CN107916301A - A kind of Bov IFN α biological activity detection methods - Google Patents
A kind of Bov IFN α biological activity detection methods Download PDFInfo
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Abstract
The invention discloses a kind of Bov IFN α biological activities detection method and its application.This method comprises the following steps：The genetic fragment of the Mxp of ox Mx albumen is obtained using PCR amplification；Remove the pCMV of pEGFP N1 vector plasmids；The genetic fragment of the Mxp of the ox Mx albumen obtained with PCR amplification replaces the pCMV in 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, Bov IFN α is added to be incubated altogether with the stable transfected cells strain after colonized culture, the expression that Mx gene promoter activities promote intracellular EGFP can be activated, cell sends the intensity of fluorescence after being irradiated by excitation source and the biological activity of Bov IFN α is proportionate, therefore can be with the biological activity of quantitative assessment Bov IFN α.
Description
Technical field
The present invention relates to a kind of Bov IFN 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 promotees 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 types interferon includes IFN-α, IFN-β,With IFN- τ；II types are done
Disturb element i.e. IFN-γ；III interferon, that is, IL-28A, IL-28B and IL-29.
IFN-α has four major functions.First, the cell and its peripheral cell that can make virus infection resist into endogenous
Virus State, the propagation of limiting virus；Second, maintain innate immune reaction to be in equilibrium state, promote antigen submission and NK thin
Suppress the generation of pro-inflammatory signals path and cell factor while born of the same parents' function；3rd, acquired immune system is activated, 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 suppressing virus multiplication
With.
The mechanism of action of IFN-α illustrated before 25 years, IFN JAK1s and TYK2 related to acceptor combination activated receptor,
Make STAT1 and STAT2 tyrosine phosphorylations, the STAT1 and STAT2 of phosphorylation form dimer and be transferred to core, assemble 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) combine, ISGs transcriptions are directly activated, host cell is entered antiviral state.ISG suppresses the approach master of virus
Have, suppress transcription, translation and the nucleic acid replication of virus；Degraded viral nucleic acid；Change host cell lipid metaboli.Therefore, as long as
The biological activity of IFN-α can directly be reacted by detecting the promoter activity increase of ISGs.
IFN-α can activate Mx promoters (Mx promoter, Mxp) through JAK-STAT signal transduction pathways, promote Mx's
Expression, Mx can suppress minus-stranded rna virus duplication.There are two kinds of albumen of Mx and Mx2, wherein Mx in ox to play a major role.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 suppresses, plaque reduces analysis and cell
Lesion suppresses.But this 3 kinds of methods all easily produce large error, and repeatability is bad, and accuracy is low.Nearest Mxp-Luciferase
(luciferase) system has begun to be used for the detection of I types interferon biological activity, which is IFN activation Mxp
Ability, there is no false positive；Whole process avoids the use of the live viruses such as VSV completely, without the misgivings of bio-safety；Detect journey
Sequence simplifies, and the time greatly shortens, and about 6h can complete to detect 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, need 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 in common laboratory, and need to use luciferase substrate,
Add use cost.
The content of the invention
In view of the problems of the above-mentioned prior art, the object of the present invention is to provide one kind to utilize green fluorescent protein
(EGFP) Bov IFN α biological activity detection methods, this method are not required internal reference plasmid, can simplify detection process,
It need not repeat plasmid transfection, improve accuracy and repeatability, practicality are stronger.
The purpose of the present invention is achieved by the following technical programs：
A kind of Bov IFN α biological activity detection methods, it comprises the following steps：
The genetic fragment of the Mxp of ox Mx albumen is obtained using PCR amplification；
The genetic fragment of the Mxp of the ox 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 Bov IFN α standard items of gradient dilution, is determined between interferon potency and fluorescent value
Mathematical logic relation；
Bov IFN α samples to be checked are added in the cell after colonized culture, then fluorescence intensity, combined standard
The potency of curve evaluation Bov IFN α samples to be checked.
