CN108815203A - The method for building up of animal model based on influenza luciferase reporter virus and application - Google Patents
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Abstract
The invention discloses the method for building up of the animal model based on influenza luciferase reporter virus and applications.This method is mainly using a kind of recombinant influenza infecting mouse for carrying Gluc gene, and the gradient of infection of the expression evaluation mouse by detection mouse lung tissue luciferase.The method based on influenza luciferase reporter virus technology screening and evaluation Tamiflu that the invention also discloses a kind of.This method mainly passes through the influence for detecting antiviral processing to the expression of infecting mouse lung tissue luciferase, screens the Tamiflu with preventing/treating effect.The present invention proved by the detection to positive drug Ribavirin, Oseltamivir phosphate, method disclosed by the invention the screening of novel Tamiflu, research and development and in terms of be with a wide range of applications.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of animal model based on influenza luciferase reporter virus technology
Method for building up, and the method screened using the animal model and evaluate Tamiflu.
Background technique
Influenza virus is to cause one of important pathogenic original of respiratory tract infection, is easy provincialism or global quick-fried in winter
Hair.These great outbursts are usually as caused by H1N1, H3N2 hypotype and influenza B virus of influenza A virus.A type stream
The main reason for Influenza Virus infection is Global influenza morbidity and is dead.
Currently, there are mainly two types of modes for anti influenza infection：Antiviral drugs and vaccine.The drug of anti influenza is according to its effect
The difference of mechanism can be classified as following several types：Act on the drug of neuraminidase (NA), Oseltamivir, zanamivir
Deng；Act on the drug of M2 ion channel, amantadine, Rimantadine etc.；Act on the drug and work of other viral albumen
For the drug of host, Ribavirin etc..What wherein current clinical use was most is Oseltamivir, but is continued due to the medicine
It uses, influenza virus is to which create more serious drug resistances.Influenza virus is every year all in the influenza for changing, and listing every year
Viral vaccine is prediction of the expert based on WHO to viral species that may be popular.But this prediction is not always pair,
Once prediction error vaccine would not tell on.Therefore, the research and development of new type influenza vaccine and Tamiflu are one important
Project, and animal model plays an important role in influenza vaccines and Tamiflu effect assessment.
The reporter virus constructed based on virus reverse genetics systems plays in external virological investigation in vivo
Important facilitation, it is quick, sensitive, intuitive the features such as make based on reporter virus technology establish animal model novel
Have great importance in the research and development and evaluation of vaccine and antiviral drugs.The present invention provides a kind of to stablize carrying Gaussia
The method and evaluation of influenza infection animal model are established based on the recombinant influenza of luciferase gene.The present invention will be stream
The application studies such as research and development, the evaluation of Influenza Virus new generation vaccine and antiviral drugs provide effective technology platform, can effectively take
It is engaged in the medical and health care system of the mankind.
Summary of the invention
The purpose of the present invention is to provide the method for building up of the animal model based on influenza luciferase reporter virus and answer
With the present invention facilitates anti-influenza vaccine/drug/therapeutic strategy exploitation and effect assessment.
In order to achieve the above object, the technical scheme adopted by the invention is that being：
1. a kind of rescue and amplification of the recombinant influenza for carrying luciferase reporter gene.
It is big by Chicago as the conventional eight plasmid reverse genetics systems of background using influenza virus A/Puerto Rico/8/34
Balaji doctor Manicassamy is learned to provide.Form insertion of the reporter gene Gluc to be merged with influenza NS1
The segment NS, and be building up to corresponding two-way startup plasmid, obtains recombinant plasmid pDZ-NS1-Gluc (attached drawing 1), the plasmid equally by
Balaji doctor Manicassamy provides.According to the strategy of traditional eight plasmid reverse genetics system of influenza virus, rescue is taken
Recombinant influenza (PR8-NS1-Gluc) with Gluc reporter gene, and expanded in instar chicken embryo on 8th.
2. a kind of method for establishing influenza infection animal model based on influenza luciferase reporter virus technology, special
Sign is to follow the steps below：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000TCID50PR8-NS1-Gluc virus；
(2) 4-6 week old female BAl BIc/c mouse is taken into the 30 diluted PR8-NS1- of μ l using isoflurane breathing anesthesia
Gluc virus liquid collunarium infecting mouse；
(3) 1 to 8 day after infection, 3 mouse are put to death daily and are dissected, lung tissue is taken, 500ul PBS is added to be homogenized
Processing；
(4) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS, 20 μ l is therefrom taken to utilize BioLux Gaussia
Luciferase Assay Kit (NEB) instructs measurement uciferase activity to specifications；
(5) the Gluc signal value measured represents the expression of mouse lung tissue luciferase, according to Gluc signal value
Height evaluates the modeling effect of animal model.
