CN107841530A - Chemical-activated luciferase gene expression chicken interferon α biological activity detection methods - Google Patents
Chemical-activated luciferase gene expression chicken interferon α biological activity detection methods Download PDFInfo
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Abstract
The invention discloses a kind of chemical-activated luciferase gene expression chicken interferon α biological activities detection method and its application.This method comprises the following steps：The genetic fragment for the pMx1 for obtaining chicken Mx1 albumen is expanded using PCR；PMx1 genetic fragment is inserted to the 5' ends of pGL3 basic carrier luc genes, then expands using PCR to obtain pMx1 luc fusion fragments；The genetic fragment of the pCMV and EGFP in pEGFP N1 carriers are substituted with pMx1 luc fusions fragment, builds pMx1 luc plasmids, transfectional cell, and select stable transfected cells strain；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 incubated altogether in cell chicken interferon α samples to be checked added after colonized culture, then fluorescence intensity, the potency of combined standard curve evaluation chicken interferon α samples to be checked.
Description
Technical field
The present invention relates to a kind of chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, belong to interference
Plain Activity determination technical field.
Background technology
Interferon-' alpha ' is widely used in the disease treatment in terms of viral infection resisting and immune dysfunction, and it is as a kind of thin
Intracellular cytokine causes the activation of intracellular series of signals transduction pathway by being combined with the special receptor that target cell shows, shows to resist
Virus function or immunoregulation effect.The comparison that the signal transduction pathway that interferon-' alpha ' works in the cell at present has been studied
Thoroughly, mainly realized by activating JAK-STAT signal cascades.Interferon-' alpha ' JAK1 related to acceptor combination activated receptor
And TYK2, make STAT1 and STAT2 TYR phosphorylations, the STAT1 and STAT2 of phosphorylation form dimer and are transferred to core, fill
The interferon stimulating factor 3 (ISGF3) of 3 aggressiveness is formed with interferon regulatory factor 9 (IRF9).ISGF3 and its homologous DNA sequence
(interferon stimulate the reaction original paper, ISREs) is combined, and is directly activated interferon-stimulated gene (ISGs) transcription, is entered host cell
Enter antiviral state.The approach that ISG suppresses virus mainly has, and suppresses transcription, translation and the nucleic acid replication of virus；Degrade viral core
Acid；Change host cell lipid metaboli.Therefore, as long as detecting that ISGs promoter activity increase can directly reacts interferon
α biological activity.
Mx albumen (Mx prorein) is main ISGs, and interferon-' alpha ' can activate through JAK-STAT signal transduction pathways
Mx promoters (Mx promoter, pMx), promote Mx expression, and Mx can suppress minus-stranded rna virus duplication.Exist in chicken
Two kinds of albumen of Mx1 and Mx2, wherein Mx1 play a major role.PMx has preferable selectivity, and it can not be by interleukins
(Interleukin), other cell factors such as TNF (Tumor Necrosis Factor, TNF) start.
Detection interferon biological activity mainly has 3 kinds of methods at present, and virus replication suppresses, plaque reduces analysis and cell
Lesion suppresses.But this 3 kinds of methods all easily produce larger error, and repeatability is bad, and accuracy is low.As an improvement, then use table
Restructuring VSV viruses up to GFP substitute wild type VSV viruses, and viral infection duplication can be reflected by the expression of fluorescin
Quantitative degree, and then judge that IFN significantly improves to the inhibition level of virus replication, sensitiveness and repeatability, but still exist
Detection process take it is long, be not easy to that the detection of big throughput sample, sensitiveness is not ideal enough and live virus bio-safety hidden danger etc. is all
It is more insufficient.
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 luciferase reporter gene
(luc) detect chicken interferon α biological activities method, detection process can be simplified, it is not necessary to repeat Virus culture or
The troublesome operation of plasmid transfection, accuracy and repeatability are improved, avoid bio-safety harm that may be present.
The purpose of the present invention is achieved by the following technical programs：
A kind of chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it comprises the following steps：
The genetic fragment for the pMx1 for obtaining chicken Mx1 albumen is expanded using PCR；
The 5' of the pMx1 for the chicken Mx1 albumen that PCR amplifications are obtained genetic fragment insertion pGL3-basic carrier luc genes
End, then expand using PCR to obtain pMx1-luc fusion fragments；
The genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers, structure are substituted with pMx1-luc fusions fragment
PMx1-luc plasmids；
With pMx1-luc 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, combined standard
The potency of curve evaluation chicken interferon α samples to be checked.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, by chicken interferon α to be checked
, it is necessary to be incubated the regular hour in cell after sample addition colonized culture, general 6 hours or so.
