CN108753918A - Chemical-activated luciferase gene expression Bov IFN α biological activity detection methods - Google Patents

Chemical-activated luciferase gene expression Bov IFN α biological activity detection methods Download PDF

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CN108753918A
CN108753918A CN201810641987.3A CN201810641987A CN108753918A CN 108753918 A CN108753918 A CN 108753918A CN 201810641987 A CN201810641987 A CN 201810641987A CN 108753918 A CN108753918 A CN 108753918A
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pmx1
luc
biological activity
bov ifn
gene expression
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单雪芹
凡玉芳
许高涛
李雅森
徐文俊
刘家炉
蒋敏之
赵雨
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ANHUI JIUCHUAN BIOTECHNOLOGY Co Ltd
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ANHUI JIUCHUAN BIOTECHNOLOGY Co Ltd
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    • G01N2333/555—Interferons [IFN]
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Abstract

The invention discloses a kind of chemical-activated luciferase gene expression Bov IFN α biological activities detection method and its applications.This approach includes the following steps：The genetic fragment of the pMx1 of ox Mx1 albumen is obtained using PCR amplification；The genetic fragment of pMx1 is inserted into the ends 5' of pGL3-basic carrier luc genes, then pMx1-luc fusion segments are obtained using PCR amplification；The genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers are substituted with pMx1-luc fusion segments, build 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 Bov IFN α standard items of gradient dilution, is incubated altogether in cell Bov IFN α samples to be checked being added after colonized culture, then fluorescence intensity, the potency of combined standard curve evaluation Bov IFN α samples to be checked.

Description

Chemical-activated luciferase gene expression Bov IFN α biological activity detection methods
Technical field
The present invention relates to a kind of chemical-activated luciferase gene expression Bov IFN 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, 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, it is mainly realized by activating JAK-STAT signal cascades.Interferon-' alpha ' JAK1 related to receptor combination activated receptor And TYK2, so that STAT1 and STAT2 tyrosine phosphorylations, the STAT1 and STAT2 of phosphorylation is formed dimer and is 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) combine, directly activate interferon-stimulated gene (ISGs) transcribe, make host cell into Enter antiviral state.ISG inhibits the approach of virus mainly to have, and inhibits transcription, translation and the nucleic acid replication of virus；The viral core of degradation Acid；Change host cell lipid metaboli.Therefore, as long as detecting that interferon is directly reacted in the promoter activity increase of ISGs The biological activity of α.
Mx albumen (Mx prorein) is main ISGs, and interferon-' alpha ' can be activated through JAK-STAT signal transduction pathways Mx promoters (Mx promoter, pMx), promote the expression of Mx, Mx minus-stranded rna virus can be inhibited to replicate.Exist in ox Two kinds of albumen of Mx1 and Mx2, wherein Mx1 play a major role.PMx has preferable specificity, it cannot be by interleukins (Interleukin), other cell factors such as tumor necrosis factor (Tumor Necrosis Factor, TNF) start.
Detection interferon biological activity mainly has 3 kinds of methods at present, and virus replication inhibits, plaque reduces analysis and cell Lesion inhibits.But this 3 kinds of methods all easy to produce large error, and repeatability is bad, and accuracy is low.As an improvement, then using table Recombination 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 IFN to the inhibition level of virus replication, sensibility and repeatability significantly improve, but still exist Detection process take it is long, be not easy to that the detection of big throughput sample, sensibility is not ideal enough and live virus bio-safety hidden danger etc. is all It is mostly insufficient.
Invention content
In view of the problems of the above-mentioned prior art, luciferase reporter gene is utilized the object of the present invention is to provide a kind of (luc) detect Bov IFN α biological activities method, detection process can be simplified, need not repeat Virus culture or The troublesome operation of plasmid transfection improves accuracy and repeatability, avoids 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 Bov IFN α biological activity detection methods comprising following steps：
The genetic fragment of the pMx1 of ox Mx1 albumen is obtained using PCR amplification；
The genetic fragment of the pMx1 for the ox Mx1 albumen that PCR amplification is obtained is inserted into the 5' of pGL3-basic carrier luc genes End, then pMx1-luc fusion segments are obtained using PCR amplification；
The genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers, structure are substituted with pMx1-luc fusion segments 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 Bov IFN α standard items of gradient dilution, is determined between interferon potency and fluorescent value Mathematical logic relationship；
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 chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, by Bov IFN α to be checked In cell after sample addition colonized culture, the incubation regular hour is needed, general 6 hours or so.
