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Application of ANP32 protein to maintain influenza virus polymerase activity in host

An influenza virus, polymerase technology, applied in antisense single-stranded RNA viruses, viruses, viral peptides, etc., can solve problems such as influenza virus mutation

Active Publication Date: 2019-09-10
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

At present, the research on anti-influenza virus drugs has achieved preliminary results. However, due to the wide clinical application of existing drugs, influenza viruses are constantly mutating and have different degrees of drug resistance to these drugs. Anti-flu drugs gaining importance

Method used

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  • Application of ANP32 protein to maintain influenza virus polymerase activity in host
  • Application of ANP32 protein to maintain influenza virus polymerase activity in host
  • Application of ANP32 protein to maintain influenza virus polymerase activity in host

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] The construction of embodiment 1.ANP32 protein expression vector

[0149] The nucleotide sequences of ANP32 proteins such as chicken, human, zebra finch, duck, turkey, pig, mouse, and horse are as follows:

[0150] Chicken ANP32A (chANP32A) (Gallus gallus, XM_413932.5), human ANP32A (huANP32A) (Homosapiens, NM_006305.3), zebra finch ANP32A (zfANP32A) (Taeniopygia guttata, XM_012568610.1), duck ANP32A2 (dk Anas platyrhynchos, XM_005022967.1), turkey ANP32A (tyANP32A) (Meleagris gallopavo, XM_010717616.1), pig ANP32A (pgANP32A) (Sus scrofa, XM_003121759.6), mouse ANP32A (muANP3030) (Mus mus , horse ANP32A (eqANP32A) (Equus caballus, XM_001495810.5), chicken ANP32B (chANP32B) (Gallus gallus, NM_001030934.1), human ANP32B (huANP32B) (Homo sapiens, NM_006401.2).

[0151] The amino acid sequences of ANP32 proteins such as chicken, human, zebra finch, duck, turkey, pig, mouse, and horse are as follows:

[0152] Chicken ANP32A (chANP32A) (Gallus gallus, XP_413932.3), human AN...

Embodiment 2

[0160] Example 2: Construction of Cell Lines

[0161] We utilize CRISPR-Cas9 technology for cell line construction. According to the NCBI published reference nucleotide sequences human ANP32A (NM_006305.3) and human ANP32B (NM_006401.2), using online software http: / / crispr.mit.edu / sgRNAs targeting these two proteins were designed (see Table 1 for the sequences).

[0162] Table 1: sgRNA sequences

[0163]

[0164] The pMD18T-U6 recombinant plasmid was constructed as follows, which contains human U6 promoter sequence + sgRNA sequence (huANP32A or huANP32B) + sgRNA scaffold sequence + TTTTTT.

[0165] First, a gene fragment is synthesized, which contains human U6 promoter sequence + huANP32A-sgRNA sequence + sgRNA scaffold sequence + TTTTTT, and the sequence is SEQ ID NO: 8. The synthesized fragment was directly ligated into the pMD-18T vector (TaKaRa, product number D101A), and the pMD18T-U6-huANPsgRNA-1 recombinant plasmid (containing huANP32AsgRNA) was successfully ...

Embodiment 3

[0174] Embodiment 3: detection of influenza polymerase activity

[0175] The influenza polymerase reporter system involved in the present invention includes influenza polymerase PB2, PB1 and PA protein, and nucleoprotein NP. These proteins were derived from human influenza H1N1 subtype A / Sichuan / 01 / 2009 (H1N1 SC09 ) and A / WSN / 1933 (WSN), human influenza H7N9 subtype A / Anhui / 01 / 2013 (H7N9 AH13 ), canine influenza H3N2 subtype A / canine / Guangdong / 1 / 2011 (H3N2 GD11 ), avian influenza H9N2 subtype A / chicken / Zhejiang / B2013 / 2012 (H9N2 ZJ12 ) and H7N9 subtype A / chicken / Zhejiang / DTID-ZJU01 / 2013 (H7N9 ZJ13 ), Equine Influenza A / equine / Jilin / 1 / 1989 (H3N8 JL89 ) and A / equine / Xinjiang / 3 / 2007 (H3N8 XJ07 ). The sequences of PB2, PB1, PA and NP proteins of these influenza subtypes are shown in Table 5.

[0176] Table 5. Nucleotide sequences of PB2, PB1, PA and NP proteins

[0177]

[0178]

[0179] Construction of plasmids containing the above proteins for each influenza virus s...

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Abstract

The invention provides recombination sequence information of a key host factor ANP32A / B necessary for replication of an influenza virus in a host. More specifically, the invention relates to a 129-130motif and a 149 site of host factor ANP32A / B protein, which are key active sites for their promotion of influenza virus replication and are potential targeting sites for anti-influenza drugs.

Description

technical field [0001] The present invention relates to key host factors necessary for the replication of influenza virus in hosts. More specifically, it involves the key host factors ANP32A and ANP32B necessary for the replication of influenza virus in the host. Background technique [0002] Influenza is one of the most serious human infectious diseases caused by influenza virus. A sudden global influenza epidemic will infect about 20%-40% of the population (Basler C F, et al.Sequence of the 1918 pandemic influenza virus nonstructural gene( NS) segment and characterization of recombinant viruses bearing the 1918 NS genes [J]. Proc Natl Acad Sci U S A, 2001, 98 (5): 2746-2751.), form a huge threat to human life, health and social life. Influenza virus is an enveloped RNA virus belonging to the Orthomyxoviridae family, and the virus is spherical or polymorphic particles with a diameter of 80-120 nm. Influenza viruses can be divided into Type A (Type A), Type B (Type B), Typ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C12N15/11C12N15/85G01N33/68
CPCC07K14/005G01N33/68C12N2760/16122C07K14/46A61P31/16C12Q1/18G01N2333/11C07K14/4702C12Y207/07048C12N9/127A61K38/00C07K14/435C12N15/1132C12N15/87
Inventor 王晓钧张海丽张振宇
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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