3. a kind of screen anti-current using the influenza infection animal model established based on influenza luciferase reporter virus technology
The method of Influenza Virus infection medicine, follows the steps below：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000TCID50PR8-NS1-Gluc virus；
(2) 4-6 week old female BAl BIc/c mouse is taken into the 30 diluted PR8-NS1- of μ l using isoflurane breathing anesthesia
Gluc virus liquid collunarium infecting mouse；
(3) it 2 days after infection, puts to death mouse and dissects, take lung tissue, add 500ul PBS homogenized；
(4) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS, 20 μ l is taken to utilize BioLux Gaussia
Luciferase Assay Kit (NEB) instructs measurement uciferase activity to specifications；
(5) drug for reducing the Gluc signal value of mouse lung tissue luciferase is screened, to obtain anti influenza infection
Positive drug；
It also include dosing step in the above method, the dosing step is divided into prevention administration and therapeutic administratp two ways,
One way in which is selected to carry out, operating method is：Prevention administration：The initial administration time is first 2 hours of infection, daily administration
Twice, it is spaced 12 hours, is administered continuously 2 days, administration mode is stomach-filling；Therapeutic administratp：The initial administration time is 6 hours after infection
And/or 24 hours, it is taken twice daily, is spaced 12 hours, continued administration terminates to experiment, and administration mode is stomach-filling；At PBS
Reason group does negative control.
4. evaluating Tamiflu/therapeutic strategy effect method based on influenza luciferase reporter virus.
The present invention select two kinds known to antiviral drug 'Libaweilin ' and Oseltamivir phosphate detect both drugs
To the therapeutic effect of influenza.
4-6 week old female BAl BIc/c mouse is selected, after isoflurane is anaesthetized, to contain 1000 TCID50PR8-NS1-Gluc
30 μ l PBS collunarium infecting mouses.Two days after infection, dissects mouse and acquire its lung tissue, measure its luciferase expression water
It is flat.And using the reduction of Gluc signal value as the index of evaluation medication effect.
Drug-treated scheme：(1) prevention group.It was administered in infection first 2 hours, administration mode is stomach-filling.Ribavirin concentration
For 80mg/kg/day, Oseltamivir phosphate selects two concentration, respectively 50mg/kg/day and 20mg/kg/day.It gives daily
Medicine twice, continues two days.(2) treatment group.It is administered within 6 hours and 24 hours after first 2 hours of infection, infection, administration mode
For stomach-filling.Medicament selection Oseltamivir phosphate, drug concentration 20mg/kg/day.It is taken twice daily, two after infection
It.
Compared with prior art, the present invention having the advantage that：
1. reducing experimental animal dosage.Most of traditional influenza infection animal models (lethal dose) generally require 10-
12 every group of mouse, to guarantee the reliability of experimental result.And this model only needs every group of 4-5 mouse that can must have statistics
The experimental result of meaning.
2. reducing experimental period.Most of traditional influenza infection animal models (lethal dose) need persistently to observe 10-
14 days.This model only needs after infection two days.
3. uciferase activity measurement method is simple.Uciferase activity measurement can be completed by kit in 10 minutes
Measurement.And traditional lung virus carrying capacity measurement then needs plaque assays or TCID50 to measure, and needs 3-5 days time.
4. mouse lung luciferase expression can sensitively react protection of the drug to mouse in second day after infection
Effect.And this point is not accomplished if the other parameters such as variation of lung virus carrying capacity, the variation of Lung Exponent, the variation of weight.
5. economical.Due to advantage 1 and advantage 2, this model experimental animal quantity and in terms of compare saving,
Further for the dosage also scaled-back of drug.Cost needed for effectively reducing drug purchase or synthesis.
Therefore, it based on the present invention, will be mentioned for the research and development of new type influenza vaccine, Tamiflu or treatment of influenza strategy
For the evaluation of programme and index of optimization.The present invention has highly important theory significance and application value.
