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Japanese encephalitis virus like particles as well as preparation method and application thereof

A Japanese encephalitis virus, virus-like technology, applied in the direction of antiviral agents, viral antigen components, inactivation/attenuation, etc., can solve problems such as toxicity and protein instability, and achieve good immunogenicity and reactivity , good stability and controllability, high sensitivity effect

Inactive Publication Date: 2012-01-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although Japanese encephalitis virus-like particles have been reported at home and abroad, there are certain problems due to the instability or toxicity of the protein at the 5' end of the recognized flavivirus.

Method used

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  • Japanese encephalitis virus like particles as well as preparation method and application thereof
  • Japanese encephalitis virus like particles as well as preparation method and application thereof
  • Japanese encephalitis virus like particles as well as preparation method and application thereof

Examples

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Embodiment 1

[0041] Example 1 Construction of recombinant plasmids pTRE-Tight-JME and pTRE-Tight-EGFP

[0042] Extract the total RNA of Japanese encephalitis virus (JEV) vaccine strain (product of China Animal Husbandry Co., Ltd., production batch number 1101001-1), reverse transcribe and synthesize cDNA, and use this as a template, SEQ ID NO: 2~3 The sequence shown is the PrM / E gene of primer amplification JEV vaccine strain (such as image 3 ). restriction endonuclease Bam h and Eco R Perform double enzyme digestion, insert the gene into the expression plasmid pTRE-Tight of the Tet-on advance system (product of Clontech Company) that has also undergone double enzyme digestion, and carry out enzyme digestion identification of the recombinant plasmid (such as Figure 4 ), the plasmid with the correct identification result was named pTRE-Tight-JME.

[0043] Plasmid pTRE-Tight-JME was transiently transfected into Hela Tet-on advanced cells by Xfect method (Xfect reagent protocol ...

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Abstract

The invention discloses Japanese encephalitis virus like particles as well as a preparation method and an application thereof. The Japanese encephalitis virus like particles are prepared by assembling a PrM / E gene of a Japanese Encephalitis virus (JEV) and a JEVRNA replicor with a nucleotide sequence as shown in SEQ ID NO: 1. The preparation method of the Japanese encephalitis virus like particles comprises the steps of: connecting the JEV PrM / E gene amplified by an RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) to a plasmid pTRE-Tight of a Tet-on advance expression system to form a recombination vector, transfecting a Hela Tet-on advanced cell by using an Xfect method, after screening and identifying through HygB, obtaining a Hela cell line for controllably and stably expressing the PrM / E gene through a limiting dilution process, inducing for expressing a PrM / E protein, and packaging to obtain the conventional virus like particles; and transfecting the Hela cell line by using a JEV replicor RNA vector and packaging to obtain the virus like particles with a capacity of transient infection. The Japanese encephalitis virus like particles can be used for preparing a vaccine or used as a detection agent, has better immunogenicity and reactivity, and is safe and reliable.

Description

technical field [0001] The invention relates to a Japanese encephalitis virus-like particle, a preparation method and application thereof. Background technique [0002] Japanese encephalitis, also known as Japanese encephalitis (referred to as Japanese encephalitis), is caused by Japanese encephalitis virus (Japanese Encephalitis Virus, JEV). It is widely spread and is mainly transmitted by mosquitoes. It is an acute infectious disease that seriously threatens the health of humans and animals. It is also one of the main infectious diseases that are prevalent in summer and autumn in my country and Asia. [0003] JEV is a single-stranded positive-strand RNA with a length of about 11kb and a molecular weight of about 4.2×10 6 D, the floating density in CsCl is 1.24~1.25g / ml. The virus is wrapped by a lipid bilayer to cover the nucleocapsid, and the cap structure m7GpppAmp at the 5' end of the genome has no effect on infectivity and stability, followed by a conserved AG sequen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/85A61K39/12A61P31/14G01N33/543
CPCY02A50/30
Inventor 亓文宝臧富玉廖明陈孝明刘国乾
Owner SOUTH CHINA AGRI UNIV
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