CN106957826A - A kind of virus inactivator and its application - Google Patents

A kind of virus inactivator and its application Download PDF

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CN106957826A
CN106957826A CN201710148704.7A CN201710148704A CN106957826A CN 106957826 A CN106957826 A CN 106957826A CN 201710148704 A CN201710148704 A CN 201710148704A CN 106957826 A CN106957826 A CN 106957826A
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virus
extract
inactivator
virus inactivator
application
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CN106957826B (en
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张建文
李莹
黄茜
王萍
任超
李宁
丛茜
杨兆祥
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KPC Pharmaceuticals Inc
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Abstract

The invention discloses a kind of virus inactivator and its application.Described virus inactivator is 0.5 ~ 1mg/ml of final concentration cinnamomum cassia extract.Using the application for described virus inactivator in inactivation paramyxovirus, herpesviral, retrovirus and influenza virus medicine is prepared.The invention discloses a kind of cinnamomum cassia extract(KPC‑rg1)Inactivation of virus effect, research has shown that, this kind of extract can occur at normal temperatures with the envelope protein of virus coat it is rapid, strongly be combined so that inactivation of virus, losing infection and amplification ability, but possess antigenicity.

Description

A kind of virus inactivator and its application
Technical field
The invention belongs to pharmaceutical technology field, and in particular to a kind of virus inactivator and its application.
Background technology
The main vaccine of current domestic and international application includes attenuated vaccine and inactivated vaccine.Due to the production technology of inactivated vaccine Simply, it is safe, easy to use, be easy to preserve, can stimulating animal produce high titre antibody and significant vaccine protecting effect etc. it is excellent Point, therefore the high-quality and efficient inactivated vaccine of exploitation is the most effective means of the various virus infections of prevention and control.Inactivated vaccine, turns round and look at name Think justice to seek to the pathogen with infective work with appropriate either physically or chemically inactivation, make its forfeiture pathogenic, But still retain its immunogenicity, the vaccine manufactured with it.
Traditional virus inactivator formaldehyde, is a kind of carcinogen for having an intense irritation.It both may act on virus and had contained The nucleotide base of amino, may act on virus coat protein again.When formaldehyde acts on virus coat protein, easily hand over protein Connection or virion aggregation, so that the neutralization immunogenicity of pathogen protein is seriously damaged.And use the formalin-inactivated time It is long, generally require and more than 24h or longer time are handled at 37~39 DEG C;The free formaldehyde of residual, if with vaccine implanter After body, irritative response can be produced.Therefore, on the premise of ensureing that inactivation is perfectly safe, when improving inactivation speed, shortening inactivation Between, the loss of Effective Antigens is reduced, is always the development goal of new virus inactivator.
Chinese cassia tree is China's parts of generic medicinal plants, first recorded in《Sheng Nong's herbal classic》, top grade is classified as, the successive dynasties, important book on Chinese herbal medicine had note Carry.Chinese cassia tree and its contained composition have many pharmacological actions, common reporter include antibacterial, it is anti-oxidant, hypoglycemic, anti- Aldose reductase, anti-inflammatory, anticomplement, antitumor activity, and to digestive system, cardiovascular system, immune system, maincenter god Adjustment effect through, internal system.The report that Chinese cassia tree is used to prevent and treat virus infection is few.
The content of the invention
The first object of the present invention is to provide a kind of virus inactivator;Second purpose is the inactivation of virus described in offer The application of agent.
The first object of the present invention is achieved in that the Chinese cassia tree that described virus inactivator is 0.5 ~ 1mg/ml of final concentration Extract.
The second object of the present invention is achieved in that described virus inactivator is preparing inactivation paramyxovirus, blister Application in exanthema virus, retrovirus and influenza virus medicine.
The invention discloses a kind of cinnamomum cassia extract(KPC-rg1)Inactivation of virus effect, research has shown that, this kind extract Thing can occur at normal temperatures with the envelope protein of virus coat it is rapid, strongly be combined, so that inactivation of virus, losing infection And amplification ability, but possess antigenicity.