In above-mentioned Bov IFN α biological activity detection methods, Bov IFN α samples to be checked are added into colonized culture
, it is necessary to be incubated the regular hour in cell afterwards, it is general 6 it is small when or so.
In above-mentioned Bov IFN α biological activity detection methods, Bov IFN α is added in stable transfected cells strain,
The expression that Mx gene promoter activities promote intracellular EGFP can be activated, cell sends the strong of fluorescence after being irradiated by excitation source
Spend and be proportionate with the biological activity of Bov IFN α, therefore can be with the biological activity of quantitative assessment Bov IFN α.
In the present invention, Mxp refers to ox Mx gene promoter regions, and pCMV refers to the CMV promoter area of pEGFP-N1 plasmids
Domain.
In above-mentioned Bov IFN α biological activity detection methods, it is preferred that obtain ox Mx albumen using PCR amplification
The step of genetic fragment of Mxp, includes：
According to the gene order for the ox Mx albumen announced in Genebank, 5' ends are selected to contain opening for ISRE response elements
Sub-area, designs PCR primer, carries out PCR amplification；
Then by double digestion after, then by gel extraction purify obtain Mxp genetic fragment.
In above-mentioned Bov IFN α biological activity detection methods, the method for designing PCR primer is conventional.
In above-mentioned Bov IFN α biological activity detection methods, it is preferred that the ox 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, shakes bacterium, recombinant plasmid is extracted, recombinant plasmid is obtained by DNA sequencing
Mxp fragments DNA sequence dna, select the recombinant plasmid of correct sequence；
The sequence is as shown in sequence 1.
In above-mentioned Bov IFN 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 stable transfected cells strain is filtered out by neomycin include：
By transfection reagent by after pEGFP-N1-Mxp plasmid transfections to cell, transfection 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, is that stable transfection is thin
Born of the same parents' strain.
In above-mentioned Bov IFN 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, so as to obtain the clone of individual cells formation.
In above-mentioned Bov IFN α biological activity detection methods, it is preferred that in the stable transfected cells strain to filtering out
Further included in the step of carrying out colonized culture and the cell after clone is detected, by being in PCR identification of cell genomes
It is no to contain Mxp genes.
In above-mentioned Bov IFN α biological activity detection methods, it is preferred that the gene fragment order of Mxp is the institute of sequence 1
Show.
In above-mentioned Bov IFN α biological activity detection methods, it is preferred that remove the pCMV of pEGFP-N1 vector plasmids
Method be double digestion method.
In above-mentioned Bov IFN α biological activity detection methods, it is preferred that (ox kidney is thin for MDBK cells for the cell
Born of the same parents).
In above-mentioned Bov IFN α biological activity detection methods, it is preferred that the Bov IFN α standard items are announcement
Number for CN106319006A a kind of restructuring that is prepared of the revealed preparation method of recombinant bovine alpha interferon standard substance of patent
Ox alpha interferon.
The present invention also provides above-mentioned Bov IFN α biological activities detection method to natural or recombinant bovine interferon-' alpha '
Carry out the application in biological activity detection.
The present invention constructs the reporter plasmid for starting EGFP expression using ox Mx gene promoters as promoter, transfection
MDBK cells, screen stable transfected cells strain, with the recombinant bovine 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 recombinant bovine interferon-' alpha ' standard items
The biological activity of Bov IFN α in sample.The detection method mainly has following characteristics：1. greatly shortened compared with conventional method
Detection time (by foreshorten within traditional 3-7 days 6-8 it is small when), eliminate Virus culture or to repeat plasmid transfection etc. 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 structure Mxp-EGFP Reporter Systems using green fluorescent protein (EGFP), this is
Internal reference plasmid is not required in system, 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 detected in cell.Only need to cultivate the cell every time, and with interferon-' alpha ' handle after 6h
Fluorescence intensity can complete interferon activity detection under specific instrument, greatly simplifie detection process, shorten detection
Time, improves accuracy, with more practicality, is adapted to apply in large-scale production.