In the present invention, pMx1 refers to ox Mx1 gene promoter regions, and pCMV refers to the CMV promoter of pEGFP-N1 plasmids Region.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, preferably：
The gene fragment order of pMx1 is shown in sequence 1；
PMx1-luc fusion fragment sequences are shown in sequence 2.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that use PCR Amplification obtain ox Mx1 albumen pMx1 genetic fragment the step of include：
According to the gene order for the ox Mx1 albumen announced in Genebank, the ends 5' is selected to contain ISRE response elements Promoter region designs PCR primer, carries out PCR amplification, obtains amplified production pMx1 genetic fragments；
Amplified production is subjected to double digestion, then purifies the genetic fragment for obtaining pMx1 by gel extraction.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that use PCR In the step of amplified production is carried out double digestion by the genetic fragment that amplification obtains the pMx1 of ox Mx1 albumen the enzyme selected be KpnI and HindIII。
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that expand PCR The genetic fragment for increasing the pMx1 of the ox Mx1 albumen obtained is inserted into the ends 5' of pGL3-basic carrier luc genes, then is expanded using PCR Increasing the step of obtaining pMx1-luc fusion segments includes：
The genetic fragment of pMx1 is connected into the ends 5' of pGL3-Basic plasmid luc genes by T4DNA ligases；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, filters out extraction recombination pGL3-basic matter after positive colony Grain is obtained the DNA sequence dna of the pMx1 segments of recombination pGL3-basic plasmids by DNA sequencing, selects the recombination containing correct sequence PGL3-basic plasmids；
Using the DNA sequence dna of the recombination pGL3-basic plasmids containing correct sequence as template, PCR primer is designed, PCR expansions are carried out Increase, obtains product；
Product is subjected to double digestion, then is purified by gel extraction and obtains pMx1-luc fusion segments.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that expand PCR The genetic fragment for increasing the pMx1 of the ox Mx1 albumen obtained is inserted into the ends 5' of pGL3-basic carrier luc genes, then is expanded using PCR It is Ase I and Xba I to increase the enzyme for obtaining selecting in the step of product is carried out double digestion by pMx1-luc fusions segment.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that use pMx1- Luc fusion segments substitute the genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers, build 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 fusion segments replace original pEGFP-N1 vector plasmids in pCMV with The genetic fragment of EGFP builds pMx1-luc plasmids；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, extracts plasmid, the pMx1- of plasmid is obtained by DNA sequencing The DNA sequence dna of luc fusion segments, selects the plasmid of correct sequence；
The correct sequence is：The gene fragment order of pMx1 is pMx1-luc fusion fragment sequences shown in sequence 1 Shown in sequence 2.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, the side of PCR primer is designed Method is conventional.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that use pMx1- Luc plasmid-transfected cells, and the step of filtering out stable transfected cells strain by neomycin includes：
PMx1-luc plasmid transfections to cell are changed into after transfecting 96h containing the complete of 2 μ g/mL neomycins by transfection reagent Full culture medium is persistently cultivated 14, and the cell with neomycin resistance, as stable transfected cells strain are filtered out.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that the clone Change cultivates the stable transfected cells strain for that will filter out by limiting dilution cultivation culture, to obtain individual cells formation Clone.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that screening The stable transfected cells strain gone out carries out in the step of colonized culture further including being detected the cell after clone, passes through PCR Whether contain pMx1-luc fusions in identification of cell genome.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that removal 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 Bov IFN α biological activity detection methods, it is preferred that the cell For MDBK cells.
In above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, it is preferred that the ox Interferon-' alpha ' standard items are the revealed a kind of system of recombinant bovine alpha interferon standard substance of patent that publication No. is CN106319006A The recombinant bovine alpha interferon that Preparation Method is prepared.
The present invention also provides above-mentioned chemical-activated luciferase gene expression Bov IFN α biological activity detection methods to day So or recombinant bovine interferon-' alpha ' carries out the application in biological activity detection.
