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Recombinant H5N1 (Hemagglutinin 5 Neuraminidase 1) avian influenza virus cell vaccine and application thereof

An avian influenza virus and cell vaccine technology, applied in the field of animal infectious diseases, can solve problems such as shortage of chicken embryos, shortage of vaccine resources, etc., and achieve the effect of good safety

Active Publication Date: 2012-07-04
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it has safety problems for humans; at the same time, if bird flu breaks out and there is a shortage of chicken embryos, the production of this vaccine will face a shortage of resources

Method used

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  • Recombinant H5N1 (Hemagglutinin 5 Neuraminidase 1) avian influenza virus cell vaccine and application thereof
  • Recombinant H5N1 (Hemagglutinin 5 Neuraminidase 1) avian influenza virus cell vaccine and application thereof
  • Recombinant H5N1 (Hemagglutinin 5 Neuraminidase 1) avian influenza virus cell vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Cloning of avian influenza virus-related genes

[0021] 1. Extraction of avian influenza virus RNA

[0022] Refer to OMEGA Bioteck RNA-SOLV The instructions of the Reagent RNA Isolation Solvent kit were used to extract RNA from avian influenza virus. The specific method is as follows: take 300 μL of fresh allantoic fluid of H9N2 subtype avian influenza virus strain A / duck / Hubei / W1 / 2004 (Xu, X. et al., 2008), and add it to diethylpyrocarbonate containing 1000 μL RNA-Solv In a 1.5mL EP tube treated with DEPC, mix it upside down, let it stand at room temperature for 8min, add 200μL of chloroform, ice bath for 10min, centrifuge at 12000r / min at 4℃ for 15min, take 500-600μL of the supernatant and transfer it to another EP tube. Add an equal volume of isopropanol, mix well, place at room temperature for 10min, centrifuge at room temperature 12000r / min for 10min, discard the supernatant, add 1mL 80% ethanol, vortex mix, centrifuge at room temperature 7000r / m...

Embodiment 2

[0088] Example 2: Rescue and biological characteristics of recombinant H5N1 avian influenza virus

[0089] 1. Extraction of endotoxin-free plasmid

[0090] Extract according to the steps of the kit Endo-free Plasmid Mini Kit II provided by OMEGA (operate according to the instructions of the kit), the specific steps are as follows:

[0091] (1) Escherichia coli carrying the target plasmids (PHW-PB2, PHW-PB1, PHW-PA, PHW-NP, PHW-M, PHW-NS, PHW-ΔHA and PHW-NA) were inoculated into 5ml LB / Ampicillin 10-20ml culture tube, cultured on a shaker at 37°C for 12-16h to amplify the plasmid. Use a 10-20ml culture tube or flask with at least 4 times the volume of the culture medium.

[0092] (2) Take 5.0ml of the bacterial solution and centrifuge at 10,000xg for 1min at room temperature to precipitate the bacterial species.

[0093] (3) Pour out or suck out the above-mentioned LB medium and discard it. Add 250 μl Solution I / RNaseA mixed solution (included in the above kit Endo-free P...

Embodiment 3

[0119] Embodiment 3: Preparation and application of recombinant H5N1 avian influenza virus cell vaccine

[0120] 1. Proliferation of recombinant H5N1 avian influenza virus rC4 / W1 strain on MDCK cells

[0121] The recombinant H5N1 avian influenza virus rC4 / W1 strain was used in 10 2 TCID 50 On the MDCK cells (purchased from the Chinese Type Collection Center in Wuhan University, Wuhan City, Hubei Province) infected with the dose of 100 mg, washed twice with DMEM medium (purchased from GIBCO company) after incubation for 1 hour, and added with 2ml containing Subculture in DMEM containing 2.5 μg / ml TPCK-trypsin (purchased from Shanghai Sangon Bioengineering Co., Ltd.), at 37°C, 5% CO 2 Incubator for 72 hours. Passage until the hemagglutination value reaches 1:1028.

[0122] 2. Inactivation and emulsification of recombinant H5N1 avian influenza virus rC4 / W1 strain

[0123] Add 0.8% formaldehyde solution to the recombinant H5N1 avian influenza virus rC4 / W1 obtained in step 1, ...

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Abstract

The invention relates to the technical field of animal infectious diseases, in particular to a recombinant H5N1 (Hemagglutinin 5 Neuraminidase 1) avian influenza virus cell vaccine and an application thereof. The recombinant H5N1 avian influenza virus cell vaccine comprises a recombination H5N1 avian influenza virus rC4 / W1 strain which is collected in the China Center for Type Culture Collection with the collection number of CCTCC NO:V201029; and a genome of the recombinant H5N1 avian influenza virus rC4 / W1 strain contains fragments shown as SEQ ID NO:1-8 of eight relevant genes of an avian influenza virus. The recombinant H5N1 avian influenza virus is saved by using a reverse genetics method, and the recombination virus is passaged to stable on an MDCK (Madin Darby Canine Kidney) cell and is taken as an oil emulsion inactivated vaccine after being inactivated. H5N1 subtype avian influenza virus HA (Hemagglutinin) and NA (Neuraminidase) genes are replaced on the background of an H9N2 avian influenza virus, and all gene fragments in a recombinant virus come from the avian influenza virus, so that higher safety is achieved. The recombinant H5N1 avian influenza virus can be subcultured on the MDCK cell to obtain a strain with high proliferation titer, so that the problem of the lack of a large quantity of chicken embryos during explosion of avian influenza is solved.

Description

technical field [0001] The invention relates to the technical field of animal infectious diseases. In particular, it relates to a recombinant H5N1 avian influenza virus cell vaccine and its application. Background technique [0002] Avian Influenza (AI) is a severe infectious disease of poultry caused by type A influenza virus. Since it was first discovered in Italy in 1878, there have been outbreaks and epidemics of bird flu caused by specific strains all over the world. Highly pathogenic avian influenza (Highly Pathogenic Avian Influenza, HPAI) is distributed worldwide and is extremely harmful. Its outbreaks are often devastating. List of infectious zoonotic diseases of the Arms Convention. In particular, the H5N1 subtype avian influenza virus that directly infected and killed humans in Hong Kong in 1997 and Southeast Asia in 2003-2004 aroused the shock and concern of the world. Since my country first reported the isolation of the H5N1 subtype highly pathogenic avian i...

Claims

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Application Information

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IPC IPC(8): A61K39/145A61K48/00A61P31/16C12N7/01C12R1/93
Inventor 金梅林涂加钢周红波徐晓娟陈焕春
Owner HUAZHONG AGRI UNIV
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