Brief 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 EGFP reporter genes method and the few cells lesion of embodiment 2 suppress the correlation of testing result.
Embodiment
In order to which technical characteristic, purpose and the beneficial effect of the present invention is more clearly understood, now to the skill of the present invention
Art scheme carry out it is described further below, but it is not intended that 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 way
Footpath obtains.
Material used is as follows in the following embodiments of the present invention：
MDBK cells (ATCC No.CCL-22) are purchased from ATCC, and VSV viruses are carried by Wuhan Virology Institute,Chinan academy of Sciences
For carrier for expression of eukaryon pEGFP-N1 is purchased from Clontech companies, various restriction enzymes, Ex-Taq enzymes, T4DNA ligases
Takara companies are purchased from plasmid extraction kit, transfection reagent Fugen6 is purchased from Promega companies, Opti-MEM, DMEM training
Support base and FBS is purchased from Invitrogen companies, various specifications Tissue Culture Plate is purchased from Corning companies, multi-function microplate reader purchase
From MD companies, Luciferase Assay Reagent box is purchased from Promega companies；
Bov IFN α standard items (107IU/mL it is) that revealed one kind of patent that publication No. is CN106319006A recombinates
The recombinant bovine alpha interferon (mode of selection embodiment 1 or 2) that the preparation method of ox alpha interferon standard substance is prepared.
Embodiment 1
The present embodiment provides a kind of Bov IFN α biological activity detection methods, it is the biology to recombinating Bov IFN α
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 Bov IFN α
Property detection, it includes the following steps：
According to the gene order for the ox Mx albumen announced in Genebank, 5' ends are selected to contain opening for ISRE response elements
Sub-area, designs PCR primer, extracts DNA from MDBK 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)；
The product that PCR amplification obtains purifies that (PCR draws in upstream by gel extraction again after Ase I and Age I double digestions
Thing 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 it is as shown in Figure 1 that it builds schematic diagram.
With pEGFP-N1-Mxp plasmid-transfected cells, and stable transfected cells strain sample is filtered out by neomycin：Using
Go endotoxin plasmid extraction kit to extract pEGFP-N1-Mxp plasmids, take 1 μ g plasmids to be placed in the sterile EP pipes of 1.5mL and use Opti
MEM culture mediums supply volume to 500 μ l, soft to mix, incubation at room temperature 5min；1.5mL sterilizing EP pipes are taken, add 5 μ l Fugen6
It is dissolved in 500 μ l serum-free Opti-MEM culture mediums, it is soft to mix, it is incubated at room temperature 5min；Plasmid solution and transfection reagent is molten
Liquid softly mixes, and is incubated at room temperature 20min；With serum-free Opti-MEM culture medium secondary culture MDBK cells into 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；It is secondary
Day, culture medium is removed, instead DMEM (containing Sodium Pyruvate and nonessential amino acid) complete medium continues to cultivate 96h；Afterwards
Use the complete medium screening and culturing 14 days containing 2 μ g/mL neomycins instead, replace once fresh culture medium within every 2 days；Period regards thin
Intracellular growth situation decides whether to pass on；The cell survived after neomycin acts on 14 is stable transfected cells；
The cell that acquisition, which will be screened, neomycin resistance is diluted to carefully with the complete medium containing 0.25 μ g/mL puromycins
Born of the same parents' concentration is 10/mL；0.1mL cell suspensions are added per hole into 96 orifice plates, contain 5%CO in 37 DEG C2Cultivated in incubator；Often
Day observation, selects the hole of single clone, when cultivating to cell covering aperture surface area 1/3, cell is transferred in 6 orifice plates and is passed through
Limiting dilution cultivation expands culture, and by whether containing Mxp genes in PCR identification of cell genomes, freezes conservation, names
For MDBK-pEGFP-N1-Mxp.
