Application of EFTUD2 and Epro-LUC-HepG2 modeling method

A technology of lv6-epro0.5-luc, which is applied in the field of genetics, can solve the problems such as the unsatisfactory cure rate of chronic hepatitis B

Pending Publication Date: 2020-12-22
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The cure rate of chronic hepatitis B (CHB) is still unsatisfactory for two drugs, interferon (IFN)

Method used

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  • Application of EFTUD2 and Epro-LUC-HepG2 modeling method
  • Application of EFTUD2 and Epro-LUC-HepG2 modeling method
  • Application of EFTUD2 and Epro-LUC-HepG2 modeling method

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Embodiment 1

[0089] Embodiment 1: The present invention proposes the application of an EFTUD2 protein as a substance for inhibiting HBV replication. Further, it is proposed that EFTUD2 protein regulates the production of key signaling molecules RIG-1 and MDA5 mRNA in the innate immune signaling pathway through splicing, thereby affecting the production of interferon-stimulated genes ISGs to inhibit the application of HBV replication.

Embodiment 2

[0090] Example 2: The present invention proposes the application of the EFTUD2 target to establish a stable cell model for compound screening. By using EFTUD2 as a target to establish a stable cell model for compound screening, it can be used for research and discovery of new molecularly targeted drugs. Screening compounds that can upregulate EFTUD2 gene expression on the basis of this model will provide more treatment options for patients with chronic HBV infection, especially those with poor clinical response to IFN-α therapy.

Embodiment 3

[0091] Embodiment 3: The present invention proposes a method for establishing an Epro-LUC-HepG2 monoclonal cell model with EFTUD2 as a target, comprising the following steps:

[0092] 1. Construction of LV6-Epro0.5-LUC lentiviral vector

[0093] The hEFTUD2pro-0.5kb promoter sequence with the strongest promoter activity screened out in previous experiments was integrated into a fusion gene with the firefly luciferase reporter gene by fusion PCR technology, and the fusion gene was homologously recombined into LV6 by XbaI and BamHI double enzyme digestion The LV6-Epro0.5-LUC shuttle plasmid was constructed in the vector, and the results of restriction enzyme digestion and identification of the recombinant plasmid were shown in the following pages. After the recombinant plasmids were verified to be correct by sequencing, they were transfected into 293T cells together with the packaging plasmids (pGag / Pol, pRev, pVSV-G) for lentivirus packaging and titer detection. The obtained l...

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Abstract

The invention relates to application of EFTUD2 and an Epro-LUC-HepG2 modeling method. The EFTUD2 protein can adjust the generation of key signal molecules RIG-1 and MDA5 mRNA in an innate immune signal path through a splicing effect, so that the generation of interferon stimulating genes (ISGs) is influenced to inhibit HBV replication. According to the application, a stable compound screening cellmodel is established by taking EFTUD2 as a target spot so that a new molecular targeted drug can be researched and explored. On the basis of the model, compounds capable of up-regulating EFTUD2 geneexpression are screened, and more treatment options are provided for HBV chronic infection patients, especially patients with poor clinical IFN-alpha treatment response.

Description

technical field [0001] The invention relates to the field of genes, in particular to the application of EFTUD2 as a target and the method for establishing the Epro-LUC-HepG2 monoclonal cell model. Background technique [0002] Hepatitis B virus (HBV) is a partially circular double-stranded DNA virus with high species specificity and tissue specificity. Its infection can cause abnormal liver function and hepatitis, and some patients will progress to liver cirrhosis or even liver cancer later. my country is a country with severe HBV infection. There are about 90 million HBV chronically infected people, 28 million of whom are in urgent need of antiviral treatment, and 7 million people are suffering from the pain of advanced liver disease and the risk of liver cancer transformation. Currently, two drugs, interferon (IFN) and nucleotide analogues (NAs), which are currently used to treat chronic hepatitis B virus (HBV) infection, have an unsatisfactory cure rate for chronic hepat...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P1/16A61P31/20C12N5/10C12N15/12C12N15/62C12N15/867C12N15/66C12Q1/66
CPCA61K38/1709A61P1/16A61P31/20C12N5/0693C12N5/067C12N15/86C12N15/66C07K14/47C12Q1/66C07K2319/61C12N2740/15043
Inventor 朱传龙李毓雯徐瑞瑞宁琴罗小平李军胡平平
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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