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Influenza luciferase reporter virus-based animal model building method and application

An animal model, luciferase technology, applied in the biological field, achieves the effects of saving feeding time, saving the number of experimental animals, and reducing costs

Inactive Publication Date: 2018-11-16
SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this kind of prediction is not always correct, once the prediction is wrong, the vaccine will not be effective

Method used

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  • Influenza luciferase reporter virus-based animal model building method and application
  • Influenza luciferase reporter virus-based animal model building method and application
  • Influenza luciferase reporter virus-based animal model building method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A reverse genetics system was used to rescue and amplify a recombinant influenza virus PR8-NS1-Gluc carrying the Gaussia luciferase gene with a replication ability similar to that of the wild type. The steps are as follows:

[0046] Eight plasmids (0.5 μg / plasmid) required by the influenza reverse genetics system (provided by Dr. Balaji Manicassamy, University of Chicago) carrying the Gaussia luciferase gene were co-transfected into co-cultured 293T / MDCK cells, and after 12 hours of transfection, The culture medium was discarded and replaced with Opti-MEM medium containing 2 μg / ml TPCK-Trypsin (purchased from Sigma, USA). After culturing for 72 hours, the supernatant was collected and inoculated into eight-day-old chicken embryos. After 48 hours, the allantoic fluid of chicken embryos was collected, which was the required recombinant influenza virus.

[0047] The amplified virus and the wild-type virus were infected with MDCK cells at 0.01 MOI respectively, and samples...

Embodiment 2

[0049] Determination of virulence of influenza luciferase reporter virus PR8-NS1-Gluc in mice. The steps are:

[0050] (1) Dilute the recombinant virus PR8-NS1-Gluc virus liquid with PBS to contain 10 2 -10 6 TCID 50 virus.

[0051] (2) 4-6 weeks old female BALB / c mice were divided into random groups, 10 in each group.

[0052] (3) Mice were inhaled and anesthetized with isoflurane, and 30 μl of serially diluted virus solution was taken to instill nasally to infect the mice.

[0053] (4) The body weight change and survival of the mice were monitored daily for 14 days.

[0054] as attached image 3 , using the recombinant virus to infect 4-6 week-old female BALB / c mice, the mice showed obvious weight loss and infection dose-dependent death. This result shows that the obtained recombinant reporter virus can effectively infect mice and has high virulence, and can be used to establish mouse models of influenza sublethal infection and lethal dose infection.

Embodiment 3

[0056] Detection of influenza luciferase reporter virus PR8-NS1-Gluc specific for infected tissues in mice. The specific steps are:

[0057] (1) Dilute the virus liquid with PBS to contain 1000TCID per 30μl PBS 50 virus.

[0058] (2) Five 4-6 week old female BALB / c mice were anesthetized by breathing with isoflurane, and 30 μl of diluted virus liquid was taken to infect the mice nasally.

[0059] (3) Six days after infection, the mice were sacrificed and dissected. For 3 of them, lung tissue, liver tissue, spleen, etc. were collected, respectively, and 500 μl of PBS were added for homogenization treatment. For the other two, lung tissues were taken and fixed in formalin. In each experiment, tissues from healthy mice were used for the same treatment as controls.

[0060] (5) Take 50 μl of lung tissue homogenate and dilute to 1 ml with PBS. Take 20 μl and use BioLux Gaussia Luciferase Assay Kit (NEB) to measure luciferase activity according to the instructions.

[0061] (...

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Abstract

The invention discloses an influenza luciferase reporter virus-based animal model building method and application. In the method, a mouse is mainly infected with Gaussia luciferase gene-carrying recombinant influenza virus, and the infection degree of the mouse is evaluated by detecting the expression level of luciferase in a lung tissue of the mouse. The invention further discloses an influenza luciferase reporter virus technology-based method for screening and evaluating an anti-influenza drug. According to the method, anti-influenza drugs with prevention / treatment effects are screened by mainly detecting the influence of antiviral treatment on the expression level of the luciferase in the lung tissue of the mouse. Detection of positive drugs ribavirin and oseltamivir phosphate shows that the method disclosed by the invention has a wide application value in the aspect of screening, researching, developing and evaluating the novel anti-influenza drug.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for establishing an animal model based on influenza luciferase reporter virus technology, and a method for screening and evaluating anti-influenza drugs using the animal model. Background technique [0002] Influenza virus is one of the important pathogens causing respiratory infections, and it is prone to regional or global outbreaks in winter. These large outbreaks are usually caused by the H1N1, H3N2 subtypes of influenza A virus and influenza B viruses. Influenza A virus infection is the main cause of influenza morbidity and mortality worldwide. [0003] Currently, there are two main ways to fight influenza infection: antiviral drugs and vaccines. Anti-influenza drugs can be divided into the following types according to their different mechanisms of action: drugs acting on neuraminidase (NA), oseltamivir, zanamivir, etc.; drugs acting on M2 ion channels , amantadine, ri...

Claims

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Application Information

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IPC IPC(8): A61K35/76A61K49/00
CPCA61K35/76A61K49/0008
Inventor 杜瑞坤崔清华杨勇荣立军张颖颖李萍
Owner SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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