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Reformation of high-pathogenicity avian influenza virus H5N1-HA gene and recombinant adeno-associated virus vaccine thereof

A technology of avian influenza virus and recombinant adenovirus, applied in the field of biomedicine, can solve the problems of increasing virus threat and high lethality of virus

Active Publication Date: 2010-09-08
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the total number of infected people has been relatively low in the past few years, it cannot be ignored that the lethality of the virus is still greater than 50%
In addition, although the limited human-to-human transmission of the virus within the family has been found in only a few countries, the rapid genetic evolution and virulence of the avian influenza virus H5N1 in recent years have changed from time to time, and in wild birds and poultry Continued transmission, suggesting that the threat of the virus to people's health is increasing

Method used

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  • Reformation of high-pathogenicity avian influenza virus H5N1-HA gene and recombinant adeno-associated virus vaccine thereof
  • Reformation of high-pathogenicity avian influenza virus H5N1-HA gene and recombinant adeno-associated virus vaccine thereof
  • Reformation of high-pathogenicity avian influenza virus H5N1-HA gene and recombinant adeno-associated virus vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Optimization of HA gene

[0031] 1.1 The optimization of HA gene refers to the principle of human dominant codon usage ( http: / / www.kazusa.or.jp / codon / ), without changing the amino acid sequence, for H5N1 (A / Anhui / 1 / 2005) (see, Shu YL, Yu HJ, Li DX. Lethal avian influenza A(H5N1) infection in a pregnant woman in Anhui Province, China .N Eng1 J Med 2006; 354(13): 1421-1422.) The nucleotide sequence (1704bp) of the full-length coding region of the HA gene (GI: 87137936) was codon optimized, and then the designed sequence was checked by software: amino acid The sequence alignment was consistent, and it was confirmed that the sequence did not contain restriction sites (EcoRI and BamHI) required for cloning. The final sequence was entrusted to Beijing Lijia Fucheng Biotechnology Co., Ltd. for synthesis. After being verified by TaKaRa company sequencing, the optimized HA gene sequence was finally obtained, named Mod.HA, and its nucleotide sequence is shown in SE...

Embodiment 2

[0037] Example 2 Obtaining of recombinant AdV virus containing Mod.HA gene

[0038] Construction of recombinant AdV virus containing optimized HA gene.

[0039] 2.1 Packaging of recombinant AdV virus containing Mod.HA gene

[0040] Divide 293 cells into 4×10 5 Seed cells per well into a six-well plate, each well containing 2ml of cell suspension, at 37°C in CO 2 Cultivate overnight in the incubator to make the density reach more than 70%-90%. According to the instructions of FuGENE HD (purchased from Roche), each 2 μg of the plasmid pDC315-Mod.HA prepared in 1.2 above and the backbone plasmid pBHGloxΔE1, 3Cre (purchased from Microbix) and 12 μl of transfection reagent were co-transfected into each well of cells . Cells were harvested 7-10 days after transfection. Freezing and thawing were repeated three times at -80°C and 37°C. The supernatant was collected after centrifugation. Take a small amount of supernatant and re-infect 293 cells, observe the lesions in the cells...

Embodiment 3

[0044] Example 3 Experiment of Immunizing Mice with Recombinant Virus rAdV-Mod.HA

[0045] 3.1 Immunization and grouping of mice

[0046] BALB / c mice were randomly divided into 2 groups, 8 mice in each group, and were immunized twice on day 0 and day 28 by intramuscular injection. The immune response was detected on day 35. See Table 1 for specific immunization contents and doses.

[0047] Table 1: Grouping of mice and immunization dose

[0048]

[0049] 3.2 Detection of humoral immune response to H5N1HA in immunized mice

[0050] The venous blood of the mice was collected on the 35th day after primary immunization, and the serum was separated. Refer to the detection of hemagglutination inhibition in the diagnostic criteria for human infection with highly pathogenic avian influenza (WS284-2008), take 10 μl of each serum, add 4 times the volume (40 μl) of receptor destroying enzyme (RDE) and put it in a water bath at 37°C for 18 hours , and then placed in a 56°C water b...

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Abstract

The invention belongs to the technical field of biomedicine and provides a reformed and optimized high-pathogenicity avian influenza virus HA gene and a recombinant adeno-associated virus vaccine containing same. The reformed and optimized HA gene not only obviously provides an expression level, but also reduces toxicity. Experiments prove that the recombinant adeno-associated virus vaccine constructed by the reformed and optimized high-pathogenicity avian influenza virus HA gene can simultaneously induce and generate strong body fluid and cell immune responses and can effectively prevent and treat high-pathogenicity avian influenza virus infections.

Description

technical field [0001] The invention belongs to the field of biomedicine. The present invention relates to a recombinant vaccine of avian influenza virus, specifically, the present invention provides a modified and optimized H5N1-HA gene, and a recombinant vaccine of avian influenza virus H5N1 constructed on this basis, that is, a recombinant adenovirus containing the above two genes and recombinant adeno-associated virus vaccines. Background of the invention [0002] Avian influenza virus (AIV) belongs to Orthomyxoviridae, Influenzavirus genus, and Influenza A virus. The natural host is poultry, and there is a species barrier to humans. In the first human infection with avian influenza virus H5N1 reported in Hong Kong in 1997, 6 of 18 infected persons died. Afterwards, the number of people infected and the scope of transmission continued to expand. As of September 24, 2009, WHO confirmed a total of 442 cases of human infection with H5N1 in 15 countries, including 262 dea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/145C12N15/44A61P31/16
Inventor 曾毅马晶张晓光舒跃龙
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT