Applications of novel antiviral protein C19orf66 in preparing medicines capable of resisting Zika virus

A Zika virus, anti-virus technology, applied in the field of protein engineering, to achieve the effect of inhibiting proliferation

Inactive Publication Date: 2017-08-18
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no studies have proved that C19orf66 has the function of resisting Zika virus infection

Method used

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  • Applications of novel antiviral protein C19orf66 in preparing medicines capable of resisting Zika virus
  • Applications of novel antiviral protein C19orf66 in preparing medicines capable of resisting Zika virus
  • Applications of novel antiviral protein C19orf66 in preparing medicines capable of resisting Zika virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Type I interferon IFN-α, IFN-β stimulates A549 cells, identification of endogenous C19orf66 expression level in cells

[0031] 1. Using A549 as a cell model, stimulate the cells with two type I interferons, IFN-α and IFN-β, at a concentration of 500U / ml.

[0032] 2. Collect cells at time points of 0.5h, 1h, 3h, 6h, 24h, and 48h, and extract total cellular RNA.

[0033] The specific operation of the Trizol method for total RNA extraction from adherent cells is as follows:

[0034] (1) RNase removal treatment: the glassware and consumables used for RNA extraction need to be soaked in 0.1% DEPC water overnight, and then sterilized by high temperature and high pressure steam twice for RNase inactivation treatment. Reagents required for RNA manipulation are prepared with high-pressure sterilized 0.1% DEPC water;

[0035] (2) After cell culture, the original culture medium was discarded, and the cells were washed twice with PBS;

[0036] (3) Add 1 ml TRlzol to t...

Embodiment 2

[0062] Example 2: Construction, identification and extraction of pMSCV-C19orf66 recombinant plasmid

[0063] 1. Primer design: Download the C19orf66 gene sequence from the GenBank database, and use PrimerPremier 5.0 software to design a pair of primers to amplify the full-length C19orf66 gene. The primers were synthesized by Shanghai Handsome Biotechnology Company. The primer sequences are as follows:

[0064] Forward: GGAAGATCTGCCATGTCTCAGGAAGGTGTGGAGCTGG

[0065] Reverse: CCGCTCGAGCTACTCCCTGGGCCCGCCCTC

[0066] 2. Obtain the target fragment: obtain the C19orf66 gene fragment by reverse transcription reaction, carry out PCR amplification with its cDNA, and electrophoresis all amplified products with 1.5% agarose gel (containing EB with a final concentration of 0.5 μg / ml) and Gel cutting recovery and purification.

[0067] 3. Extract pMSCV plasmid with QIAprep spin miniprep kit

[0068] 4. Double enzyme digestion reaction of pMSCV plasmid DNA and C19orf66 gene fragment PCR ...

Embodiment 3

[0073] Example 3: Establishment and identification of a cell line stably and highly expressing exogenous C19orf66

[0074] 1. Preparation of retrovirus by calcium phosphate transfection

[0075] The recombinant plasmids pMSCV-puro, pMSCV-C19orf66 and packaging plasmid pIK were transiently co-transfected in 293T cells by calcium phosphate method.

[0076] The specific experimental steps are as follows:

[0077] (1) Take the 293T cell line growing in the logarithmic phase, digest and count the cells, inoculate in a 100mm culture dish at a density of 1×106 cells per well, add 10ml DMEM complete medium, place at 37°C, 5 %CO 2 Culture in a cell incubator (the number of cells prepared depends on the specific situation);

[0078] (2) After 24 hours, mix 20 μg of various recombinant plasmids, 20 μg packaging plasmid pIK, 380 μl 1×TE, 60 μl 2M CaCl2 in a 1.5ml centrifuge tube, and then slowly add 480 μl 2×HEPES drop by drop, at room temperature Stand still for 30 minutes;

[0079]...

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Abstract

The invention discloses applications of novel antiviral protein C19orf66 in preparing medicines capable of resisting Zika virus. For the novel antiviral protein C19orf66, through interferon stimulation and Zika virus infection, the increase of the expression level of novel antiviral protein C19orf66 can be effectively stimulated, meanwhile, the C19orf66 with stable and high expression can specifically inhibit the infection of the Zika virus, and moreover, the result also shows that the silent endogenous C19orf66 can promote the infection of the Zika virus. The scheme proves that C19orf66 has the efficient activity in resisting Zika virus infection and proliferation, and thus a novel thought and direction are provided for the development of the medicines capable of resisting the Zika virus.

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 in the preparation of anti-Zika virus drugs. Background technique [0002] Zika virus belongs to the Flavivirus genus of the family Flaviviridae, and the virus particles are spherical in shape with a diameter of about 40-70nm. Zika virus is a single-stranded positive-sense RNA virus with a genome length of about 10.8kb and a single open reading frame. The viral protein consists of a single polyprotein precursor, which is digested by host protease and viral protease, including three structures Protein (C, prM / M, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5), the structural protein is located at the amino terminal, and the non-structural protein is located at the carboxy terminal, with serine protease, RNA Helicase and RNA-dependent RNA polymerase (RdRP) function. Among them...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P31/14
CPCA61K38/1709Y02A50/30
Inventor 黎孟枫朱勋何振健吴珏珩林嘉捷章诗豪胡忆文陈嘉慧安树董信怀于暕辰袁洁李隽朱显忠古锦敏杨毅
Owner SUN YAT SEN UNIV
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