The luciferase report that above-mentioned Bov IFN α biological activities detection method is built based on ox Mx1 gene promoters It accuses gene and its transfected into MDBK cells is screened into the cell strain of stable transfection.As Bov IFN α (IFN-α) and stable transfection fluorescence When the MDBK cells of plain enzyme reporter gene are incubated jointly, ox IFN-α can enhance Mx1 gene promoter activities, promote fluorescein The expression of enzyme detects luminous value after substrate is added and the biological activity of ox IFN-α is proportionate, to realize to ox IFN-α The quantitative detection of biological activity.
The present invention constructs ox Mx1 gene promoter sequences and the fusion of luc, and is inserted into eukaryon expression plasmid, turns Contaminate MDBK cells, screen stable transfected cells strain, with the recombinant bovine interferon-' alpha ' standard items and measuring samples of gradient dilution respectively with After cell is incubated altogether, luciferase substrate is added after lytic cell, detects luminous value, is prepared with recombinant bovine interferon-' alpha ' standard items Standard curve detects the biological activity of Bov IFN α in measuring samples.The detection method mainly has following characteristics：1. significantly Shorten detection time (being foreshortened 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.
The present invention uses ox Mx1 compared with existing I types interferon biological activity luciferase reporter gene detection method Gene promoter sequence and bovine kidney cells MDBK, biological activity of the special optimization for ox IFN-α detects, and is turned using stablizing Dye cell strain saves plasmid and repeats transfection procedure, simplifies detection process, improves accuracy.It is given birth to traditional I type interferon Object activity test method divides as virus replication inhibits (Virus Yield-reduction Assay, VYRA), plaque to reduce Analyse (Plaque ReductionAssay, PRA), cytopathic effect inhibition (Cytopathic Effect ReductionAssay, The methods of) CPERA compared to only cell need to be cultivated, substrate fluorescence intensity is added after interferon-' alpha ' processing cell 6h is used in combination Antiviral activity of interferon detection is completed, detection process is greatly simplified, improves accuracy.Coordinate Bov IFN standard items Use, the standardization of Bov IFN α biological activities may be implemented in application study and industrialization production and measure.
The present invention protrusion effect be：
The present invention quantitatively detects Bov IFN α using the chemical-activated luciferase gene expression of luciferase reporter gene (luc) Biological activity can simplify detection process, need not 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 Bov IFN α, the detection of interferon-' alpha ' biological activity can be completed in fluorescence intensity, Accuracy and repeatability are improved, bio-safety harm that may be present is avoided.
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 inhibits the correlation ratio of testing result with few cells lesion Compared with figure.
Specific implementation mode
In order to which technical characteristic, purpose and the advantageous effect to the present invention are more clearly understood, now to the skill of the present invention Art scheme carry out it is described further below, but should not be understood as to the present invention can practical range restriction.Institute in following embodiments Experimental method is stated, is conventional method unless otherwise specified；The reagent and material unless otherwise specified can be from business ways Diameter obtains.
Present invention material as used in the following examples is as follows：
MDBK cells (ATCC No.CCL-22) are purchased from ATCC, the plasmid pGL3-Basic purchases containing luciferase reporter gene From Promega companies, carrier for expression of eukaryon pEGFP-N1 is 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 various specifications 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；
Bov IFN α standard items (107IU/mL) it is the revealed a kind of recombination of patent that publication No. is CN106319006A 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 chemical-activated luciferase gene expression Bov IFN α biological activity detection methods, are pair The biological activity of recombinant bovine 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 the Activity determination of natural Bov IFN α comprising following steps：
According to the gene order for the ox Mx1 albumen announced in Genebank, the ends 5' is selected to contain ISRE response elements Promoter region (ifn response element is shown in runic character segment) designs PCR primer), Kpn I digestions are introduced in sense primer Site, downstream primer introduces Hind III digestions site (underscore part is upstream and downstream primer position), different by phenol-chloroform- Amylalcohol method extracts DNA as template from MDBK cells, carries out PCR amplification using above-mentioned PCR primer and Ex-Taq enzymes, is expanded Increase production object.
