Application of IFITM3 (interferon induced transmembrane protein 3) packaging exosome to preparation of dengue virus infection prevention medicine

A technology of transmembrane protein, dengue virus, applied in the field of protein engineering

Inactive Publication Date: 2015-05-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Vif protein encoded by HIV can antagonize the deaminase effect of APOBEC3G, and the two form a dynamic balance, so HIV can finally escape the antiviral effect of APOBEC3G

Method used

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  • Application of IFITM3 (interferon induced transmembrane protein 3) packaging exosome to preparation of dengue virus infection prevention medicine
  • Application of IFITM3 (interferon induced transmembrane protein 3) packaging exosome to preparation of dengue virus infection prevention medicine
  • Application of IFITM3 (interferon induced transmembrane protein 3) packaging exosome to preparation of dengue virus infection prevention medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Construction, identification and extraction of pMSCV-IFITM recombinant plasmid

[0030] Download the IFITM1, IFITM2, and IFITM3 gene sequences from the GenBank database, and use the Primer Premier 5.0 software to design a pair of primers to amplify the full-length IFITM1 / 2 / 3 genes. The primers were synthesized by Shanghai Handsome Biotechnology Company. The primer series are as follows: the forward primer sequence is as follows : GGAAGATCTGCCATGAATCACACTGTCCAAACCTTC; reverse primer sequence is as follows: CCGGAATTCCTAAGCGTAGTCTGGGACGTCGTATGGGTATCCATAGGCCTGGAAGATCAG.

[0031]After the total cellular RNA was extracted by the Trizol method, the RNA concentration was measured with a nucleic acid protein quantifier. According to the measured concentration, all the RNA was diluted with RNase-free water to a final concentration of 1 μg / μl. We used the extracted cellular RNA to obtain the cDNA products of IFITM1 / 2 / 3 genes, and then amplified the products by PCR. T...

Embodiment 2

[0034] Example 2: Establishment of Stable and Highly Expressed Exogenous IFITM Cell Lines

[0035] 1. Calcium phosphate transfection to prepare retrovirus: Take dengue virus-susceptible cells HEK-293T and U937 in the logarithmic phase, respectively mix 20 μg of various recombinant plasmids, 20 μg of packaging plasmid pIK, 380 μl 1×TE, After mixing 60 μl 2M CaCl2, slowly add 480 μl 2×HEPES drop by drop, add the mixture evenly into HEK-293T medium, add 10 μl chloroquine (100 μM) at the same time and mix gently, place the cells in Cultured in a 37°C, 5% CO2 cell incubator.

[0036] 2. Collect the virus and infect U937 cells: collect the virus liquid at 8:00, 12:00, 16:00, 20:00, and 24:00 the next day, and filter it with a 0.22 μm pore size filter. Among them, the virus liquid collected at 8:00, 12:00, 16:00, and 20:00 was added to Polybrane (40 μg / ml) to infect U937 cells immediately, and the virus liquid collected at 24:00 was frozen and stored at -80 ℃ for later use. The resu...

Embodiment 3

[0039] Embodiment 3: Flow cytometry detection virus infection situation

[0040] Fix: remove the culture supernatant from the cells, wash 3 times with 1×PBS, then add 1×PBS to the cells, scrape the cells from the culture dish with a cell scraper, blow the cells gently and repeatedly with a pipette, and make a single cell suspension , transferred to a centrifuge tube, centrifuged at 1500 rpm, 4°C for 3 min. Discard the supernatant, wash the cells twice with pre-cooled 1×PBS, and centrifuge at 1500 rpm for 3 min. Remove the supernatant, add 1 ml 4% paraformaldehyde to resuspend the cells, fix at 37 °C for 10 min, and place on ice for 1 min.

[0041] Permeabilization treatment: After the cells are fixed, centrifuge to remove paraformaldehyde, add pre-cooled 90% methanol, mix well, and place on ice for 30 min.

[0042] Immunostaining: After cell permeabilization, centrifuge at 1500 rpm for 3 min at 4°C to remove methanol, wash 3 times with 1×PBS, then resuspend cells in 1×PBS co...

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Abstract

The invention discloses application of IFITM3 (interferon induced transmembrane protein 3) packaging exosome to preparation of a dengue virus infection prevention medicine. A preparation method of the exosome comprises the following steps: collecting the supernatant of an HUVEC (Human Umbilical Vein Endothelial Cell) culture medium with high-expressed IFITM3, and performing sucrose density gradient ultracentrifugation on the supernatant to prepare the exosome. The action mechanism is that the exosome with high-expressed IFITM3 can strongly inhibit dengue virus from adsorbing and penetrating host cells. The invention builds a new strategy for resisting dengue virus with the IFITM3 packaging exosome. Whether the antiviral protein packaging exosome can replace interferon to be directly applied to research and development of antiviral medicines opens up a new vision and new application of humanized antiviral protein in the field of antiviral prevention and treatment, thus providing new ideas and directions for development of novel antiviral medicines.

Description

technical field [0001] The present invention relates to the technical field of protein engineering, and more specifically, relates to the application of exosomes packaged by transmembrane protein 3 in the preparation of anti-dengue virus infection drugs. Background technique [0002] Exosomes, also known as extracellular bodies, are a kind of membranous vesicles (vesicles) released into the extracellular matrix after the multivesicular body (MVB) in the cell fuses with the cell membrane. The term exosome was first proposed and named by the report of Johnstone et al. They isolated and purified this small vesicle-like substance from the supernatant of sheep reticulocytes cultured in vitro during the study of the transition from reticulocytes to mature erythrocytes. It can take away plasma membrane proteins that are not needed by mature red blood cells, such as transferrin receptor and acetylcholinesterase. Exosomes originate from the endosome in the process of endocytosis. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K35/44A61K48/00A61P31/12C12N15/85C12N15/66
CPCY02A50/30
Inventor 黎孟枫朱勋何振健文维韬胡忆文陈嘉慧袁洁吴珏珩于暕辰周睿王小群苏扬帆袁杰豪潘静李燃邓海静
Owner SUN YAT SEN UNIV
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