Chinese cassia tree of the present invention includes:Chinese cassia tree, great Ye Qinghua osmanthus, Ceylon cinnamon, south China osmanthus, cinnamomum obtusifoliumNees, burmannii, India Osmanthus, Cinnamomum mairei Levl., fragrant osmanthus, bavin osmanthus, river osmanthus;It is preferred that, the Chinese cassia tree includes:Chinese cassia tree, great Ye Qinghua osmanthus and Ceylon cinnamon;More preferably , the Chinese cassia tree includes:Chinese cassia tree and great Ye Qinghua osmanthus.
The plant parts of the Chinese cassia tree are bark.
The method of inactivator inactivation of viruses of the present invention is by virus liquid to be inactivated virus of the present invention Inactivator carries out 4 ~ 37 DEG C of inactivation.
Brief description of the drawings
Fig. 1 is virus inactivator cinnamomum cassia extract of the present invention(KPC-rg1)The inhibitory action signal bred to vero cells Figure;
Influence schematic diagram after the HSV-1 virus infected cells that Fig. 2 pre-processes for HSV-1 and KPC-rg1 to cellular morphology;
Fig. 3 is HSV-1 virus titer change schematic diagrams after KPC-rg1 is handled;
Fig. 4 attacks the situation of change schematic diagram of Mouse Weight after poison for H1N1;
Fig. 5 is the blood clotting suppression potency schematic diagram in mice serum for H1N1 viruses;
Fig. 6 is the 3rd day and experimental endpoints after infection(28 days)Each group mouse lung H1N1 virus M gene RNA copy number schematic diagrames.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but the present invention is not subject in any way Limitation, based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Virus inactivator of the present invention is 0.5 ~ 1mg/ml of final concentration cinnamomum cassia extract.
Described virus inactivator also includes the cinnaldehydrum of cinnamomum cassia extract percentage by weight 1 ~ 5%;
Described cinnamomum cassia extract is the Chinese cassia tree Aqueous extracts prepared under conditions of pH value is 1 ~ 5.
Described cinnamomum cassia extract is to be prepared by raw material of Chinese cassia tree through pre-treatment, extraction, post-processing step, specific bag Include following steps:
A, pre-treatment:The material a of the broken sieving of raw material ground cinnamon is standby;
B, extraction:The water of 3 ~ 30 times of material a weight is added in material a, in extracting 1 ~ 3 time at 4 ~ 80 DEG C of temperature, 1 ~ 24h every time, Merge extract solution and obtain extract solution b;
C, post processing:Extract solution b is concentrated into relative density to obtain adding acidity in concentrate c, concentrate c for 1.01 ~ 1.05 In phosphate buffer, with salt acid for adjusting pH value to 1 ~ 5,8 ~ 24h is stood, it is described cinnamomum cassia extract to take sediment fraction; Add the well mixed as object virus inactivator of cinnaldehydrum of sediment fraction percentage by weight 1 ~ 5%.
Described cinnamomum cassia extract is to be prepared by raw material of Chinese cassia tree through pre-treatment, extraction, post-processing step, specific bag Include following steps:
A, pre-treatment:The material a of the broken sieving of raw material ground cinnamon is standby;
B, extraction:The water of 3 ~ 12 times of material a weight is added in material a, is extracted 1 ~ 3 time at 4 ~ 40 DEG C of temperature, 1 ~ 8h, is closed every time And extract solution obtains extract solution b;
C, post processing:Extract solution b is concentrated into relative density to obtain adding pH value 3 in concentrate c, concentrate c for 1.01 ~ 1.05 In ~ 6 acidic phosphate buffer, with salt acid for adjusting pH value to 2 ~ 4,8 ~ 24h is stood, it is described Chinese cassia tree to take sediment fraction Extract;Add the well mixed as object virus inactivator of cinnaldehydrum of sediment fraction percentage by weight 1 ~ 5%.
Described cinnamomum cassia extract is to be prepared by raw material of Chinese cassia tree through pre-treatment, extraction, post-processing step, specific bag Include following steps:
A, pre-treatment:The material a of the broken sieving of raw material ground cinnamon is standby;
B, extraction:The water of 10 times of material a weight is added in material a, is extracted 3 times at 20 DEG C of temperature, each 2h merges extract solution Obtain extract solution b;
C, post processing:Extract solution b is concentrated into relative density to obtain adding pH value 6 in concentrate c, concentrate c for 1.01 ~ 1.05 Acidic phosphate buffer in, with salt acid for adjusting pH value to 3, stand 12h, it is that described Chinese cassia tree is extracted to take sediment fraction Thing;Add the well mixed as object virus inactivator of cinnaldehydrum of sediment fraction percentage by weight 1 ~ 5%.