Amplified production is purified by gel extraction again after EcoR I and XhoI double digestions, obtains the gene piece of pMx1 The gene fragment order of section, pMx1 is shown in sequence 1；
The genetic fragment of pMx1 is connected into the ends 5' of pGL3-Basic plasmid luc genes by T4DNA ligases；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, filters out extraction recombination pGL3-basic matter after positive colony Grain is obtained the DNA sequence dna of the pMx1 segments of recombination pGL3-basic plasmids by DNA sequencing, selects the recombination containing correct sequence PGL3-basic plasmids；
Using the DNA sequence dna of the recombination pGL3-basic plasmids containing correct sequence as template, PCR primer is designed
(sense primer：5 '-attaatcggggtaccgccaggaacttcagaagg-3 ',
Downstream primer：5 '-tctagattacacggcgatctttccgcccttc-3 ' introduce Ase I in sense primer Restriction enzyme site, downstream primer introduce Xba I restriction enzyme sites), PCR amplification is carried out with above-mentioned primer and Ex-Taq enzymes, obtains product；
By product after Ase I and Xba I double digestions, then is purified by gel extraction and obtain pMx1-luc fusion pieces Section, pMx1-luc fusion fragment sequences 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 digestion methods；
By T4DNA ligases with pMx1-luc fusion segments replace original pEGFP-N1 vector plasmids in pCMV with The genetic fragment of EGFP builds 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 for obtaining pMx1-luc plasmids, selects the plasmid of correct sequence；
The correct sequence is：The gene fragment order of pMx1 is shown in sequence 1, and pMx1-luc gene orders are sequence 2 It is shown.
The structure schematic diagram of entire 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, and 1 μ g plasmids is taken to be placed in 1.5mL sterile EP tubes with Opti MEM cultures Base supplies volume to 500 μ l, and soft mixing is incubated at room temperature 5min；1.5mL sterilizing EP pipes are taken, 5 μ l Fugen6 are added and are dissolved in 500 In μ l serum-free Opti-MEM culture mediums, soft mixing 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 in serum-free Opti-MEM culture medium secondary culture MDBK cells to 6 orifice plates, per hole 5 × 105It is a Cell；Plasmid-transfection reagent compound is added to every hole, contains 5%CO in 37 DEG C2Overnight incubation in incubator；Next day removes training Base is supported, instead DMEM (containing Sodium Pyruvate and nonessential amino acid) complete medium continues to cultivate 96h；It is used instead later containing 2 μ g/ The complete medium screening and culturing of mL neomycins 14 days replaces primary fresh culture medium for 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, which is obtained, neomycin The cell of resistance is diluted to a concentration of 10/mL with the complete medium containing 0.25 μ g/mL puromycins, is added with the holes 0.1mL/ In 96 orifice plates, contain 5%CO in 37 DEG C2It is cultivated in incubator；Daily observation, selects the hole individually cloned, and culture to cell is covered When cap bore surface area 1/3, cell is transferred in 6 orifice plates to expand and is cultivated, conservation is frozen, is named as MDBK-pMx1-luc.
Bov IFN α standard items 1mL are taken, according to 1:10,1:100,1:103…1:107Gradient complete medium it is dilute It releases；
MDBK-pMx1-luc is seeded in 24 porocyte culture plates makes cell density reach 90% overnight, discards culture Base；Recombinant bovine interferon-' alpha ' standard items after being diluted to every hole addition 1mL with complete medium, 3 parallel holes of each dilution, It is arranged and is not added with the hole of recombinant bovine interferon-' alpha ' as a contrast, contains 5%CO in 37 DEG C2Continue to cultivate 6h in incubator, takes out culture Plate detects each hole fluorescence intensity according to the operation of Luciferase Assay Reagent box specification, and testing result is as shown in Fig. 2, marked As a result directrix curve shows fluorescence intensity and the negative logarithm of the dilution of recombinant bovine interferon-' alpha ' standard items linearly related R2= 0.9917, detection range 0.1-108IU/mL。
It is incubated altogether in cell Bov IFN α samples to be checked being added after colonized culture, then fluorescence intensity, knot Standardization curve evaluation obtains the potency of Bov IFN α samples to be checked.
Embodiment 2
The present embodiment provides the inspections of the chemical-activated luciferase gene expression Bov IFN α biological activity detection methods of the present invention Survey result inhibits the comparison correlation of method testing result to test with few cells lesion.
20 parts of recombinant bovine interference of method detection while detection are inhibited using chemical-activated luciferase gene expression and few cells lesion Plain α samples show whether the result of two methods has correlation by linear regression analysis.
Chemical-activated luciferase gene expression testing process is as described in Example 1.
