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Infectious bursal disease virus VP2 gene expressed recombinant newcastle disease LaSota attenuated vaccine strain

An attenuated vaccine, Newcastle disease technology, applied in genetic engineering, virus antigen components, plant genetic improvement, etc., can solve problems such as not achieving widespread application

Active Publication Date: 2006-12-20
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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  • Infectious bursal disease virus VP2 gene expressed recombinant newcastle disease LaSota attenuated vaccine strain
  • Infectious bursal disease virus VP2 gene expressed recombinant newcastle disease LaSota attenuated vaccine strain
  • Infectious bursal disease virus VP2 gene expressed recombinant newcastle disease LaSota attenuated vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Expression of the VP2 gene of infectious bursal virus (IBDV)

[0030] Construction of Recombinant Newcastle Disease LaSota Attenuated Vaccine Strain

[0031] cells and viruses

[0032] BHK-21 cells (milk hamster kidney cells ATCC CCL-10), the medium is DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (Hyclone); NDV Lasota vaccine strain AV1615 (purchased from China Center for Veterinary Culture Collection (CVCC). The allantoic cavity of chicken embryos inoculated with 9-10 days old SPF was amplified and the half infection dose (EID) of chicken embryos was titrated 50 ) 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 embryo and SPF chicks were provided by the SPF Experimental Animal Center of Harbin Veterinary Research Institute. The IBDV supervirulent strain vvIBDV GX w...

Embodiment 2

[0041] Example 2 Immunofluorescence Analysis of Antigen Protein Expressed by Recombinant Newcastle Disease Virus rLasota-VP2

[0042] The NDV LaSota vaccine strain can transiently infect mammalian cells cultured in vitro. In order to prove the replication of rLasota-VP2 virus in BHK-21 cells and the expression of virus antigen, rLasota-VP2 chicken embryos were subcultured F2 to infect BHK-21 cells (3A & 3C) and uninfected BHK-21 cells (3B & 3D), Chicken anti-Newcastle disease hyperimmune serum (3A & 3B) and chicken anti-IBDV hyperimmune serum (3C& 3D) were used as primary antibodies for indirect immunofluorescence detection. The results showed that a strong positive reaction was observed under the fluorescence microscope of virus-infected cells ( image 3 A & 3C), while the fluorescent staining of SPF chicken control serum was negative ( image 3 B & 3D).

Embodiment 3

[0043] Example 3 Western-Blot identification of recombinant NDV expressing IBDV VP2 protein

[0044] Take the amplified rLaSota-VP2 BHK-21 cells, discard the culture medium, add 1 / 10 volume of PBS, suspend the cells, add an equal volume of 2×SDS lysis buffer for boiling water lysis for 10 minutes, centrifuge at 12000g for 10 minutes, and take The supernatant was subjected to SDS-PAGE (Bio-Rad); the protein was electrotransferred (Bio-Rad) onto a nylon membrane (Ameresco), blocked overnight with 10% skim milk, washed with PBST (0.05% Tween20) and added 1:50 PBST diluted Primary antibody (chicken anti-IBDV hyperimmune serum). The secondary antibody was horseradish peroxidase (HRP)-labeled rabbit anti-chicken goat anti-mouse IgG (Sigma), diluted 1:2500 times in PBST. DAB (diaminobenzidine, Sigma) developed color for 3-5 minutes and then terminated the reaction with deionized water. The result is as Figure 5 , proving that the infectious bursal virus VP2 antigen is effectively...

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Abstract

The invention discloses a VP2 gene restructuring Newcastle disease LaSota weak virus strain rLasota-VP2 of Infectious bursal disease virus, IBDV and appliance in the Newcastal disease virus,NDV and IBDV vaccine.

Description

technical field [0001] The invention relates to the field of recombinant virus vaccines, more specifically, the invention relates to a recombinant Newcastle disease LaSota attenuated vaccine strain expressing infectious bursal virus VP2 gene. The invention also relates to a preparation method of the recombinant Newcastle disease LaSota attenuated vaccine strain and its application as a vaccine. 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: NDV attenuated vaccines including LaSota strains have been used for poultry epidemic prevention for a long time, and their safety and effectiveness have been fully proved; NDV is relatively stable genetically, with only one serotype, the virus The possibility of recombination and virulence re...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/33A61K39/12
Inventor 步志高王笑梅陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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