Application of novel antiviral protein C19orf66 in targeted Zika virus non-structural protein NS3 antiviral drugs

A non-structural protein, Zika virus technology, applied in the application field of antiviral drugs, can solve the problem of unknown precise drug target of C19orf66

Active Publication Date: 2020-04-14
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A patent we have applied for also declares the claim that the antiviral protein C19orf66 is used in the preparation of anti-Zika virus infection drugs, but the precise drug target of C19orf66 in the preparation of anti-Zika virus infection drugs is still unknown

Method used

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  • Application of novel antiviral protein C19orf66 in targeted Zika virus non-structural protein NS3 antiviral drugs
  • Application of novel antiviral protein C19orf66 in targeted Zika virus non-structural protein NS3 antiviral drugs
  • Application of novel antiviral protein C19orf66 in targeted Zika virus non-structural protein NS3 antiviral drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Cellular colocalization of C19orf66 and Zika virus nonstructural protein NS3.

[0023] 1. Experimental method

[0024] 1. Cell transfection.

[0025] (1) Using hNPC as a cell model, 24 hours before transfection, inoculate an appropriate amount of cells on a circular cover slip treated with ultraviolet sterilization;

[0026] (2) Add 200 ng each of the plasmid pCEDF-C19orf66-myc with high expression of C19orf66 and the plasmid pCDEF-NS3-flag with high expression of NS3 into 25 μl Opti-MEM, add 1 μl of P3000TM, and mix gently;

[0027] (3) Add 2 μl LipofectamineTM 3000 reagent to 25 μl Opti-MEM, mix gently, and place at room temperature for 5 minutes;

[0028] (4) Mix the above (2) and (3) liquids, flick the tube wall to mix, and let stand at room temperature for 5 minutes;

[0029] (5) adding (4) into the culture medium of hNPC cells, and culturing in a 37°C incubator;

[0030] (6) Change the medium after 4 to 6 hours and continue culturing.

[0031] 2. Ce...

Embodiment 2

[0048] Example 2 Interaction between C19orf66 and Zika virus non-structural protein NS3.

[0049] 1. Experimental method

[0050] 1. Spread an appropriate amount of cells into a cell culture dish P100. After 24 hours, infect Zika virus (1 MOI). After 48 hours, the cells are used for co-immunoprecipitation, and the corresponding proteins are detected by Western blotting.

[0051] 2. The method of co-immunoprecipitation is as follows:

[0052] (1) Take out the treated cells, discard the original medium, and wash twice with pre-cooled 1×PBS;

[0053] (2) Add pre-cooled 6ml 1×PBS, scrape the cells from the culture dish, transfer the cell solution to a 15ml centrifuge tube, and centrifuge at 2000rpm 4°C for 5min;

[0054] (3) Discard the supernatant, add 500μl 1×IP lysis buffer (containing protease inhibitors) to the cell pellet, transfer the cell suspension to a 1.5ml centrifuge tube, place it on ice, shake it every 10min, Each time for 30s, maintain for 30min to ensure that th...

Embodiment 3

[0062] Example 3 Effect of C19orf66 on the content of Zika virus non-structural protein NS3 in cells.

[0063] 1. Experimental method

[0064] 1. The method of exogenous high expression is as described in Step 1 of Example 1. The amounts of the C19orf66 plasmid were 0, 0.2, 0.4, 0.6, 0.8, and 1 ug, respectively, the amount of the plasmid NS3-Flag was 2 ug, and the amounts of P3000TM and LipofectamineTM were changed accordingly.

[0065] 2. After 48 hours of transfection, the protein was collected, and the corresponding protein was detected by immunoblotting.

[0066] 3. The immunoblotting steps are the same as those in Example 2.

[0067] see results image 3 As shown, when the expression level of C19orf66 in cells increases, the content gradient of NS3 decreases, showing that C19orf66 promotes the degradation of Zika virus non-structural protein NS3.

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Abstract

The invention discloses an application of a novel antiviral protein C19orf66 as a Zika virus non-structural protein NS3 inhibitor in antiviral drugs. The novel antiviral protein C19orf66 provided by the invention has a new application of efficiently targeting Zika virus non-structural protein NS3 and promoting lysosome pathway degradation of NS3 so as to inhibit proliferation of Zika virus in a host. The invention develops a new clinical application field of the humanized antiviral protein in the field of antiviral prevention and treatment, thereby providing a new thought and direction for thedevelopment of drugs targeting Zika virus non-structural protein NS3, providing a scientific basis for the development of anti-Zika-virus drugs, and providing a new thought for clinical antiviral treatment.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, and more specifically relates to the application of novel antiviral protein C19orf66 as Zika virus non-structural protein NS3 inhibitor in antiviral drugs. Background technique [0002] Zika virus (ZIKV) belongs to the Flaviviridae family and is mainly transmitted by Aedes mosquitoes. In recent decades, mosquito-borne flaviviruses, including dengue virus (dengue virus, DENV), West Nile virus (WNV), yellow fever virus (yellow fever virus, YFV), and the recent epidemic of Zika Virus (Zika virus, ZIKV) has broken out a major outbreak and is prevalent in some parts of the world. At the end of 2013 or early 2014, Zika virus disease began to spread on a large scale in the Americas, especially Brazil. According to statistics in 2018, 84 countries and regions have reported cases of ZIKV infection. Although most people are infected with ZIKV, the main clinical symptoms are self-limited feve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P31/14
CPCA61K38/1709A61P31/14Y02A50/30
Inventor 黎孟枫朱勋何振健吴珏珩吴云董信怀谭姹辉陈德林袁洁李隽
Owner SUN YAT SEN UNIV
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