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Application of mst4 gene diagnosis and cell therapy for infectious diseases and related drugs

A therapeutic drug, MST4 technology, applied in the field of infectious diseases, can solve the problems such as the poor understanding of MST4 biological functions

Active Publication Date: 2020-01-07
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other kinases of the GCKIII family have been reported to function in the TNFα-induced NF-κB signaling pathway, but the biological function of MST4 is currently poorly understood

Method used

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  • Application of mst4 gene diagnosis and cell therapy for infectious diseases and related drugs
  • Application of mst4 gene diagnosis and cell therapy for infectious diseases and related drugs
  • Application of mst4 gene diagnosis and cell therapy for infectious diseases and related drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] The relationship between embodiment 1MST4 expression and bacterial infection

[0140] 1. Purpose of the experiment

[0141] The purpose of this example is to study the correlation between the MST4 gene and MST family homologous genes and bacterial infection.

[0142] 2. Experimental method

[0143] 2.1 Detection of gene expression levels in human monocyte THP-1 and mouse bone marrow-derived macrophage BMM cells

[0144] The method for extracting total RNA from THP-1 nuclei and BMM cells is as described in Basic Experimental Method 12. AMV reverse transcriptase synthesizes the first strand, and uses it as a template to perform PCR amplification with corresponding primers under the action of DNA taq enzyme; See Table 1 for THP-1 cell sample amplification PCR primers, BMM cell sample amplification PCR primers in Table 2, reverse transcription system in Table 3, reverse transcription reaction program in Table 4, Realtime-PCR reaction system in Table 5, Realtime -See Tabl...

Embodiment 2

[0166] Example 2 Changes in the expression level of MST4 after bacterial infection

[0167] 1. Purpose of the experiment

[0168] The purpose of this example is to study the effect of the expression level of MST4 on the expression level of inflammatory cytokines after bacterial infection, and the change of the expression level of MST4 in immune-related tissues or cells.

[0169] 2. Experimental method

[0170] 2.1 Detection of gene expression levels in THP-1 cells after LPS stimulation

[0171] The method for extracting total RNA is as described in Basic Experimental Method 12, and the method for sample amplification and detection is as described in Example 1. The PCR primers for MST4 gene amplification are shown in Table 1, and the PCR primers for TNFα and IL-6 amplification are shown in Table 7.

[0172] Table 7 Primer sequences

[0173]

[0174] 2.2 The experimental method for the detection of gene expression levels in mouse arterial vascular epithelial cells (AVEC),...

Embodiment 3

[0183] Example 3 MST4 negatively regulates the TLR signaling pathway induced by LPS or bacteria

[0184] 1. Purpose of the experiment

[0185] The purpose of this example is to study the effect of MST4 on the expression of immune-related pro-inflammatory cytokines and the regulation of signaling pathways.

[0186] 2. Experimental method

[0187] 2.1 Preparation of lentiviral recombinant shMST4

[0188] Design a 21-base target sequence (shRNA) to knock out the coding region and non-coding region of MST4 respectively. This sequence can form a reverse complementary hairpin-like structure and silence the expression of related genes. MST4 shRNA and control shRNA (scramble) were connected to the lentiviral vector pLKO.1-puro. shRNA (scramble) was used as a control.

[0189] Human shMST4oligo-1 (targets MST4 coding region)

[0190] f

[0191] 5'-CCGGCAGCAAGTCGTTGCTATTAAAATCTCGAGATTTTAATAGCTTCGACTTGCTGTTTTTG-3' (SEQ ID NO: 63)

[0192] R

[0193] 5'-AATTCAAAAACAGCAAGTCGTTGCTAT...

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Abstract

The invention discloses application of an isolated MST4 gene and a homologous sequence thereof in preparing or screening drugs for treating infectious diseases. The MST4 directly interacts with TRAF6 and is effective on phosphorylating T463 and T486 of the TRAF6, and the MST4 is capable of inhibiting antoubiquitination of a K63Ub chain thereof as well as the activation of a downstream signal path and is capable of regulating an excessive immune response. Immune injury of an MST4 knock-out mouse in septic shock is significantly exacerbated, showing intensified macrophage infiltration and a lower mouse survival rate. Furthermore, MST4 gene expression in macrophage is subjected to in vitro reconstruction and related cell therapy means in a mouse body are inputted once again, so that the excessive immune response of the mouse with sepsis can be effectively affected. The MST4 gene disclosed by the invention can be used as an index for the early diagnosis of the sepsis, bacillary dysentery and malaria; the excessive immune response in a host can be improved by regulating and controlling MST4 level, so that the MST4 gene is applicable to the therapy of such infectious diseases as the sepsis; and the study can offer new technical means to the clinical related therapy of the infectious diseases; therefore, the application has a considerable market value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of MST4 gene in diagnosis and treatment of infectious diseases such as sepsis and related medicines. Background technique [0002] Excessive inflammatory response can adversely affect the body and even cause death. Toll-like receptor (TLR) signaling plays a key role in both bacterial infection and tissue damage. In a variety of pathological conditions, such as sepsis, the disturbance of TLR signaling pathway will lead to excessive inflammatory response and early field damage to the body. Therefore, fine regulation is required between immune inflammation and immune tolerance. After detecting the pathogen-associated molecular pattern, most Toll-like receptors will activate the signal transduction intermediate molecule TRAF6, and then activate the downstream NF-kB pathway to produce pro-inflammatory cytokines. Thus, the activation of TRAF6 needs to be finely regulated to main...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/113C12N15/867A61K31/713A61P29/00A61P31/04A61P33/06A61P1/12A61P7/00
CPCY02A50/30
Inventor 周兆才焦石
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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