The application of virus inactivator of the present invention is that described virus inactivator is preparing inactivation paramyxovirus, blister Application in exanthema virus, retrovirus and influenza virus medicine.
Described virus inactivator is preparing inactivation paramyxovirus, herpesviral, retrovirus and influenza virus Application in vaccine.
The described preparation method bag for inactivating paramyxovirus, herpesviral, retrovirus and influenza virus vaccine Include following steps:
A, prepare antigen liquid;
After B, the cinnamomum cassia extract that addition concentration is 0.5 ~ 1mg/ml in antigen liquid are fully mixed, mixed liquor is positioned over temperature 5 ~ 10min, which is incubated, for concussion under conditions of 20 ~ 30 DEG C obtains object inactivated vaccine;Wherein cinnamomum cassia extract and antigen liquid is matched somebody with somebody It is 1 than ratio:2.5(Wherein cinnamomum cassia extract is in terms of mass unit, and virus is in terms of blood coagulation unit).
The described antigen liquid for preparing is to be seeded in virus in host cell by the inoculum concentration of percentage by volume 1% ~ 3%, when When host cell growth is to 40% ~ 90%, nutrient solution is replaced by maintaining liquid, maintains collection virus after 48 ~ 72h of observation to obtain antigen Liquid.
Described nutrient solution is that described nutrient solution is according to the different corresponding selections of virus host cells(Virus host cells Difference, culture medium is also different);Described maintaining liquid is the nutrient solution containing 2% hyclone.
So that case is embodied, the present invention will be further described below:
The preparation of the virus inactivator of embodiment 1
Step one:Cinnamon bark is crushed, with 3 times of amount 4 DEG C of water soak extractions 1 time, extracted 1 hour.Step 2:Filtration, takes filter Liquid, the dregs of a decoction are standby.Step 3:Filtrate adds ethanol to ethanol content up to 65%, stands.Step 4: centrifugation, supernatant is standby simultaneously Preserve precipitation.Step 5:The supernatant in step 4 is taken, is concentrated under reduced pressure, is dried in vacuo.Step 6:It is dissolved in water, filters.Step Rapid seven:Filtrate adjusts pH value to 5 with watery hydrochloric acid.Step 8:Filtering, takes precipitation, dries.By step 4 and step 8 and commercially available cassia bark Aldehyde presses 10:10:0.5 ratio mixing both the virus inactivator.
The preparation of the virus inactivator of embodiment 2
Step one:Cinnamon bark is crushed, with 8 times of amount water normal temperature(20 ℃)Stirring 3 times, every time two hours.Step 2:Filtration, Filtrate is taken, the dregs of a decoction are standby.Step 3:Filtrate adds ethanol to ethanol content up to 80%, stands.Step 4: centrifugation, supernatant is standby With reservation sediment fraction.Step 5:By the supernatant in step 4, it is concentrated under reduced pressure, is dried in vacuo.Step 6:It is dissolved in water, Filtering.Step 7:Filtrate adjusts pH value to 2 with watery hydrochloric acid.Step 8:Filtering, takes precipitation, dries.By step 4 and step 8 and Commercially available cinnaldehydrum presses 10:10:0.5 ratio mixing both the virus inactivator.
The cinnamon bark total starches of embodiment 3, the preparation of total polyphenols
The preparation of cinnamon bark total starches:Step one:Cinnamon bark is crushed, extraction 3 times is stirred with 30 times of 80 DEG C of amount water, often Secondary 24 hours.Step 2:Filtration, takes filtrate, the dregs of a decoction are standby.Step 3:Filtrate adds ethanol to ethanol content up to 90%, stands. Step 4: centrifugation, supernatant is standby, retain sediment fraction.Step 5:By the supernatant in step 4, it is concentrated under reduced pressure, vacuum is done It is dry.Step 6:It is dissolved in water, filters.Step 7:Filtrate adjusts pH value to 1 with watery hydrochloric acid.Step 8:Filtering, takes precipitation, dries. Step 4 and step 8 and commercially available cinnaldehydrum are pressed 10:10:0.5 ratio mixing both the virus inactivator.
