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Avian influenza virus marking vaccine, preparation process and application thereof

A kind of technology of avian influenza virus and avian influenza, applied in H5N1 subtype avian influenza virus labeled vaccine strain H5N1/PR8-5B19, the field of preparing said H5N1 subtype avian influenza virus labeled vaccine

Inactive Publication Date: 2007-05-30
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, the marked vaccine of this strategy can only be used in the case of a single virus epidemic background. If the virus epidemic situation in an area is complicated and various subtypes of avian influenza viruses coexist, it is difficult to select a suitable NA as a natural marker. Therefore, a better strategy is to molecularly modify the influenza virus gene fragments, add exogenous tags, and distinguish vaccine immunity from poultry naturally infected with avian influenza by tag detection

Method used

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  • Avian influenza virus marking vaccine, preparation process and application thereof
  • Avian influenza virus marking vaccine, preparation process and application thereof
  • Avian influenza virus marking vaccine, preparation process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Production of Molecularly Attenuated H5N1 Subtype Avian Influenza Virus Marker Vaccine Strain H5N1 / PR8-5B19 1 Materials and Methods

[0070] 1.1 Virus strains

[0071] A / Goose / Guangdong / 1 / 96(H5N1)(GS / GD / 1 / 96)(purchased from Harbin Veterinary Research Institute)

[0072] 1.2 SPF Chicken and SPF Chicken Embryo

[0073] 9-11-day-old SPF chicken embryos and 4-8-week-old SPF chickens were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute. All SPF chicken trials were performed in negative pressure isolators.

[0074] 1.3 Plasmid vector and transfection plasmid

[0075] pBD vector (see Figure 1 for map) (Zejun Li, Hualan Chen, Peirong Jiao, Guohua Deng, GuobinTian, ​​Yanbing Li, Erich Hoffmann, Robert G.Webster, Yumiko Matsuoka, and KangzhenYu. Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian MouseModel.J.Virol.79:12058-12064.) was constructed by Dr. Chen Hualan, and the unidirectional transcription ...

Embodiment 2

[0119] The biological characteristic of embodiment 2 H5N1 / PR8-5B19 vaccine strain

[0120] 1. Materials and Methods

[0121] 1.1 Pathogenicity of H5N1 / PR8-5B19 vaccine strain to chickens

[0122] In the intravenous inoculation pathogenicity index (IVPI) determination test, select 2 groups of 10 6-week-old SPF chickens, and each chicken was intravenously inoculated with 0.2ml 1:10 times diluted H5N1 / PR8-5B19 or H5N1 / PR8 virus allantoic fluid , observe the morbidity and mortality within 10 days after inoculation, and calculate the IVPI.

[0123] In the nasal infection test, 2 groups of 10 6-week-old SPF chickens were selected, and each chicken was inoculated with 0.1ml10 by nasal route. 6 EID 50 Allantoic fluid of H5N1 / PR8-5B19 or H5N1 / PR8 virus. Throat swabs and cloacal swabs were collected 1, 3, 5, 7, and 9 days after infection for virus isolation, and the incidence was observed. Twenty-one days after infection, serum was collected to determine hemagglutination inhibitory...

Embodiment 3

[0150] Example 3 Preliminary establishment of an ELISA detection method capable of distinguishing H5N1 / PR8-5B19 labeled vaccine immune serum from non-labeled vaccine immune serum or naturally infected chicken serum

[0151] 1. Materials and methods

[0152] 1.1 ELISA operating procedures

[0153] In order to be able to use serological methods to distinguish marked vaccine immunized chickens from non-marked vaccine immunized chickens or chickens naturally infected with H5N1 subtype avian influenza virus, an indirect polypeptide enzyme-linked immunosorbent assay method was established. The specific steps are as follows:

[0154] 1. Antigen coating: 16 amino acids of the murine hepatitis virus 5B19 epitope ("SPLLGCIGSTCAEDGN") were artificially synthesized as the antigen for coating. After the peptide is synthesized, it is dissolved in double distilled water, and the peptide is diluted to a certain concentration with a carbonate buffer solution of pH 9.2. Add 100 μl of polypept...

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Abstract

The invention discloses a vaccine strain H5N1 / PR8-5B19 marked by H5N1 hypotype poultry influenza virus, which also provides the application to prevent and monitor poultry influenza.

Description

technical field [0001] The present invention relates to the field of recombinant virus vaccines, specifically, the present invention relates to a H5N1 subtype avian influenza virus labeled vaccine in which the dominant B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein is inserted into the neck of the NA gene, more specifically , which is H5N1 subtype avian influenza virus marker vaccine strain H5N1 / PR8-5B19. The invention also discloses a method for preparing the H5N1 subtype avian influenza virus marker vaccine and the application of the marker vaccine in preparing vaccines for preventing avian influenza and in epidemiological monitoring of avian influenza. Background technique [0002] 1. Reverse Genetic Technology of Type A Influenza Virus [0003] In recent years, the molecular biology research of influenza virus has progressed rapidly, mainly in the fields of molecular epidemiology, influenza virus replication and regulation, influenza virus vector transfor...

Claims

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Application Information

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IPC IPC(8): A61K39/145C12N15/44A61P31/16
Inventor 陈化兰田国彬姜永萍步志高于康震
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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