CN108795982A - A kind of chicken interferon α biological activity detection methods - Google Patents
A kind of chicken interferon α biological activity detection methods Download PDFInfo
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Abstract
The invention discloses a kind of chicken interferon α biological activities detection method and its applications.This approach includes the following steps：The genetic fragment of the Mxp of chicken Mx albumen is obtained using PCR amplification；Remove the pCMV of pEGFP-N1 vector plasmids；The genetic fragment of the Mxp of the chicken Mx albumen obtained with PCR amplification replaces the pCMV of original pEGFP-N1 vector plasmids by T4DNA ligases, builds pEGFP-N1-Mxp plasmids；With pEGFP-N1-Mxp plasmid-transfected cells, and stable transfected cells strain is filtered out by neomycin；Colonized culture is carried out to the stable transfected cells strain filtered out, chicken interferon α is added to be incubated altogether with the stable transfected cells strain after colonized culture, Mx gene promoter activities can be activated to promote the expression of intracellular EGFP, cell sends out the intensity of fluorescence after being irradiated by excitation light source and the biological activity of chicken interferon α is proportionate, therefore can be with the biological activity of quantitative assessment chicken interferon α.
Description
Technical field
The present invention relates to a kind of chicken interferon α biological activity detection methods, belong to interferon activity detection technique field.
Background technology
Interferon (IFN) is that cell and body are infected with the virus, or by nucleic acid, bacterial endotoxin and promote cell division
After the effect of element etc., by a kind of broad-spectrum antiviral glycoprotein of recipient cell secretion.According to the source of interferon and physicochemical property,
It can be classified as I types, II types and III interferon.I type interferon includes IFN-α, IFN-β,With IFN- τ；II types are dry
Disturb element i.e. IFN-γ；III interferon, that is, IL-28A, IL-28B and IL-29.
There are four major functions for IFN-α.First, the cell of virus infection and its peripheral cell can be made to enter endogenous anti-
Virus State, the propagation of limiting virus；Second, it maintains innate immune reaction to be in equilibrium state, promotes antigen submission and NK thin
Inhibit the generation of pro-inflammatory signals access and cell factor while born of the same parents' function；Third activates acquired immune system, promotes high parent
With the generation of power T cells with antigenic specificity, promote B cell reaction and immunological memory；4th, there is the work for inhibiting virus multiplication
With.
The mechanism of action of IFN-α illustrated before 25 years, IFN JAK1s and TYK2 related to receptor combination activated receptor,
So that STAT1 and STAT2 tyrosine phosphorylations, the STAT1 and STAT2 of phosphorylation is formed dimer and is transferred to core, assembles IFN tune
Save the IFN stimulating factors 3 (ISGF3) that the factor 9 (IRF9) forms 3 aggressiveness.ISGF3 and its homologous DNA sequence (IFN stimulate the reaction
Element, ISREs) it combines, ISGs transcriptions are directly activated, host cell is made to enter antiviral state.ISG inhibits the approach master of virus
Have, inhibits transcription, translation and the nucleic acid replication of virus；Degradation viral nucleic acid；Change host cell lipid metaboli.Therefore, as long as
Detect that the promoter activity increase of ISGs can directly react the biological activity of IFN-α.
IFN-α can activate Mx promoters (Mx promoter, Mxp) through JAK-STAT signal transduction pathways, promote Mx's
Expression, Mx can inhibit minus-stranded rna virus to replicate.There are two kinds of albumen of Mx and Mx2, wherein Mx to play a major role in chicken.Mxp
There is preferable specificity, it cannot be started by other cell factors such as interleukins and tumor necrosis factor, can be accurately anti-
Answer IFN-α biological function.
Detection interferon biological activity mainly has 3 kinds of methods at present：Virus replication inhibits, plaque reduces analysis and cell
Lesion inhibits.But this 3 kinds of methods all easy to produce large error, and repeatability is bad, and accuracy is low.Nearest Mxp-Luciferase
(luciferase) system has begun to detect for I type interferon biological activities, which is IFN activation Mxp
False positive is not present in ability；Whole process avoids the use of the live virus such as VSV completely, not the misgivings of bio-safety；Detect journey
Sequence simplifies, and the time greatly shortens, and detection can be completed in about 6h after IFN handles cell, and detection speed is fast；The expression of luciferase
Precise quantification can be detected by instrument, not only increase the quantitative accuracys of IFN, the high-throughput behaviour for a large amount of samples of being more convenient for
Make.But this method is based on based on plasmid transient transfection, needs to prepare internal reference plasmid every time, and ensure plasmid transfection efficiency one
Cause can just access accurate testing result, therefore be difficult to carry out, and need to use luciferase substrate in common laboratory,
Increase use cost.
