CN106546747B - A kind of detection method of interferon biological activity - Google Patents
A kind of detection method of interferon biological activity Download PDFInfo
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- CN106546747B CN106546747B CN201610928699.7A CN201610928699A CN106546747B CN 106546747 B CN106546747 B CN 106546747B CN 201610928699 A CN201610928699 A CN 201610928699A CN 106546747 B CN106546747 B CN 106546747B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/555—Interferons [IFN]
- G01N2333/57—IFN-gamma
Abstract
The present invention relates to the detection methods of interferon biological activity, and using porcine kidney cell (IBRS-2) as measurement cell, vesicular stomatitis virus (VSV) detects interferon activity as challenge virus.The method of the present invention be used to can effectively solve when porcine IFN γ titration it is existing when detecting porcine IFN γ potency in existing method be not easy to observe, subjective error it is big, the problems such as sensibility is poor, because that can not detect activity caused by species specificity.But the method for the present invention is not limited to the titration of porcine IFN γ, is also suitable for other pig interferon titrations, such as pig IFN-α；Also it is suitable for non-II type human interferon, such as humanIFN-α simultaneously.
Description
Technical field
The invention belongs to immunologys and microbiological art, are related to a kind of biological activity detection method of biological products,
More particularly to a kind of detection method of interferon biological activity.
Background technique
China have long pig raising history, be the world pig raising big country, pig year livestock on hand about 1,000,000,000.With pig breeding industry scale
Change and intensive development, the viral disease of swine disease especially pig is brought to pig breeding industry to be seriously threatened, in recent years pig circular ring virus 2
The appearance of the virosis such as malicious (PCV2) disease and porcine reproductive and respiratory syndrome (PRRS), especially exempts from caused by these virus infections
Epidemic disease inhibits, and causes swinery that the mixed infection of multiple pathogens occurs, the death rate of pig reaches 8%-10% or higher, annual therefore to make
At huge economic loss.At present although China is inoculated with various swine disease vaccines, but since virus variation is fast, new virus and variation disease
Poison continuously emerges, and the control effect of vaccine is barely satisfactory.
Interferon (Interferon, IFN) is the multi-functional secreted protein that a kind of animal body generates, and is had disease-resistant
The biological functions such as malicious, antitumor, immunological regulation.Therefore pig interferon all has the infectious disease virus with significant threat anti-
Imperial and inhibiting effect.A series of in vitro tests show: pig IFN-α can effectively inhibit the vigor of foot and mouth disease virus；Pig is infected to infect
The piglet of property marcy agent (TGEV), can generate rapidly the pig IFN-α of anti-TGEV in gut epithelium tissue；Use porcine IFN γ
The pig macrophage of processing infection porcine reproductive and respiratory syndrome virus (PRRSV) can inhibit PRRSV proliferation；At IFN-γ
After managing MARC-145 cell, PRRSV street strain and cell adaptation strain proliferation can inhibit；Porcine IFN γ can inhibit infection and infect
Virus replication in the pig epithelial cell and pulmonary macrophage of property marcy agent；Porcine IFN γ may also suppress infection swine fever virus
Monocyte and the virus replication of pulmonary macrophage etc..When preventing and treating viral disease using interferon, need to utilize genetic engineering
Technology production recombination pig interferon, establishes the system of efficiently production pig interferon, it can just pushed away in large-scale cultivation production
It is wide to use, generate good disease prevention and cure effect.
Currently, the pig interferon for uniquely obtaining the Ministry of Agriculture " novel chiral synthon certificate " and " production approval number " is " pig leucocyte
Interferon ", it is the activated protein mixture for stimulating cell to generate by virus, based on pig IFN-α.In its quality standard still
The Determination of biological activity method of human interferon is so continued to use, i.e., determination of activity is carried out using the WISH-VSV system of pharmacopeia.Specifically
Are as follows: using human amniotic cell (WISH) as measurement cell, vesicular stomatitis virus (VSV) is used as challenge virus；Human interferon
After acting on WISH cell, can inducing cell establish antiviral state, power and the human interferon of cell anti-virus ability
Active size is directly proportional, and the active size of human interferon can be reflected according to the degree of cytopathy.
