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Preparation method and applications of H5N1 subtype bird flu pseudovirion

A technology of avian influenza virus and pseudovirus, which is applied in the preparation and application of H5N1 subtype avian influenza pseudovirus, can solve the problems of high fatality rate and harsh experimental conditions, and achieve the effect of reducing risks and ensuring biological safety

Inactive Publication Date: 2011-09-21
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

[0009] It is known that the highly pathogenic avian influenza virus of the H5N1 subtype is highly contagious and has a high fatality rate. It is quite dangerous to conduct research directly with the highly pathogenic avian influenza virus of the H5N1 subtype and requires harsh experimental conditions , so it is very necessary and urgent to develop a pseudovirus infection model of H5N1 subtype highly pathogenic avian influenza virus

Method used

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  • Preparation method and applications of H5N1 subtype bird flu pseudovirion
  • Preparation method and applications of H5N1 subtype bird flu pseudovirion
  • Preparation method and applications of H5N1 subtype bird flu pseudovirion

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preparation example Construction

[0070] Therefore, the preparation method of the avian influenza pseudovirus of the present application may comprise:

[0071] (1) Provide HA gene and NA gene of highly pathogenic avian influenza virus;

[0072] (2) co-linking the HA gene and the NA gene into the eukaryotic cell expression vector;

[0073] (3) co-transfecting the host cell with the eukaryotic cell expression vector and packaging plasmid obtained in step (2) comprising the HA gene and the NA gene; and

[0074] (4) Cultivate the transfected host cells, and recover the pseudoviruses from the cell supernatant.

[0075] The HA gene and the NA gene can be respectively provided from a plasmid containing the HA gene and the NA gene of the pathogenic avian influenza virus, and then co-ligated into the eukaryotic cell expression vector.

[0076] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, known...

Embodiment 1

[0077] Embodiment 1: Cloning of H5N1 subtype highly pathogenic avian influenza virus HA and NA protein gene

[0078] 1.1 The DNA sequence of NA was amplified by PCR method

[0079] The PCR primer design of cloning NA gene, according to the pGEMT-NA gene [Influenza Avirus (A / Chicken / Henan / 12 / 2004 (H5N1))] sequence that has been sequenced, according to the primer design principle, use Primer premier5.0 software to design primers, Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences are:

[0080] Sp 5'-CCC AAGCTT ATGAATCCAAATCAGAAG-3' (SEQ ID NO: 1)

[0081] As 5'-GC TCTAGA CTACTTGTCAATGGTGAATGG-3' (SEQ ID NO: 2)

[0082] The X underlines in the primer sequences represent the restriction endonuclease HindIII and XbaI sites, respectively.

[0083] 1.2 The PCR product was connected to the pBudCE4.1 vector (purchased from Invitrogen)

[0084] After digestion with restriction endonucleases HindIII and Xba I, insert it into the pBudCE4.1 vector th...

Embodiment 2

[0095] Embodiment 2: the preparation of H5N1 subtype avian influenza pseudovirus, adopt liposome transfection method (purchased from Invitorgen lipofectamine2000)

[0096] 2.1 Plasmid pNL4-3Luc that will need to be transfected + Env - Vpr -("Conserved amino acids W423and N424 in receptor-binding domain of SARS-CoV are potential targets for therapeutic monoclonal antibody", Chao Bian et al., Virology 383(2009) 39-46; "Analysis of hemagglutinin-mediated entry tropism of H5N1 avian za", Ying Guo et al., Virology Journal 2009, 6:39; Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type-1 inMononuclear Phagocytes, Connor et al., Vigology 206, 935-944(1995)), pBudCE4.1-NA-HA, green Fluorescent protein expression plasmid pGFP (“Hemagglutinin pseudotypedlentiviral particles: Characterization of a new method for avian H5N1 influenza sero-diagnosis”, Isabelle Nefkens et al., Journal of Clinical Virology 39(2007) 27-33), vesicular stomatitis virus G protein exp...

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Abstract

The invention relates to a preparation method and applications of an H5N1 subtype bird flu pseudovirion. Specifically, the application relates to a bird flu pseudovirion with packaging plasmid and the HA gene and NA gene of the highly virulent form of the bird flu virus and a preparation method and applications thereof; and the application also relates to a system for preparing the pseudovirion.

Description

technical field [0001] The invention relates to a preparation method and application of H5N1 subtype avian influenza pseudovirus. Background technique [0002] Avian influenza (AI) is a disease syndrome infected by poultry and wild birds caused by type A influenza virus. The pathogenic avian influenza virus (avian influenza virus, AIV) belongs to the Orthomyxoviridae family, the genus Influenza virus, and is a single-stranded negative-sense RNA virus. For type A influenza virus, according to the antigenicity of its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), influenza virus is further divided into different subtypes. Currently, Type A virus has 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9), all of which are present in avian influenza viruses. [0003] Avian influenza viruses are divided into two categories based on viral toxicity: highly pathogenic avian influenza virus (HPAI), which can cause a systemic fatal infection, killing birds within 24 hou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/85C12Q1/70C12Q1/66C12Q1/02G01N33/577G01N33/53G01N21/64C12R1/93
Inventor 孙兵徐莹
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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