Novel in-vitro and in-vivo infection model based on Ebola virus-like particles

An Ebola virus, virus-like technology, applied in the biological field, can solve the problems of hindering the formation of virus-like particles, high price, and low throughput of β-lactamase activity measurement

Inactive Publication Date: 2017-05-10
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, β-lactamases hinder the formation of virus-like particles [1]
More importantly, the low throughput of β-lactamase activity assays and the expensive substrates (CCF2-AM or CCF4-AM) used for the detection prevent this system from being widely used

Method used

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  • Novel in-vitro and in-vivo infection model based on Ebola virus-like particles
  • Novel in-vitro and in-vivo infection model based on Ebola virus-like particles
  • Novel in-vitro and in-vivo infection model based on Ebola virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0164] Preparation and infection experiment of EBOVLP

[0165] To prepare EBOVLP / Fluc, pcDNA-EBOV GP, pcDNA-EBOV VP40, pcDNA-EBOV NP and pcDNA-Fluc were co-transfected into 293T cells (purchased from the Cell Bank of Chinese Academy of Sciences) using PEI, cultured at 37°C for 4 hours, and the medium was changed to It was complete DMEM containing 10 μM sodium butyrate. After continuing to culture for 8 hours, the medium was replaced with FS293. After 48 hours, the supernatant was harvested and centrifuged at high speed to remove cell debris. The same method was used to prepare EBOVLP / Fluc / GP-, that is, the VLP without GP protein was used as a negative control.

[0166] 24 hours before the EBOVLP infection experiment, in a 96-well plate, add 1×10 4 Vero or Huh7.5.1 cells. After quantifying the VP40 contained in the supernatant VLP by Western blot, add 150ng of VP40-containing VLP to each well, and after incubation for a certain period of time, discard the supernatant, wash th...

Embodiment 1

[0185] Example 1 EBOVLP packaging of reporter gene protein

[0186] In order to express EBOVLP containing Fluc (EBOVLP / Fluc), the inventors constructed plasmids encoding Fluc, VP40, NP and GP ( figure 1 A), and these plasmids were co-transfected into 293T cells in different combinations, and the expression of VLP in the supernatant was detected by Western blot. Such as figure 1 As shown in B, co-transfected cells (lane 1) with plasmids encoding VP40, NP, GP and Fluc, all three viral proteins were expressed in the cells and released into the cell supernatant, and Fluc was detected in the supernatant However, when Fluc was expressed alone, Fluc could not be secreted into the supernatant (lane 8), which indicated that Fluc protein was successfully packaged into VLP and released out of the cell along with VLP. Since VP40 can be assembled into virus-like particles alone, so long as VP40 and Fluc (lane 5) are expressed at the same time, no matter whether it is co-expressed with NP...

Embodiment 2

[0187] Example 2 EBOVLP introduces the encapsulated reporter gene protein into cells through infection

[0188] Although VP40 alone could also encapsulate Flu in VLPs, when the inventors infected cells with supernatants containing intact granular EBOVLP / Fluc (lane 1) and granular EBOVLP / Fluc / GP-(lane 2) lacking the GP protein When , EBOVLP / Fluc can infect cells and release Fluc into the cells, resulting in the intracellular Fluc signal peaking at 6h after infection, while EBOVLP / Fluc / GP- is not infectious due to the lack of GP protein ( figure 1 C). Although the Fluc released into the cells was rapidly degraded after 6h, obvious Fluc signals could still be detected in EBOVLP / Fluc-infected cells at 48h and 72h after infection, which was significantly different from that in EBOVLP / Fluc / GP-infected cells. difference.

[0189] Then the inventors detected the activity of Fluc in 293T cells after transfection, hoping to determine the relationship between the amount of Fluc express...

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Abstract

The invention provides a novel infection model based on EBOV (Ebola Virus) virus-like particles (VLP) and taking firefly luciferase (Fluc) as a reporter protein. The model enters cells with the help of an EBOV envelope protein (GP); the Fluc can be introduced into the cells after the model enters the cells, so that cell infection can be quantified according to signal intensity of the Fluc in the cells; mouse infection can be quantified through biological imaging according to signal intensity of bioluminescence in a mouse body. Therefore, the inventor can utilize the model to carry out in-vitro and in-vivo pre-screening experiments on a neutralizing antibody and an entered inhibitor of the GP outside a BSL-4 (Biosafety Level-4) laboratory.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a novel infection model in vitro and in vivo based on Ebola virus-like particles. Background technique [0002] Ebola virus disease (EVD) is an acute hemorrhagic infectious disease caused by Ebola virus (EBOV), and it is one of the most deadly infectious diseases. EBOV belongs to the Ebolavirus genus of the filoviridae family, and there are currently five known species; among them, the Zaire Ebola virus has the highest lethality and has caused The 2013-2015 outbreak in West Africa was the largest Ebola outbreak since EBOV was first discovered in 1976. It is estimated that the case fatality rate of this epidemic is as high as 70.8%. According to the statistics of the World Health Organization (WHO), it has caused more than 25,000 infections and more than 11,000 deaths. Pay attention to. [0003] While a number of candidate vaccines and drugs against Ebola are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N5/10C12N15/85C12Q1/70C12Q1/66C12Q1/02C12R1/93
Inventor 黄忠陈探李大鹏
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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