CN108707642A - Chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method - Google Patents
Chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method Download PDFInfo
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Abstract
The invention discloses a kind of chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method and its applications.This approach includes the following steps：The genetic fragment of the pMx1 of pig Mx1 albumen is obtained using PCR amplification；The genetic fragment of pMx1 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 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 porcine interferon alpha standard items of gradient dilution, is incubated altogether in cell porcine interferon alpha sample to be checked being added after colonized culture, then fluorescence intensity, the potency of combined standard curve evaluation porcine interferon alpha sample to be checked.
Description
Technical field
The present invention relates to a kind of chemical-activated luciferase gene expression porcine interferon alpha 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 pig
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 porcine interferon alpha biological activity 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 porcine interferon alpha biological activity detection method comprising following steps：
The genetic fragment of the pMx1 of pig Mx1 albumen is obtained using PCR amplification；
The genetic fragment of the pMx1 for the pig Mx1 albumen that PCR amplification is obtained is inserted into the 5&apos 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 porcine interferon alpha standard items of gradient dilution, is determined between interferon potency and fluorescent value
Mathematical logic relationship；
Porcine interferon alpha sample to be checked is added in the cell after colonized culture, then fluorescence intensity, combined standard
The potency of curve evaluation porcine interferon alpha sample to be checked.
In above-mentioned chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method, by porcine interferon alpha 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 pig Mx1 gene promoter regions, and pCMV refers to the CMV promoter of pEGFP-N1 plasmids
Region.
In above-mentioned chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method, 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 porcine interferon alpha biological activity detection method, it is preferred that use PCR
Amplification obtain pig Mx1 albumen pMx1 genetic fragment the step of include：
According to the gene order for the pig Mx1 albumen announced in Genebank, 5&apos is selected;Contain ISRE response elements in end
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 porcine interferon alpha biological activity detection method, 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 pig Mx1 albumen the enzyme selected be KpnI and
HindIII。
In above-mentioned chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method, it is preferred that expand PCR
The genetic fragment for increasing the pMx1 of the pig Mx1 albumen obtained is inserted into the 5&apos of pGL3-basic carrier luc genes;End, then expanded using PCR
Increasing the step of obtaining pMx1-luc fusion segments includes：
The genetic fragment of pMx1 is connected into the 5&apos of pGL3-Basic plasmid luc genes by T4DNA ligases;End；
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 porcine interferon alpha biological activity detection method, it is preferred that expand PCR
The genetic fragment for increasing the pMx1 of the pig Mx1 albumen obtained is inserted into the 5&apos of pGL3-basic carrier luc genes;End, then 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 porcine interferon alpha biological activity detection method, 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 porcine interferon alpha biological activity detection method, the side of PCR primer is designed
Method is conventional.
In above-mentioned chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method, 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 porcine interferon alpha biological activity detection method, 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 porcine interferon alpha biological activity detection method, 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 porcine interferon alpha biological activity detection method, 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 porcine interferon alpha biological activity detection method, it is preferred that the cell
For pk-15 cells.
In above-mentioned chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method, it is preferred that the pig
Interferon-' alpha ' standard items are the revealed a kind of system of recombinant porcine alpha interferon standard substance of patent that notification number is CN101736062B
The recombinant porcine alpha interferon that Preparation Method is prepared.
The present invention also provides above-mentioned chemical-activated luciferase gene expression porcine interferon alpha biological activity detection methods to day
So or recombination porcine interferon alpha carries out the application in biological activity detection.
The luciferase report that above-mentioned porcine interferon alpha biological activity detection method is built based on pig Mx1 gene promoters
Gene is accused, pk-15 cells is transfected, screens the cell strain of stable transfection.When porcine interferon alpha (IFN-α) and stable transfection are glimmering
When the pk-15 cells of light element enzyme reporter gene are incubated jointly, pig IFN-α can enhance Mx1 gene promoter activities, promote fluorescence
The expression of plain enzyme detects luminous value after substrate is added and the biological activity of pig IFN-α is proportionate, to realize to pig IFN-
The quantitative detection of α biological activities.