Embodiment 6
The inhibitory action that KPC-rg1 breeds to vero cells
The orifice plate of vero plating cells 96, in 37 DEG C, 5% C02Incubator, which is stayed overnight, to be covered with after individual layer, and the Chinese cassia tree for adding various concentrations carries Take thing(kpc-rg1), set each with the detection of CCK8 kits after experimental group, cell controls group, blank control group, 48h respectively OD values under concentration, cell survival rate is calculated according to formula.As a result show, when kpc-rg1 concentration is less than 0.05mg/ml, Vero cells acellular poison is acted on.Specific data are shown in Fig. 1.
Embodiment 7
Suppression of the KPC-rg1 to HSV-1 virus activities
40ug/ml KPC-rg1 and Mol=0.1 HSV-1 virus liquids are incubated 24 hours jointly at 4 degree, vero is then added to In 96 orifice plates of plating cells, while setting virus control and cell controls (being not added with virus, only add culture medium), culture 24 is small When after observation of cell form under microscope, and calculate the TCID50 values of viral suspension.As a result show, KPC-rg1 is to HSV-1 diseases The deactivation of poison significantly, and plays a very good protection to vero cells.Specific experiment result is shown in Fig. 2, Fig. 3.
Embodiment 8
KPC-rg1 is tested the Anti-viral activity in vitro of RSV viruses and HIV-1 viruses
Sample preparation:KPC-rg1 is dissolved with sterile water-soluble heated to 8 mg/ml and in 80 DEG C of water-baths 15 minutes.Solution in 2000 RPM take supernatant to be stored in 4 DEG C as KPC-rg1 mother liquors after centrifuging 2 minutes.In EC50 determination experiments, KPC-rg1 and Reference compound tests 8 concentration points, each concentration determination duplicate hole by 3 times of doubling dilutions.Highest test concentrations are 1000 μ g/ml.DMSO ultimate densities in cell culture medium are 0.5%.And the method for according to the form below carries out antiviral activity test:
The Antiviral breeding method list of table 1
RSV Along pathological changes caused by virus is tested:Cell is with certain density(Table 1)It is inoculated into microwell plate and in 37 DEG C, 5% Overnight incubation in CO2 incubators.Second day, the compound and virus of various concentrations(Table 1)Premixed liquid add cell plates in 37 DEG C, it is complete that 5% CO2 cultivates the 3-5 days cytopathies into virus control wells.Cytoactive then uses CCK8 cell viabilities Detection kit is detected according to the explanation of detection reagent.Antiviral activity under compound various concentrations to virus by causing The inhibitory action of cytopathic effect determine.All data are through blank infection control calibration.EC50 is by GraphPad Prism softwares carry out analysis calculating.
HIV-1 pathological changes caused by virus is tested:With virus after test compound and reference compound doubling dilution(Table 1)It is mixed Close, mixed liquor and the MT-4 cells of subsequent compound and virus add microwell plate and in 33 DEG C or 37 DEG C, 5% CO2 incubators Cultivate the 2-6 days cytopathies into virus control wells complete.Cytoactive then uses CCK8 or CellTiter Glo cells Viability detection kit is detected according to the explanation of detection reagent.Antiviral activity of the compound under various concentrations is by disease The inhibitory action of cytopathic effect caused by malicious is determined.EC50 carries out analysis calculating by GraphPad Prism softwares.
As a result show, the antiviral activity of the reference compound of all antiviral activity experiments of this experiment meets with expection, Prove that this experimental result is reliable.KPC-rg1 antiviral activity is similar with positive control drug, has preferable activity to two kinds of viruses Inhibitory action, the results are shown in Table 2.
The cinnamomum cassia extract antiviral activity list of table 2.
Embodiment 9
Checking KPC-rg1 resisiting influenza virus is acted on and produced due to " in-situ solidifying immunological effect " that KPC-rg1 is induced Animal protection effect
Experiment material and animal packet
Test sample:KPC-rg1
Experimental virus strain:H1N1 (A/Puerto Rico/8/1934).Virus is bred by MDCK.Strain- Its HA titer is measured before 70 DEG C of storages(HAU)For 256;Experiment is 250HAU with virus.
