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Recombinant Infectious Cloning Plasmid of Japanese Encephalitis Virus Attenuated Strain and Its Application

A Japanese encephalitis virus, infectious cloning technology, applied in the field of bioengineering, can solve the problems of insertion, instability of JEV genome cDNA clone, difficulty in establishing virus infectious clone, etc., achieves broad application prospects, and is conducive to prevention and control Effect

Inactive Publication Date: 2016-08-10
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of using infectious cloning technology to study JEV, there is a major obstacle that the JEV genome cDNA clone is unstable in the host bacteria, and it is prone to mutations such as nucleotide deletion and insertion, which makes it difficult to establish infectious clones of the virus

Method used

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  • Recombinant Infectious Cloning Plasmid of Japanese Encephalitis Virus Attenuated Strain and Its Application
  • Recombinant Infectious Cloning Plasmid of Japanese Encephalitis Virus Attenuated Strain and Its Application
  • Recombinant Infectious Cloning Plasmid of Japanese Encephalitis Virus Attenuated Strain and Its Application

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1 Screening of Japanese Encephalitis Virus (JEV) HEN0701 Passage Attenuated Strain

[0045] The JEV GI strain HEN0701 was continuously passaged on BHK-21 cells for 100 passages, and was continuously purified 10 times with plaques, and 6 clones were selected to inoculate mice subcutaneously and intracranially to screen attenuated strains. The specific process is as follows:

[0046] 1.1 Virus passage

[0047] BHK-21 cells grown in a confluent monolayer in a 35 mm cell culture dish were washed once with 1 mL of PBS, and the HEN0701 strain was inoculated into BHK-21 cells. After incubating at 37°C for 1 h, wash once with 1 mL of PBS, add 2.5 mL of MEM containing 2% FBS to each plate to maintain culture medium, and place at 37°C containing 5% CO 2 incubator. When 70-80% of the BHK-21 cells inoculated with HEN0701 were damaged, the cell supernatant was harvested, divided into 1.5mL centrifuge tubes, and frozen at -70°C. This batch of viruses was called HEN0701P1. ...

Embodiment 210

[0053] Cloning of embodiment 210S3 whole genome

[0054] According to the whole genome sequence of JEV HEN0701 strain with accession number FJ495189 in Genbank, 4 pairs of primers were designed and synthesized as RT and PCR primers, respectively. Viral genomic RNA was extracted from the infected supernatant of the attenuated JEV strain 10S3, cDNA was synthesized by reverse transcription, and used as a PCR template, PCR amplification was performed using 4 pairs of primers to obtain 4 cDNA fragments, and the 4 cDNA fragments were respectively cloned into In the pCR-Blunt II-TOPO vector, select positive clones and determine the cloned cDNA sequence. Using the SeqMan software in DNAStar, the measured sequences were spliced ​​to obtain the full-length viral genome sequence. And use the MegAlign software in DNAStar to compare the measured 10S3 genome sequence with HEN0701. The specific process is as follows:

[0055] 2.1 Primer design

[0056] According to the whole genome seque...

Embodiment 3

[0073] Construction of embodiment 3 attenuated JEV strain 10S3 full-length infectious clone

[0074] Design synthetic primers, add T7 promoter sequence at the 5' end of 10S3 genome by PCR method, add hepatitis D virus ribozyme sequence (delta hepatitis virus ribozyme, HDVr) sequence at the 3' end, and use site-directed mutagenesis PCR method, The genome 9291 base C was mutated to G, thus forming a SacII site at this site. Through specific restriction sites, the cDNA fragments modified at both ends and the two cDNA fragments in the middle were constructed in the pBR322 vector to construct the full-length cDNA clone pAHEN of 10S3 strain. pAHEN was linearized by enzyme digestion, RNA was transcribed in vitro and transfected into BHK-21 cells to obtain infectious virus, which was called vAHEN. vAHEN infects BHK-21 cells and can be specifically recognized by JEV NS3 monoclonal antibody. The vAHEN cDNA fragment amplified by PJEF8791 / PJER10965 can be cut by SacII. The specific pro...

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Abstract

The invention discloses a restructured infectious cloning plasmid of a low virulent strain of a Japanese encephalitis virus, which contains a full-length genome cDNA sequence of the low virulent strain 10S3 of the Japanese encephalitis virus, or a full-length cDNA sequence of a marked cleavage site introduced in the full-length genome cDNA sequence of the low virulent strain 10S3 of the Japanese encephalitis virus. The invention also discloses an infectious cloning low virulent strain of the Japanese encephalitis virus. The low virulent strain is a cloning strain of the low virulent strain 10S3 of the Japanese encephalitis virus, and the full-length genome cDNA sequence of the low virulent strain 10S3 is shown in SEQ ID NO.1. The restructured infectious cloning plasmid of the low virulent strain of the Japanese encephalitis virus and the infectious cloning low virulent strain of the Japanese encephalitis virus rescued from cells can provide complete immunoprotection for immunized animals resisting Japanese encephalitis viruses, thereby extremely facilitating the prevention and control of Japanese encephalitis viruses.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Japanese encephalitis virus attenuated strain recombinant infectious cloning plasmid and application thereof. Background technique [0002] Japanese Encephalitis Virus (JEV) is an important zoonotic pathogen, belonging to the Flavivirus genus (Flavivirus), important members of this genus also include Yellow Fever Virus (YFV), Dengue virus (Dengue Virus, DENV) and West Nile virus (West nile virus, WNV), etc. The JEV genome is a single-stranded positive-sense RNA with a 5' cap and no 3' polyadenosine tail, with a total length of about 11kb. The genome encodes a single long open reading frame flanked by a 5'-untranslated region (5'-UTR) and a 3'-untranslated region (3'-UTR). Translation of a single long open reading frame produces a polyprotein, which is cleaved into 10 proteins during or after translation. The N-terminal quarter of the polyprotein encodes a structural p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N7/04C12Q1/70C12Q1/68A61K39/12A61P31/14A61P25/00
CPCY02A50/30
Inventor 郑浩童光志郑旭晨童武马志永郭亦非
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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