The present invention constructs pig Mx1 gene promoter sequences and the fusion of luc, and is inserted into eukaryon expression plasmid, turns
Pk-15 cells are contaminated, stable transfected cells strain is screened, are distinguished with the recombination porcine interferon alpha standard items and measuring samples of gradient dilution
After being incubated altogether with cell, luciferase substrate is added after lytic cell, luminous value is detected, to recombinate porcine interferon alpha standard items system
Standby standard curve detects the biological activity of porcine interferon alpha in measuring samples.The detection method mainly has following characteristics：1. big
Shorten detection time (being foreshortened to 6-8 hours by traditional 3-7 days) greatly；Repeat Virus culture or plasmid turn 2. eliminating
The cumbersome operations such as dye, 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 pig Mx1 compared with existing I types interferon biological activity luciferase reporter gene detection method
Gene promoter sequence and porcine kidney cell pk-15, biological activity of the special optimization for pig IFN-α detects, and uses and stablize
Transfection cell strain saves plasmid and repeats transfection procedure, simplifies detection process, improves accuracy.With traditional I type interferon
Biological activity detection method, as virus replication inhibits (Virus Yield-reduction Assay, VYRA), plaque to reduce
Analyze (Plaque Reduction Assay, PRA), cytopathic effect inhibition (Cytopathic Effect Reduction
Assay, CPERA) the methods of compared to only cell need to be cultivated, be used in combination after interferon-' alpha ' processing cell 6h that substrate detection fluorescence is added is strong
Antiviral activity of interferon detection can be completed in degree, greatly simplifies detection process, improves accuracy.Coordinate pig interferon
The use of standard items may be implemented porcine interferon alpha biological activity standardization in application study and industrialization production and measure.
The present invention protrusion effect be：
The present invention quantitatively detects porcine interferon alpha 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 porcine interferon alpha, 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：
Pk-15 cells are purchased from ATCC, and the plasmid pGL3-Basic containing luciferase reporter gene is purchased from Promega companies,
Carrier for expression of eukaryon pEGFP-N1 be purchased from Clontech companies, various restriction enzymes, Ex-Taq enzymes, T4DNA ligases and
Plasmid extraction kit is purchased from Takara companies, and transfection reagent Fugen6 is purchased from Promega companies, DMEM culture mediums and FBS purchases
From Invitrogen companies, various specifications tissue culture plate is purchased from Corning companies；VSV viruses are by Chinese Academy of Sciences's Wuhan disease
Malicious research institute provides, and multi-function microplate reader is purchased from MD companies, and Luciferase Assay Reagent box is purchased from Promega companies；
Porcine interferon alpha standard items (1,000,000 IU/mL) are that the patent that notification number is CN101736062B is revealed a kind of heavy
The recombinant porcine alpha interferon (selection embodiment mode) that the preparation method of group porcine alpha interferon standard substance is prepared.
Embodiment 1
The present embodiment provides a kind of chemical-activated luciferase gene expression porcine interferon alpha biological activity detection methods, are pair
The biological activity of recombination porcine 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 porcine interferon alpha comprising following steps：
According to the gene order for the pig Mx1 albumen announced in Genebank, 5&apos is selected;Contain ISRE response elements in end
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 pk-15 cells, carries out PCR amplification using above-mentioned PCR primer and Ex-Taq enzymes, obtains
Amplified production.
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 5&apos of pGL3-Basic plasmid luc genes by T4DNA ligases;End；
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'-attaatcggGgtaccctgaatagcagcaggaactcc-3 ',
Downstream primer：5'-tctagaTtacacggcgatctttccgcccttc-3 ' introduces 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 pk-15 cells to 6 orifice plates, per hole 5 × 105
A cell；Plasmid-transfection reagent compound is added to every hole, contains 5%CO in 37 DEG C2Overnight incubation in incubator；Next day removes
Culture medium, instead DMEM (containing Sodium Pyruvate and nonessential amino acid), complete medium continued to cultivate 96h；It is used instead later containing 2 μ
The complete medium screening and culturing of g/mL neomycins 14 days replaces primary fresh culture medium for every 2 days；Period visual cell grows feelings
Condition decides whether to pass on；The cell survived after neomycin acts on 14 is stable transfected cells；Screening acquisition is had new mould
The cell of plain 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/
Enter in 96 orifice plates, contains 5%CO in 37 DEG C2It is cultivated in incubator；The hole individually cloned, culture to cell are selected in daily observation
When covering aperture surface area 1/3, cell is transferred in 6 orifice plates to expand and is cultivated, conservation is frozen, is named as pk-15-pMx1-luc.