In the present invention, pMx1 refers to chicken Mx1 gene promoter regions, and pCMV refers to the CMV promoter of pEGFP-N1 plasmids
Region.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, preferably：
PMx1 gene fragment order is shown in sequence 1；
PMx1-luc fusions fragment sequence is shown in sequence 2.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that using PCR
The step of pMx1 of amplification acquisition chicken Mx1 albumen genetic fragment, includes：
According to the gene order for the chicken Mx1 albumen announced in Genebank, 5' ends are selected to contain ISRE response elements
Promoter region, PCR primer is designed, enter performing PCR amplification, obtain amplified production pMx1 genetic fragments；
Amplified production is subjected to double digestion, then the genetic fragment for obtaining pMx1 is purified by gel extraction.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that using PCR
In the step of amplified production is carried out double digestion by the genetic fragment that amplification obtains the pMx1 of chicken Mx1 albumen the enzyme selected for KpnI and
HindIII。
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that expand PCR
Increase the 5' ends of the pMx1 of the chicken Mx1 albumen obtained genetic fragment insertion pGL3-basic carrier luc genes, then expanded using PCR
The step of increasing obtains pMx1-luc fusion fragments includes：
PMx1 genetic fragment is connected into the 5' ends of pGL3-Basic plasmid luc genes by T4DNA ligases；
Bacillus coli DH 5 alpha competent cell is converted, shakes bacterium, filters out extraction restructuring pGL3-basic matter after positive colony
Grain, the DNA sequence dna of the pMx1 fragments of restructuring pGL3-basic plasmids is obtained by DNA sequencing, selects the restructuring containing correct sequence
PGL3-basic plasmids；
Using the DNA sequence dna of the restructuring pGL3-basic plasmids containing correct sequence as template, PCR primer is designed, enters performing PCR expansion
Increase, obtain product；
Product is subjected to double digestion, then is purified by gel extraction and obtains pMx1-luc fusion fragments.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that expand PCR
Increase the 5' ends of the pMx1 of the chicken Mx1 albumen obtained genetic fragment insertion pGL3-basic carrier luc genes, then expanded using PCR
It is Ase I and Xba I to increase the enzyme for obtaining being selected in the step of product is carried out double digestion by pMx1-luc fusions fragment.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that use pMx1-
Luc fusions fragment substitutes the genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers, builds the step of pMx1-luc plasmids
Suddenly include：
Remove the genetic fragment of the pCMV and EGFP in pEGFP-N1 vector plasmids；
By T4DNA ligases with pMx1-luc fusions fragment replace pCMV in former pEGFP-N1 vector plasmids and
EGFP genetic fragment, build pMx1-luc plasmids；
Bacillus coli DH 5 alpha competent cell is converted, shakes bacterium, extracts plasmid, the pMx1- of plasmid is obtained by DNA sequencing
The DNA sequence dna of luc fusion fragments, select the plasmid of correct sequence；
The correct sequence is：PMx1 gene fragment order is pMx1-luc fusion fragment sequences shown in sequence 1
For shown in sequence 2.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, the side of PCR primer is designed
Method is conventional.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that use pMx1-
Luc plasmid-transfected cells, and include the step of filter out stable transfected cells strain by neomycin：
By transfection reagent by pMx1-luc 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.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that the clone
The stable transfected cells strain for changing culture to filter out is by limiting dilution cultivation culture, so as to obtain individual cells formation
Clone.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that to screening
The stable transfected cells strain gone out carries out also including detecting the cell after clone in the step of colonized culture, passes through PCR
Whether contain pMx1-luc fusions in identification of cell genome.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that remove
The method of the genetic fragment of pCMV and EGFP in pEGFP-N1 vector plasmids is double digestion method.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that the cell
For CEF cells.
In above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it is preferred that described chicken
Interferon-' alpha ' standard items are the system that publication No. is a kind of recombined chicken alpha interferon standard items disclosed in CN105039474A patent
The recombined chicken alpha interferon that Preparation Method is prepared.
The present invention also provides above-mentioned chemical-activated luciferase gene expression chicken interferon α biological activity detection methods to day
So or recombination chicken interferon-' alpha ' carries out the application in biological activity detection.
The luciferase report that above-mentioned chicken interferon α biological activities detection method is built based on chicken Mx1 gene promoters
Gene is accused, CEF cells is transfected, screens the cell line of stable transfection.As chicken interferon α (IFN-α) and stable transfection fluorescence
When the CEF cells of plain enzyme reporter gene are incubated jointly, chicken IFN-α can strengthen Mx1 gene promoter activities, promote luciferase
Expression, add substrate after detect luminous value and chicken IFN-α biological activity be proportionate, so as to realize to chicken IFN-α life
The quantitative detection of thing activity.
The present invention constructs chicken Mx1 gene promoter sequences and luc fusion, and inserts eukaryon expression plasmid, 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, luciferase substrate is added after cell lysis, detects luminous value, is prepared with recombination chicken interferon-' alpha ' standard items
Standard curve detects the biological activity of chicken interferon α in measuring samples.The detection method mainly has following characteristics：1. significantly
Shorten detection time (foreshortening to 6-8 hours by traditional 3-7 days)；Repeat Virus culture or plasmid transfection 2. eliminating
Etc. cumbersome operation, it is only necessary to which cell is determined in culture, avoids bio-safety harm that may be present, improves repetition
Property.
It is of the invention compared with existing I types interferon biological activity luciferase reporter gene detection method, employ chicken Mx1
Gene promoter sequence and Chicken kidney cell CEF, specially optimize the biological activity detection for chicken IFN-α, and turned using stable
Dye cell line saves plasmid and repeats transfection procedure, simplifies detection process, improves accuracy.Given birth to traditional I types interferon
Thing activity test method, as virus replication suppresses (Virus Yield-reduction Assay, VYRA), plaque is reduced and divided
Analyse (Plaque ReductionAssay, PRA), cytopathic effect inhibition (Cytopathic Effect ReductionAssay,
CPERA) the methods of, which is compared, need to only cultivate cell, and add substrate fluorescence intensity after handling cell 6h with interferon-' alpha '
Antiviral activity of interferon detection is completed, detection process is greatly simplified, improves accuracy.Coordinate chicken interferon standard items
Use, it is possible to achieve in application study and industrialization production chicken interferon α biological activities standardization measure.
The present invention protrusion effect be：
The present invention quantitatively detects chicken interferon α using the chemical-activated luciferase gene expression of luciferase reporter gene (luc)
Biological activity, detection process can be simplified, it is not necessary to repeat the troublesome operation of Virus culture or plasmid transfection, only need to train
Specific cells are supported, and after being incubated 6h altogether with chicken interferon α, fluorescence intensity can complete the detection of interferon-' alpha ' biological activity,
Accuracy and repeatability are improved, avoids bio-safety harm that may be present.
Brief description of the drawings
Fig. 1 is the pMx1-luc plasmid construction schematic diagrames of embodiment 1；
Fig. 2 is the chemical-activated luciferase gene expression standard curve and cut-off value figures of embodiment 1；
Fig. 3 is that the chemical-activated luciferase gene expression of embodiment 2 suppresses the correlation ratio of testing result with few cells lesion
Compared with 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 way
Footpath obtains.
Material used is as follows in the following embodiments of the present invention：
CEF cells be laboratory according to cellar culture, the plasmid pGL3-Basic containing luciferase reporter gene is purchased from
Promega companies, carrier for expression of eukaryon pEGFP-N1 are purchased from Clontech companies, various restriction enzymes, Ex-Taq enzymes,
T4DNA ligases and plasmid extraction kit are purchased from Takara companies, and transfection reagent Fugen6 is purchased from Promega companies, DMEM
Culture medium and FBS are purchased from Invitrogen companies, and all size Tissue Culture Plate is purchased from Corning companies；VSV viruses are by China
Wuhan institute of viruses of the academy of sciences provides, and multi-function microplate reader is purchased from MD companies, and Luciferase Assay Reagent box is purchased from Promega
Company；
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 chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, and it is pair
The biological activity of recombination chicken interferon-' alpha ' carries out a kind of application of quantitative detection, and the detection method of the present embodiment can also correspond to
Ground is used for natural chicken interferon α Activity determination, and it comprises the following steps：
According to the gene order for the chicken Mx1 albumen announced in Genebank, 5' ends are selected to contain ISRE response elements
Promoter region (ifn response element is shown in runic character segment), design PCR primer), Kpn I digestions are introduced in sense primer
Site, anti-sense primer introduce Hind III digestions site (underscore part is upstream and downstream primer position), by phenol-chloroform-different
Amylalcohol method extracts DNA as template from CEF cells, enters performing PCR using above-mentioned PCR primer and Ex-Taq enzymes and expands, is expanded
Increase production thing.
Amplified production is purified by gel extraction again after EcoR I and XhoI double digestions, obtains pMx1 gene piece
Section, pMx1 gene fragment order is shown in sequence 1；
PMx1 genetic fragment is connected into the 5' ends of pGL3-Basic plasmid luc genes by T4DNA ligases；
Bacillus coli DH 5 alpha competent cell is converted, shakes bacterium, filters out extraction restructuring pGL3-basic matter after positive colony
Grain, the DNA sequence dna of the pMx1 fragments of restructuring pGL3-basic plasmids is obtained by DNA sequencing, selects the restructuring containing correct sequence
PGL3-basic plasmids；
Using the DNA sequence dna of the restructuring pGL3-basic plasmids containing correct sequence as template, PCR primer is designed
(sense primer：5 '-attaatcggggtacctcggtgtcacatccacacggt-3 ',
Anti-sense primer：5 '-tctagattacacggcgatctttccgcccttc-3 ', Ase I are introduced in sense primer
Restriction enzyme site, anti-sense primer introduce Xba I restriction enzyme sites), enter performing PCR with above-mentioned primer and Ex-Taq enzymes and expand, obtain product；
By product after Ase I and Xba I double digestions, then purified by gel extraction and obtain pMx1-luc fusion pieces
Section, pMx1-luc fusions fragment sequence are shown in sequence 2；
The genetic fragment of the pCMV and EGFP in pEGFP-N1 vector plasmids are removed using Ase I and Xba I double digestions method；
By T4DNA ligases with pMx1-luc fusions fragment replace pCMV in former pEGFP-N1 vector plasmids and
EGFP genetic fragment, build pMx1-luc plasmids；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, extracts plasmid after filtering out positive colony, pass through DNA sequencing
The DNA sequence dna of pMx1-luc plasmids is obtained, selects the plasmid of correct sequence；
The correct sequence is：PMx1 gene fragment order is that pMx1-luc gene orders are sequence 2 shown in sequence 1
It is shown.
The structure schematic diagram of whole structure pMx1-luc plasmids is as shown in Figure 1.
With pMx1-luc plasmid-transfected cells, and stable transfected cells strain sample is filtered out by neomycin：Using in going
Toxin plasmid extracts kit extracts pMx1-luc plasmids, takes 1 μ g plasmids to be placed in the sterile EP pipes of 1.5mL and is cultivated with Opti MEM
Base supplies volume to 500 μ l, soft to mix, incubation at room temperature 5min；1.5mL sterilizing EP pipes are taken, 5 μ l Fugen6 is added and is dissolved in 500
It is soft to mix in μ l serum-free Opti-MEM culture mediums, it is incubated at room temperature 5min；Plasmid solution and transfection reagent solution are softly mixed
It is even, it is incubated at room temperature 20min；With serum-free Opti-MEM culture medium Secondary Culture CEF cells into 6 orifice plates, per hole 5 × 105It is individual
Cell；Plasmid-transfection reagent compound is added to every hole, contains 5%CO in 37 DEG C2Overnight incubation in incubator；Next day, remove training
Base is supported, instead DMEM (containing Sodium Pyruvate and nonessential amino acid) complete medium continues to cultivate 96h；Use instead afterwards containing 2 μ g/
The complete medium screening and culturing of mL neomycins 14 days, change once fresh culture medium within every 2 days；Period visual cell's growing state
Decide whether to pass on；The cell survived after neomycin acts on 14 is stable transfected cells；Screening acquisition there is into neomycin
It is 10/mL that the cell of resistance is diluted to concentration with the complete medium containing 0.25 μ g/mL puromycins, is added with 0.1mL/ holes
In 96 orifice plates, contain 5%CO in 37 DEG C2Cultivated in incubator；Daily observation, selects the hole of single clone, cultivates to cell and cover
During cap bore surface area 1/3, cell is transferred in 6 orifice plates to expand and cultivated, conservation is frozen, is named as CEF-pMx1-luc.
Chicken interferon α standard items 1mL are taken, according to 1:10、1:100、1:103…1:107Gradient complete medium it is dilute
Release；
CEF-pMx1-luc is seeded in 24 porocyte culture plates makes cell density reach 90% overnight, discards culture
Base；Recombination chicken interferon-' alpha ' standard items after being diluted to every hole addition 1mL with complete medium, each 3 parallel holes of dilution factor,
The hole for being not added with recombination chicken interferon-' alpha ' is set to contain 5%CO in 37 DEG C as control2Continue to cultivate 6h in incubator, take out culture
Plate, each hole fluorescence intensity is detected according to the operation of Luciferase Assay Reagent box specification, testing result is as shown in Fig. 2 marked
Directrix curve, as a result show the negative logarithm of fluorescence intensity and the dilution factor of recombination chicken interferon-' alpha ' standard items linearly related R2=
0.991, detection range 0.1-109IU/mL。
It is incubated altogether in cell chicken interferon α samples to be checked added after colonized culture, then fluorescence intensity, knot
Standardization curve evaluation obtains the potency of chicken interferon α samples to be checked.
Embodiment 2
The present embodiment provides the inspection of the chemical-activated luciferase gene expression chicken interferon α biological activity detection methods of the present invention
Survey the contrast correlation experiment that result suppresses method testing result with few cells lesion.
20 parts of recombination chicken interference of method detection detection simultaneously are suppressed using chemical-activated luciferase gene expression and few cells lesion
Whether plain α samples, the result for showing two methods by linear regression analysis have correlation.
Chemical-activated luciferase gene expression testing process is as described in Example 1.
Few cells lesion suppresses method by following operation：Recombination chicken interferon-' alpha ' sample 1mL is taken, with complete medium 1:100
Diluted again with complete medium doubling dilution after dilution；CEF Secondary Cultures are seeded to 96 porocyte culture plates, are inoculated with per hole
100 μ l cell suspensions (2 × 105Individual/mL)；The μ l of recombination chicken Interferon α1 00 of different dilution factors are added per hole, virus control
100 μ l culture mediums are added with cell controls, contain 5%CO in 37 DEG C2Continue to cultivate 16h in incubator；Culture medium is discarded, except cell
Outside control wells, 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 contain 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.Two kinds
The correlation analysis result of detection method is as shown in figure 3, result shows R2＞ 0.9, show that both have very high correlation.The knot
Fruit shows that the chemical-activated luciferase gene expression chicken interferon alpha active detection method that the present invention establishes is simple, quick, testing result
Reliably, stably, few cells lesion can be substituted and suppress this traditional detection method of method applied to the interference of natural or recombination chicken
Plain alpha active measure.
In summary, the embodiment of the present invention utilizes the chemical-activated luciferase gene expression chicken of luciferase reporter gene (luc)
Interferon-' alpha ' biological activity detection method, can simplify detection process, it is not necessary to repeat Virus culture or plasmid transfection
Troublesome operation, specific cells only need to be cultivated, and after being incubated 6h altogether with chicken interferon α, fluorescence intensity can complete interferon-' alpha '
Biological activity detects, and improves accuracy and repeatability, avoids bio-safety harm that may be present, with more practicality,
It is 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 all be included within the scope of the present invention.
Sequence table
<110>Anhui Jiuchuan Biotechnology Co., Ltd.