Experimental animal and packet:The mouse at 28-30 days ages.
A) PBS control group:6 mouse.
B) viral group(PR8 attacks malicious group):8 mouse(250 HAU/ are only).
C)KPC-rg1 administration groups, totally 8 mouse.
Experimental method
The KPC-rg1 of prescribed dose(100ug/only)With virus(250 HAU/ are only)At room temperature training is pre-mixed with appropriate PBS Support 5-10 min.KPC-rg1/ viral solutions or viral solution that the mouse inoculation at 28-30 days ages has been pre-mixed.KPC- Cumulative volume of the rg1/ viruses in PBS is 50 ul/ mouse.Anaesthetized before inoculation.PBS is as blank control.Observe mouse 28 days Survival level.And in experimental endpoints(28 days)Mice serum is collected in eyeball blood sampling, carries out erythrocyte agglutination Inhibition test(HI)Inspection Survey in mice serum and suppress potency for the blood clotting of H1N1 viruses(HI titer).It is positive effectively HI that blood clotting, which suppresses Jie Guo≤20, Potency.n=3.Simultaneously in the 3rd day after infection and experimental endpoints(28 days)Mouse lung is obtained, lung total serum IgE, RT-PCR detection each groups is extracted Mouse lung H1N1 virus M gene RNA copy numbers.
Experimental result
28 days survival conditions of mouse
Table 3 each group mouse, 28 days survival levels
Attack poison group mouse and death occur within the 3rd day after poison is attacked, attack dead more than half in malicious one week after, survival rate is 37.5%(It is 8 small Mouse amounts to dead 5).KPC-rg1 groups do not occur death after poison is attacked, and survival rate is absolutely.Body weight and existence result are disclosed Medicine has notable protectiveness effect to mouse.
Mouse Weight situation of change is shown in Fig. 4
Compare PBS group Mouse Weight steady growths, and be related to virus attack attack malicious group, KPC-rg1 groups Mouse Weight attacking Decline after poison, body weight is gradually recovered and risen after one week.
Mouse blood clotting suppresses potency(Antibody level)See Fig. 5
28 days after infection, malicious group attacked, can detect that 128 blood clotting suppresses potency in KPC-rg1 group mice serums, show virus Attack to mouse makes mouse immune system generate corresponding antibodies and protectiveness.
Fig. 6 is shown in the detection of mouse lung virus load
3 days detection virus infection pulmonary conditions after poison are attacked, each group mouse lung virus load reaches 10 every 50 mg of 7 powers Lung, shows virus in mouse lung infection development situation.During experimental endpoints 28 days, attack poison group mouse lung virus load and detected Less than, show virus removed by mouse immune system.The above results disclose virus from infection again to the pulmonary infection of removing Process.
Summary result, virus is infected each group mouse lung after poison is attacked, and is caused to attack poison group mouse and is existed Infect dead more than half in one week after.And KPC-rg1 groups mouse does not occur the phenomena of mortality, show medicine to H1N1 influenza viruse attacks Mouse has notable protecting effect.Attack the poison group and medicine group mouse survived for 28 days after infection has significant antibody to produce, Virus has also been removed by body immune system.

Claims (10)

1. a kind of virus inactivator, it is characterised in that described virus inactivator is extracted for 0.5 ~ 1mg/ml of final concentration Chinese cassia tree Thing.
2. virus inactivator according to claim 1, it is characterised in that described virus inactivator also includes Chinese cassia tree and extracted The cinnaldehydrum of thing percentage by weight 1 ~ 5%;Described cinnamomum cassia extract is the Chinese cassia tree prepared under conditions of pH value is 1 ~ 5 Aqueous extracts.
3. virus inactivator according to claim 1 or 2, it is characterised in that described cinnamomum cassia extract is using Chinese cassia tree as original Material is prepared through pre-treatment, extraction, post-processing step, specifically includes following steps:
A, pre-treatment:The material a of the broken sieving of raw material ground cinnamon is standby;
B, extraction:The water of 3 ~ 30 times of material a weight is added in material a, in extracting 1 ~ 3 time at 4 ~ 80 DEG C of temperature, 1 ~ 24h every time, Merge extract solution and obtain extract solution b;
C, post processing:Extract solution b is concentrated into relative density to obtain adding acidity in concentrate c, concentrate c for 1.01 ~ 1.05 In phosphate buffer, with salt acid for adjusting pH value to 1 ~ 5,8 ~ 24h is stood, it is described cinnamomum cassia extract to take sediment fraction; Add the well mixed as object virus inactivator of cinnaldehydrum of sediment fraction percentage by weight 1 ~ 5%.