Porcine interferon alpha standard items 1mL is taken, according to 1:10,1:100,1:103…1:107Gradient complete medium it is dilute
It releases；
Pk-15-pMx1-luc is seeded in 24 porocyte culture plates makes cell density reach 90% overnight, discards culture
Base；Recombination porcine 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 for recombinating porcine 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 the linear related R of negative logarithm of fluorescence intensity and the dilution for recombinating porcine interferon alpha standard items2=
0.9908, detection range is 0.1IU-1 × 107IU/mL。
It is incubated altogether in cell porcine interferon alpha sample to be checked being added after colonized culture, then fluorescence intensity, knot
Standardization curve evaluation obtains the potency of porcine interferon alpha sample to be checked.
Embodiment 2
The present embodiment provides the inspections of the chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method of the present invention
Survey result inhibits the comparison correlation of method testing result to test with few cells lesion.
20 parts of Recombinant Swine 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：Recombination porcine interferon alpha sample 1mL is taken, with complete medium 1:100
Complete medium doubling dilution is used to dilute again after dilution；Pk-15 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 Swine 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 pk-15 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 porcine interferon alpha activity test 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 Swine
Plain alpha active measures.
In conclusion the embodiment of the present invention utilizes the chemical-activated luciferase gene expression pig 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 porcine interferon alpha, 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
<110>Anhui Jiuchuan Biotechnology Co., Ltd.
<120>Chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 591
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cggggtaccc tgaatagcag caggaactcc tacagcaggt tttgtttgtt tgttttgttt 60
ttttaattgt tatttcccca atacaatttt ttttttctac tggacagcat ggtgacccag 120
ttacacatgc atgtacagat tctcatgctc catcataagc gactagacac agttcccagg 180
gctacacagc aggatctcac tgctgatcca ttccaaagga cagcagggtt ttaagacacc 240
caaatggcgc ttgcatctat tcagaagacc aggggtgaag ggatcggcta agggttcaaa 300
gctaccttca gcttttggat gacctggggc ttgttagagc cgcggttctc acagtgccag 360
aaccttcaga agggcccgac accactcagg tagctgggcc ctgtgcccga gcagcaggtc 420
tgggcggacc caagaatctg catttcccgc gagctcccaa ggatgctgtg ggtgcaggaa 480
gctccccccg acccccactc gctcgcgcca gacgcctcta ggtttcgttt ctaacgcgca 540
gccctccttc ccgggcagtt ccggggagtt tcgtttcccc gcaagcttgg g 591
<210> 2
<211> 2285
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
attaatcggg gtaccctgaa tagcagcagg aactcctaca gcaggttttg tttgtttgtt 60
ttgttttttt aattgttatt tccccaatac aatttttttt ttctactgga cagcatggtg 120
acccagttac acatgcatgt acagattctc atgctccatc ataagcgact agacacagtt 180
cccagggcta cacagcagga tctcactgct gatccattcc aaaggacagc agggttttaa 240
gacacccaaa tggcgcttgc atctattcag aagaccaggg gtgaagggat cggctaaggg 300
ttcaaagcta ccttcagctt ttggatgacc tggggcttgt tagagccgcg gttctcacag 360