Invention content
In view of the problems of the above-mentioned prior art, green fluorescent protein is utilized the object of the present invention is to provide a kind of
(EGFP) chicken interferon α biological activity detection methods, this method do not need internal reference plasmid, can simplify detection process,
It need not repeat plasmid transfection, improve accuracy and repeatability, practicability are stronger.
The purpose of the present invention is achieved by the following technical programs：
A kind of chicken interferon α biological activity detection methods comprising following steps：
The genetic fragment of the Mxp of chicken Mx albumen is obtained using PCR amplification；
The genetic fragment of the Mxp of the chicken Mx albumen obtained with PCR amplification is replaced original pEGFP-N1 by T4DNA ligases and is carried
The genetic fragment of pCMV in constitution grain builds pEGFP-N1-Mxp plasmids；
With pEGFP-N1-Mxp plasmid-transfected cells, and stable transfected cells strain is filtered out by neomycin；
Colonized culture is carried out to the stable transfected cells strain filtered out；
Standard curve is prepared with the chicken interferon α standard items of gradient dilution, is determined between interferon potency and fluorescent value
Mathematical logic relationship；
Chicken interferon α samples to be checked are added in the cell after colonized culture, then fluorescence intensity, combined standard
The potency of curve evaluation chicken interferon α samples to be checked.
In above-mentioned chicken interferon α biological activity detection methods, colonized culture is added in chicken interferon α samples to be checked
In cell afterwards, the incubation regular hour is needed, general 6 hours or so.
In above-mentioned chicken interferon α biological activity detection methods, chicken interferon α is added in stable transfected cells strain,
Mx gene promoter activities can be activated to promote the expression of intracellular EGFP, cell sends out the strong of fluorescence after being irradiated by excitation light source
It spends and is proportionate with the biological activity of chicken interferon α, therefore can be with the biological activity of quantitative assessment chicken interferon α.
In the present invention, Mxp refers to chicken Mx gene promoter regions, and pCMV refers to the CMV promoter area of pEGFP-N1 plasmids
Domain.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that obtain chicken Mx albumen using PCR amplification
The step of genetic fragment of Mxp includes：
According to the gene order for the chicken Mx albumen announced in Genebank, the ends 5' is selected to contain opening for ISRE response elements
PCR primer is designed in sub-area, carries out PCR amplification；
Then by double digestion after, then pass through gel extraction purifying obtain Mxp genetic fragment.
In above-mentioned chicken interferon α biological activity detection methods, the method for designing PCR primer is conventional.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the chicken Mx albumen obtained with PCR amplification
The genetic fragment of Mxp replaces the genetic fragment of the pCMV in original pEGFP-N1 vector plasmids, structure by T4DNA ligases
The step of pEGFP-N1-Mxp plasmids includes：
Remove the genetic fragment of the pCMV of pEGFP-N1 vector plasmids；
The gene piece of the pCMV in original pEGFP-N1 vector plasmids is replaced with the genetic fragment of Mxp by T4DNA ligases
Section, construction recombination plasmid pEGFP-N1-Mxp；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, recombinant plasmid is extracted, recombinant plasmid is obtained by DNA sequencing
Mxp segments DNA sequence dna, select the recombinant plasmid of correct sequence；
The sequence is as shown in sequence 1.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that thin with pEGFP-N1-Mxp plasmid transfections
Born of the same parents, and the step of filtering out stable transfected cells strain by neomycin includes：
By transfection reagent by pEGFP-N1-Mxp plasmid transfections to cell, after transfecting 96h or so, change into left containing 2 μ g/mL
The complete medium of right neomycin is persistently cultivated 14, filters out the cell with neomycin resistance, as stable transfection is thin
Born of the same parents' strain.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the colonized culture will filter out
Stable transfected cells strain is by limiting dilution cultivation culture, to obtain the clone of individual cells formation.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that in the stable transfected cells strain to filtering out
Further include being detected to the cell after clone in the step of carrying out colonized culture, by being in PCR identification of cell genomes
It is no to contain Mxp genes.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the gene fragment order of Mxp is 1 institute of sequence
Show.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the pCMV of removal pEGFP-N1 vector plasmids
Method be double digestion method.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the cell is that (chicken embryo is at fibre for CEF cells
Tie up cell).