Detailed description of the invention
Fig. 1 is a kind of construction strategy of recombinant influenza for carrying Gluc gene；
Fig. 2 is growth curve and Gluc signal and virus titer, primary infection agent of the PR8-NS1-Gluc in mdck cell
Measure dependency diagram；
Fig. 3 is that PR8-NS1-Gluc causes mouse weight to decline with different infective doses infection BALB/c female mice respectively
Situation and survival condition；
Fig. 4 is special for expression of the reporter gene in different tissues (lung, liver, spleen) after PR8-NS1-Gluc infecting mouse
Property；
Fig. 5 is that PR8-NS1-Gluc infecting mouse its lung tissue HE dyes schematic diagram；
Fig. 6 is PR8-NS1-Gluc respectively with 103With 105TCID50Lung tissue Gluc signal and virus after infecting mouse
Carrying capacity with infection time variation dynamic；
Fig. 7 is that influence of the positive drug prevention administration to PR8-NS1-Gluc infecting mouse lung tissue Gluc expression is shown
It is intended to；
Fig. 8 is that influence of the positive drug therapeutic administratp to PR8-NS1-Gluc infecting mouse lung tissue Gluc expression is shown
It is intended to；
Fig. 9 is that positive drug handles PR8-NS1-Gluc infecting mouse, 2 day datas and 4,6 day datas after infection after infection
Comparison；
Figure 10 is that positive drug is handled to PR8-NS1-Gluc infecting mouse, lung tissue Gluc expression and other indexs
The comparison of lung virus carrying capacity, Lung Exponent, changes of weight.
Specific embodiment
Experimental method in the present embodiment, unless otherwise specified, be that conventional method is exemplified below is only of the invention
Several specific embodiments.It is clear that the invention is not restricted to following embodiment, acceptable there are many deformations.The common skill of this field
All deformations that art personnel directly can export or associate from present disclosure, are considered as protection model of the invention
It encloses.
Embodiment 1：
It is saved using reverse genetics system and amplifies one plant of carrying with replication capacity close with wild type
The recombinant influenza PR8-NS1-Gluc of Gluc gene, step are：
The influenza reverse genetics system of Gluc gene will be carried (by Chicago University Balaji
Doctor Manicassamy provide) needed for eight plasmids (0.5 μ g/ plasmid) jointly transfection co-culture 293T/MDCK cell,
After transfection 12 hours, discards culture solution and be changed to containing 2 μ g/ml TPCK-Trypsin's (being purchased from Sigma Co., USA)
Opti-MEM culture medium.After culture 72 hours, supernatant is collected, and be inoculated with instar chicken embryo on the eight.After 48 hours, chick embryo allantois is collected
Liquid, as required recombinant influenza.
Amplification gained virus with wild-type virus is infected into mdck cell respectively with 0.01MOI, respectively at 0,12,24,36,
48, sampling in 60 hours carries out titer determination and growth curve is drawn, and shows that two strain virus have similar amplification curve (Fig. 2 a).
Uciferase activity measurement is carried out simultaneously to each point in time sampling, shows the expression (Gluc signal value) and disease of luciferase
(R is had good correlation between malicious titre2=0.807, p<0.0001；Fig. 2 b, c).And Gluc signal and virus initially sense
Stain amount is related (Fig. 2 d).
Embodiment 2：
Measurement of the influenza luciferase reporter virus PR8-NS1-Gluc to mouse virulence.Its step is：
(1) recombinant virus PR8-NS1-Gluc virus liquid is contained with PBS gradient dilution into every 30 μ l PBS respectively
102-106TCID50Virus.
(2) 4-6 week old female BAl BIc/c mouse is grouped at random, every group 10.
(3) anesthetized mice is breathed using isoflurane, the virus liquid of 30 μ l gradient dilutions is taken to distinguish collunarium infecting mouse.
(4) mouse weight variation and survival condition are monitored day by day, continue 14 days.
Such as attached drawing 3, using recombinant virus infection 4-6 week old female BAl BIc/c mouse, mouse in obvious weight loss and
The death that infective dose relies on.This result shows that show gained recombination reporter virus can effectively infecting mouse and have it is very high
Virulence, can be used to establish the mouse model of influenza Asia lethal infection and mortal infection.
Embodiment 3：
Detection of the influenza luciferase reporter virus PR8-NS1-Gluc to mouse infection tissue specificity.Specific steps
For：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000TCID50Virus.
