Target for resisting mycobacterium tuberculosis and applications thereof

A Mycobacterium tuberculosis, domain technology, applied in medical preparations containing active ingredients, antibacterial drugs, peptide/protein components, etc., can solve the problems of unclear specific molecular mechanism, decreased viability, etc. effect, inhibition of intracellular survival

Active Publication Date: 2019-03-29
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
View PDF3 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Target for resisting mycobacterium tuberculosis and applications thereof
  • Target for resisting mycobacterium tuberculosis and applications thereof
  • Target for resisting mycobacterium tuberculosis and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 The TPR domain of the 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 in the 506th 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 the autophagy flow of cells and promote the intracellular survival process of Mycobacterium tuberculosis through the interaction between the TPR domain and host proteins Rab14 (Gene ID: 612673), Akt (Gene ID: 207). The sequence of the pknG 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; ultrasonically break the bacteria, and 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), and concentrate by centrifugation 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] GS...

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 pEGFP-N1 plasmids; the recombinant plasmids were transfected into HeLa cells laid on coverslips with Lipofectamine 2000 (Invitrogen, Carlsbad, CA), and simultaneously transfected The pEGFP-N1 empty vector was used as a control; after 6 hours of transfection, replace the fresh DMEM medium containing 10% FBS to continue the culture; after 24 hours of culture, discard the medium, wash the cells twice with PBS, and then fix the cells with 4% paraformaldehyde 10min, wash the cells three times with PBS, permeate the cells with 0.5% Triton X-100 for 10min, wash the cells three times with PBS, block the cells with 1% BSA for 30min; Incubate with Alexa594 red fluorescent secondary antibody for 1 h at room temperature, then wash the cells three times with PBS and seal with a mounting medium contai...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a target for resisting mycobacterium tuberculosis and applications thereof, and belongs to the field of cell biology. The invention discloses a mycobacterium tuberculosis secretory protein PknG capable of regulating host cell autophagy flux and promoting the intracellular survival of mycobacterium tuberculosis, and a key structural domain-TPR structural domain with the function of regulating the autophagy flux. The interaction of the structural domain with proteins such as Rab14 and Akt can promote the intracellular survival process of mycobacterium tuberculosis. The invention can provide new targets and new ideas for the development of clinical tuberculosis drugs, and can also be directly applied to the field of scientific research to guide the development of commercial products such as autophagy flux regulators based on the PknG TPR structural domain.

Description

technical field [0001] The invention relates to an anti-tuberculosis mycobacterium target and application thereof, belonging 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. 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 bioi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap