CN107858450A - A kind of chicken interferon α biological activity detection methods - Google Patents
A kind of chicken interferon α biological activity detection methods Download PDFInfo
- Publication number
- CN107858450A CN107858450A CN201711264355.1A CN201711264355A CN107858450A CN 107858450 A CN107858450 A CN 107858450A CN 201711264355 A CN201711264355 A CN 201711264355A CN 107858450 A CN107858450 A CN 107858450A
- Authority
- CN
- China
- Prior art keywords
- chicken
- mxp
- interferon
- pegfp
- biological activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
Abstract
The invention discloses a kind of chicken interferon α biological activities detection method and its application.This method comprises the following steps：The genetic fragment for the Mxp for obtaining chicken Mx albumen is expanded using PCR；Remove the pCMV of pEGFP N1 vector plasmids；The Mxp of the chicken Mx albumen obtained with PCR amplifications genetic fragment replaces the pCMV of former 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, 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 chicken interferon α biological activity is proportionate, therefore can be with quantitative assessment chicken interferon α biological activity.
Description
Technical field
The present invention relates to a kind of chicken interferon α biological activity detection methods, belong to interferon activity detection technique neck
Domain.
Background technology
Interferon (IFN) is that cell and body are infected with the virus, or by nucleic acid, bacterial endotoxin and promotees cell point
After the effect for splitting element etc., by a kind of broad-spectrum antiviral glycoprotein of recipient cell secretion.According to the source of interferon and physics and chemistry
Matter, I types, II types and III interferon can be classified as.I types interferon includes IFN-α, IFN-β,With IFN- τ；II
Type interferon is IFN-γ；III interferon 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 enter endogenous
Antiviral state, the propagation of limiting virus；Second, maintain innate immune reaction to be in poised state, promote antigen submission and NK
Suppress the generation of pro-inflammatory signals path and cell factor while cell function；3rd, acquired immune system is activated, is promoted
The generation of high-affinity T cells with antigenic specificity, promote B cell reaction and immunological memory；4th, have and suppress virus multiplication
Effect.
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 TYR phosphorylations, the STAT1 and STAT2 of phosphorylation form dimer and be transferred to core, assemble IFN
Regulatory factor 9 (IRF9) forms the IFN stimulating factors 3 (ISGF3) of 3 aggressiveness.(IFN stimulates anti-ISGF3 with its homologous DNA sequence
Answer element, ISREs) combine, ISGs transcriptions are directly activated, host cell is entered antiviral state.ISG suppresses the way of virus
Footpath mainly has, and suppresses transcription, translation and the nucleic acid replication of virus；Degraded viral nucleic acid；Change host cell lipid metaboli.Therefore,
As long as detect that ISGs promoter activity increase can directly reacts 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 suppress minus-stranded rna virus duplication.Two kinds of albumen of Mx and Mx2 in chicken be present, wherein Mx plays a major role.
Mxp has preferable selectivity, and it can not be started by other cell factors such as interleukins and TNF, Neng Gouzhun
Really reaction IFN-α biological function.
Detection interferon biological activity mainly has 3 kinds of methods at present：Virus replication suppresses, plaque reduces analysis and thin
Born of the same parents' lesion suppresses.But this 3 kinds of methods all easily produce larger error, and repeatability is bad, and the degree of accuracy is low.Nearest Mxp-
Luciferase (luciferase) system has begun to be used for the detection of I types interferon biological activity, and the system detectio is
IFN activates Mxp ability, in the absence of false positive；Whole process avoids the use of the live viruses such as VSV completely, without biology peace
Full misgivings；Program simplification is detected, the time greatly shortens, and about 6h can complete to detect after IFN handles cell, detection speed
It hurry up；The expression of luciferase can detect precise quantification by instrument, not only increase the quantitative accuracys of IFN, be more convenient for big
Measure the high flux operation of sample.But this method is based on based on plasmid transient transfection, need to prepare internal reference plasmid every time, and
Ensure that plasmid transfection efficiency unanimously can just access accurate testing result, therefore be difficult to carry out in common laboratory, Er Qiexu
Luciferase substrate is used, adds use cost.
The content of the invention
In view of the defects of above-mentioned prior art is present, it is an object of the invention to provide one kind to utilize green fluorescent protein
(EGFP) chicken interferon α biological activity detection methods, this method do not need internal reference plasmid, can simplify detection process,
Plasmid transfection need not be repeated, accuracy is improved and repeatability, practicality 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, it comprises the following steps：
The genetic fragment for the Mxp for obtaining chicken Mx albumen is expanded using PCR；
The Mxp of the chicken Mx albumen obtained with PCR amplifications genetic fragment replaces former pEGFP-N1 by T4DNA ligases
The genetic fragment of pCMV in vector plasmid, build 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 relation；
Chicken interferon α samples to be checked are added in the cell after colonized culture, then fluorescence intensity, with reference to mark
Directrix curve evaluates the potency of chicken interferon α samples to be checked.
In above-mentioned chicken interferon α biological activity detection methods, chicken interferon α samples to be checked are added into cloning training
, it is necessary to be incubated the regular hour in cell after supporting, 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,
The expression that Mx gene promoter activities promote intracellular EGFP can be activated, cell sends fluorescence after being irradiated by excitation source
Intensity and chicken interferon α biological activity are proportionate, therefore can be with quantitative assessment chicken interferon α biological activity.
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 expanded using PCR and obtain chicken Mx albumen
The step of Mxp genetic fragment, includes：
According to the gene order for the chicken Mx albumen announced in Genebank, 5' ends are selected to contain ISRE response elements
Promoter region, PCR primer is designed, enter performing PCR amplification；
Then by double digestion after, then by gel extraction purify 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 of acquisition is expanded with PCR
Mxp genetic fragment replaces the genetic fragment of the pCMV in former pEGFP-N1 vector plasmids, structure by T4DNA ligases
The step of pEGFP-N1-Mxp plasmids, includes：
Remove the pCMV of pEGFP-N1 vector plasmids genetic fragment；
The gene piece of the pCMV in former pEGFP-N1 vector plasmids is replaced with Mxp genetic fragment 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 chicken interferon α biological activity detection methods, it is preferred that thin with pEGFP-N1- Mxp plasmid transfections
Born of the same parents, and include the step of filter out stable transfected cells strain by neomycin：
By transfection reagent by after pEGFP-N1-Mxp plasmid transfections to cell, transfection 96h or so, change into containing 2 μ g/mL
The complete medium of the neomycin of left and right is persistently cultivated 14, filters out the cell with neomycin resistance, as stable transfection
Cell line.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the colonized culture is to filter out
Stable transfected cells strain by limiting dilution cultivation culture, so as 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 to filtering out
Strain carries out also including detecting the cell after clone in the step of colonized culture, by PCR identification of cell genomes
Whether Mxp genes are contained.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that Mxp gene fragment order is sequence 1
It is shown.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that remove pEGFP-N1 vector plasmids
PCMV method is double digestion method.
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the cell be CEF cells (chicken embryo into
Fibrocyte).
In above-mentioned chicken interferon α biological activity detection methods, it is preferred that the chicken interferon α standard items are announcement
Number for a kind of recombined chicken alpha interferon standard items disclosed in CN105039474A patent the weight that is prepared of preparation method
Group chicken alpha interferon.
The present invention also provides above-mentioned chicken interferon α biological activities detection method 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, turns
Contaminate CEF cells, screen stable transfected cells strain, with the recombination chicken interferon-' alpha ' standard items and measuring samples of gradient dilution respectively with
After cell is incubated altogether, the luminous value of EGFP in cell is detected, standard curve is prepared to detect with recombination chicken interferon-' alpha ' standard items
Chicken interferon α biological activity in measuring samples.The detection method mainly has following characteristics：It is 1. big compared with conventional method
Detection time (foreshortening to 6-8 hours by traditional 3-7 days) is shortened greatly, Virus culture is eliminated or repeats plasmid turn
The cumbersome operations such as dye, it is only necessary to cultivate cell, avoid bio-safety harm that may be present, improving to weigh
Renaturation.2. substrate need not be used compared with luciferase reporter gene detecting system, cost is reduced.
The present invention protrusion effect be：
The present invention replaces luciferase structure Mxp-EGFP Reporter Systems using green fluorescent protein (EGFP), this
System does not need internal reference plasmid, therefore can stably be expressed in Mxp-EGFP Reporter Systems in cell, and due to
The progress of detection technique, EGFP can be quantified and detected in cell.The cell need to be only cultivated every time, and handles 6h with interferon-' alpha '
Fluorescence intensity can complete interferon activity detection under specific instrument afterwards, greatly simplifie detection process, shorten
Detection time, accuracy is improved, with more practicality, be 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 the EGFP reporter genes method of embodiment 2 compared with the correlation that few cells lesion suppresses testing result
Figure.
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
Approach obtains.
Material used is as follows in the following embodiments of the present invention：
CEF cells are that laboratory is provided according to cellar culture, VSV viruses 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
Base and FBS are purchased from Invitrogen companies, and all size Tissue Culture Plate is purchased from Corning companies, and multi-function microplate reader is purchased from
MD companies, Luciferase Assay Reagent box are purchased from Promega companies；
Chicken interferon α standard items (108U/mL it is) that publication No. is a kind of restructuring disclosed in CN105039474A patent
The recombined chicken alpha interferon (mode of selection embodiment 1) that the preparation method of chicken alpha interferon standard items is prepared.
Embodiment 1
The present embodiment provides a kind of chicken interferon α biological activity detection methods, and it is the life to recombinating chicken interferon α
A kind of application that thing activity is detected, the detection method of the present embodiment can also be correspondingly used for natural chicken interferon α's
Activity determination, it comprises the following steps：
According to the gene order for the chicken Mx albumen announced in Genebank, 5' ends are selected to contain ISRE response elements
Promoter region, designs PCR primer, and DNA is extracted from CEF cells by phenol-chloroform-isoamyl alcohol method and is used as template, in use
State PCR primer and Ex-Taq enzymes enter performing PCR amplification (underscore part is upstream and downstream primer position)；
The product that PCR amplifications obtain is purified (in upstream PCR by gel extraction again after Ase I and Age I double digestions
Primer introduces Ase I restriction enzyme sites, and downstream PCR primer introduces Age I restriction enzyme sites, wherein the ifn response element included is shown in
Runic character segment), Mxp genetic fragment is obtained, Mxp gene fragment order is shown in sequence 1；
The pCMV of pEGFP-N1 vector plasmids genetic fragment 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 former pEGFP-N1 plasmids
PCMV genetic fragment, DH5 α competent cells are converted, bacterium is shaken, extracts plasmid after filtering out positive colony, pass through DNA sequencing
Mxp DNA sequences are obtained, it is pEGFP-N1-Mxp plasmids to select the correct plasmid of sequence, and it builds schematic diagram such as Fig. 1 institutes
Show.
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 is dissolved in 500 μ l serum-free Opti-MEM culture mediums, soft to mix, and is incubated at room temperature 5min；By plasmid solution and transfection
Reagent solution softly mixes, and is incubated at room temperature 20min；With serum-free Opti-MEM culture mediums Secondary Culture CEF cells to 6 orifice plates
In, per hole 5 × 105Individual cell；Plasmid-transfection reagent compound is added to every hole, contains 5%CO in 37 DEG C2Cultivated in incubator
Overnight；Next day, culture medium is removed, instead DMEM (containing Sodium Pyruvate and nonessential amino acid) complete medium continues to cultivate
96h；Use the complete medium screening and culturing 14 days containing 2 μ g/mL neomycins instead afterwards, change once fresh culture within every 2 days
Base；Period visual cell's growing state decides whether to pass on；The cell survived after neomycin acts on 14 is stable transfection
Cell；
The cell that acquisition, which will be screened, neomycin resistance is diluted to the complete medium containing 0.25 μ g/mL puromycins
Cell concentration is 10/mL；0.1mL cell suspensions are added per hole into 96 orifice plates, contain 5%CO in 37 DEG C2Trained in incubator
Support；Daily observation, the hole of single clone is selected, when cultivating to cell covering aperture surface area 1/3, cell is transferred to 6 orifice plates
In expanded by limiting dilution cultivation and cultivate, and by whether containing Mxp genes in PCR identification of cell genomes, freeze
Conservation, it is named as 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
Release；
0.1mL stable transfected cells strain CEF-pEGFP-N1-Mxp is seeded in 96 porocyte culture plates and trained overnight
Supporting makes cell density reach 90%, discards culture medium；The recombination chicken added to every hole after 0.1mL is diluted with complete medium is done
Plain α standard items are disturbed, each 3 parallel holes of dilution factor, set the hole for being not added with recombination chicken 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, as a result show with
The gradient dilution fluorescence intensity of recombination chicken interferon-' alpha ' gradually reduces, fluorescence intensity and the dilution of recombination chicken interferon-' alpha ' standard items
The negative logarithm of degree 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, with reference to mark
Directrix curve is evaluated to obtain 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 result and few cells disease of chicken interferon α biological activity detection methods of the present invention
Become 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 recombination chicken interferon-' alpha 's simultaneously
Sample, whether there is 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：Chicken interferon α sample 1mL are taken, with complete medium 1:100 is dilute
Complete medium doubling dilution is used after releasing again；CEF Secondary Cultures are seeded to 96 porocyte culture plates, it is thin that 100 μ l are inoculated with per hole
Born of the same parents' suspension (2 × 105Individual/mL)；The μ l of recombination chicken Interferon α1 00 of different dilution factors are added per hole, virus control and cell
Control adds 100 μ l culture mediums, contains 5%CO in 37 DEG C2Continue to cultivate 16h in incubator；Culture medium is discarded, except cell controls
Outside hole, the VSV (24h can make the VSV virus titers that CEF cells are completely fallen off) of 100 μ l appropriate titers is added per hole, in 37 DEG C
Containing 5%CO2Continue in incubator cultivate 24h, micro- Microscopic observation virus control wells clasmatosis up to 100% and cell controls
When normal, you can with violet staining, record is visually observed after washing, cell is in completely purple in cell control well, and virus is right
Show that system is set up according to non-coloring (100%CPE++++), observe coloring case in different holes on its basis, record CPE feelings
Condition, the extension rate of half cytopathic effect inhibition is calculated by Reed-Muench methods, so as to obtain the potency of interferon.