(2) 5 4-6 week old female BAl BIc/c mouse are taken into the diluted viral drop of 30 μ l using isoflurane breathing anesthesia
Nose infecting mouse.
(3) it 6 days after infection, puts to death mouse and dissects.To wherein 3, lung tissue, hepatic tissue, spleen etc. are taken respectively, point
Not plus 500 μ l PBS homogenizeds.For other 2, lung tissue, formalin fixing process are taken.Each experiment takes health respectively
Mouse tissue does same treatment and compares.
(5) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS.20 μ l are taken to utilize BioLux Gaussia
Luciferase Assay Kit (NEB) instructs measurement uciferase activity to specifications.
(6) it for fixed lung tissue, is dyed by embedding, slice and HE, observes pathological change.
Uciferase activity is measured to infecting mouse lung homogenate, can be detected very high gluc signal, and liver,
It then can't detect apparent uciferase activity (Fig. 4) in spleen.Sense is shown to infecting mouse lung tissue section HE coloration result
It contaminates mouse lung tissue and the relevant lesion of obvious influenza, such as alveolar wall thickening, cell infiltration, apparent group damage (figure occurs
5).The above results show that recombinant virus PR8-NS1-Gluc being capable of specific infection mouse lung tissue.
Embodiment 4：
The side of mouse influenza infection model is established based on the recombinant influenza for carrying Gluc gene
Method.Its step is：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 105Or 103 TCID50Virus.
(2) 4-6 week old female BAl BIc/c mouse is grouped at random, one group 18, another group 24.
(2) anesthetized mice is breathed using isoflurane, takes the diluted virus liquid collunarium infecting mouse of 30 μ l.Wherein 105TCID50
Dosage infection 18,103TCID50Dosage infects 24.
(3) since after infection 1 day, every infective dose group is put to death 3 mouse daily and is dissected, and takes lung tissue, adds 500ul
PBS homogenized.Wherein 105TCID50Dosage infected group continues 6 days, and 103TCID50Dosage infected group continues 8 days.
(4) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS.20 μ l are taken to utilize BioLux Gaussia
Luciferase Assay Kit (NEB) instructs measurement uciferase activity to specifications.
(5) titer determination is carried out to each group lung homogenate sample respectively, and compares virus titer and Gluc signal value
Correlation.
Present invention selection low (103TCID50) and height (105TCID50) mouse nuclei is established for two kinds of infective doses,
And using the expression of infecting mouse lung tissue luciferase (Gluc signal value) as the finger for judging mouse infection severity
Mark.For low dosage infected group, 1 to 4 day after infection, mouse lung Luciferase expression levels were gradually risen up to peak value,
Hereafter the expression quantity of reporter gene gradually decreases, the effect (figure that reaction virus is gradually removed under the action of mouse immune system
6a).Mouse lung virus load also shows identical dynamic change, and having the same dynamic between Gluc signal and virus titer
State variation, correlation significant (R2=0.809, p<0.0001) (Fig. 6 b, c).
For high dose infected group, mouse lung tissue Luciferase expression levels and virus load arrive rapidly reach to peak value
(after infection 1 day) hereafter shows downward trend (Fig. 6 d, e).Its Gluc signal value same as virus titer is in significant phase
Closing property (R2=0.367, p<0.01).
Show that mouse lung tissue Luciferase expression levels can be used as the index for effectively judging mouse infection degree.However
Compared with the measurement of virus titer, Gluc determination of activity has many advantages, such as quick, sensitive, convenient.
Embodiment 5：
Antiviral treatment effect is evaluated based on the recombinant influenza for carrying Gluc gene
Method.
The present invention select two kinds known to antiviral drug 'Libaweilin ' and Oseltamivir phosphate detect both drugs
To the therapeutic effect of influenza.Its step is：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000TCID50Virus.
(2) 4-6 week old female BAl BIc/c mouse is grouped at random, every group of 4-5 is only.
(3) anesthetized mice is breathed using isoflurane, takes the diluted virus liquid collunarium infecting mouse of 30 μ l.
(4) it 2 days after infection, puts to death mouse and dissects, take lung tissue, add 500ul PBS homogenized.
(5) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS.20 μ l are taken to utilize BioLux Gaussia
Luciferase Assay Kit (NEB) instructs measurement uciferase activity to specifications.