Few cells lesion inhibits method by following operation：Recombinant bovine interferon-' alpha ' sample 1mL is taken, with complete medium 1:100 Complete medium doubling dilution is used to dilute again after dilution；MDBK secondary cultures are seeded to 96 porocyte culture plates, are inoculated with per hole 100 μ l cell suspensions (2 × 105A/mL)；Add the every holes 00 μ l of recombinant bovine Interferon α1 of different dilutions, 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, cell is removed Outside control wells, the VSV (the VSV virus titers that MDBK cells can for 24 hours completely fallen off) of 100 μ l appropriate titers is added per hole, in 37 DEG C contain 5%CO2Continue in incubator culture for 24 hours, the clasmatosis of microscopically observation virus control wells up to 100% and cell pair According to it is normal when, you can use violet staining, visually observe record after washing, cell is in completely purple in cell control well, viral Control non-coloring (100%CPE++++) shows that system is set up, and observes coloring case in different holes on its basis, records CPE feelings Condition calculates the extension rate of half cytopathic effect inhibition by Reed-Muench methods, to obtain the potency of interferon.Two kinds The results are shown in Figure 3 for the correlation analysis of detection method, as a result shows R2> 0.9 shows that the two has very high correlation.The knot Fruit shows that the chemical-activated luciferase gene expression Bov IFN alpha active detection method that the present invention establishes is simple, quick, testing result Reliably, stablize, few cells lesion can be substituted, this traditional detection method of method is inhibited to be applied to the interference of natural or recombinant bovine Plain alpha active measures.
In conclusion the embodiment of the present invention utilizes the chemical-activated luciferase gene expression ox of luciferase reporter gene (luc) Interferon-' alpha ' biological activity detection method, can simplify detection process, need not repeat Virus culture or plasmid transfection Troublesome operation only need to cultivate specific cells, and after being incubated 6h altogether with Bov IFN α, interferon-' alpha ' can be completed in fluorescence intensity Biological activity detects, and improves accuracy and repeatability, avoids bio-safety harm that may be present, has more practicability, It is suitble to apply in large-scale production.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto, Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
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<120>Chemical-activated luciferase gene expression Bov IFN α biological activity detection methods
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cggggtaccg ccagcagcag gtctgggtgc gggccgagaa tctgcatttc ccgcaagctc 60
ctagggatgc tgttgatgcg gggcgcacct agaatgcctt tgggagaata cagttgagtg 120
tgcggggcgc agcgacctcg ggaggccccg gtgcggaagt gcgccacccg ttcggtttcg 180
gtttcctttc cgatccagca gccctgaaaa ctttctgagt ttcgtttctc cgaggctggg 240
taggagatga gggacgggga ggcggggcag cgagctaggg gcggcgttag cgctgaataa 300
agccgaggag ggtgagcgct gggagcctgc cggtctctgc gctggggacg ggtaagtgta 360
gcgctcctgg gtctgggcac tctccgcggg tcggggcatt tgggcgttgg ctgggagttg 420
agcgctctgg ggtgccggta ttgcgcagac taggagaagg cgatggcacc ccactccagt 480
actcttgcct ggaaaatccc atggacggag gagcctggta ggctgcagtc catggagtct 540
cgaaaagtcg gacaccacag aagcgactta gcagcagcag cagcagtaat agtaaaaatg 600
aaaatgatta atagtgatgg caacccagta ttcttgcctg gagaatgcca tggacagagg 660
aacctggtgg gctgcagtcc atggggtctc aaagagtcgg acacgactga agcgacttag 720
cacaagcttg gg 732
<210> 2
<211> 2486
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
attaatcggg gtaccgccag gaacttcaga agggccggat aaagctcggg tagctgggtc 60