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the chicken interferon α standard items are to announce
Number be CN105039474A a kind of revealed recombination that the preparation method of recombined chicken alpha interferon standard items is prepared of patent
Chicken alpha interferon.
The present invention also provides above-mentioned chicken interferon α biological activities detection methods to natural or recombination chicken interferon-' alpha '
Carry out the application in biological activity detection.
The present invention constructs the reporter plasmid for starting EGFP expression using chicken Mx gene promoters as promoter, transfection
CEF cells screen stable transfected cells strain, with the recombination chicken interferon-' alpha ' standard items and measuring samples of gradient dilution respectively with carefully
After born of the same parents are incubated altogether, the luminous value of EGFP in cell is detected, it is to be checked to detect to prepare standard curve with recombination chicken interferon-' alpha ' standard items
The biological activity of chicken interferon α in sample.The detection method mainly has following characteristics：1. being greatly shortened compared with conventional method
Detection time (being foreshortened to 6-8 hours by traditional 3-7 days) eliminates Virus culture or repeats plasmid transfection etc. and is more numerous
Trivial operation, it is only necessary to cultivate cell, avoid bio-safety harm that may be present, improve repeatability.2. with
Luciferase reporter gene detecting system reduces cost compared to that need not use substrate.
The present invention protrusion effect be：
The present invention replaces luciferase to build Mxp-EGFP Reporter Systems using green fluorescent protein (EGFP), this is
System does not need internal reference plasmid, therefore can stablize Mxp-EGFP Reporter Systems and be expressed in cell, and due to inspection
The progress of survey technology, EGFP can be quantified and be detected in cell.Every time only need to cultivate the cell, be used in combination interferon-' alpha ' handle 6h after
Interferon activity detection can be completed in fluorescence intensity under specific instrument, greatly simplifies detection process, shortens detection
Time improves accuracy, has more practicability, is suitble to apply in large-scale production.
Description of the drawings
Fig. 1 is the pEGFP-N1-Mxp plasmid construction schematic diagrames of embodiment 1；
Fig. 2 is the EGFP reporter gene method canonical plottings of embodiment 1；
Fig. 3 is figure compared with the EGFP reporter genes method of embodiment 2 inhibits the correlation of testing result with few cells lesion.
Specific implementation mode
In order to which technical characteristic, purpose and the advantageous effect to the present invention are more clearly understood, now to the skill of the present invention
Art scheme carry out it is described further below, but should not be understood as to the present invention can practical range restriction.Institute in following embodiments
Experimental method is stated, is conventional method unless otherwise specified；The reagent and material unless otherwise specified can be from business ways
Diameter obtains.
Present invention material as used in the following examples is as follows：
CEF cells be laboratory according to routine culture, VSV viruses are provided by Wuhan Virology Institute,Chinan academy of Sciences, very
Nuclear expression carrier pEGFP-N1 is purchased from Clontech companies, various restriction enzymes, Ex-Taq enzymes, T4DNA ligases and matter
Grain extracts kit is purchased from Takara companies, and transfection reagent Fugen6 is purchased from Promega companies, Opti-MEM, DMEM culture medium
Invitrogen companies are purchased from FBS, various specifications tissue culture plate is purchased from Corning companies, and multi-function microplate reader is purchased from MD
Company, Luciferase Assay Reagent box are purchased from Promega companies；
Chicken interferon α standard items (108U/mL) it is the revealed a kind of recombination of patent that publication No. is CN105039474A
The recombined chicken alpha interferon (mode of selection embodiment 1) that the preparation method of chicken alpha interferon standard items is prepared.
