Recombinant Newcastle disease LaSota low virulent vaccine strain expressing infectious bursal disease virus VP2 gene

An attenuated vaccine, Newcastle disease technology, applied in vaccines, viruses, viral peptides, etc., can solve problems such as not being widely used

Inactive Publication Date: 2006-05-17
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the shortcomings and defects of the above-mentioned expression system, the li

Method used

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  • Recombinant Newcastle disease LaSota low virulent vaccine strain expressing infectious bursal disease virus VP2 gene
  • Recombinant Newcastle disease LaSota low virulent vaccine strain expressing infectious bursal disease virus VP2 gene
  • Recombinant Newcastle disease LaSota low virulent vaccine strain expressing infectious bursal disease virus VP2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of recombinant Newcastle disease LaSota attenuated vaccine strain expressing the VP2 gene of infectious bursal virus (IBDV)

[0029] cells and viruses

[0030] 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 chicken embryos inoculated with SPF was expanded 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. The IBDV supervirulent strain vvIBDV GX was purchased from the Harbin Veterinary Research Institute of the Chinese Acade...

Embodiment 2

[0039] Example 2 Recombinant NDV expresses IBDV VP2 protein indirect immunofluorescence assay (IFA) test

[0040] NDV LaSota vaccine strain can transiently infect weak animal 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 allantoic virus infected about 70-80% of monolayer BHK-21 cells at a MOI of 1, and 24 hours after infection Early CPE (cytopathic) phenomenon appeared in the cells, and indirect immunofluorescence staining was performed immediately with the positive serum of NDV high-immunity SPF chicken as the detection antibody. As a result, a strong positive reaction was observed under the fluorescence microscope of virus-infected cells (Figure 3A), while the control serum of SPF chicken Fluorescent staining was negative (Figure 3B). More specifically, the experimental steps are as follows:

[0041] Chicken embryos were inoculated with allantoic virus liquid rLasota-VP2 and ...

Embodiment 3

[0042] Example 3 Western-Blot identification of recombinant NDV expressing IBDV VP2 protein

[0043] 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 Clearances were subjected to SDS-PAGE (Bio-Rad). Afterwards, the protein was electrotransferred (Bio-Rad) onto a nylon membrane (Ameresco), blocked overnight with 10% skim milk, washed with PBST (0.05% Tween20) and then added with a 1:50 dilution of the primary antibody (chicken anti-IBDV or chicken anti-NDV) . 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 shown in Figure 5.

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Abstract

The present invention relates to one kind of recombinant Newcastle disease Lasota low virulent vaccine strain expressing infectious bursal disease virus (IBDV) VP2 gene, and is especially recombinant Newcastle disease Lasota low virulent vaccine strain rLasota-VP2. The present invention also discloses 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 IBDV caused diseases.

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): C12N7/01A61K39/17A61P31/14C12N15/33C12N15/63
CPCA61K31/12A61K31/14A61K39/12A61K39/17A61K2039/522A61K2039/523A61K2039/5254A61K2039/5256A61K2039/552A61P31/14C07K14/005C12N15/86C12N2720/10022C12N2720/10034C12N2760/18134C12N2760/18143C12N2760/18161
Inventor 步志高王笑梅陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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