG interacts with Akt and Rab14 through the TPR domain

[0024] The preparation method of the PknG TPR domain protein in the examples: construct the gene encoding PknG or its truncated body on the pGEX-6P-1 plasmid, construct the Akt and Rab14 genes on pET30a respectively, and then transform the plasmids into the large intestine In Bacillus BL21, use IPTG to induce protein expression at 30°C; sonicate the bacteria, collect the supernatant after high-speed centrifugation; slowly flow the supernatant through the packed Glutathione-Sepharose beads (GE), and then add it to the column Wash the column fully with washing buffer, and finally add 2-3ml of eluent to elute the protein; add the collected eluate to a 10KD protein concentration tube (millipore), centrifuge and concentrate at 3500rpm for 20 minutes; add 2ml of PBS to the protein concentration tube Mix well and centrifuge; aliquot the protein and store at -80°C until use.

[0025] Wild-type PknG with GST t...

Embodiment 3

[0026] Example 3 PknG promotes autophagy by interacting with Akt

[0027] The PknG gene encoding PknG and the PknG gene encoding the deletion of the TPR domain were respectively constructed on the pEGFP-N1 plasmid; the recombinant plasmid was transfected into the HeLa cells laid on the coverslip with Lipofectamine 2000 (Invitrogen, Carlsbad, CA), and transfected at the same time The pEGFP-N1 empty vector was used as a control; after 6 hours of transfection, the fresh DMEM medium containing 10% FBS was replaced to continue the culture; after 24 hours of culture, the medium was discarded, the cells were washed twice with PBS, and then sequentially washed with 4% paraformaldehyde Fix the cells for 10 min, wash the cells three times with PBS, penetrate the cells with 0.5% Triton X-100 for 10 min, wash the cells three times with PBS, block the cells with 1% BSA for 30 min; After washing the cells three times, they were incubated with Alexa594 red fluorescent secondary antibody for ...

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Abstract

The invention discloses an anti-tuberculosis mycobacterium target and application thereof, belonging to the field of cell biology. The present invention has discovered a Mycobacterium tuberculosis secretory protein PknG that can regulate the autophagy flow of host cells and promote the intracellular survival of Mycobacterium tuberculosis and its key domain-TPR domain that plays the function of autophagy flow regulation. It can pass the The interaction of the domain with proteins such as Rab14 and Akt promotes the intracellular survival process of Mycobacterium tuberculosis. The achievements of the present invention can provide new targets and new ideas for the development of clinical tuberculosis drugs, and can also be directly applied in the field of scientific research to guide the development of commercial products such as autophagy flux regulators based on the PknG TPR domain.

Description

technical field [0001] An anti-tuberculosis mycobacterium target and application thereof belong to the field of cell biology. Background technique [0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis ( Mycobacterium tuberculosis , Mtb), which is still one of the infectious diseases that seriously threaten human health around the world. Previous research results have shown that the eukaryotic-like serine / threonine kinase PknG secreted by Mycobacterium tuberculosis can promote the intracellular survival of pathogenic bacteria by regulating the host's natural immune response after entering cells. Compared with wild-type Mtb, the survival ability of PknG-knockout Mtb in macrophages is significantly reduced, but the specific molecular mechanism of PknG regulating host innate immunity is still unclear. Contents of the invention [0003] The present invention finds a conserved TPR domain in the Mycobacterium tuberculosis PknG protein through bioinformatics analysi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12G01N33/68A61K38/45A61P31/06
CPCA61K38/00A61P31/06C12N9/12C12Y207/11G01N33/68
Inventor 刘翠华汪静
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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