4. virus inactivator according to claim 3, it is characterised in that described cinnamomum cassia extract is using Chinese cassia tree as raw material Prepared through pre-treatment, extraction, post-processing step, specifically include following steps:
A, pre-treatment:The material a of the broken sieving of raw material ground cinnamon is standby;
B, extraction:The water of 3 ~ 12 times of material a weight is added in material a, is extracted 1 ~ 3 time at 4 ~ 40 DEG C of temperature, 1 ~ 8h, is closed every time And extract solution obtains extract solution b;
C, post processing:Extract solution b is concentrated into relative density to obtain adding pH value 3 in concentrate c, concentrate c for 1.01 ~ 1.05 In ~ 6 acidic phosphate buffer, with salt acid for adjusting pH value to 2 ~ 4,8 ~ 24h is stood, it is described Chinese cassia tree to take sediment fraction Extract;Add the well mixed as object virus inactivator of cinnaldehydrum of sediment fraction percentage by weight 1 ~ 5%.
5. virus inactivator according to claim 3, it is characterised in that described cinnamomum cassia extract is using Chinese cassia tree as raw material Prepared through pre-treatment, extraction, post-processing step, specifically include following steps:
A, pre-treatment:The material a of the broken sieving of raw material ground cinnamon is standby;
B, extraction:The water of 10 times of material a weight is added in material a, is extracted 3 times at 20 DEG C of temperature, each 2h merges extract solution Obtain extract solution b;
C, post processing:Extract solution b is concentrated into relative density to obtain adding pH value 6 in concentrate c, concentrate c for 1.01 ~ 1.05 Acidic phosphate buffer in, with salt acid for adjusting pH value to 3, stand 12h, it is that described Chinese cassia tree is extracted to take sediment fraction Thing;Add the well mixed as object virus inactivator of cinnaldehydrum of sediment fraction percentage by weight 1 ~ 5%.
6. the application of any described virus inactivator of a kind of claim 1 ~ 5, it is characterised in that described virus inactivator exists Prepare the application in inactivation paramyxovirus, herpesviral, retrovirus and influenza virus medicine.
7. the application of virus inactivator according to claim 6, it is characterised in that described virus inactivator is gone out in preparation Application in paramyxovirus living, herpesviral, retrovirus and influenza virus vaccine.
8. the application of virus inactivator according to claim 7, it is characterised in that described inactivation paramyxovirus, bleb The preparation method of virus, retrovirus and influenza virus vaccine comprises the following steps:
A, prepare antigen liquid;
After B, the cinnamomum cassia extract that addition concentration is 0.5 ~ 1mg/ml in antigen liquid are fully mixed, mixed liquor is positioned over temperature 5 ~ 10min, which is incubated, for concussion under conditions of 20 ~ 30 DEG C obtains object inactivated vaccine;Wherein cinnamomum cassia extract and antigen liquid is matched somebody with somebody It is 1 than ratio:2.5.
9. the application of virus inactivator according to claim 8, it is characterised in that the described antigen liquid for preparing is by virus It is seeded in by the inoculum concentration of percentage by volume 1% ~ 3% in host cell, when host cell growth is to 40% ~ 90%, by nutrient solution Maintaining liquid is replaced by, maintains collection virus after 48 ~ 72h of observation to obtain antigen liquid.
10. the application of virus inactivator according to claim 9, it is characterised in that described nutrient solution is according to viral place Chief cell difference corresponding selection;Described maintaining liquid is the nutrient solution containing 2% hyclone.
CN201710148704.7A 2017-03-14 2017-03-14 Virus inactivating agent and application thereof Active CN106957826B (en)

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CN112294868B (en) * 2019-07-31 2023-11-21 昆明扬添生物科技有限公司 Medicine for inactivating virus, sterilizing and activating immunity of organism

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