Inventor is not having found obvious problem using above-mentioned WISH-VSV system detection pig IFN-α, but with the system pair
Porcine IFN γ carries out determination of activity and is just no longer applicable in.Show as being not easy to observe, subjective error it is big, sensibility is poor, practical potency is inclined
It is low.In addition, though it has been reported that porcine kidney cell (PK-15)-vesicular stomatitis virus (VSV) system can be used as pig interferon effect
Valence measures system, but there is also centainly not with observation, it is difficult to the problem of embodying interferon true potency.
Summary of the invention
It is not easy to observe, lead present invention seek to address that existing interferon titer detection method is existing when detecting porcine IFN γ
See the problems such as error is big, and sensibility is poor, because that can not detect activity caused by species specificity.
By a large number of experiments analysis and literature research, it is since II type interferes why inventor's discovery, which has the above problem,
The biological effect of element such as IFN-γ has the species specificity of height, i.e. people's IFN-γ does not have biology living zooblast
Property, porcine IFN γ is to people's cell (such as WISH) also without biological activity；And interferon type Ⅰ such as IFN-α is without species specificity.Base
In this, the people's cell (WISH) in existing detection architecture is replaced with pig cell by inventor, is made using porcine kidney cell (IBRS-2)
To measure cell, vesicular stomatitis virus (VSV) detects interferon activity as challenge virus.
The detection method of above-mentioned interferon biological activity, specifically includes the following steps:
(1), cultured IBRS-2 cell monolayer is diluted to cell density 2-3 × 10 in advance5The cell suspension of a/mL,
Culture；
Etc. (2), after cells are adherent, interferon is subjected to 4 times of gradient dilutions, does 12 gradients；Interferon one after dilution
96 adherent porocyte plates of secondary addition carry out changing liquid, 100 holes μ L/, culture length to uniform monolayers cell；
(3), supernatant is abandoned, VSV virus liquid is added into every hole, cell control well adds maintaining liquid；Culture is about for 24 hours；
(4), cytopathy is observed, counts the hole each dilution CPE and without CPE hole count；
(5), pig interferon potency is calculated by Reed-Muench Liang Shi method.
The above method is deposited when being used to can effectively solve existing method detection porcine IFN γ potency when porcine IFN γ titration
The problem of, but be not limited to the titration of porcine IFN γ, be also suitable for other pig interferon titrations, such as pig IFN-
α；Also it is suitable for non-II type human interferon, such as humanIFN-α simultaneously, but is not suitable for II type human interferon such as people's IFN-γ, this
It is not have species specificity based on interferon type Ⅰ, and interferon type Ⅱ has the species specificity of height.
Found through comparative studies of the present invention, the detection system of IBRS-2 combination VSV, cytopathy significantly easily observation, can be most
Big degree reduces subjective judgement bring error；The pig interferon biological activity of detection is high, sensibility is strong, can more reflect that pig is dry
Disturb the anti-swine disease cytotoxic activity of reality of element；The influence of species specificity is eliminated, the true anti-swine disease poison of reality for reflecting porcine IFN γ is living
Property.Therefore, it compared with common WISH-VSV interferon detection system and PK-15-VSV interferon detection system, uses
IBRS-2-VSV system carries out the measurement of pig interferon biological activity, and testing result is more scientific, true, effective, and being based on should
The activity determination method of system is a kind of method for being more suitable for pig interferon Determination of biological activity.
Detailed description of the invention
The active cytopathy observation of Fig. 1: IBRS-2-VSV system measurement pig interferon, left figure are as follows: cell control well, it is right
Figure are as follows: virus control wells.
The active cytopathy observation of Fig. 2: PK-15-VSV system measurement pig interferon, left figure are as follows: cell control well, it is right
Figure are as follows: virus control wells.
The active cytopathy observation of Fig. 3: WISH-VSV system measurement pig interferon, left figure are as follows: cell control well, right figure
Are as follows: virus control wells.
Specific embodiment
Instrument that the following example uses, material information are as follows:
One, instrument:
| Title | Model | Manufacturer |
| Biohazard Safety Equipment | BSC-1300ⅡA2 | Su Jing is safe and sound |
| CO2Incubator | MCO-20AIC | Sanyo |
| Biomicroscope | IX73 | Olympus |
| Hitachi's centrifuge | CF5RE | Hitachi |
| Refrigerator | SC-390 | Haier |
| Autoclave | SX-700 | Tomy |
| Eddy blending machine | IP40 | IKA |
Two, reagent/reagent:
Three, material:
| Title | Source |
| 1 age in days SPF hatching egg | Cimmeria dimension in Beijing is logical |
| IBRS-2 | Preclinical medicine cell centre |
| WISH | Preclinical medicine cell centre |
| PK-15 | China Veterinery Drug Inspection Office |
| VSV | Beijing animal and veterinary research institute |
The culture of 1 VSV of embodiment and malicious valence titrate
The embodiment is only used for for example, being not used as limiting the present invention, and all conventional VSV cultures and malicious valence measure
Method is used equally for the present invention.
