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Gene type I and gene type III Japanese encephalitis virus infectious clone as well as construction method and application thereof

An infectious cloning, encephalitis virus technology, applied in the field of molecular biology, to achieve the effect of improving efficiency, having cross protection and overcoming genetic instability

Pending Publication Date: 2022-04-26
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Simultaneously, in view of the actual problem that my country currently lacks genotype I JEV vaccine, the genotype I JEV infectious cloning platform of the present invention can be used to develop safe and effective novel genotype I JEV genetically engineered vaccines

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  • Gene type I and gene type III Japanese encephalitis virus infectious clone as well as construction method and application thereof
  • Gene type I and gene type III Japanese encephalitis virus infectious clone as well as construction method and application thereof
  • Gene type I and gene type III Japanese encephalitis virus infectious clone as well as construction method and application thereof

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Embodiment Construction

[0044] The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0045] 1. Materials

[0046] 1.1 Viruses, plasmids, cells

[0047] The genotype I strain YZ-1 (NCBI accession number: MZ540901) and the genotype III strain HSY-1 (NCBI accession number: MZ540902) were preserved by this laboratory; the vector plasmid pYES1L was purchased from ThermoFisher Scientific and preserved by this experiment; BHK- 21 cell lines were preserved by this experiment.

[0048] 1.2 Main reagents

[0049] Anti-JEV NS3 protein antibody was purchased from Genetex; Q5 high-fidelity DNA polymerase, dNTP MIX (100mM), restriction enzymes (Bsu36I, SacII, XmaI, BsrGI, BbvCI), T7 in vitro transcription kit, m7G (5' ) ppp(5') ARNA cap structure mimics were purchased from NEB Company; gel recovery kit, RNA extraction reagent (TRIzol) were purchased from Nanjing Novizan Company; Lipofectamine3000 tran...

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Abstract

The invention discloses a gene type I and gene type III Japanese encephalitis virus infectious clone as well as a construction method and application thereof, and belongs to the technical field of molecular biology. According to the invention, four pairs of conservative primers are designed, fragments covering the full length of gene type I and gene type III JEV genomes are amplified once in four segments, and based on the homology of the tail ends of DNA fragments, homologous recombination is carried out on the segmented viral genome fragments and a linearized vector by using a transformation coupling recombination technology in saccharomycetes, so that the gene type I and gene type III JEV genomes are obtained. And recombinant plasmids carrying gene type I and gene type III JEV genome full lengths are respectively constructed at one time. After the recombinant plasmid is subjected to linearization treatment, virus RNA is transcribed in vitro by using a T7 promoter, and the infectious cloned virus can be obtained after the RNA transfects a BHK-21 cell. The method disclosed by the invention can be stably passed in saccharomycetes for more than 10 generations, and mutation and deletion of a genome sequence or insertion of an exogenous sequence do not exist.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a one-step construction method and application of full-length genome infectious clones of two genotypes (type I and type III) of Japanese encephalitis virus. Background technique [0002] Japanese encephalitis (JE) is an acute zoonotic disease caused by Japanese encephalitis virus (JEV) and is transmitted by mosquitoes. It is mainly prevalent in Asia, and my country has a high incidence of JE. one of the districts. JEV has a wide range of hosts in nature, and its main targets are humans and pigs. Humans are mainly susceptible to children under the age of ten. The clinical symptoms after infection are high fever, convulsions, disturbance of consciousness, pathological reflexes, and meningeal irritation. Critical cases may die or have neurological sequelae in varying degrees after recovery; pigs are mainly manifested as Reproductive disorder, abortion, stillbirth or mummified fetuse...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12N15/64C12N15/40C12N7/01C12R1/93C12R1/865
CPCC12N15/81C07K14/005C07K7/00C12N2770/24122C12N2770/24152C12N2770/24121Y02A50/30
Inventor 李晨曦李燕华陈萱
Owner YANGZHOU UNIV
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