Application of interferon-induced transmembrane protein 3 (IFITM 3) for preparing medicament against hepatitis B virus (HBV) infection

A hepatitis B virus and protein technology, applied in antiviral agents, drug combinations, peptide/protein components, etc.

Inactive Publication Date: 2011-09-14
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Application of interferon-induced transmembrane protein 3 (IFITM 3) for preparing medicament against hepatitis B virus (HBV) infection
  • Application of interferon-induced transmembrane protein 3 (IFITM 3) for preparing medicament against hepatitis B virus (HBV) infection
  • Application of interferon-induced transmembrane protein 3 (IFITM 3) for preparing medicament against hepatitis B virus (HBV) infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Intracellular overexpression of IFITMs protein molecules inhibits HBV replication

[0043] Materials and Methods

[0044] 1. Cell culture

[0045] The cells used were hepatoma cells HepG2, which were cultured in DMEM containing 10% fetal bovine serum (FBS, Gibco) at 37°C and 5% CO 2 cultivated under conditions.

[0046] 2. Plasmid

[0047] The pAAVMCS plasmid (Stratagene Company) was preserved by our laboratory. Primers were designed according to the CDS region sequences of each protein of IFNa and IFITMs published by NCBI, and inserted into the pAAVMCS plasmid by amplification and enzyme digestion. After positive clone PCR identification, sequencing, and BLAST comparison, the results showed that pAAVMCS-IFNa, pAAVMCS- IFITM1, pAAVMCS-IFITM2 and pAAVMCS-IFITM3 were constructed successfully. The HBV replication plasmid pHBV1.31 contains 1.31 copies of HBV DNA and has a complete replication unit, which can complete virus replication in transfected HepG2 cel...

Embodiment 2

[0060] Example 2 Intracellular overexpression of IFITMs protein molecules inhibits HBV promoter transcriptional activity

[0061] Materials and Methods

[0062] 1. Cell culture

[0063] Method is the same as embodiment one.

[0064] 2. Plasmid

[0065] Gaussia secreted luciferase expression plasmids pENI / Xp-Gluc, pENII / Cp-Gluc, pSp1-Gluc and pSp2-Gluc, which are transcriptionally regulated by different promoters of HBV, were constructed and preserved by our laboratory.

[0066] 3. Cell Transfection

[0067] Basic method is the same as embodiment one. Co-transfect HepG2 cells with pAAVMCS, pAAVMCS-IFNa, pAAVMCS-IFITM1, pAAVMCS-IFITM2, and pAAVMCS-IFITM3, respectively, with equal amounts of pENI / Xp-Gluc, pENII / Cp-Gluc, pSp1-Gluc, and pSp2-Gluc, and transfect After 48 hours, the cell supernatant was collected for immediate detection or stored at -20°C for later use.

[0068] 4. Gaussia luciferase activity detection

[0069] NEB company's Gaussia secreted luciferase detecti...

Embodiment 3

[0074] Example 3 Overexpression of IFITM3 protein molecules in mice significantly inhibits the expression of HBV genes

[0075] Materials and Methods

[0076] 1. Animal modeling

[0077] SPF grade C57 / BL6 mice, 18±2g, were purchased from Beijing Weitong Lihua Co., Ltd. The model was established by rapid injection of large-capacity plasmids into the tail vein of mice, and 10 μg of pAAVMCS, pAAVMCS-IFNα, pAAVMCS-IFITM1, pAAVMCS-IFITM2, pAAVMCS-IFITM3 and 10 μg of pHBV1.18 (containing 1.18 copies of HBV DNA) were co-infected. Dissolve in PBS of 1 / 10 volume of mouse body weight, and inject at a uniform speed within 5-8 seconds.

[0078] 2. Detection of HBsAg and HBeAg

[0079] On the 1st, 3rd, 5th, and 7th day after modeling, blood was taken from the tail vein of the mouse, and after appropriate dilution, HBsAg and HBeAg were detected according to the operation steps of the ELISA quantitative detection kit (Zhengzhou Antu Lvke Bioengineering Co., Ltd.). Take 50 μl Serum at a s...

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Abstract

The invention relates to new application of interferon-induced transmembrane proteins (IFITMs) in pharmaceutical engineering, in particular to application of IFITM 3 in preparing a medicament for treating diseases related to hepatitis B virus (HBV) infection. Eukaryotic expression vectors of IFITM 1, IFITM2 and the IFITM 3 are respectively constructed by a molecular biological method in a lab; and in vivo-in vitro tests prove that the IFITM 1, the IFITM2 and the IFITM 3 can obviously inhibit HBV replication, wherein, the IFITM 3 has the most remarkable effect. Therefore, the IFITM 3 and an encoding gene thereof are expected to become novel medicaments for treating HBV-related diseases and reducing hazard of HBV-related diseases.

Description

technical field [0001] The present invention relates to a new application of biochemical substances in genetic engineering, protein engineering and pharmaceutical engineering, more specifically interferon-induced transmembrane protein 3 (IFITM3) and its nucleotide coding sequence are used in the preparation and treatment of hepatitis B Application in drugs for diseases related to virus (HBV) infection. Background technique [0002] Hepatitis B virus (HBV) infection seriously threatens human health and is an important cause of hepatitis, liver cirrhosis and liver cancer. There are 350 million hepatitis B virus carriers in the world, and there are about 120 million HBV carriers in China, of which more than 20 million are chronic hepatitis B patients. The direct treatment cost of chronic viral hepatitis in my country exceeds 50 billion yuan every year. HBV infection not only causes huge economic and spiritual burdens to patients and their families, but also causes huge labor a...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00C07K14/435C12N15/12C12N15/63C12N5/10C12N1/21C12N1/19A61P1/16A61P31/20
Inventor 王升启王学军李丹丹赵海峰李英杨静
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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