Bov IFN α standard items 1mL are taken, according to 1:10、1:100、1:103…1:107Gradient complete medium it is dilute
Release；
The stable transfected cells strain MDBK-pEGFP-N1-Mxp of 0.1mL is seeded in 96 porocyte culture plates and is trained overnight
Supporting makes cell density reach 90%, discards culture medium；The recombinant bovine added to every hole after 0.1mL is diluted with complete medium disturbs
Plain α standard items, each 3 parallel holes of dilution factor, set the hole for being not added with recombinant bovine interferon-' alpha ' to contain 5% in 37 DEG C as control
CO2Continue to cultivate 6h in incubator, take out culture plate, be placed on multi-function microplate reader monitor station, set excitation light wave a length of
488nm, it is 597nm to receive wavelength, and back light source is irradiated.Testing result as shown in Fig. 2, obtain standard curve, the results show with
The gradient dilution fluorescence intensity of recombinant bovine interferon-' alpha ' gradually reduces, fluorescence intensity and the dilution factor of recombinant bovine interferon-' alpha ' standard items
Negative logarithm be in significant exponential relationship R2=0.9943, detection range 0.1IU-107IU/mL。
Bov IFN α samples to be checked are added in the cell after colonized culture, then fluorescence intensity, combined standard
Curve evaluation obtains the potency of Bov IFN α samples to be checked.
It is reproducible it can be seen that internal reference plasmid is not required 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 result and few cells lesion of Bov IFN α biological activity detection methods of the present invention
The contrast correlation experiment of suppression method testing result.
Method detection is suppressed using EGFP reporter genes method and few cells lesion and detects 20 parts of recombinant bovine interferon-' alpha ' marks at the same time
Whether this, have correlation by two methods of the result of linear regression analysis.
EGFP reporter gene methods testing process is as described in Example 1.
Few cells lesion suppresses method by following operation：Bov IFN α sample 1mL are taken, with complete medium 1:100 dilutions
Use complete medium doubling dilution again afterwards；MDBK secondary cultures are seeded to 96 porocyte culture plates, 100 μ l cells are inoculated with per hole
Suspension (2 × 105A/mL)；The 00 μ l of recombinant bovine Interferon α1 of different dilution factors are added per hole, 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 (24h can make the VSV virus titers that MDBK cells are completely fallen off) of 100 μ l appropriate titers is added per hole, contains 5% in 37 DEG C
CO2Continue to cultivate 24h in incubator, micro- Microscopic observation virus control wells clasmatosis up to 100% and during normal cell controls,
I.e. available violet staining, visually observes record after washing, cell is in completely purple in cell control well, virus control non-coloring
(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, so as 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 both have very high phase
Guan Xing.Should the result shows that, the interferon activity detection method established of the present invention is simple, quick, and testing result is reliable, stablizes, can
Suppress this traditional detection method of method applied to natural or recombinant bovine interferon-' alpha ' determination of activity to substitute few cells lesion.
In conclusion the embodiment of the present invention replaces luciferase structure Mxp-EGFP reports using green fluorescent protein (EGFP)
Genic system is accused, internal reference plasmid is not required in the system, 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 detected in cell.Only it need to cultivate the cell every time, and with doing
Disturb after element-α processing 6h that fluorescence intensity can complete antiviral activity of interferon detection under specific instrument, it is greatly simple
Detection process is changed, has shortened detection time, improve accuracy, with more practicality, be adapted to apply in large-scale production.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
<110>Wuhu Co., Ltd of Ying Tefeier biological products industrial research institute
<120>A kind of Bov IFN α biological activity detection methods
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 786
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
attaatgcca ggaacttcag aagggccgga taaagctcgg gtagctgggt cctgcgcccg 60
