A kind of chimeric virus and its preparation method and application

A technology of chimeric viruses and uses, applied in biochemical equipment and methods, antiviral agents, virus antigen components, etc., can solve the problems that cells cannot be produced with human vaccines, the duration is short, and there is no cross-protection weak, etc., to achieve good Application prospect, strong immunogenicity and high titer effect

Active Publication Date: 2016-01-06
CHENGDU INST OF BIOLOGICAL PROD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is cross-reaction between the four serotypes, there is no cross-protection or the cross-protection is very weak, and the duration is very short, only a few months
Since the patented product can only reproduce on mosquito cell C6 / 36, this cell cannot be used for the production of human vaccines, and it is only a chimeric virus of JE / Dengue 2, which cannot be used for vaccine production. defect

Method used

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  • A kind of chimeric virus and its preparation method and application
  • A kind of chimeric virus and its preparation method and application
  • A kind of chimeric virus and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Construction, identification and recovery of recombinant virus containing JEV vaccine strain SA14-14-2 full-length infectious full-length cDNA clone

[0078] 1. Transformation of the carrier

[0079] (1) Modification of low copy plasmid pACNR

[0080] Dissolve primer Oligo-1 (5'-CGCGCCATTA GGCGCC TTAT AGATCT AATG TCCGGA TTAT GGATCC TGAT TGATCA TTAT TCTAGA ATTAC-3', underlined are KasI, BglII, BspEI, BamHI, BclI, XbaI recognition sites, artificially synthesized) and Oligo-2 (5'-TCGAGTAAT TCTAGA ATAA TGATCA ATCA GGATCC ATAA TCCGGA CATT AGATCT ATAA GGCGCC TAATGG-3', after forming complementary double strands with Oligo-1, the 5' and 3' ends respectively form sticky ends after Ascl and Xhol enzyme digestion, artificially synthesized) in 100 μl sterile double distilled water, mix 10 μl each, and heat to Cool naturally at 100°C, take 2 μl and connect with pACNR double-digested with AscI and Xhol at 4°C overnight, transform competent cell TOP...

Embodiment 2

[0139] Embodiment 2: Preparation and identification of Japanese encephalitis / dengue type 1 chimeric virus

[0140] 1. Preparation of JE / dengue type 1 chimeric virus

[0141] 1. Synthesis of dengue virus type 1 antigen gene prM-E fragment

[0142] Take the gene fragment prME (as shown in SEQ ID NO.4) encoding the E protein of the dengue virus type 1 ThD1000881 strain, and use the Japanese encephalitis virus SA14-14-2 strain to encode the 9 nucleotides of the carboxy-terminal protein of the E protein in the fragment After nucleotide substitution at the corresponding site, it is fused with 204 nucleotides at the amino terminal of the NS1 protein gene of the Japanese encephalitis virus SA14-14-2 strain.

[0143] Then insert the fragment into the pcDNA3.1 vector to obtain the plasmid clone pcDNA3.1-D1PrME of the dengue virus type 1 antigen gene prM-E.

[0144] 2. Construction of JE / dengue type 1 5' half molecule

[0145] The above-mentioned pcDNA3.1-D1prM-E plasmid containing th...

Embodiment 3

[0179] Example 3 Construction of full-length cDNA plasmid of Japanese encephalitis / dengue type 2 chimeric virus, virus recovery and identification of important biological characteristics

[0180] 1. Construction of full-length cDNA plasmid of Japanese encephalitis / dengue type 2 chimeric virus

[0181] 1. Synthesis of DEN2prM / E protein coding gene

[0182] Get the gene fragment prM-E (as shown in SEQ ID NO.5) of the DEN2PUO-218 strain encoding the E protein, and use the 9 nucleotides of the carboxy-terminal protein of the E protein in the fragment to use the Japanese encephalitis virus SA14-14-2 strain After nucleotide replacement at the corresponding site, it was fused with 204 nucleotides at the 5' end of the JE virus SA14-14-2NS1 protein gene, and inserted into the vector pMD18-T for the construction of a chimeric full-length clone.

[0183] 2. Construction of JE / DEN2 type 2 chimeric fragment 5' semi-molecular plasmid pJE / DEN2-5'

[0184] The synthetic gene fragment prM / E ...

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Abstract

The invention discloses an infectious Japanese encephalitis virus clone which is a recombinant vector including full-length cDNA of genome of a Japanese encephalitis virus attenuated strain SA14-14-2. The invention further discloses a chimeric virus and application thereof and a bacterial artificial chromosome as represented by SEQ ID No. 1. The chimeric virus provided by the invention can grow and proliferate on a plurality of cells used for production of human vaccines and show similar growth characteristics, is applicable to production of four serotype Dengue vaccines and has the advantages of strong immunogenicity, high antibody titer, good application prospects and a high industrial application value.

Description

technical field [0001] The invention relates to a chimeric virus, in particular to a Japanese encephalitis virus infectious clone chimerized with dengue virus protective antigen. Background technique [0002] Dengue fever is one of the most serious neglected tropical and subtropical infectious diseases hailed by WHO. According to WHO statistics, since the disease was discovered in 1955, the number of dengue cases reported by WHO has increased by about 30 times. At present, nearly half of the world's population (3.5 billion) live in dengue fever-endemic areas, with about 50 million cases occurring each year, but there is currently no vaccine available worldwide. [0003] The key difficulty in the development of dengue vaccines is that dengue viruses are divided into four types according to serotypes, namely dengue 1, dengue 2, dengue 3, and dengue 4, which appear alternately in endemic areas. Although there is cross-reactivity among the four serotypes, there is no cross-pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N7/01A61K39/12A61P31/14C12R1/93
CPCY02A50/30
Inventor 李玉华吴永林杨会强李竹石杨健林华赵宇俞永新董关木
Owner CHENGDU INST OF BIOLOGICAL PROD
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