(6) dosage regimen.
Prevention group：The initial administration time is first 2 hours of infection.It is taken twice daily, is spaced 12 hours.Continued administration 2
It.Administration mode is stomach-filling.Dosage is：Ribavirin 80mg/kg/day；Oseltamivir 50mg/kg/day and 20mg/kg/
day。
Treatment group：The initial administration time is 6 hours and 24 hours after infection.It is taken twice daily, is spaced 12 hours.Continue
Being administered to experiment terminates.Administration mode is stomach-filling.Dosage is：Oseltamivir 20mg/kg/day.
Negative control is done with PBS processing group.
The present invention selects 1000TCID50Mouse influenza infection model is established as infective dose, and with 2 days mouse after infection
The reduction degree of the expression (Gluc signal value) of lung tissue luciferase is as the index for judging medication effect.As a result
Show positive drug Ribavirin (80mg/kg/day) and Oseltamivir (50mg/kg/day and 20mg/kg/day) prevention to
The expression (Fig. 7) of 2 days mouse lung tissue luciferases after infection can be effectively reduced in medicine.Oseltamivir (20mg/kg/
Day) treatment group the result shows that, start within 6 hours and 24 hours after infection administration can effectively inhibit mouse lung tissue luciferase
Expression, however 24 hours beginning drug treatment effects are substantially reduced, this result is also consistent with it, i.e. Ao Sita
Wei reduces or loses (Fig. 8) for influenza infection Late curative effect.
Embodiment 6：
Antiviral treatment effect is evaluated based on the recombinant influenza for carrying Gluc gene
In method, 2 days acquisition data are compared with 4,6 days acquisition data after infection after infection.Its step is：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000TCID50Virus.
(2) 4-6 week old female BAl BIc/c mouse is grouped at random, every group 12.
(3) anesthetized mice is breathed using isoflurane, takes the diluted virus liquid collunarium infecting mouse of 30 μ l.
(4) detection mouse weight variation day by day.
(5) 2 after infection, 4,6 days, 4 mouse of every group of execution are simultaneously dissected, and lung tissue is taken, and 500ul PBS is added to be homogenized
Processing.
(7) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS.20 μ l are taken to utilize BioLux Gaussia
Luciferase Assay Kit (NEB) instructs measurement uciferase activity to specifications.
(8) dosage regimen is prevention administration.
The initial administration time is first 2 hours of infection.It is taken twice daily, is spaced 12 hours.It is administered continuously 5 days or until locates
Dead mouse.Administration mode is stomach-filling.Dosage is：Ribavirin 80mg/kg/day；Oseltamivir 50mg/kg/day and 20mg/
kg/day.Negative control is done with PBS processing group.
Such as Fig. 9, after infection 4 days with the expression of 2 days Gluc after infection can it is sensitive, accurately react antiviral agent
The protection or therapeutic effect of object.And for Oseltamivir processing group, 6 days Gluc levels can not significant reaction treatment group after infection
With the difference (Fig. 9) of untreated fish group, this may be due to infection after 4 days mouse lung virus quantities i.e. in the effect of immune system
Under gradually remove, corresponding Gluc expression is gradually lowered (Fig. 6).
Embodiment 7：Comparative example.
Luciferase expression levels index and other conventional indexs such as lung tissue virus load, Lung Exponent, body in the present invention
The comparison changed again.Its step is：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000TCID50Virus.
(2) 4-6 week old female BAl BIc/c mouse is grouped at random, every group 12.
(3) anesthetized mice is breathed using isoflurane, takes the diluted virus liquid collunarium infecting mouse of 30 μ l.
(4) detection mouse weight variation day by day.
(5) 2 after infection, 4,6 days, 4 mouse of every group of execution are simultaneously dissected, and are taken lung tissue, after weighing, are added 500ul
PBS homogenized.
(6) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS.20 μ l are taken to utilize BioLux Gaussia
Luciferase Assay Kit (NEB) instructs measurement uciferase activity to specifications.
(7) each group mouse Lung Exponent is calculated.And with healthy mice Lung Exponent be control.
(8) dosage regimen is prevention administration.
The initial administration time is first 2 hours of infection.It is taken twice daily, is spaced 12 hours.It is administered continuously 5 days or until locates
Dead mouse.Administration mode is stomach-filling.Dosage is：Ribavirin 80mg/kg/day；Oseltamivir 50mg/kg/day and 20mg/
kg/day.Negative control is done with PBS processing group.
