Recombinant avian influenza virus carrying NanoLuc gene and application thereof in living imaging mouse model

An avian influenza virus and recombinant virus technology, which is applied in the fields of molecular biology and genetic engineering, can solve the problem of not carrying recombinant H9N2 subtype avian influenza virus, etc., and achieves the effect of good stability

Active Publication Date: 2019-08-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no research on the recombinant H9N2 subtype avian influenza virus carrying the NanoLuc luciferase gene and related applications

Method used

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  • Recombinant avian influenza virus carrying NanoLuc gene and application thereof in living imaging mouse model
  • Recombinant avian influenza virus carrying NanoLuc gene and application thereof in living imaging mouse model
  • Recombinant avian influenza virus carrying NanoLuc gene and application thereof in living imaging mouse model

Examples

Experimental program
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Effect test

Embodiment 1

[0059] The construction of the NS gene recombinant plasmid pHW2000-V-NS-NanoLuc, the steps are as follows: The schematic diagram of the recombinant plasmid construction is as follows figure 1 As shown, the sequence of the V-NS-NanoLuc fragment is shown in SEQ ID NO.1: the protective base of the head-end restriction endonuclease Aar I and its recognition site (position 1-18), the NS segment of V virus 3' end non-coding region sequence (position 19-44), sequence of V virus NS1 segment coding region (excluding terminator) (position 45-695), Linker (GSGG) sequence (position 696-707), NanoLuc luciferase gene sequence (without terminator) (708-1220), PTV-1 virus 2A peptide coding sequence (1221-1286), V virus NEP segment coding sequence (1287-1652 ), the sequence of the 5' non-coding region of the NS segment of the V virus (position 1653-1678), the protective base of the terminal restriction endonuclease Aar I and its recognition site (position 1679-1697). The V-NS-NanoLuc fragment...

Embodiment 2

[0063] Rescue the recombinant H9N2 subtype avian influenza virus V-NS1-NanoLuc carrying the NanoLuc luciferase gene by reverse genetics, the steps are:

[0064]293T cells were inoculated in 6-well cell plates and cultured with DMEM cell culture medium containing 10% fetal bovine serum (293T cells were purchased from ATCC; fetal bovine serum (FBS) was purchased from Israel Biological Industries; DMEM cell culture medium was purchased from In Gibco Company), the next day, the eight plasmids pHW2000-V-PB2, pHW2000-V-PB1, pHW2000-V-PA, pHW2000-V-HA required for the influenza reverse genetic system carrying the NanoLuc luciferase gene , pHW2000-V-NP, pHW2000-V-NA, pHW2000-V–M and pHW2000-V-NS-NanoLuc (0.6 μg / plasmid) were co-transfected into 6-well cell plates, and after 4-6 hours of transfection, discard The culture medium was removed and replaced with Opti-MEM medium containing 0.2 μg / mL TPCK trypsin and a final concentration of 0.2% BSA (bovine serum albumin). After 48 hours of...

Embodiment 3

[0066] The recombinant H9N2 subtype avian influenza virus carrying the NanoLuc luciferase gene is passed on the SPF chicken embryo, and the steps are as follows:

[0067] The allantoic fluid of HA-positive chicken embryos obtained in the previous generation (i.e. V-NS1-NanoLuc recombinant virus) was diluted 100 times with PBS, and then 100 μL of the allantoic fluid was inoculated into 9-day-old SPF chicken embryos, incubated at 37°C for 72 hours, and HA was collected Positive chick embryo allantoic fluid was stored in a -80°C refrigerator. This operation was repeated 4 times, and the obtained virus generations were named P1-P4 in order.

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Abstract

The invention discloses a recombinant avian influenza virus carrying NanoLuc gene and application thereof in a living imaging mouse model. The invention provides a DNA fragment of which the nucleotidesequence is as shown in SEQ ID NO.1, and the DNA fragment can be used to construct a recombinant plasmid, a recombinant virus and a mouse model. In the invention, a recombinant avian influenza viruswhich is similar to the wild type virus in the replication ability and pathogenicity is constructed. In the SPF chick embryo passage, the NanoLuc luciferase gene with good stability can be detected inthe collected 4 generations of virus solutions. In the invention, a living imaging mouse model which has a visualization function is successfully constructed, and the distribution position of the influenza virus in a mouse can be detected without dissecting the mouse. Longitudinal assessment can be performed to observe the dynamic process of influenza virus infection in the same mouse for a continuous period of time, and a visualization tool is supplied for the study of influenza virus.

Description

technical field [0001] The invention belongs to the field of molecular biology and genetic engineering, in particular to a recombinant avian influenza virus carrying NanoLuc gene and its application in live imaging mouse model. Background technique [0002] The H9N2 subtype avian influenza virus is a type of influenza A virus, which is widely prevalent in various regions of the world. It not only brings great harm to the poultry industry, but also can infect humans across the host barrier, which is of great significance in public health [1] . [0003] The mouse model is a commonly used animal model to study the infection, pathogenicity and transmission ability of influenza virus in vivo [2,3] . However, in the mouse model, the distribution of the virus in the body and the dynamic process of infection cannot be observed in real time. In addition, the evaluation of the viral load requires the mice to be killed, which excludes the longitudinal evaluation. In vivo imaging of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/66C12N15/63C12N7/01C12N15/86A01K67/027
CPCA01K67/0275A01K2227/105A01K2267/03C12N9/0069C12N15/66C12N15/86C12N2760/16143C12Y113/12
Inventor 亓文宝劳光杰廖明马凯雄邱子雯
Owner SOUTH CHINA AGRI UNIV
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