Embodiment 1
It is the biology to recombinating chicken interferon α the present embodiment provides a kind of chicken interferon α biological activity detection methods
A kind of application that activity is detected is learned, the detection method of the present embodiment can also be correspondingly used for the work of natural chicken interferon α
Property detection comprising following steps：
According to the gene order for the chicken Mx albumen announced in Genebank, the ends 5' is selected to contain opening for ISRE response elements
Sub-area designs PCR primer, extracts DNA from CEF cells by phenol-chloroform-isoamyl alcohol method and be used as template, use is above-mentioned
PCR primer and Ex-Taq enzymes carry out PCR amplification (underscore part is upstream and downstream primer position)；
By gel extraction purifying, (in upstream, PCR draws the product that PCR amplification obtains again after Ase I and Age I double digestions
Object introduces Ase I restriction enzyme sites, and downstream PCR primer introduces Age I restriction enzyme sites, and ifn response element wherein included is shown in slightly
Body character segment), the genetic fragment of Mxp is obtained, the gene fragment order of Mxp is shown in sequence 1；
The genetic fragment of the pCMV of pEGFP-N1 vector plasmids is removed using double digestion method；
The genetic fragment for the Mxp for being obtained above-mentioned gel extraction by T4DNA ligases is replaced in original pEGFP-N1 plasmids
The genetic fragment of pCMV converts DH5 α competent cells, shakes bacterium, extract plasmid after filtering out positive colony, obtained by DNA sequencing
The DNA sequence dna of Mxp is obtained, it is pEGFP-N1-Mxp plasmids to select the correct plasmid of sequence, and structure schematic diagram is as shown in Figure 1.
With pEGFP-N1-Mxp plasmid-transfected cells, and stable transfected cells strain sample is filtered out by neomycin：Using
It goes endotoxin plasmid extraction kit to extract pEGFP-N1-Mxp plasmids, takes 1 μ g plasmids to be placed in 1.5mL sterile EP tubes and use Opti
MEM culture mediums supply volume to 500 μ l, and soft mixing is incubated at room temperature 5min；1.5mL sterilizing EP pipes are taken, 5 μ l Fugen6 are added
It is dissolved in 500 μ l serum-free Opti-MEM culture mediums, soft mixing, is incubated at room temperature 5min；Plasmid solution and transfection reagent is molten
The soft mixing of liquid is incubated at room temperature 20min；With in serum-free Opti-MEM culture medium secondary culture CEF cells to 6 orifice plates, per hole 5
×105A cell；Plasmid-transfection reagent compound is added to every hole, contains 5%CO in 37 DEG C2Overnight incubation in incubator；Next day,
Culture medium is removed, instead DMEM (containing Sodium Pyruvate and nonessential amino acid) complete medium continues to cultivate 96h；It uses instead later
Complete medium screening and culturing containing 2 μ g/mL neomycins 14 days replaces primary fresh culture medium for every 2 days；Period visual cell gives birth to
Long situation decides whether to pass on；The cell survived after neomycin acts on 14 is stable transfected cells；
Screening, which is obtained, has the cell of neomycin resistance to be diluted to carefully with the complete medium containing 0.25 μ g/mL puromycins
A concentration of 10/mL of born of the same parents；0.1mL cell suspensions are added per hole into 96 orifice plates, contain 5%CO in 37 DEG C2It is cultivated in incubator；Often
Day observation selects the hole individually cloned and cell is transferred in 6 orifice plates and is passed through when culture to cell covers aperture surface area 1/3
Limiting dilution cultivation expands culture, and by whether containing Mxp genes in PCR identification of cell genomes, freezes conservation, name
For CEF-pEGFP-N1-Mxp.
Chicken interferon α standard items 1mL are taken, according to 1:10,1:100,1:103…1:107Gradient complete medium it is dilute
It releases；
The stable transfected cells strain CEF-pEGFP-N1-Mxp of 0.1mL is seeded in 96 porocyte culture plates and is incubated overnight
So that cell density is reached 90%, discards culture medium；The recombination chicken interferon after 0.1mL is diluted with complete medium is added to every hole
α standard items, 3 parallel holes of each dilution, setting are not added with the hole of recombination chicken interferon-' alpha ' as a contrast, contain 5%CO in 37 DEG C2
Continue to cultivate 6h in incubator, take out culture plate, be placed on multi-function microplate reader monitor station, a length of 488nm of excitation light wave is set,
It is 597nm, the irradiation of back light source to receive wavelength.As a result testing result is shown with recombination chicken as shown in Fig. 2, obtain standard curve
The gradient dilution fluorescence intensity of interferon-' alpha ' continuously decreases, negative pair of the dilution of fluorescence intensity and recombination chicken interferon-' alpha ' standard items
Number is in significant exponential relationship R2=0.99, detection range 0.1U-108U/mL。
Chicken interferon α samples to be checked are added in the cell after colonized culture, then fluorescence intensity, combined standard
Curve evaluation obtains the potency of chicken interferon α samples to be checked.