As a result as shown in figure 3, the Correlation analysis showed R of two kinds of detection methods2＞ 0.9, show that both have very high phase
Guan Xing.The result shows that the interferon activity detection method that the present invention establishes is simple, quick, and testing result reliably, stably, can
Suppress this traditional detection method of method applied to natural or recombination chicken interferon-' alpha ' determination of activity to substitute few cells lesion.
In summary, the embodiment of the present invention replaces luciferase structure Mxp-EGFP using green fluorescent protein (EGFP)
Reporter System, the system does not need internal reference plasmid, therefore can stably be expressed in Mxp-EGFP Reporter Systems
In cell, and due to the progress of detection technique, EGFP can be quantified and detected in cell.The cell only need to be cultivated every time, and
With fluorescence intensity can complete antiviral activity of interferon detection, pole under specific instrument after interferon-' alpha ' processing 6h
The earth simplifies detection process, shortens detection time, improves accuracy, with more practicality, be adapted in large-scale production
Using.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited to
This, 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 all be included within the scope of the present invention.
Sequence table
<110>Anhui Jiuchuan Biotechnology Co., Ltd.
<120>A kind of chicken interferon α biological activity detection methods
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 470
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
attaattcgg tgtcacatcc acacggtcct tgaacactcc tagagatgac tccaccacct 60
ctctgggcag cctgtgccac tgcctcacca ctctttcggg gaataaatgt ttcctgatat 120
ccaacctgaa aaggaaaggg ctgttgctca taagcagtat tacctagcac ctgtgccatc 180
tgcccgctga ttcctctgca ggagaaggga gaaaccacag gacaaggagg gtagtagtgc 240
attggagtgg ttttgttaca gggttctgca aagaactggg acgaaaattg gctaatgatg 300
aggaatttga gtgaaacaca catcaggata ctgttttcaa taatgaaagc attttagttt 360
cgtttctcct tgtttatgtc atgtaggtgg agtctgtgta tagaaaagca ttcagagcgg 420
ctgaatgtag ttaattgttt ctccttgctg tgtgactctg gcagaccggt 470
Claims (10)
1. a kind of chicken interferon α biological activity detection methods, it comprises the following steps：
The genetic fragment for the Mxp for obtaining chicken Mx albumen is expanded using PCR；
The Mxp of the chicken Mx albumen obtained with PCR amplifications genetic fragment replaces former pEGFP-N1 carriers matter by T4DNA ligases
The genetic fragment of pCMV in grain, build 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, it is characterised in that obtained using PCR amplifications
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 5' ends to contain ISRE response elements
Region, PCR primer is designed, enter performing PCR amplification；
Then by double digestion after, then by gel extraction purify obtain Mxp genetic fragment.
3. chicken interferon α biological activity detection methods according to claim 1, it is characterised in that expanded and obtained with PCR
The Mxp genetic fragment of chicken Mx albumen pass through the gene that T4DNA ligases replace the pCMV in former pEGFP-N1 vector plasmids
Fragment, build pEGFP-N1-Mxp plasmids the step of include：
Remove the pCMV of pEGFP-N1 vector plasmids genetic fragment；
The genetic fragment of the pCMV in former pEGFP-N1 vector plasmids, structure are replaced with Mxp genetic fragment 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, select 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, it is characterised in that use pEGFP-N1-
Mxp plasmid-transfected cells, and include the step of filter out stable transfected cells strain by neomycin：
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, as stable transfected cells strain.
