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T7RNA polymerase-based positive-strand RNA virus rapid rescue system and construction method and application thereof

A RNA virus and rescue system technology, which is applied in the field of positive-strand RNA virus rapid rescue system, achieves efficient and stable rescue efficiency, avoids tedious virus rescue, and is easy to operate

Active Publication Date: 2021-06-04
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the defects and problems existing in the construction and virus rescue process of the current positive-strand RNA virus rescue system, the purpose of the present invention is to provide a Dow virus as a model, T7 RNA polymerase-based positive-strand RNA virus rapid rescue system and its construction method and application

Method used

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  • T7RNA polymerase-based positive-strand RNA virus rapid rescue system and construction method and application thereof
  • T7RNA polymerase-based positive-strand RNA virus rapid rescue system and construction method and application thereof
  • T7RNA polymerase-based positive-strand RNA virus rapid rescue system and construction method and application thereof

Examples

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Embodiment

[0051] Embodiment: This embodiment provides a kind of establishment method of enterovirus EV71 rescue system based on T7 RNA polymerase

[0052] In this example, a mammalian cell line HEK293FT expressing T7 RNA polymerase and an EV71 infectious clone containing a T7 promoter were first constructed. The EV71 infectious clone was then transfected into T7 RNA polymerase expressing cells to obtain rescued infectious virus (see figure 1 ).

[0053] Materials and Methods:

[0054] Virus strains: A clinical EV71 strain (GenBank No. HQ891927) was isolated from a patient with HFMD.

[0055] Cell lines: human embryonic kidney cells HEK293FT were preserved by the laboratory; rhabdomyosarcoma cell lines (Rhabdomyosarcoma, RD) were purchased from the Cell Bank of the Chinese Academy of Sciences; human neuroblastoma cells (SH-SY5Y) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0056] Plasmid: pCDH-CMV-EF1α-puro was purchased from System Bioscience (CD510B-1); th...

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Abstract

The invention provides a T7RNA polymerase-based positive-strand RNA virus rapid rescue system and a construction method and application thereof, and mainly relates to the technical field of genetic engineering. The system comprises a mammalian cell line capable of efficiently expressing T7RNA polymerase, and an RNA virus whole genome cDNA infectious clone containing a T7 promoter and taking EV71 as an example, wherein a used vector is efficiently matched with a cell expressing the T7RNA polymerase. Compared with an RNA virus rescue system based on in-vitro transcription, the infectious positive-strand RNA virus can be simply and efficiently rescued, and the generated virus can be continuously passed by taking EV71 as an example and has consistent biological characteristics with a parent virus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a T7 RNA polymerase-based rapid rescue system for positive-strand RNA viruses and its construction method and application. Background technique [0002] T7 RNA polymerase (T7pol) is a DNA-dependent 5'→3' RNA polymerase that highly specifically recognizes the T7 promoter sequence. Because T7pol has strong transcription efficiency and strict promoter recognition specificity, the virus rescue system based on T7pol is widely used. [0003] Enterovirus 71 (Enterovirus 71, EV71) is a non-enveloped positive-strand RNA virus and is a member of the Enterovirus genus in the Picornaviridae family. The virus infection mainly causes hand-foot-and-mouth disease (HFMD) in infants and young children, and may also lead to neurological complications and even death. The full length of the EV71 virus genome is about 7.4kb, which can be used as mRNA to encode a small upstream protein and a large polypr...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N7/02
CPCC12N15/86C12N15/65C12N7/00C12N2740/15043C12N2770/32352C12N2800/107C12N2800/40Y02A50/30
Inventor 龙健儿傅美贤
Owner FUDAN UNIV
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