Reverse genetic operation system of New castle disease LaSota vaccine strain and its applciation

An operating system and reverse genetic technology, applied in the field of reverse genetic operating system of Newcastle disease LaSota vaccine strain, can solve the problems of inability to prevent virus replication and emission, poor replication ability, etc.

Active Publication Date: 2006-05-17
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

But because B1 itself is highly attenuated, its replication ability in immunized chickens is poor, so the ability to induce immune chickens to form effective immune protection is also relatively weak. Tests have shown that the NDV B1 strain expressing the H7 subtype HA gene is resistant to NDV and The survival protection of H7 subtype highly pathogenic avian influenza lethal challenge is only 60% and 40%, respectively, and cannot prevent virus replication and shedding in the body (12)

Method used

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  • Reverse genetic operation system of New castle disease LaSota vaccine strain and its applciation
  • Reverse genetic operation system of New castle disease LaSota vaccine strain and its applciation
  • Reverse genetic operation system of New castle disease LaSota vaccine strain and its applciation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 The construction of the reverse genetics operating system of Newcastle disease LaSota attenuated vaccine strain

[0032] cells and viruses

[0033] 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 / m1 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.

[0034] Construction of transcription vector

[0035] The genomic RNA transcription vector pBTRT is based on the low-copy cloning vector pBR322 (Invitrogen) ...

Embodiment 2

[0042] Example 2 Indirect Immunofluorescence Assay (IFA) Test

[0043] NDV LaSota vaccine strain can transiently infect weak animal cells cultured in vitro. In order to prove the replication of rLaSota-GFP virus in BHK-21 cells and the expression of virus antigens, rLaSota-GFP allantoic virus infected about 70-80% of monolayer BHK-21 cells at an 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:

[0044] Chicken embryos were inoculated with allantoic virus liquid rLaSota-GFP diluted with DMEM at an appropriate multiple,...

Embodiment 3

[0045] Example 3 Expression of GFP protein

[0046] Allantoxin rLaSota-GFP infected about 80% of monolayer BHK-21 cells at an MOI of 1. Early CPE appeared in the cells 24 hours after infection, and the results were observed under a fluorescent microscope (Leica DMIRES2) after the lesions appeared. Green fluorescence appeared in positive samples, wild-type NDV was used as negative control, and the result was negative (Fig. 4).

[0047] In order to prove that the replication of rLaSota-EGFP virus in BHK-21 cells and the insertion of exogenous reporter gene GFP can still maintain stable expression during the passage process, rLaSota-EGFP chicken embryos were inoculated into the allantoic cavity for 9 consecutive passages, and each generation of chicken embryos The allantoic virus liquid was serially diluted 10 times and serially diluted in 100 ul volume to inoculate 24-well plates to culture chicken embryo primary cells (CEF). After 24 hours, the results were directly observed wi...

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Abstract

The present invention is reverse genetic operation system of Newcastle disease Lasota low virulent vaccine strain and its application. The system includes one transcription plasmid including the genome cDNA sequence of the low virulent vaccine strain; one or several transcription aiding plasmids including the cDNA sequence coding the nucleoprotein of the low virulent vaccine strain, the cDNA sequence coding the phosphoprotein of the low virulent vaccine strain and the cDNA sequence coding the large polymerase protein of the low virulent vaccine strain; and the host cell of the Newcastle disease Lasota low virulent vaccine strain. Wild viral strain is obtained by means of the reverse genetic operation system. The present invention lays firm foundation for further development of Newcastle disease virus live carrier vaccine and Newcastle disease virus related research.

Description

technical field [0001] The invention relates to the field of virus genetic manipulation, more specifically, the invention relates to a reverse genetic operating system of Newcastle disease LaSota vaccine strain and its application. 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 reversion between strains is extremely small; the replication process is completed in the cytoplasm, from RNA to RNA, there is no possibility of DNA stage and cell genom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N7/01C12N15/85C12N15/867
Inventor 步志高陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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