Low virulent strain of recombinant newcastle disease lasota vaccine expressing HA protein of avian influenza-H5 virus

A technology of avian influenza virus and attenuated vaccine, which is applied to a method and the recombinant Newcastle disease LaSota attenuated vaccine strain in the field of preparation and prevention of poultry, HA and rLa, which can solve the problems of inconvenient use, high manufacturing cost, and unrealized wide application.

Active Publication Date: 2011-06-15
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

From the end of 2003 to the beginning of 2004, Asian countries such as South Korea, Japan, Vietnam, Thailand, Indonesia, Cambodia, Laos, and mainland China have successively broken out H5 subtype highly pathogenic avian influenza. Currently, inactivated avian influenza vaccines and live fowl pox vectors are available. Vaccines have the advantages of safety and good immune protection effect, but they still have the disadvantages of high manufacturing cost and relatively inconvenient use
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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  • Low virulent strain of recombinant newcastle disease lasota vaccine expressing HA protein of avian influenza-H5 virus
  • Low virulent strain of recombinant newcastle disease lasota vaccine expressing HA protein of avian influenza-H5 virus
  • Low virulent strain of recombinant newcastle disease lasota vaccine expressing HA protein of avian influenza-H5 virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of recombinant Newcastle disease LaSota attenuated vaccine strain expressing wild-type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein

[0037] Cells, viruses and test materials

[0038] 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 / Goose / Guangdong / 96 / 1 / H5N...

Embodiment 2

[0047] Embodiment 2 Recombinant NDV expresses AVI HA protein indirect immunofluorescence assay (IFA) test

[0048] The NDV LaSota vaccine strain can transiently infect mammalian cells cultured in vitro. In order to prove the replication of rLasota-H5wtHA and rLasota-H5mutHA 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 load ( 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...

Embodiment 3

[0051] Example 3 Western-Blot identification of recombinant NDV expressing H5 subtype avian influenza virus HA protein

[0052] Take virus-infected chicken embryo fibroblast (CEF) lysate (after discarding the culture medium, add 1 / 10 volume of PBS, suspend the cells, add an equal volume of 2×SDS lysis buffer to lyse in boiling water for 10 min, centrifuge at 12000 g for 10 min , harvest the supernatant) or virus-inoculated SPF chicken embryo allantoic stock solution, carry out SDS-PAGE (Bio-Rad). Protein electrotransfer (Bio-Rad) to nylon membrane (Ameresco), 10% skimmed milk blocked overnight, PBST (0.05% Tween20) was added after washing, 1:50 diluted DNA was added for immunization to prepare chicken anti-H5 subtype avian influenza virus HA Antigen hyperimmune serum was the primary antibody, horseradish peroxidase (HRP)-labeled rabbit anti-chicken goat anti-mouse IgG (Sigma) was the secondary antibody, diluted 1:2500 times in PBST, DAB (diaminobenzidine, Sigma) After 3-5 min...

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Abstract

Low virulent strain of recombinant newcastle disease LaSota vaccine expressing wild or mutant hemagglutinin(HA)protein of avian influenza-H5 virus is provided. Particularly, Low virulent strains of recombinant newcastle disease LaSota vaccine are rLasota-H5wtHA and rLasota-H5mutHA. The present invention also provides method for preparing said low virulent strain, and the use of such 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 Specifically, the recombinant Newcastle disease LaSota attenuated vaccine strains are rLasota-H5wtHA and rLasota-H5mutHA. 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 l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/09C12N15/63C12N15/33C12N5/06A61K39/145A61P31/16C12N5/10
Inventor 步志高陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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