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Recombination VSV virus carrier and recombination VSV virus and its preparation method and use

A virus vector and virus genome technology, applied in the direction of virus/bacteriophage, biochemical equipment and methods, virus antigen components, etc., can solve problems such as safety doubts

Inactive Publication Date: 2005-03-09
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because DNA viruses may be integrated into the host cell genome, although some DNA virus vector vaccines can provide certain immune protection, their safety is questioned when applied

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 constructs VSV vector and helper plasmid

[0049] 1. The VSV Indiana strain comes from the School of Life Sciences, Peking University

[0050] 2. The construction of the recombinant VSV vector refers to the method described in PNAS (1995) Vol.92p4477-81. In order to facilitate genetic manipulation, NheI site was introduced into the 3' untranslated region of G protein through site-directed mutagenesis, and a Linker and XhoI site with minimal transcription initiation and termination units were inserted between G and L:

[0051] GCTAGG TATGAAAAAAA CT AACAG AT ATCACG CTCGAG AATTAATT GCTAG

[0052] Stop / poly(A) Transcription initiation XhoI NheI

[0053] In a similar way, MluI was introduced into the upstream untranslated region of the G protein to facilitate the replacement of the G protein of different strains in the future. This plasmid is called pVSV1, and its basic structure is:

[0054] T7 promoter-N-P-M-G-L-HDV ribozyme sequence-T7 t...

Embodiment 2

[0056] Example 2 transfection and recovery of recombinant VSV virus

[0057] BHK-21 cells in a 1.10cm dish grew to 70% full, and were infected with vTF7-3 at an MOI of 10 for 1 hour. vTF7-3 was derived from PNAS (1986) Vol.83p8122-26;

[0058] 2. After 1 hour, the liposome method was used to co-transfect the four plasmids pVSV1, pSK-N, pSK-P, and pSK-L, and the ratio of the four plasmids was 10 μg: 3 μg: 5 μg: 2 μg;

[0059] 3. After 48 hours of culture at 37 degrees, the cells were repeatedly frozen and thawed three times (-70 degrees → 37 degrees), the lysate was collected, and the cell debris was removed by centrifugation at 12,000 rpm for 5 minutes;

[0060] 4. Infect fresh BHK cells with half of the lysate, and at the same time add 25 μg / μl AraC to inhibit poxvirus;

[0061] 5. After 48 hours, the supernatant was collected, centrifuged at 12,000 rpm for 10 minutes, and filtered with a 0.22 μm filter (Millipore) to completely remove the poxvirus to obtain the recombinant VS...

Embodiment 3

[0063] The amplification of embodiment 3 recombinant VSV virus

[0064] 1. Determination of virus titer by plaque assay;

[0065] 2. Infect fresh BHK cells with the viral supernatant at an MOI of 0.1, collect the supernatant after 16-24 hours, and store at -70°C.

[0066] 4

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PUM

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Abstract

A recombinant VSV carrier including VSV genom and carried exogenous gene is created. The recombinant VSV particles with duplicating function and infectivity and able to reproduce and express said exogenous gene in the infected host cell is prepared. Said carrier and virus can be used to infect the experimental animal for obtaining the antibody of exogenous gene product, which can be used to develop relative vaccine.

Description

technical field [0001] The invention relates to a recombinant VSV (vesicular stomatitis virus) virus vector, a recombinant VSV virus, and a preparation method of the virus vector and the recombinant virus. The invention also relates to the use of the recombinant VSV virus vector in the expression of foreign protein, the preparation of antibody and the development of vaccine. Background technique [0002] Vaccines are highly effective against diseases caused by viruses, bacteria, parasites, etc. The traditional way to prepare vaccines is to use inactivated or attenuated pathogens, but the process of inactivation and attenuation often destroys the immunogenicity of pathogens, affects the immune effect, and is difficult to guarantee safety. Although subunit vaccines and DNA vaccines are relatively safe, they have low immunogenicity and poor immune effects. [0003] Recombinant viral vectors have become a hot spot in current vaccine research due to their high expression effici...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61K39/42C12N5/10C12N7/01C12N15/11C12N15/86
Inventor 邓宏魁丁明孝聂玉春袁菲王在郭延平胡建军李锦全易凌
Owner PEKING UNIV
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