(1), the hatching of chicken embryo:
1, temperature: most suitable incubation temperature is 37.8 DEG C.
2, humidity: generally 40%~70%.
3, placing hatching egg should be greatly head-up small to lay flat upside down, small can not head-up hatch.Check and approve incubator
Temperature and humidity, record puts the date and hour of egg.Hatching process is both it is noted that temperature, humidity, will also carry out ventilation and turn over
Egg work (is generally turned over 5 times daily；Egg-turning angle is bent forward and backward each 45 degree with horizontal position to be advisable；When egg-turning movement it is light, it is steady,
Slowly).
4, transillumination is carried out per daily candler, rejects dead germ.
(2), chicken Embryo is for fibroblastic preparation:
1, take 9 days instar chicken embryo 1, wipe chick embryo air sac exochorion respectively with the tincture of iodine and cotton ball soaked in alcohol.
2, two sterilized petri dishes are taken out, and are marked 1. and 2., for use.
3, in RPMI-1640 and Hank ' with 1% amount penicillin and streptomysin is added in sterile working respectively in s liquid, it is sucked out
10~15mlLHank ' s liquid is spare into sterilized petri dishes.
4, then carefully remove gas chamber exochorion at the top of big tweezers tapping chorion.
5, egg membrane is carefully torn after disinfecting forceps, 1. two people, which work in coordination to take out chicken embryo and be placed in, fills the plate of Hank ' s liquid
In, careful decaptitating, internal organ, pawl and mucus and blood cell, washing be placed on fill the plate of Hank ' s liquid 2. in, then sufficiently washing.
6, it takes out clean tissue block to be placed in big penicillin bottle, be cut into about in flame vicinity with Sterile ophthalmic scissors
0.5~1mm3Fritter (about 250 times), volume about 0.5ml at this time；It is washed twice with Hank s ' liquid.
7,1% trypsase of 1mL is added, being adjusted with appropriate Hank ' s liquid makes its working concentration 0.25%, and 5.6%
NaHCO3It adjusts pH to about 7.3~8.0 (it is flesh pink that naked eyes, which are seen, and 1mL suction pipe about 3 drips) and covers bottle stopper, finish writing label.
8, above-mentioned cell bottle is placed in 37 DEG C of water-baths, starts timing, about 15~30min slowly shakes up with abundant therebetween
Digestion.The mark that digestion stops is: shaking gently rear cell and hang, in cloud, assembles agglomerating quickly, show cell viability
It is good.
9, it after digesting, takes out, slightly precipitates, supernatant is slowly sucked out, carefully wash 2~3 times with Hank ' s liquid, Ke Yijin
Amount reduces the residual quantity of trypsase, and RPMI-1640 liquid about 5mL is then added, is sufficiently blown and beaten with small head straw, keeps cell abundant
Dispersion.It sucts clear；Repetitive operation 3~4 times, collect cell conditioned medium.
10, through four layers of sterile gauze or 200 mesh stainless steel steel mesh filtering and collecting filter liquids, cell count, and cell is adjusted
Density about 1 × 106/mL；
11, the above-mentioned cell suspension prepared is evenly distributed in T25 cell bottle, 7mL/ bottles, is stoppered screw socket plug, pays attention to
Growth down, marks, including Cell Name, the inoculated and cultured time, name etc. on non-aufwuchsplate.
12, cell is uniformly placed in specified 37 DEG C in this room, 5%CO2It is cultivated in incubator.
(3), malicious valence titration:
1, after chicken embryo fibroblasts culture to single layer, 1:100 dilutes VSV kind venom, is diluted with maintaining liquid 5%FBS.
Each 25cm2Cell bottle adds the virus liquid after 7mL dilution.
2,37 DEG C are set, 5%CO2Cultivated in incubator, until 90% appears above cytopathy, sealing, by cell bottle freeze in-
20℃。
3, natural thaw is taken out after freezing, is shaken in course of defrosting for several times, so that cell falls off from bottle wall safely.