5. chicken interferon 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 as to obtain gram of individual cells formation
It is grand；
Preferably, also include 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. chicken interferon α biological activity detection methods according to claim 1 or 2, it is characterised in that：Mxp gene
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 pCMV of vector plasmid method is double digestion method.
8. the 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：Described chicken interference
Plain α standard items are the preparation side that publication No. is a kind of recombined chicken alpha interferon standard items disclosed in CN105039474A patent
The recombined chicken alpha interferon that method is prepared.
10. the chicken interferon α biological activities detection method described in claim any one of 1-9 is to the interference of natural or recombination chicken
Plain α carries out the application in biological activity detection.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711264355.1A CN107858450A (en) | 2017-12-05 | 2017-12-05 | A kind of chicken interferon α biological activity detection methods |
| CN201810642051.2A CN108795982A (en) | 2017-12-05 | 2018-06-21 | A kind of chicken interferon α biological activity detection methods |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711264355.1A CN107858450A (en) | 2017-12-05 | 2017-12-05 | A kind of chicken interferon α biological activity detection methods |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107858450A true CN107858450A (en) | 2018-03-30 |
Family
ID=61704665
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711264355.1A Pending CN107858450A (en) | 2017-12-05 | 2017-12-05 | A kind of chicken interferon α biological activity detection methods |
| CN201810642051.2A Withdrawn CN108795982A (en) | 2017-12-05 | 2018-06-21 | A kind of chicken interferon α biological activity detection methods |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810642051.2A Withdrawn CN108795982A (en) | 2017-12-05 | 2018-06-21 | A kind of chicken interferon α biological activity detection methods |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN107858450A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
-
2017
- 2017-12-05 CN CN201711264355.1A patent/CN107858450A/en active Pending
-
2018
- 2018-06-21 CN CN201810642051.2A patent/CN108795982A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108795982A (en) | 2018-11-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107841530A (en) | Chemical-activated luciferase gene expression chicken interferon α biological activity detection methods | |
| Philipson et al. | Molecular biology of adenoviruses | |
| CN108004270B (en) | Method for detecting activity of duck IFN-beta promoter by using dual-luciferase reporter gene | |
| CN107858450A (en) | A kind of chicken interferon α biological activity detection methods | |
| CN108707642A (en) | Chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method | |
| CN107858449A (en) | A kind of dog interferon alpha biological activity detection method | |
| Soubies et al. | Propagation and titration of infectious bursal disease virus, including non-cell-culture-adapted strains, using ex vivo-stimulated chicken bursal cells | |
| CN104911152B (en) | One 09 plant of plant weight group infectious hematopoietic necrosis poison rIHNV HLJ and its construction method and application | |
| Rascati et al. | Characterization of Fv-1 gene-product-mediated resistance transfer | |
| Naylor et al. | Avian metapneumovirus SH gene end and G protein mutations influence the level of protection of live-vaccine candidates | |
| CN107937602A (en) | A kind of porcine interferon alpha biological activity detection method | |
| CN108753919A (en) | Chemical-activated luciferase gene expression dog interferon alpha biological activity detection method | |
| CN108753918A (en) | Chemical-activated luciferase gene expression Bov IFN α biological activity detection methods | |
| CN108732153A (en) | A kind of Bov IFN α biological activity detection methods | |
| Corredor et al. | Fowl adenovirus-based vaccine platform | |
| CN114672460B (en) | Preparation method and application of CD 44-targeted heterogeneous CIC cell model | |
| Bodakuntla et al. | Knocking out multiple genes in cultured primary neurons to study tubulin posttranslational modifications | |
| CN110129272A (en) | Stablize the PK-15 cell strain for expressing MAP3K8 albumen and its building and application | |
| CN102994550B (en) | The method of expression alien gene in zooblast or animal tissues | |
| McAllister et al. | Childhood sarcomas and lymphomas. Characterization of new cell lines and search for type‐C virus | |
| CN107746854A (en) | A kind of sheep interferon-tau biological activity detection method | |
| CN109423522B (en) | Identification and application of molecular breeding marker of functional mutation site of porcine CD4 gene | |
| Ferreira et al. | Infectious bronchitis virus replication in the chicken embryo related cell line | |
| CN107937601A (en) | Chemical-activated luciferase gene expression sheep interferon-tau biological activity detection method | |
| KR102154380B1 (en) | Method for generating high-titer hepatitis e virus stocks and titration assay for hepatitis e virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180330 |