ctgcgcccga gtttctgagg cagcaggtct gggtgcgggc cgagaatctg catttcccgc 120
aagctcctag ggatgctgtt gatgcggggc gcacctagaa tgcctttggg agaatacagt 180
tgagtgtgcg gggcgcagcg acctcgggag gccccggtgc ggaagtgcgc cacccgttcg 240
gtttcggttt cctttccgat ccagcagccc tgaaaacttt ctgagtttcg tttctccgag 300
gctgggtagg agatgaggga cggggaggcg gggcagcgag ctaggggcgg cgttagcgct 360
gaataaagcc gaggagggtg agcgctggga gcctgccggt ctctgcgctg gggacgggta 420
agtgtagcgc tcctgggtct gggcactctc cgcgggtcgg ggcatttggg cgttggctgg 480
gagttgagcg ctctggggtg ccggtattgc gcagactagg agaaggcgat ggcaccccac 540
tccagtactc ttgcctggaa aatcccatgg acggaggagc ctggtaggct gcagtccatg 600
gagtctcgaa aagtcggaca ccacagaagc gacttagcag cagcagcagc agtaatagta 660
aaaatgaaaa tgattaatag tgatggcaac ccagtattct tgcctggaga atgccatgga 720
cagaggaacc tggtgggctg cagtccatgg ggtctcaaag agtcggacac gactgaagcg 780
acttagcaca agcttggggg cattccggta ctgttggtaa agccaccatg gaagacgcca 840
aaaacataaa gaaaggcccg gcgccattct atccgctgga agatggaacc gctggagagc 900
aactgcataa ggctatgaag agatacgccc tggttcctgg aacaattgct tttacagatg 960
cacatatcga ggtggacatc acttacgctg agtacttcga aatgtccgtt cggttggcag 1020
aagctatgaa acgatatggg ctgaatacaa atcacagaat cgtcgtatgc agtgaaaact 1080
ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca gttgcgcccg 1140
cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gggcatttcg cagcctaccg 1200
tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa aagctcccaa 1260
tcatccaaaa aattattatc atggattcta aaacggatta ccagggattt cagtcgatgt 1320
acacgttcgt cacatctcat ctacctcccg gttttaatga atacgatttt gtgccagagt 1380
ccttcgatag ggacaagaca attgcactga tcatgaactc ctctggatct actggtctgc 1440
ctaaaggtgt cgctctgcct catagaactg cctgcgtgag attctcgcat gccagagatc 1500
ctatttttgg caatcaaatc attccggata ctgcgatttt aagtgttgtt ccattccatc 1560
acggttttgg aatgtttact acactcggat atttgatatg tggatttcga gtcgtcttaa 1620
tgtatagatt tgaagaagag ctgtttctga ggagccttca ggattacaag attcaaagtg 1680
cgctgctggt gccaacccta ttctccttct tcgccaaaag cactctgatt gacaaatacg 1740
atttatctaa tttacacgaa attgcttctg gtggcgctcc cctctctaag gaagtcgggg 1800
aagcggttgc caagaggttc catctgccag gtatcaggca aggatatggg ctcactgaga 1860
ctacatcagc tattctgatt acacccgagg gggatgataa accgggcgcg gtcggtaaag 1920
ttgttccatt ttttgaagcg aaggttgtgg atctggatac cgggaaaacg ctgggcgtta 1980
atcaaagagg cgaactgtgt gtgagaggtc ctatgattat gtccggttat gtaaacaatc 2040
cggaagcgac caacgccttg attgacaagg atggatggct acattctgga gacatagctt 2100
actgggacga agacgaacac ttcttcatcg ttgaccgcct gaagtctctg attaagtaca 2160
aaggctatca ggtggctccc gctgaattgg aatccatctt gctccaacac cccaacatct 2220
tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg tgaacttccc gccgccgttg 2280
ttgttttgga gcacggaaag acgatgacgg aaaaagagat cgtggattac gtcgccagtc 2340
aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa gtaccgaaag 2400
gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat cctcataaag gccaagaagg 2460
gcggaaagat cgccgtgtaa tctaga 2486

Claims (10)

1. a kind of chemical-activated luciferase gene expression Bov IFN α biological activity detection methods comprising following steps：
The genetic fragment of the pMx1 of ox Mx1 albumen is obtained using PCR amplification；
The genetic fragment of the pMx1 for the ox Mx1 albumen that PCR amplification is obtained is inserted into the ends 5' of pGL3-basic carrier luc genes, PMx1-luc fusion segments are obtained using PCR amplification again；
The genetic fragment of the pCMV and EGFP in pEGFP-N1 carriers are substituted with pMx1-luc fusion segments, build 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 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. chemical-activated luciferase gene expression Bov IFN α biological activity detection methods according to claim 1, feature It is：
The gene fragment order of pMx1 is shown in sequence 1；
PMx1-luc fusion fragment sequences are shown in sequence 2.