4, virus liquid is collected in centrifuge tube, and 3000rpm 4 DEG C, 10min, removes cell fragment, it is (i.e. viral to collect supernatant
Liquid).It is spare to dispense cryopreservation.
5, about 2-3 × 10 are diluted to growth-promoting media after IBRS-2/WISH/PK-15 cell dissociation5A/96 orifice plate of mL bed board,
The hole 100uL/, 37 DEG C, 5%CO2Culture to cell grows up to single layer.
6, virus liquid is made into continuous 10 times of dilutions in centrifuge tube, from 10-1~10-10。
7, by the poison disease vaccination diluted into 96 porocyte plates, each dilution is inoculated with 8 holes, the hole 100uL/.
8, two tandem of normal cell controls is set, that is, is changed to 5%FBS culture solution (dilution).
9, it observes and records day by day as a result, about observing 3~5d.Lesion degree " ++ " or more is judged to the hole CPE, is otherwise judged to
Without the hole CPE.Lesion degree is indicated with following symbol: "-" indicates cell without lesion；"+" indicates that 25% cell below has lesion；
" ++ " indicates that the cell of 25%-50% has lesion；" +++ " indicates that the cell of 50%-75% has lesion；" ++++" indicate 75%-
100% cell has lesion.
10, TCID is calculated by Reed-Muench method50
Attached: (no CPE hole count listed in Table, the numerical value of CPE hole count occur be only this example explanation to Reed-Muench Liang Shi method
Random value, not test specific value)
CPE hole count when the specific dilution of virus liquid: it is specific that the CPE hole count occurred from minimum dilution is added to this one by one
CPE hole count when dilution, if dilution is 10-1CPE hole count be dilution be 10-6、10-5、10-4、10-3、10-2、10-1When
CPE hole count add up one by one, i.e. 0+1+3+7+8+8=27；Dilution is 10-2CPE hole count be dilution be 10-6、10-5、
10-4、10-3、10-2When CPE hole count add up one by one, 0+1+3+7+8=19.
When the specific dilution of virus liquid without CPE hole count: it is specific dilute without CPE hole count to be added to this one by one from highest dilution
When degree of releasing is 10 without CPE hole count, such as dilution-2Be dilution without CPE hole count be 10-1、10-2When it is tired without CPE hole count
Add, i.e. 0+0=0；If dilution is 10-3Be dilution without CPE hole count be 10-1、10-2、10-3When it is tired without CPE hole count
Add, i.e. 0+0+1=1.
CPE:Cytopathic effect cytopathy
Distance proportion=(higher than the percentage -50% of 50% lesion rate)/(percentage-higher than 50% lesion rate is lower than
The percentage of 50% lesion rate)=(91.6-50)/(91.6-40)=0.8
lgTCID50The logarithm of the dilution of 50% lesion rate of difference between=distance proportion × dilution logarithm+be higher than=
0.8 × (- 1)+(- 3)=- 3.8
TCID50=10-3.8/0.1ml.Meaning: by the viral dilution 103.8100 μ L of inoculation can be such that 50% cell occurs again
Lesion.
2 pig interferon sample of embodiment Determination of biological activity in different cell line detection systems:
(1), method
1, preparatory cultured IBRS-2/WISH/PK-15 cell monolayer, with 10% after being digested with 0.05%Trypsin
FBS MEM/DMEM (IBRS-2/WISH MEM culture medium, PK-15 DMEM culture medium) is diluted to suitable cell density (about
2-3×105A/mL) cell suspension, spread 96 porocyte plates, 100 hole μ L/, 37 DEG C of 5%CO24h or so is cultivated, it is adherent to its.
Etc. 2, after cells are adherent, porcine interferon alpha/γ sample is first subjected to 4 times of gradient dilutions, is 12 gradients, Mei Geti
Degree repeats 3 holes, dilution 7%FBS MEM/DMEM culture medium.Pig interferon sample after dilution is once added adherent
96 porocyte plates carry out changing liquid, 100 holes μ L/, and 100 μ L dilutions, 37 DEG C of 5%CO are respectively added in cell controls and virus control wells2
It is incubated overnight 18-24h, it is long to uniform monolayers cell.
