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Infectious clone of Zika virus MR766 strain and application thereof

A technology of MR766 and Zika virus, which is applied in the field of genetic engineering and medicine, can solve the problems of unstable reproduction and amplification, weakened virulence, and difficult cloning of virus sequences, and achieve the effect of reducing the infectivity and replication level of offspring viruses

Inactive Publication Date: 2019-08-16
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 2016,1(5):e00246-16), some mutations may lead to weakening of virulence (Shan, et al.CellHost Microbe.2016,19(6):891-900)
One of the challenges in constructing infectious clones of flavivirus family members is that the viral sequences are difficult to clone, and the clones that are successfully constructed may also be unstable during the reproduction and amplification process

Method used

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  • Infectious clone of Zika virus MR766 strain and application thereof
  • Infectious clone of Zika virus MR766 strain and application thereof
  • Infectious clone of Zika virus MR766 strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the construction of the infectious cDNA clone of Zika virus MR766 strain

[0054] Such as figure 1 As shown in A, we adopted the strategy of de novo synthesis of the whole genome sequence of the virus. According to the sequence information of the Zika virus MR766 strain published in the public database, we first divided into five segments and synthesized the sequence of the Zika virus MR766 strain (AY632535.2) (Kuno G et al.Arch Virol.2007,152( 4):687-96). Firstly, the synthesized F3 fragment was digested with the restriction endonuclease NotI / AfeI, and then ligated with the pACNR vector digested with the same restriction endonuclease to obtain the pACNR-F3 plasmid. The F1 fragment synthesized in vitro was digested by restriction enzyme NotI / AgeI and ligated in vitro with the F2 fragment digested by restriction endonuclease AgeI / SbfI, and the successfully ligated F1+F2 fragment was recovered by agarose gel electrophoresis. Then NotI / SbfI digestion was p...

Embodiment 2

[0058] Embodiment 2: Zika virus MR766 strain infectious cDNA clone produces viral replication ability and infectivity

[0059] Similar to the above method, the plasmids pZikaMR766-C7, pZikaMR766-C7-Gluc and pZikaMR766-C7-Venus were digested with AfeI, linearized, and then used in vitro transcription kit. 3 g of RNA transcribed in vitro was transferred into Vero cells by electrotransduction. The cytopathic changes were observed at different time points after electroporation. Such as figure 2 As shown in A, after the RNA transduction of pZikaMR766-C7 (C7), pZikaMR766-C7-Gluc (C7-Gluc) and pZikaMR766-C7-Venus (C7-Venus) transfected Vero cells, the cells showed obvious cytopathic changes (CPE). Among them, C7 showed obvious CPE on the 3rd day after electroporation (3dpe); C7-Gluc and C7-Venus showed obvious CPE on the 5th day. In C7-Venus, cells expressing green fluorescent protein can be seen on the 3rd day after electroporation, and then increase. Cell supernatants were col...

Embodiment 3

[0060] Embodiment 3: the stability of the recombinant virus expression reporter gene containing reporter gene

[0061] It has been reported that foreign gene segments inserted in the genome of flaviviruses are easily deleted during virus replication (Schoggins, et al. Proc Natl Acad Sci. 2012, 109(36): 14610). Such as image 3 As shown, in order to verify the stability of the virus expression reporter gene with the reporter gene we constructed, we took the recombinant virus C7-Venus containing the reporter gene Venus as an example, and the cell supernatant (P1 ) to re-infect new Vero cells at a dilution of 1:10, and observe the cells with a fluorescent microscope three days after infection; the new cell supernatant (P2) containing the C7-Venus recombinant virus was the same as above and re-infected new Vero cells at a dilution of 1:10 Cells, three days after infection the cells were visualized with a fluorescence microscope. The infection was subcultured sequentially as abov...

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Abstract

The invention belongs to the fields of genetic engineering and medicine, and relates to a series of stable cDNA clones based on a Zika virus MR766 strain. The cDNA includes a nucleic acid sequence ofthe Zika virus MR766 strain and a low-copy plasmid skeleton; the nucleic acid sequence of the Zika virus MR766 strain includes Zika virus MR766 strain 5' to 3' positive polarity sequences, virus 5' and 3' non-coding regions and an open reading frame encoding a viral protein, wherein the 3' non-coding region does not include a sequence shown in SEQ ID NO 13; in the nucleic acid sequence of the Zikavirus MR766 strain, the 5' non-coding region, the open reading frame encoding the viral protein and the 3' non-coding region are arranged sequentially. The clones also include derivative clones and mutant clones; the invention provides various vectors, recombinant viruses and subunit viral particles produced by the clones, and an application of the viruses in vaccine development and diagnostic reagents.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and medicine, and relates to the construction of an infectious cDNA clone of Zika virus MR766 strain, and the application of the cDNA clone and its derivative clones in virus treatment, vaccine research and development, and virus diagnosis. Background technique [0002] The prior art discloses that Zika virus (Zika virus) is a member of the flavivirus family of Flaviviridae (Flaviviridae), which was first isolated and identified from monkeys in Uganda in 1947, and was subsequently found to infect humans. The virus was mainly concentrated in the African continent until it was discovered in Southeast Asia in the 1980s, then it was discovered in Micronesia (Federated States of Micronesia) in 2007, and it was discovered in the Americas in 2014 and has a global spreading trend (Saiz et al.FrontMicrobiol.2016,7 :496). Zika virus is highly related to human infantile microcephaly (Driggers et al. N En...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N7/00C07K16/10G01N33/569A61K39/12A61P31/14A61K49/00
CPCC07K14/005C12N7/00C07K16/10G01N33/56983A61K39/12A61P31/14A61K49/0008C12N2770/24122C12N2770/24123C12N2770/24121C12N2770/24134A61K2039/5254Y02A50/30
Inventor 易志刚袁正宏
Owner FUDAN UNIV
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