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Recombination newcastle disease LaSota weak virus vaccine for expressing poultry influenza virus H5 sub type HA protein

An avian influenza virus, attenuated vaccine technology, applied in the direction of virus, antiviral agent, virus antigen components, etc., can solve the problem of not achieving widespread application

Active Publication Date: 2009-05-13
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the shortcomings and defects of the above-mentioned expression system, the live virus vector itself, and the cost of use, it has not been widely used in actual production.

Method used

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  • Recombination newcastle disease LaSota weak virus vaccine for expressing poultry influenza virus H5 sub type HA protein
  • Recombination newcastle disease LaSota weak virus vaccine for expressing poultry influenza virus H5 sub type HA protein
  • Recombination newcastle disease LaSota weak virus vaccine for expressing poultry influenza virus H5 sub type HA protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of recombinant Newcastle disease LaSota attenuated vaccine strain expressing wild-type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein

[0043] Cells, viruses and test materials

[0044] BHK-21 cells (milk hamster kidney cells ATCC CCL-10), culture medium is DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (Hyclone) and 1 μg / ml G418; NDV Lasota vaccine Strain AV1615 (purchased from China Center for Veterinary Culture Collection (CVCC)). The allantoic cavity of 9-10-day-old SPF chicken embryos was inoculated and frozen at -70°C for later use; chicken anti-NDV hyperimmune serum was prepared by our laboratory (Chu, H.P., G.Snell, D.J.Alexander, and G.C.Schild.1982 .Avian Pathol 11:227-234); SPF chicken embryos and SPF chicks were provided by the SPF Experimental Animal Center of Harbin Veterinary Research Institute. H5 subtype highly pathogenic avian influenza virus (HPAIV) A / Bar-headed goose / Qinghai...

Embodiment 2

[0053] Example 2 Recombinant NDV expresses AVI HA protein indirect immunofluorescence assay (IFA) test

[0054] The NDV LaSota vaccine strain can transiently infect mammalian cells cultured in vitro. In order to prove the replication of rL-OHwH5 and rL-QHmH5 viruses in BHK-21 cells and the expression of virus antigens, the two allantoxins infected about 70-80% of monolayer BHK-21 cells with MOI as 1 virus amount ( image 3 A and B), while using the NDV wild-type LaSota vaccine strain infected cells as a control ( image 3 C), 20 hours after the infection, the early CPE (cytopathic) phenomenon appeared in the cells, and immediately carried out indirect immunofluorescence staining with the positive serum of NDV high-immune SPF chickens as the detection antibody. As a result, strong positive reactions were observed under the fluorescence microscope of three kinds of virus-infected cells ( image 3 A, B and C) More specifically, the experimental steps are as follows:

[0055] Ch...

Embodiment 3

[0057] Example 3 Growth characteristics and pathogenicity of rNDV in chicken embryos

[0058] In order to determine the growth characteristics of rL-QHwH5 and rL-QHmH5 chicken embryos rescued by reverse genetic manipulation and their pathogenicity to chicken embryos, the rescued virus chicken embryos were amplified by 1×10 4 EID 50Inoculate the allantoic cavity of 9-10-day-old SPF chicken embryos. The wild-type Newcastle disease virus LaSota vaccine strain (rLaSota that reverse genetic operation rescued of result uses transcription vector pBTRT and transcription auxiliary plasmid pBSNP, pBSP and pBSL promptly, the wild-type virus rescued by reverse genetic operation as described in the present invention Newcastle disease virus LaSota vaccine strain AV161) completely non-lethal SPF chicken embryos within 120 hours, allantoic fluid was harvested 24 hours, 48 ​​hours, 72 hours and 96 hours after inoculation, EID per milliliter of allantoic fluid 50 10 -8.5 、10 -86 、10 -100 a...

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Abstract

Recombinant Newcastle disease LaSota low virulent vaccine strains expressing wild or mutant avian influenza virus H5 subtype HA protein. Especially, the recombinant Newcastle disease LaSota low virulent vaccine strains are rL-QHwH5 and rL-QHmH5. The preparation process of the recombinant Newcastle disease LaSota low virulent vaccine strain and the application of the recombinant Newcastle disease LaSota low virulent vaccine strain in preparing vaccine for preventing avian influenza.

Description

technical field [0001] The present invention relates to the field of recombinant virus vaccines, more specifically, the present invention relates to a recombinant Newcastle disease LaSota attenuated vaccine strain expressing a gene encoding wild-type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein, more specifically The recombinant Newcastle disease LaSota attenuated vaccine strains are rL-QHwH5 and rL-QHmH5. The invention also discloses a method for preparing the recombinant Newcastle disease LaSota attenuated vaccine strain and the application of the recombinant Newcastle disease LaSota attenuated vaccine strain in preparing vaccines for preventing bird flu. Background technique [0002] Newcastle disease virus (NDV) is a non-segmented single-stranded negative-sense RNA virus. As an important member and model virus of the Paramyxoviridae family, it has been deeply studied. Recombinant NDV has extraordinary advantages as a live virus vaccine carrier: ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/33C12N15/45C12N7/01A61K39/17C12N15/63
CPCC12N2760/18143A61K39/145A61K2039/5256C12N2760/16134C12N15/86A61K39/12A61P31/14
Inventor 步志高陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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