3, supernatant is abandoned, 100 μ L VSV virus liquid (about 1-2 × 10 are added into every hole2TCID50/ hole, a certain cell detection
System carries out specific virus liquid dilution according to the VSV poison valence measured on corresponding cell), cell control well adds 100 μ L to maintain
Liquid (3%FBS MEM/DMEM culture medium), VSV dilution 3%FBS MEM/DMEM culture medium.Add 37 DEG C of 5%CO of postposition2Training
It supports about for 24 hours.
4, it attacks after poison about for 24 hours, observes cytopathy under inverted microscope, count the hole each dilution CPE and without the hole CPE
Number.When lesion (" ++++") occurs in 75% or more cell of virus control wells, result is determined.Lesion degree " ++ " or more is judged to
Otherwise the hole CPE is judged to no hole CPE.Lesion degree is indicated with following symbol: "-" indicates cell without lesion；"+" indicate 25% with
Under cell have lesion；" ++ " indicates that the cell of 25%-50% has lesion；" +++ " indicates that the cell of 50%-75% has lesion；
" ++++" indicate that the cell of 75%-100% has lesion.
5, pig interferon potency is calculated by Reed-Muench Liang Shi method, pig interferon active unit is indicated with U.It can protection
Half cell be immune against attacks viral subversive pig interferon highest dilution inverse, as pig interferon potency.
Citing: Reed-Muench Liang Shi method calculates sample to be tested pig interferon potency, and (no CPE hole count listed in Table occurs
The numerical value of CPE hole count is only the random value that this example illustrates, not tests specific value)
Distance is than=(higher than the percentage -50% of 50% lesion rate)/(percentage-higher than 50% lesion rate is lower than
The percentage of 50% lesion rate)=(75-50)/(75-25)=0.5
Log4(X)=distance is higher than logarithm=0.5 of the dilution of 50% lesion rate than × difference between dilution logarithm+
× (- 1)+(- 6)=- 6.5
Highest dilution (X)=4-6.5.Meaning: pig interferon dilution to 4-6.5When, half cell can be protected to be immune against attacks
Viral subversive.The 1/X reciprocal of the dilution, the as potency [pig interferon active unit (U) number] of 0.1mL sample to be tested.This
The potency of sample to be tested is 4 in example6.5× 10=8.19 × 104U/mL。
(2), measurement result compares:
1, cytopathy conspicuousness is compared with easy observation property
From from the conspicuousness of cytopathy and easily in terms of property comparison result, IBRS-2 > PK-15 >=K-15.As shown in Figure 1:
The cytopathy of IBRS-2 is significant, and the contracting of cell circle simultaneously can form significant cytopathy plaque, the cell controls with uniform ground
Easily distinguish；As shown in Figure 2: the cytopathy of PK-15 is more significant, and cell shrinkage forms dead cell agglomeration and gathers, but cell controls
Also there is a degree of living cells to assemble, therefore in the critical hole for CPE occur, the aggregation of the lesion that is not easily distinguishable dead cell with it is normal
Living cells aggregation, is easy to bring subjective error；As shown in Figure 3: the cytopathy of WISH cell is slight, only forms lesser cell
Gather spot, in the critical hole for CPE occur, CPE aggregation spot is smaller to be not easy to distinguish with the Normal aggregation of living cells, is easy
Bring subjective error.Therefore, using IBRS-2 system detection, cytopathy is significantly easy to distinguish, and subjective error is small, as a result trueer
It is real reliable.
2, sample biological activity result compares
Never the potency comparison result of homologous ray (different cells are VSV virus) same porcine interferon alpha sample of measurement is seen
(table 1), IBRS-2 > PK-15 > WISH, illustrates that IBRS-2 cell is most strong to the sensibility of porcine interferon alpha, and theoretically potency is examined
Result is surveyed closest to practical potency.Porcine kidney cell line IBRS-2 is more suitable for the Determination of biological activity of porcine interferon alpha.
The potency of the same porcine interferon alpha sample of the different system detections of table 1. compares
The potency comparison result that never homologous ray measures same Porcine interferon-gamma sample sees (table 2), IBRS-2 > PK-15,
WISH cell does not detect Porcine interferon-gamma potency.IBRS-2 cell is stronger to the sensibility of Porcine interferon-gamma, theoretically potency
Testing result is closer to practical potency, and for comparing PK-15, IBRS-2 is more suitable for the bioactivity of Porcine interferon-gamma.