3. chemical-activated luciferase gene expression Bov IFN α biological activity detection methods according to claim 1, feature Be, using PCR amplification obtain ox Mx1 albumen pMx1 genetic fragment the step of include：
According to the gene order for the ox Mx1 albumen announced in Genebank, the startup for selecting the ends 5' to contain ISRE response elements Subregion designs PCR primer, carries out PCR amplification, obtains amplified production；
Amplified production is subjected to double digestion, then purifies the genetic fragment for obtaining pMx1 by gel extraction；
Preferably, the step of genetic fragment of the pMx1 of ox Mx1 albumen is by amplified production progress double digestion is obtained using PCR amplification The enzyme of middle selection is KpnI and HindIII.
4. chemical-activated luciferase gene expression Bov IFN α biological activity detection methods according to claim 1, feature It is, the genetic fragment of the pMx1 for the ox Mx1 albumen that PCR amplification is obtained is inserted into the ends 5' of pGL3-basic carrier luc genes, The step of obtaining pMx1-luc fusion segments using PCR amplification again include：
The genetic fragment of pMx1 is connected into the ends 5' of pGL3-Basic plasmid luc genes by T4DNA ligases；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, extraction recombination pGL3-basic plasmids after positive colony is filtered out, leads to The DNA sequence dna that DNA sequencing obtains the pMx1 segments of recombination pGL3-basic plasmids is crossed, the recombination pGL3- containing correct sequence is selected Basic plasmids；
Using the DNA sequence dna of the recombination pGL3-basic plasmids containing correct sequence as template, PCR primer is designed, PCR amplification is carried out, Obtain product；
Product is subjected to double digestion, then is purified by gel extraction and obtains pMx1-luc fusion segments；
Preferably, the genetic fragment of the pMx1 of ox Mx1 albumen PCR amplification obtained is inserted into pGL3-basic carrier luc genes The ends 5', then the enzyme selected in the step of product is carried out double digestion by pMx1-luc fusions segment is obtained using PCR amplification 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 fusion segments The step of building pMx1-luc plasmids include：
Remove the genetic fragment of the pCMV and EGFP in pEGFP-N1 vector plasmids；
Pass through pCMV and EGFP of the T4DNA ligases in pMx1-luc fusion segments replacement original pEGFP-N1 vector plasmids Genetic fragment, build pMx1-luc plasmids；
Bacillus coli DH 5 alpha competent cell is converted, bacterium is shaken, extracts plasmid, plasmid pMx1-luc fusions are obtained by DNA sequencing The DNA sequence dna of genetic fragment selects the plasmid of correct sequence；
The correct sequence is：The gene fragment order of pMx1 is shown in sequence 1, and pMx1-luc fusion fragment sequences are sequence Shown in row 2.
5. chemical-activated luciferase gene expression Bov IFN α biological activity detection methods according to claim 1, feature It is, with pMx1-luc plasmid-transfected cells, and the step of filtering out stable transfected cells strain by neomycin includes：
PMx1-luc plasmid transfections to cell are changed into complete training containing 2 μ g/mL neomycins by transfection reagent after transfecting 96h Foster base is persistently cultivated 14, and the cell with neomycin resistance, as stable transfected cells strain are filtered out.
6. chemical-activated luciferase gene expression Bov IFN α biological activity detection methods according to claim 1, feature Be, the colonized culture be will the stable transfected cells strain that filter out by limiting dilution cultivation culture, to obtain The clone that individual cells are formed；
Preferably, further include to thin after clone in the step of stable transfected cells strain to filtering out carries out colonized culture Born of the same parents are detected, by whether containing pMx1-luc fusions in PCR identification of cell genomes.
7. chemical-activated luciferase gene expression Bov IFN α biological activity detection methods according to claim 4, 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 Bov IFN α biological activity detection methods according to claim 1 or 5, It is characterized in that：The cell is MDBK cells.
9. chemical-activated luciferase gene expression Bov IFN α biological activity detection methods according to claim 1, feature It is：The Bov IFN α standard items are the revealed a kind of recombinant bovine α interference of patent that publication No. is CN106319006A The recombinant bovine alpha interferon that the preparation method of plain standard items is prepared.
10. claim 1-9 any one of them chemical-activated luciferase gene expression Bov IFN α biological activity detection methods exist Application in biological activity detection is carried out to natural or recombinant bovine interferon-' alpha '.
CN201810641987.3A 2017-12-05 2018-06-21 Chemical-activated luciferase gene expression Bov IFN α biological activity detection methods Withdrawn CN108753918A (en)

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