It is reproducible it can be seen that do not need internal reference plasmid using the detection method that builds of the present invention, not by other cells because
Son influences.
Embodiment 2
The present embodiment provides the testing results of chicken interferon α biological activity detection methods of the present invention and few cells lesion
The comparison correlation of inhibition method testing result is tested.
Method detection is inhibited using EGFP reporter genes method and few cells lesion while detecting 20 parts of recombination chicken interferon-' alpha ' marks
Whether this, have correlation by two methods of the result of linear regression analysis.
EGFP reporter gene method testing processes are as described in Example 1.
Few cells lesion inhibits method by following operation：Chicken interferon α sample 1mL are taken, with complete medium 1:100 dilutions
Use complete medium doubling dilution again afterwards；CEF secondary cultures are seeded to 96 porocyte culture plates, 100 μ l cells are inoculated with per hole
Suspension (2 × 105A/mL)；Add the every holes 00 μ l of recombination chicken Interferon α1 of different dilutions, virus control and cell controls
100 μ l culture mediums are added, contain 5%CO in 37 DEG C2Continue to cultivate 16h in incubator；Culture medium is discarded, in addition to cell control well,
The VSV (the VSV virus titers that CEF cells can for 24 hours completely fallen off) of 100 μ l appropriate titers is added per hole, contains 5% in 37 DEG C
CO2Continue culture in incubator for 24 hours, the clasmatosis of microscopically observation virus control wells up to 100% and when normal cell controls,
I.e. available violet staining visually observes record after washing, cell is in completely purple, virus control non-coloring in cell control well
(100%CPE++++) shows that system is set up, and observes coloring case in different holes on its basis, records CPE situations, passes through
Reed-Muench methods calculate the extension rate of half cytopathic effect inhibition, to obtain the potency of interferon.
The results are shown in Figure 3, the Correlation analysis showed R of two kinds of detection methods2> 0.9 shows that the two has very high phase
Guan Xing.Should the result shows that, interferon activity detection method that the present invention establishes is simple, quick, and testing result is reliable, stablizes, can
This traditional detection method of method is inhibited to be applied to natural or recombination chicken interferon-' alpha ' determination of activity to substitute few cells lesion.
In conclusion the embodiment of the present invention replaces luciferase structure Mxp-EGFP to report using green fluorescent protein (EGFP)
Genic system is accused, this system does not need internal reference plasmid, therefore can stablize Mxp-EGFP Reporter Systems and be expressed in carefully
In born of the same parents, and due to the progress of detection technique, EGFP can be quantified and be detected in cell.The cell only need to be cultivated every time, is used in combination dry
Disturb after element-α processing 6h that antiviral activity of interferon detection can be completed in fluorescence intensity under specific instrument, it is greatly simple
Detection process is changed, has shortened detection time, improve accuracy, has had more practicability, be suitble to apply in large-scale production.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
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<120>A kind of chicken interferon α biological activity detection methods
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Claims (10)
1. a kind of chicken interferon α biological activity detection methods comprising following steps：
The genetic fragment of the Mxp of chicken Mx albumen is obtained using PCR amplification；
The genetic fragment of the Mxp of the chicken Mx albumen obtained with PCR amplification replaces original pEGFP-N1 carrier matter by T4DNA ligases
The genetic fragment of pCMV in grain builds pEGFP-N1-Mxp plasmids；
With pEGFP-N1-Mxp plasmid-transfected cells, and stable transfected cells strain is filtered out by neomycin；
Colonized culture is carried out to the stable transfected cells strain filtered out；
Standard curve is prepared with the chicken interferon α standard items of gradient dilution, determines the mathematics between interferon potency and fluorescent value
Logical relation；
Chicken interferon α samples to be checked are added in the cell after colonized culture, then fluorescence intensity, combined standard curve
Evaluate the potency of chicken interferon α samples to be checked.