The potency of the same Porcine interferon-gamma sample of the different system detections of table 2. compares
Different detection systems detect human interferon and pig interferon sample activity respectively, the results are shown in Table 3.Human interferon sample
Activity is measured in IBRS-2-VSV system only improves 16 times, but pig interferon sample compared to the activity that WISH-VSV system measures
Activity is measured in IBRS-2-VSV system and improves 90 times compared to the activity that WISH-VSV system measures, and pig interferon sample exists
The active increase rate measured in IBRS-2-VSV system is significantly higher than human interferon sample, shows pig interferon IBRS-2-
The sensibility of VSV system detection is stronger, and theoretically bioactivity result is closer to its practical potency.
The different detection systems of table 3. detect human interferon respectively and the potency of pig interferon sample compares
3, species specificity compares
The potency comparison result that never homologous ray measures same Porcine interferon-gamma sample sees (table 2), IBRS-2 > PK-15,
WISH cell does not detect Porcine interferon-gamma potency.Since interferon gamma has stringent species specificity, so people's cell pair
Pig interferon is insensitive, and the potency of Porcine interferon-gamma is not measured with WISH cell inspection.Should the result shows that, for pig interferon biology
The determination of activity of active measurement, especially Porcine interferon-gamma is learned, the activity monitor system (WISH- of human interferon cannot be continued to use
VSV).It is equally pig cell, because cellular sensitivity is stronger, bioactivity result is closer to its practical potency, so comparing PK-15
For, IBRS-2 is more suitable for the bioactivity of Porcine interferon-gamma.It can be seen that IBRS-2-VSV detection system is equally also suitable
In non-II type human interferon, such as the titration of humanIFN-α.
In view of the degree of recognition of interferon activity measurement system, in order to keep consistent with human interferon determination of activity system
Property and comparability, so using VSV as interferon activity measure with virus it is more suitable；And VSV can equally infect pig, be
Cause the important virus of swine pox, therefore VSV is more suitable for the active measurement of pig interferon.
In conclusion whether from the conspicuousness of cytopathy and easily from property, cellular sensitivity or species specificity
Angle considers that the system that porcine kidney cell line IBRS-2 is combined with VSV is all more suitable for the biological activity detection of pig interferon.
Claims (1)
1. the detection method of interferon biological activity, it is characterised in that: the interferon is porcine IFN γ, and thin using pig kidney
For born of the same parents IBRS-2 as measurement cell, vesicular stomatitis virus VSV detects porcine IFN γ activity as challenge virus,
Specifically includes the following steps:
(1) preparatory cultured IBRS-2 cell monolayer is diluted to cell density 2 × 105-3×105The cell suspension of a/mL,
Culture；
(2) etc. after cells are adherent, interferon is subjected to 4 times of gradient dilutions, does 12 gradients；Interferon difference after dilution is successively
96 adherent porocyte plates are added to carry out changing liquid, 100 holes μ L/, culture length to uniform monolayers cell；
(3) supernatant is abandoned, VSV virus liquid is added into every hole, cell control well adds maintaining liquid；Culture is about for 24 hours；
(4) cytopathy is observed, counts the hole each dilution CPE and without CPE hole count；
(5) pig interferon potency is calculated by Reed-Muench Liang Shi method.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1533242A (en) * | 2001-04-26 | 2004-09-29 | ��������ũҵ�� | Foot and mouth disease virus vaccine |
| CN103981192A (en) * | 2014-04-14 | 2014-08-13 | 中国农业科学院哈尔滨兽医研究所 | Recombinant adenovirus for expression of goat alpha interferon and construction method and application thereof |
-
2016
- 2016-10-31 CN CN201610928699.7A patent/CN106546747B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1533242A (en) * | 2001-04-26 | 2004-09-29 | ��������ũҵ�� | Foot and mouth disease virus vaccine |
| CN103981192A (en) * | 2014-04-14 | 2014-08-13 | 中国农业科学院哈尔滨兽医研究所 | Recombinant adenovirus for expression of goat alpha interferon and construction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Enhanced Antiviral Activity against Foot-and-Mouth Disease Virus by a Combination of Type I and II Porcine Interferons;Mauro Pires Moraes et al.;《Journal of Virology》;20070731;第81卷(第13期);第7124-7135页 |
| 猪β-干扰素的研究进展;徐蕙 等;《中国畜牧兽医》;20091231;第36卷(第12期);第120-122页 |
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