agtttctgag gcagcaggtc tgggtgcggg ccgagaatct gcatttcccg caagctccta 120
gggatgctgt tgatgcgggg cgcacctaga atgcctttgg gagaatacag ttgagtgtgc 180
ggggcgcagc gacctcggga ggccccggtg cggaagtgcg ccacccgttc ggtttcggtt 240
tcctttccga tccagcagcc ctgaaaactt tctgagtttc gtttctccga ggctgggtag 300
gagatgaggg acggggaggc ggggcagcga gctaggggcg gcgttagcgc tgaataaagc 360
cgaggagggt gagcgctggg agcctgccgg tctctgcgct ggggacgggt aagtgtagcg 420
ctcctgggtc tgggcactct ccgcgggtcg gggcatttgg gcgttggctg ggagttgagc 480
gctctggggt gccggtattg cgcagactag gagaaggcga tggcacccca ctccagtact 540
cttgcctgga aaatcccatg gacggaggag cctggtaggc tgcagtccat ggagtctcga 600
aaagtcggac accacagaag cgacttagca gcagcagcag cagtaatagt aaaaatgaaa 660
atgattaata gtgatggcaa cccagtattc ttgcctggag aatgccatgg acagaggaac 720
ctggtgggct gcagtccatg gggtctcaaa gagtcggaca cgactgaagc gacttagcac 780
accggt 786
Claims (10)
1. a kind of Bov IFN α biological activity detection methods, it comprises the following steps：
The genetic fragment of the Mxp of ox Mx albumen is obtained using PCR amplification；
The genetic fragment of the Mxp of the ox 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 Bov IFN α standard items of gradient dilution, determines the mathematics between interferon potency and fluorescent value
Logical relation；
Bov IFN α samples to be checked are added in the cell after colonized culture, then fluorescence intensity, combined standard curve
Evaluate the potency of Bov IFN α samples to be checked.
2. Bov IFN α biological activity detection methods according to claim 1, it is characterised in that obtained using PCR amplification
The step of genetic fragment for taking the Mxp of ox Mx albumen, includes：
According to the gene order for the ox Mx albumen announced in Genebank, the promoter for selecting 5' ends to contain ISRE response elements
Region, designs PCR primer, carries out PCR amplification；
Then by double digestion after, then by gel extraction purify obtain Mxp genetic fragment.
3. Bov IFN α biological activity detection methods according to claim 1, it is characterised in that obtained with PCR amplification
Ox Mx albumen Mxp genetic fragment pass through T4DNA ligases replace original pEGFP-N1 vector plasmids in pCMV gene
The step of fragment, structure pEGFP-N1-Mxp plasmids, includes：
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, shakes bacterium, extracts recombinant plasmid, the Mxp of recombinant plasmid is obtained by DNA sequencing
The DNA sequence dna of fragment, selects the recombinant plasmid of correct sequence；
The correct sequence is as shown in sequence 1.
4. Bov IFN α biological activity detection methods according to claim 1, it is characterised in that use pEGFP-N1-
Mxp plasmid-transfected cells, and the step of stable transfected cells strain is filtered out by neomycin include：
By transfection reagent by pEGFP-N1-Mxp plasmid transfections to cell, after transfecting 96h, change into containing the complete of 2 μ g/mL neomycins
Full culture medium is persistently cultivated 14, filters out the cell with neomycin resistance, is stable transfected cells strain.
5. Bov IFN α biological activity detection methods according to claim 1, it is characterised in that the cloning training
Support for by the stable transfected cells strain filtered out by limiting dilution cultivation culture so that obtain individual cells formation gram
It is grand；
Preferably, further included in the step of stable transfected cells strain to filtering out carries out colonized culture to thin after clone
Born of the same parents are detected, by whether containing Mxp genes in PCR identification of cell genomes.
6. Bov IFN 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. Bov IFN 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. the Bov IFN α biological activity detection methods according to claim 1 or 4, it is characterised in that：The cell is
MDBK cells.
9. Bov IFN α biological activity detection methods according to claim 1, it is characterised in that：The ox interference
Plain α standard items are the revealed a kind of preparation side of recombinant bovine alpha interferon standard substance of patent that publication No. is CN106319006A
The recombinant bovine alpha interferon that method is prepared.
10. claim 1-9 any one of them Bov IFN α biological activities detection methods are to the interference of natural or recombinant bovine
Plain α carries out the application in biological activity detection.
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