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Japanese encephalitis virus JEV replicon vector and application thereof

A technology of encephalitis virus and replicon, which is applied in the direction of virus/bacteriophage, antiviral agent, virus antigen component, etc., and can solve problems such as potential safety hazards

Inactive Publication Date: 2010-05-26
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used pseudovirus packaging method is to provide packaging structural proteins through helper viruses, but this packaging is easy to cause the production of recombinant viruses, resulting in potential safety hazards

Method used

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  • Japanese encephalitis virus JEV replicon vector and application thereof
  • Japanese encephalitis virus JEV replicon vector and application thereof
  • Japanese encephalitis virus JEV replicon vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the preparation process of replicon vector pCMW-2M

[0073] 1. Transformation of plasmid vector pMW118 (Nippon Gene)

[0074] Based on the low-copy pMW118 (Nippon Gene) plasmid vector, two oligonucleotide sequences D118 (SEQ ID NO: 2) and P118 (SEQ ID NO: 3) were used to construct an adapter, which was cloned into the plasmid vector pMW118 through the Sal I and EcoR I restriction sites to construct the recombinant plasmid pMW-118L.

[0075] Using the plasmid pcDNA3.1 as a template, a 607bp CMV sequence was amplified by PCR with primers pPCMV (SEQ ID NO: 4) and primers pSCMV (SEQ ID NO: 5) (see SEQ ID NO: 1 for CMV promoter). The plasmid pCMW-118L was constructed by cloning into the plasmid vector pMW-118L through SphI and SalI restriction sites.

[0076] 2. Prepare the JEV replicon vector pCMW-2M from which the JEV virus structural protein has been removed.

[0077] On the basis of the full-length infectious clone of the JEV vaccine strain SA14-14-2, th...

Embodiment 2

[0082] Example 2, Functional Identification of Replicon Vector pCMW-2M

[0083] 1. Functional identification at the cellular level

[0084] Take about 5 μg of JEV replicon DNA as a template, linearize the plasmid by single-cutting with XbaI enzyme, flatten the MBN, and purify the fragment. BHK-21 cells were transfected by liposome (LipofectamineTM 2000, Invitrogen Company). Three days after transfection, an indirect immunofluorescence assay (IFA) was performed to analyze the expression of the replicon in BHK-21 cells. Guinea pig-derived anti-JEV polyclonal antibody was used as the primary antibody, and FITC-labeled goat anti-guinea pig IgG was used as the secondary antibody. anti-Evans blue staining. See figure 1 , about 30% of the cells showed positive results, showing green fluorescence, cytoplasmic expression. The negative control group was observed under a fluorescent microscope to appear dark red.

[0085] Expression of 2 replicon vector pCMW-2M-EGFP in BHK-21 cells ...

Embodiment 3

[0092] Embodiment 3, experiment of replicon vector pCMW-2M immunization mice

[0093] 1 Grouping of experimental mice:

[0094] 3-week-old BALB / C mice, female, were selected for the experiment, 6 mice in each group, and the two groups were respectively immunized with the replicon vectors pCMW-2M and pCMW-G2-R, and a PBS group and an empty vector control group were set at the same time. The specific experimental groups are shown in Table 1.

[0095] Table 1 Experimental grouping of mice immunized with replicon plasmids

[0096]

[0097] 2 Immunization route, injection dose:

[0098] Intramuscular injection i.m.: Inject 25 μg of plasmid diluted in PBS into the quadriceps muscle of each leg, both thighs. Each mouse needs 50 μg of ultrapure plasmid for immunization.

[0099] Immediately after the injection, the injection site was electrically stimulated. The parameters of the WJ-2002 in vivo gene introduction instrument were: 100V, 50ms, after 6 pulses, reverse the polarity...

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Abstract

The invention provides a replicon vector taking Japanese encephalitis virus genome as a framework, as well as a cell line for packaging the same and a packaging system. The JEV replicon vector has the capability of efficiently expressing foreign protein. Mice are immunized with the replicon vector, and the titer of an anti-JEV antibody reaches 1:1280 after three immunizations so as to protect 75 percent of suckling mice from virus attack. The cell line provided can produce 1.6*105 U / ml of pseudovirus particles after packaging JEV replicon, and the titer of the anti-JEV antibody reaches 1:2560 after two immunizations so as to protect 73 percent of suckling mice from virus attack. The invention establishes a technical platform for a JEV replicon vector system for the first time, and explores the feasibility of researching replicon vaccines and pseudovirus vaccines through the JEV replicon vector system, thereby laying a solid foundation for developing and researching a plurality of novel vaccines used to prevent and treat tumors and viral diseases in future.

Description

technical field [0001] The invention relates to a replicon carrier with Japanese encephalitis virus genome as the backbone, and a cell line and packaging system used for packaging the replicon carrier. More specifically, the present invention relates to a JEV expression system that can encode exogenous immunogenic proteins and polypeptides that can be used as a vaccine delivery system. Vaccines can be administered in the form of DNA or virus-like particles, and the carrier system and its derivative products can be used for gene therapy and gene prevention research. Background technique [0002] JEV belongs to the genus Flaviviridae and is transmitted by mosquitoes. It is an important human pathogen and can cause permanent neurological diseases and even fatal diseases. There are more than 50,000 cases each year, the fatality rate exceeds 25%, and more than half of the survivors will have lasting neurologic sequelae. JEV is mainly distributed in Asia, from the Soviet Union t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/85C12N5/10C12N7/01A61K39/12A61K48/00A61P31/14C12R1/91C12R1/93
CPCY02A50/30
Inventor 黄莺刘珊杨鹏孙志伟俞炜源
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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