2. chicken interferon α biological activity detection methods according to claim 1, which is characterized in that obtained using PCR amplification
The step of genetic fragment for taking the Mxp of chicken Mx albumen includes：
According to the gene order for the chicken Mx albumen announced in Genebank, the promoter for selecting the ends 5' to contain ISRE response elements
PCR primer is designed in region, carries out PCR amplification；
Then by double digestion after, then pass through gel extraction purifying obtain Mxp genetic fragment.
3. chicken interferon α biological activity detection methods according to claim 1, which is characterized in that obtained with PCR amplification
Chicken Mx albumen Mxp genetic fragment pass through T4DNA ligases replace original pEGFP-N1 vector plasmids in pCMV gene
Segment, build pEGFP-N1-Mxp plasmids the step of include：
Remove the genetic fragment of the pCMV of pEGFP-N1 vector plasmids；
The genetic fragment of the pCMV in original pEGFP-N1 vector plasmids, structure are replaced with the genetic fragment of Mxp by T4DNA ligases
Build recombinant plasmid pEGFP-N1-Mxp；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, extracts recombinant plasmid, the Mxp of recombinant plasmid is obtained by DNA sequencing
The DNA sequence dna of segment selects the recombinant plasmid of correct sequence；
The correct sequence is as shown in sequence 1.
4. chicken interferon α biological activity detection methods according to claim 1, which is characterized in that use pEGFP-N1-
Mxp plasmid-transfected cells, and the step of filtering out stable transfected cells strain by neomycin includes：
PEGFP-N1-Mxp plasmid transfections to cell are changed into after transfecting 96h containing the complete of 2 μ g/mL neomycins by transfection reagent
Full culture medium is persistently cultivated 14, and the cell with neomycin resistance, as stable transfected cells strain are filtered out.
5. chicken interferon α biological activity detection methods according to claim 1, which is characterized in that the cloning training
Support for by the stable transfected cells strain filtered out by limiting dilution cultivation culture, to obtain gram of individual cells formation
It is grand；
Preferably, further include to thin after clone in the step of stable transfected cells strain to filtering out carries out colonized culture
Born of the same parents are detected, by whether containing Mxp genes in PCR identification of cell genomes.
6. chicken interferon α biological activity detection methods according to claim 1 or 2, it is characterised in that：The gene of Mxp
Fragment sequence is shown in sequence 1.
7. chicken interferon α biological activity detection methods according to claim 3, it is characterised in that：Remove pEGFP-N1
The method of the pCMV of vector plasmid is double digestion method.
8. chicken interferon α biological activity detection methods according to claim 1 or 4, it is characterised in that：The cell is
CEF cells.
9. chicken interferon α biological activity detection methods according to claim 1, it is characterised in that：The chicken interference
Plain α standard items are the revealed a kind of preparation side of recombined chicken alpha interferon standard items of patent that publication No. is CN105039474A
The recombined chicken alpha interferon that method is prepared.
10. claim 1-9 any one of them chicken interferon α biological activities detection methods are to the interference of natural or recombination chicken
Plain α carries out the application in biological activity detection.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112760291A (en) * | 2020-12-25 | 2021-05-07 | 山东晶辉生物技术有限公司 | Cell line for detecting human interferon-kappa activity and construction and detection method thereof |
| CN114324282A (en) * | 2021-12-30 | 2022-04-12 | 青岛嘉智生物技术有限公司 | Safety evaluation method of veterinary astragalus polysaccharide injection |
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| CN113046387A (en) * | 2019-12-27 | 2021-06-29 | 华南农业大学 | Method for detecting chicken IFN-alpha biological activity by using dual-luciferase reporter gene |
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2017
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112760291A (en) * | 2020-12-25 | 2021-05-07 | 山东晶辉生物技术有限公司 | Cell line for detecting human interferon-kappa activity and construction and detection method thereof |
| CN112760291B (en) * | 2020-12-25 | 2022-12-23 | 山东睿鹰制药集团有限公司 | Cell line for detecting human interferon-kappa activity and construction and detection method thereof |
| CN114324282A (en) * | 2021-12-30 | 2022-04-12 | 青岛嘉智生物技术有限公司 | Safety evaluation method of veterinary astragalus polysaccharide injection |
| CN114324282B (en) * | 2021-12-30 | 2023-06-27 | 青岛嘉智生物技术有限公司 | Veterinary astragalus polysaccharide injection safety evaluation method |
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Application publication date: 20181113 |