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Enterovirus 71 (EV 71) Fuyang strain and cDNA (deoxyribonucleic acid) infectious clone of attenuated strain of enterovirus 71 (EV 71) Fuyang strain as well as application of enterovirus 71 (EV 71) Fuyang strain

A technology of infectious clones and enteroviruses, applied in the field of RNA virus rescue

Active Publication Date: 2013-10-30
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Many studies have also proved that the genome sequences of strains with different clinical manifestations have little difference, suggesting that the virulence determination site of EV71 is not a single site, and may be jointly affected by multiple functional regions of the genome

Method used

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  • Enterovirus 71 (EV 71) Fuyang strain and cDNA (deoxyribonucleic acid) infectious clone of attenuated strain of enterovirus 71 (EV 71) Fuyang strain as well as application of enterovirus 71 (EV 71) Fuyang strain
  • Enterovirus 71 (EV 71) Fuyang strain and cDNA (deoxyribonucleic acid) infectious clone of attenuated strain of enterovirus 71 (EV 71) Fuyang strain as well as application of enterovirus 71 (EV 71) Fuyang strain
  • Enterovirus 71 (EV 71) Fuyang strain and cDNA (deoxyribonucleic acid) infectious clone of attenuated strain of enterovirus 71 (EV 71) Fuyang strain as well as application of enterovirus 71 (EV 71) Fuyang strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Genome Sequencing and Sequence Analysis of Enterovirus Type 71 FY Strain and ZD Strain

[0029]According to the conserved sequence of EV71 genome, 9 pairs of universal primers were designed to sequence the whole genome of Enterovirus 71 strain (FY) and its attenuated strain (ZD) passed down in the laboratory in Fuyang, China in 2008. Using DNAStar software, the genome sequences of FY strain and ZD strain, including the sequence of 5'-UTR region, 3'-UTR region and protein coding region, and the amino acid sequence of encoded polyprotein were analyzed and compared. The whole genome sequence identity of FY strain and ZD strain was 97%, among which the sequence identity of 5'-UTR region was 98%, the identity of 3'-UTR region was 99%, and the protein coding region was 97%. The amino acid sequence identity of the polyproteins of the two viruses is 99%, and the sites and names of the mutated amino acids are shown in Table 1. By comparing the gene sequences of the st...

Embodiment 2

[0033] Example 2 Construction of full-length cDNA infectious clones of enterovirus type 71 FY strain and ZD strain

[0034] Two pairs of primers were designed according to the measured genome sequence and the selected restriction site. The upstream primer Sal-T7-FY+ at the 5' end introduced the T7 promoter sequence and the Sal I restriction site, and the downstream primer Hind- at the 3' end. End-introduced 37 Poly(T) and HindIII restriction sites, synthesized by Sangon Company. Viral genomic RNA was extracted with Trizol. Use the long-fragment reverse transcriptase M-MLV Reverse Transcriptase (product number: M1705, purchased from Promega Company) to use the viral genome RNA as a template, and use the 3' downstream primer Hind-End- as a reverse transcription primer, and reverse at 42°C for 2 hours The full-length cDNA of the viral genome was recorded. Using it as a template, DNA polymerase PrimeSTAR (product number: DR010S, purchased from TaKaRa Company) was used to carry o...

Embodiment 3

[0036] Example 3 In vitro transcription and cell transfection

[0037] Cell culture: After the Vero cells (African green monkey kidney cells) grow into a monolayer, the cell surface is washed with PBS for 3 times. Digest the cells with 0.25% trypsin, stop the digestion when the cells become round, suck out the trypsin, immediately add high-sugar DMEM medium containing 10% FBS, blow the bottom of the bottle gently with a pipette, so that the cells are completely detached from the bottom of the bottle and dispersed as a single-cell suspension. After counting on a hemocytometer, adjust the cell concentration to 2 × 10 with medium 5 cells / ml, inoculate 2ml in each well of a six-well plate, place at 37°C, 5% CO 2 Incubator culture. After 24 hours (the cell abundance is 80%), it is used for RNA transfection.

[0038] In vitro transcription and cell transfection: Digest the 3' end of the EV71 cDNA clone with HindIII to obtain a linearized DNA template. After extraction and purifi...

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Abstract

The invention provides an enterovirus 71 (EV 71) Fuyang strain and cDNA (deoxyribonucleic acid) infectious clone of an attenuated strain of the EV 71 Fuyang strain as well as application of the EV 71 vFuyang strain. The EV 71 Fuyang strain and the cDNA infectious clone of the attenuated strain of the EV 71 Fuyang strain are constructed through a reverse genetic operation technique, and lay an important foundation for explaining the pathogenic mechanism of EV 71, searching the virulence determining locus of the EV71, researching and developing anti-EV71 virus drugs and research and developing the EV71 vaccine.

Description

technical field [0001] The invention relates to RNA virus rescue technology, in particular to cDNA infectious cloning and application of enterovirus type 71 Fuyang strain and its attenuated strain. Background technique [0002] Enterovirus type 71 (EV71) belongs to the Picornaviridae family and the genus Enterovirus. The genome is single-stranded positive-sense RNA. EV71 mainly infects children under the age of 5 and is transmitted through the fecal-oral route or droplets. Its infection mainly causes hand-foot-mouth disease (HFMD), which is clinically indistinguishable from HFMD caused by Coxsackievirus A16 infection. Severe EV71 infection can also cause aseptic encephalitis, meningoencephalitis, brainstem encephalitis, poliomyelitis-like paralysis, myocarditis and pulmonary edema and other diseases related to the nervous system, resulting in severe and even fatal cases. Since EV71 was first isolated in 1969 by Schmidt et al. from the stool samples of infants with central ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C07K14/085C12N1/21C12N7/01A61K39/125A61P31/14C12R1/93C12R1/19
Inventor 岑山张永欣李晓宇周永东
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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