Attenuated vaccinia virus Tiantan strain vector and its preparation and application

A vaccinia virus vector, vaccinia virus technology, applied in the direction of virus/bacteriophage, botany equipment and method, biochemical equipment and method, etc., can solve the problems of attenuation and non-existence

Inactive Publication Date: 2005-08-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese Journal of Veterinary Medicine 16 (6), 562-569), but there is no research and experimental proof on the toxicity of the recombinant virus to organisms and its safety as a potential vaccine
At the same time, there are no other experimental results or studies so far, showing that inactivation or deletion of HA can lead to attenuation of vaccinia virus Tiantan strain in vivo and in vitro

Method used

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  • Attenuated vaccinia virus Tiantan strain vector and its preparation and application
  • Attenuated vaccinia virus Tiantan strain vector and its preparation and application
  • Attenuated vaccinia virus Tiantan strain vector and its preparation and application

Examples

Experimental program
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Effect test

Embodiment 1

[0131] Replication kinetics and viral replication-related properties of attenuated vaccinia virus in animal cells.

[0132] 1) The replication kinetics of viruses in animal cells:

[0133] In order to understand the replication ability of attenuated vaccinia virus in animal cells, animal cells from different types and tissues, including human cell lines, were selected to conduct research experiments on virus replication kinetics. At the same time, the replication kinetics curve of the parental virus Tiantan strain in these cells was studied under the same conditions in order to be able to compare with the replication properties of the attenuated vaccinia virus.

[0134] The compared viruses were: significantly attenuated vaccinia virus pZCI and wild Tiantan vaccinia virus TT. The selected animal cells and their types, sources and cell types are listed in Table 1. Among them, the primary chicken embryonic cell CEF is a self-made cell. Although it is indicated as the primary c...

Embodiment 2

[0169] Genetic stability of foreign gene insertion and expression in significantly attenuated vaccinia virus

[0170]Safe and effective viral vectors must have stable biological properties, and the stability of foreign gene insertion and expression is particularly important. Significantly attenuated vaccinia virus (the late promoter region of HA gene has been inserted into the exogenous GFP gene) was used to infect monolayer Vero cells at 0.01 MOI, the medium was removed after 90 minutes of adsorption, and the cells were washed once with DMEM medium containing 10% NCS. Supplement fresh culture with 10% NCS based on 37°C, 5% CO 2 Cultured in an incubator for 48 hours, the virus-infected cells and supernatant were harvested, frozen and thawed three times, and the titer of the virus was determined in Vero cells. Vero cells were then infected at the same MOI. In this way, the attenuated vaccinia virus was continuously passaged in Vero cells for 6 times. The first-generation and...

Embodiment 3

[0173] Toxicity comparison of attenuated vaccinia virus vector and wild Tiantan virus in model animal mice

[0174] In order to identify the characteristics of the reduced virulence of attenuated vaccinia virus in vivo, the BALB / c mouse model system was used to measure the significantly attenuated vaccinia virus pZCI and its parent virus wild Tiantan strain TT through nasal cavity and cranial cavity respectively. Toxicity in mice. The BALB / c mouse system is an animal model system widely used in the world, and has been proven to be sensitive to vaccinia virus (Betakova et al., 2000, The vaccinia virus A14.5L gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebratepoxviruses. J Virol, 74(9), 4085-92; Lee, M.S.et al., Molecular attenuation of vaccinenia virus: mutant generation and animal characterization. J Virol, 66(5), 2617-30; Zhang.W.H.et al., Vaccinia virus F12L protein is required for actintail form...

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Abstract

The present invention discloses one attenuated vaccinia virus Tiantan plant carrier and its preparation process and application, and the attenuated vaccinia virus is obviously attenuated TT.WHU.pZCI, CCTCC: V200416. The preparation process includes the following steps: A. constituting shuttle plasmid containing flanking sequence homogenous with the late promoter sequence or two flanking sequences of the vaccinia virus Tiantan plant HA gene; and B. homogenously recombining the shuttle plasmid and the vaccinia virus Tiantan plant to deactivate vaccinia virus HA gene part for obviously attenuating the vaccinia virus Tiantan plant. The attenuated vaccinia virus Tiantan plant carrier is used in preparing vaccine for treating or preventing infectious diseases.

Description

technical field [0001] The present invention relates to a kind of attenuated vaccinia virus Tiantan strain carrier, they are derived from vaccinia virus Tiantan strain, they are characterized in that they lose or reduce the reproduction and replication ability in animal cells, and reduce the toxicity to cells; Significantly attenuated properties, especially the neurotoxicity characteristic of vaccinia virus, were significantly reduced. The invention also relates to the preparation method of the attenuated vaccinia virus Tiantan strain carrier. The significantly attenuated vaccinia virus vector of the present invention has higher safety than the wild vaccinia virus Tiantan strain, and can be used to construct live virus vector vaccines for humans or animals; the attenuated vaccinia virus vector can be further used to develop tumor gene therapy Or preventive drugs; or used as expression vectors of exogenous genes to produce target proteins on a large scale; or used to express e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/275A61P35/00C12N7/01C12N7/04C12N15/863G01N33/53
Inventor 陈志伟张林琦方清田波
Owner WUHAN UNIV
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