In the present invention, it can be accurately reflected within 2 days after earliest infection using Luciferase expression levels as index and respectively be controlled
The protecting effect (Fig. 9) of regime on mouse is treated, and is done not if conventional index such as lung virus carrying capacity, Lung Exponent, changes of weight etc.
To this point (Figure 10).This is the result shows that the index (Gluc signal value) that the present invention is used to evaluate antiviral effect has standard
Really, sensitive advantage.
Claims (4)
1. a kind of method for establishing influenza infection animal model using influenza virus.
2. a kind of method for building animal model based on influenza luciferase reporter virus technology, it is characterised in that according to following step
It is rapid to carry out：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000-100000TCID50PR8-NS1-Gluc virus；
(2) 4-6 week old female BAl BIc/c mouse is taken into the diluted PR8-NS1-Gluc virus of 30 μ l using isoflurane breathing anesthesia
Drop nose infecting mouse；
(3) 1 to 8 day after infection, 3 mouse are put to death daily and are dissected, lung tissue is taken, adds 500ul PBS homogenized；
(4) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS, 20 μ l is therefrom taken to utilize BioLux Gaussia
Luciferase Assay Kit instructs measurement uciferase activity to specifications；
(5) the Gluc signal value measured represents the expression of mouse lung tissue luciferase, according to the height of Gluc signal value
Evaluate the modeling effect of animal model.
3. a kind of susceptible using the influenza infection animal model established based on influenza luciferase reporter virus technology screening anti-current
The method of malicious infection medicine, it is characterised in that follow the steps below：
(1) virus liquid is diluted in every 30 μ l PBS with PBS and contains 1000TCID50PR8-NS1-Gluc virus；
(2) 4-6 week old female BAl BIc/c mouse is taken into the diluted PR8-NS1-Gluc virus of 30 μ l using isoflurane breathing anesthesia
Drop nose infecting mouse；
(3) it 2 days after infection, puts to death mouse and dissects, take lung tissue, add 500ul PBS homogenized；
(4) 50 μ l lung homogenates are taken, are diluted to 1ml with PBS, 20 μ l is taken to utilize BioLux Gaussia Luciferase
Assay Kit instructs measurement uciferase activity to specifications；
(5) drug for reducing the Gluc signal value of mouse lung tissue luciferase is screened, so that it is positive to obtain anti influenza infection
Drug；
It also include dosing step in the above method, the dosing step is divided into prevention administration and therapeutic administratp two ways, selects
One way in which carries out, and operating method is：Prevention administration：The initial administration time is first 2 hours of infection, daily administration two
It is secondary, it is spaced 12 hours, is administered continuously 2 days, administration mode is stomach-filling；Therapeutic administratp：The initial administration time is 6 hours after infection
And/or 24 hours, it is taken twice daily, is spaced 12 hours, continued administration terminates to experiment, and administration mode is stomach-filling；At PBS
Reason group does negative control.
4. application of the described in any item methods of claim 2-3 in terms of influenza vaccines or anti influenza infection medicine screening.
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| CN110184284A (en) * | 2019-05-22 | 2019-08-30 | 华南农业大学 | The recombinant fowl influenza virus for carrying NanoLuc gene and its application in living imaging mouse model |
| CN110305854A (en) * | 2019-07-18 | 2019-10-08 | 山东中医药大学 | A kind of recombination H3N2 subtype influenza virus, construction method and application carrying luciferase |
| CN114574521A (en) * | 2022-03-03 | 2022-06-03 | 山东中医药大学 | Balance compensation based recombinant influenza virus construction method |
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| CN110305854A (en) * | 2019-07-18 | 2019-10-08 | 山东中医药大学 | A kind of recombination H3N2 subtype influenza virus, construction method and application carrying luciferase |
| CN110305854B (en) * | 2019-07-18 | 2023-09-15 | 山东中医药大学 | Recombinant H3N2 subtype influenza virus carrying luciferase, construction method and application |
| CN114574521A (en) * | 2022-03-03 | 2022-06-03 | 山东中医药大学 | Balance compensation based recombinant influenza virus construction method |
| CN114574521B (en) * | 2022-03-03 | 2023-09-12 | 山东中医药大学 | Recombinant influenza virus construction method based on balance compensation |
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