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Japanese encephalitis/dengue chimeric virus and application thereof

A dengue virus, replicon technology, applied in the direction of antiviral agents, virus/phage, virus antigen components, etc., can solve the problem of no dengue vaccine and so on

Inactive Publication Date: 2009-10-21
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The dengue virus vaccine has always been the focus of attention of scholars from various countries. In the past 60 years, efforts to develop a dengue vaccine have never stopped, but there is still no dengue vaccine for clinical use.
However, there is no report that the JE virus SA14-14-2 strain is used to construct a virus vaccine vector, especially for constructing a dengue virus vaccine

Method used

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  • Japanese encephalitis/dengue chimeric virus and application thereof
  • Japanese encephalitis/dengue chimeric virus and application thereof
  • Japanese encephalitis/dengue chimeric virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Construction of JEV full-length cDNA clone

[0036] 1. Cultivation of JEV SA14-14-2 strain and extraction of total cellular RNA

[0037] The JEV SA14-14-2 strain is a product of Chengdu Institute of Biological Products, and it has been subcultured in this laboratory for no more than 3 generations. Inoculate the JEV SA14-14-2 virus seed solution on the adherent and overgrown BKH-21 cells (Stratagene, USA), and when the cell lesion reaches +++~++++, the total RNA of the cells is extracted by the Trizol reagent method. LS Reagent is a product of Invitrogen Corporation of the United States.

[0038] 2. Integrate the full-length JEV cDNA into the plasmid pBR322 to prepare a full-length JEV cDNA clone

[0039] Using ThermoScript TM RT-PCR System (Invitrogen, USA) reverse-transcribed JEV RNA to synthesize the first strand of cDNA. The Elongase Amplification System (Invitrogen, USA) was used to amplify the whole JEV genome in two segments (the 5' end half mo...

Embodiment 2

[0042] Embodiment 2: Construction and identification of JEV replicon

[0043] 1. Construction of JEV replicon

[0044] On the basis of the above-mentioned JEV full-length cDNA clone pBRF, the structural protein gene PrM / E was deleted and replaced by a multiple clone site (MCS) for convenient cloning of foreign genes. The principle of multiple cloning site selection: After comprehensive analysis of the full sequence of the plasmid pBR322 and JEV SA14-14-2 strain, and the prM / E sequence of the DV2NGC strain, the cleavage sequence of 6 bases was selected based on the principle of no frame shift. Age I, BSSH II, Xho I, Xma I are multiple cloning sites. The restriction site analysis software is the SeqBuilder module in Lasergene. In order to avoid lethal deletion, two replicons were constructed at the same time, one completely deleted the PrM / E gene; the other retained the C-terminal 213 bp of the E gene, because the inventors considered that this part of the sequence may be impo...

Embodiment 3

[0061] Embodiment 3: Preparation and identification of Japanese encephalitis / dengue type 2 chimeric virus

[0062] 1. Construction of JE / dengue type 2 chimera clone

[0063] Using DV2 virus cDNA as a template, the prM / E full-length gene (DV2-prM / E, 1983bp) of DV2 (NGC strain, Institute of Viral Diseases, Chinese Center for Disease Control and Prevention) and the intercepted prM / E gene were obtained by RT-PCR technology A 213nt gene fragment at the 3' end (DV2-1770, 1770bp) (amplified using Platinum Pfx DNA polymerase from Invitrogen). The primers for amplifying DV2-prM / E are: upstream primer 5' accggt TTCCATTTAACC ACA-3' (the underline is the introduced Age I restriction site), downstream primer 5'- cccggg GGCCTGCACCATGAC-3 (the underline is the introduced Xma I restriction site); the primers for amplifying DV2-1770 are: the upstream primer is 5'- accggt TTCCATTTAACCACA-3' (the underline is the introduced Age I restriction site), the downstream primer is 5'- ccccgggg G...

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Abstract

The invention provides a Japanese encephalitis / dengue chimeric virus, which is obtained by replacing a prM / E gene in Japanese encephalitis cDNA clone with the prM / E gene of dengue virus type II. IFA and Western blot confirm that the chimeric virus can express E protein of a dengue virus and can stabilize passage in C6 / 36 cells. The invention lays a foundation for developing a dengue virus vaccine. The chimeric virus can be taken as a vaccine to immunize animals so as to enable the animals to obtain immune protection against the dengue virus.

Description

technical field [0001] The invention relates to the construction of full-length cDNA clone of Japanese encephalitis virus (JEV) SA14-14-2 strain and the construction of replicon vector. And the preparation method of the Japanese encephalitis / dengue chimeric virus that replaces the main structural protein prM / E protein of the Japanese encephalitis virus with the corresponding protein of the dengue virus. Background technique [0002] Dengue virus (Dengue Virus DV) infection is the most widespread arbovirus disease in humans today. Dengue virus is transmitted by Aedes mosquitoes and is now distributed in almost all tropical and subtropical regions. According to WHO estimates, there are 2.5 to 3 billion people in the world who are at risk of being infected with dengue virus, and about 50 to 100 million cases of dengue fever (Dengue fever DF) occur every year, manifesting as high fever, headache, myalgia, joint pain and rash. Symptoms; 500,000 of them developed into more sever...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/01A61K39/12A61P31/14
CPCY02A50/30
Inventor 李德新韦艳梁米芳李川王晓芳
Owner 中国疾病预防控制中心病毒病预防控制所
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