<120>Chemical-activated luciferase gene expression chicken interferon α biological activity detection methods
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tatccaacct gaaaaggaaa gggctgttgc tcataagcag tattacctag cacctgtgcc 180
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tcctgatatc caacctgaaa aggaaagggc tgttgctcat aagcagtatt acctagcacc 180
tgtgccatct gcccgctgat tcctctgcag gagaagggag aaaccacagg acaaggaggg 240
tagtagtgca ttggagtggt tttgttacag ggttctgcaa agaactggga cgaaaattgg 300
ctaatgatga ggaatttgag tgaaacacac atcaggatac tgttttcaat aatgaaagca 360
ttttagtttc gtttctcctt gtttatgtca tgtaggtgga gtctgtgtat agaaaagcat 420
tcagagcggc tgaatgtagt taattgtttc tccttgctgt gtgactctgg cagaagcttg 480
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gaagagatac gccctggttc ctggaacaat tgcttttaca gatgcacata tcgaggtgga 660
catcacttac gctgagtact tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata 720
tgggctgaat acaaatcaca gaatcgtcgt atgcagtgaa aactctcttc aattctttat 780
gccggtgttg ggcgcgttat ttatcggagt tgcagttgcg cccgcgaacg acatttataa 840
tgaacgtgaa ttgctcaaca gtatgggcat ttcgcagcct accgtggtgt tcgtttccaa 900
aaaggggttg caaaaaattt tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat 960
tatcatggat tctaaaacgg attaccaggg atttcagtcg atgtacacgt tcgtcacatc 1020
tcatctacct cccggtttta atgaatacga ttttgtgcca gagtccttcg atagggacaa 1080
gacaattgca ctgatcatga actcctctgg atctactggt ctgcctaaag gtgtcgctct 1140
gcctcataga actgcctgcg tgagattctc gcatgccaga gatcctattt ttggcaatca 1200
aatcattccg gatactgcga ttttaagtgt tgttccattc catcacggtt ttggaatgtt 1260
tactacactc ggatatttga tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga 1320
agagctgttt ctgaggagcc ttcaggatta caagattcaa agtgcgctgc tggtgccaac 1380
cctattctcc ttcttcgcca aaagcactct gattgacaaa tacgatttat ctaatttaca 1440
cgaaattgct tctggtggcg ctcccctctc taaggaagtc ggggaagcgg ttgccaagag 1500
gttccatctg ccaggtatca ggcaaggata tgggctcact gagactacat cagctattct 1560
gattacaccc gagggggatg ataaaccggg cgcggtcggt aaagttgttc cattttttga 1620
agcgaaggtt gtggatctgg ataccgggaa aacgctgggc gttaatcaaa gaggcgaact 1680
gtgtgtgaga ggtcctatga ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc 1740
cttgattgac aaggatggat ggctacattc tggagacata gcttactggg acgaagacga 1800
acacttcttc atcgttgacc gcctgaagtc tctgattaag tacaaaggct atcaggtggc 1860
tcccgctgaa ttggaatcca tcttgctcca acaccccaac atcttcgacg caggtgtcgc 1920
aggtcttccc gacgatgacg ccggtgaact tcccgccgcc gttgttgttt tggagcacgg 1980
aaagacgatg acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa 2040
aaagttgcgc ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact 2100
cgacgcaaga aaaatcagag agatcctcat aaaggccaag aagggcggaa agatcgccgt 2160
gtaatctaga 2170
Claims (10)
1. a kind of chemical-activated luciferase gene expression chicken interferon α biological activity detection methods, it comprises the following steps：
The genetic fragment for the pMx1 for obtaining chicken Mx1 albumen is expanded using PCR；
The 5' ends of the pMx1 for the chicken Mx1 albumen that PCR amplifications are obtained genetic fragment insertion pGL3-basic carrier luc genes,
Expand to obtain pMx1-luc fusion fragments using PCR again；
The genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers are substituted with pMx1-luc fusions fragment, builds pMx1-
Luc plasmids；
With pMx1-luc 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. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 1, its feature
It is：
PMx1 gene fragment order is shown in sequence 1；
PMx1-luc fusions fragment sequence is shown in sequence 2.
3. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 1, its feature
The step of being, the genetic fragment for the pMx1 for obtaining chicken Mx1 albumen is expanded using PCR includes：
According to the gene order for the chicken Mx1 albumen announced in Genebank, the startup for selecting 5' ends to contain ISRE response elements
Subregion, PCR primer is designed, enter performing PCR amplification, obtain amplified production；
Amplified production is subjected to double digestion, then the genetic fragment for obtaining pMx1 is purified by gel extraction；
Preferably, the step of genetic fragment for the pMx1 for obtaining chicken Mx1 albumen is by amplified production progress double digestion is expanded using PCR
The enzyme of middle selection is KpnI and HindIII.
4. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 1, its feature
It is, the genetic fragment that PCR is expanded to the pMx1 of the chicken Mx1 albumen obtained inserts the 5' ends of pGL3-basic carrier luc genes,
The step of being expanded using PCR again and obtained pMx1-luc fusion fragments includes：
PMx1 genetic fragment is connected into the 5' ends of pGL3-Basic plasmid luc genes by T4DNA ligases；
Bacillus coli DH 5 alpha competent cell is converted, shakes bacterium, extraction restructuring pGL3-basic plasmids after positive colony is filtered out, leads to
The DNA sequence dna that DNA sequencing obtains the pMx1 fragments of restructuring pGL3-basic plasmids is crossed, selects the restructuring pGL3- containing correct sequence
Basic plasmids；
Using the DNA sequence dna of the restructuring pGL3-basic plasmids containing correct sequence as template, PCR primer is designed, enters performing PCR amplification,
Obtain product；
Product is subjected to double digestion, then is purified by gel extraction and obtains pMx1-luc fusion fragments；
Preferably, the genetic fragment that PCR is expanded to the pMx1 of the chicken Mx1 albumen obtained inserts pGL3-basic carrier luc genes
5' ends, then using PCR expand to obtain pMx1-luc fusions fragment will product carry out double digestion the step of in the enzyme selected
For Ase I and Xba I；
Preferably, the genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers, structure are substituted with pMx1-luc fusions fragment
The step of building pMx1-luc plasmids includes：
Remove the genetic fragment of the pCMV and EGFP in pEGFP-N1 vector plasmids；
Pass through pCMV and EGFP of the T4DNA ligases in the former pEGFP-N1 vector plasmids of pMx1-luc fusions fragment replacement
Genetic fragment, build pMx1-luc plasmids；
Bacillus coli DH 5 alpha competent cell is converted, shakes bacterium, extracts plasmid, plasmid pMx1-luc fusions are obtained by DNA sequencing
The DNA sequence dna of genetic fragment, select the plasmid of correct sequence；
The correct sequence is：PMx1 gene fragment order is that pMx1-luc fusions fragment sequence is sequence shown in sequence 1
Shown in row 2.
5. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 1, its feature
It is, includes with pMx1-luc plasmid-transfected cells, and the step of filter out stable transfected cells strain by neomycin：
By transfection reagent by pMx1-luc plasmid transfections to cell, after transfecting 96h, the complete training containing 2 μ g/mL neomycins is changed into
Foster base is persistently cultivated 14, filters out the cell with neomycin resistance, as stable transfected cells strain.
6. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 1, its feature
Be, the colonized culture for will the stable transfected cells strain that filter out by limiting dilution cultivation culture, so as to obtain
The clone that individual cells are formed；
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 pMx1-luc fusions in PCR identification of cell genomes.
7. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 4, its feature
It is：The method for removing the genetic fragment of the pCMV and EGFP in pEGFP-N1 vector plasmids is double digestion method.
8. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 1 or 5, its
It is characterised by：The cell is CEF cells.
9. chemical-activated luciferase gene expression chicken interferon α biological activity detection methods according to claim 1, its feature
It is：Described chicken interferon α standard items be publication No. be disclosed in CN105039474A patent a kind of recombination chicken alpha interference
The recombined chicken alpha interferon that the preparation method of plain standard items is prepared.
10. the chemical-activated luciferase gene expression chicken interferon α biological activity detection methods described in claim any one of 1-9 exist
Application in biological activity detection is carried out to natural or recombination chicken interferon-' alpha '.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111549029A (en) * | 2020-05-25 | 2020-08-18 | 浙江省农业科学院 | Duck Mx promoter mutant and construction method and application of recombinant plasmid of luciferase reporter gene thereof |
| CN113009156A (en) * | 2021-03-22 | 2021-06-22 | 华南农业大学 | Method for detecting dog IFN-alpha biological activity by using green fluorescent protein reporter gene |
| 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 |
| CN117126889A (en) * | 2023-10-24 | 2023-11-28 | 上海惠盾因泰生物科技有限公司 | Human ISG15 reporter gene stable transgenic cell strain and construction method and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951761A (en) * | 2019-12-24 | 2020-04-03 | 扬州大学 | Method for detecting activity of chicken OASL promoter |
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2017
- 2017-12-05 CN CN201711264212.0A patent/CN107841530A/en active Pending
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Cited By (5)
| 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 |
| CN111549029A (en) * | 2020-05-25 | 2020-08-18 | 浙江省农业科学院 | Duck Mx promoter mutant and construction method and application of recombinant plasmid of luciferase reporter gene thereof |
| CN113009156A (en) * | 2021-03-22 | 2021-06-22 | 华南农业大学 | Method for detecting dog IFN-alpha biological activity by using green fluorescent protein reporter gene |
| CN117126889A (en) * | 2023-10-24 | 2023-11-28 | 上海惠盾因泰生物科技有限公司 | Human ISG15 reporter gene stable transgenic cell strain and construction method and application thereof |
| CN117126889B (en) * | 2023-10-24 | 2024-01-12 | 上海惠盾因泰生物科技有限公司 | Human ISG15 reporter gene stable transgenic cell strain and construction method and application thereof |
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