tgccagaacc ttcagaaggg cccgacacca ctcaggtagc tgggccctgt gcccgagcag 420
caggtctggg cggacccaag aatctgcatt tcccgcgagc tcccaaggat gctgtgggtg 480
caggaagctc cccccgaccc ccactcgctc gcgccagacg cctctaggtt tcgtttctaa 540
cgcgcagccc tccttcccgg gcagttccgg ggagtttcgt ttccccgcaa gcttgggggc 600
attccggtac tgttggtaaa gccaccatgg aagacgccaa aaacataaag aaaggcccgg 660
cgccattcta tccgctggaa gatggaaccg ctggagagca actgcataag gctatgaaga 720
gatacgccct ggttcctgga acaattgctt ttacagatgc acatatcgag gtggacatca 780
cttacgctga gtacttcgaa atgtccgttc ggttggcaga agctatgaaa cgatatgggc 840
tgaatacaaa tcacagaatc gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg 900
tgttgggcgc gttatttatc ggagttgcag ttgcgcccgc gaacgacatt tataatgaac 960
gtgaattgct caacagtatg ggcatttcgc agcctaccgt ggtgttcgtt tccaaaaagg 1020
ggttgcaaaa aattttgaac gtgcaaaaaa agctcccaat catccaaaaa attattatca 1080
tggattctaa aacggattac cagggatttc agtcgatgta cacgttcgtc acatctcatc 1140
tacctcccgg ttttaatgaa tacgattttg tgccagagtc cttcgatagg gacaagacaa 1200
ttgcactgat catgaactcc tctggatcta ctggtctgcc taaaggtgtc gctctgcctc 1260
atagaactgc ctgcgtgaga ttctcgcatg ccagagatcc tatttttggc aatcaaatca 1320
ttccggatac tgcgatttta agtgttgttc cattccatca cggttttgga atgtttacta 1380
cactcggata tttgatatgt ggatttcgag tcgtcttaat gtatagattt gaagaagagc 1440
tgtttctgag gagccttcag gattacaaga ttcaaagtgc gctgctggtg ccaaccctat 1500
tctccttctt cgccaaaagc actctgattg acaaatacga tttatctaat ttacacgaaa 1560
ttgcttctgg tggcgctccc ctctctaagg aagtcgggga agcggttgcc aagaggttcc 1620
atctgccagg tatcaggcaa ggatatgggc tcactgagac tacatcagct attctgatta 1680
cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt tgttccattt tttgaagcga 1740
aggttgtgga tctggatacc gggaaaacgc tgggcgttaa tcaaagaggc gaactgtgtg 1800
tgagaggtcc tatgattatg tccggttatg taaacaatcc ggaagcgacc aacgccttga 1860
ttgacaagga tggatggcta cattctggag acatagctta ctgggacgaa gacgaacact 1920
tcttcatcgt tgaccgcctg aagtctctga ttaagtacaa aggctatcag gtggctcccg 1980
ctgaattgga atccatcttg ctccaacacc ccaacatctt cgacgcaggt gtcgcaggtc 2040
ttcccgacga tgacgccggt gaacttcccg ccgccgttgt tgttttggag cacggaaaga 2100
cgatgacgga aaaagagatc gtggattacg tcgccagtca agtaacaacc gcgaaaaagt 2160
tgcgcggagg agttgtgttt gtggacgaag taccgaaagg tcttaccgga aaactcgacg 2220
caagaaaaat cagagagatc ctcataaagg ccaagaaggg cggaaagatc gccgtgtaat 2280
ctaga 2285
Claims (10)
1. a kind of chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method comprising following steps：
The genetic fragment of the pMx1 of pig Mx1 albumen is obtained using PCR amplification；
The genetic fragment of the pMx1 for the pig Mx1 albumen that PCR amplification is obtained is inserted into the 5&apos of pGL3-basic carrier luc genes;End,
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 porcine interferon alpha standard items of gradient dilution, determines the mathematics between interferon potency and fluorescent value
Logical relation；
Porcine interferon alpha sample to be checked is added in the cell after colonized culture, then fluorescence intensity, combined standard curve
Evaluate the potency of porcine interferon alpha sample to be checked.
2. chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method 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 porcine interferon alpha biological activity detection method according to claim 1, feature
Be, using PCR amplification obtain pig Mx1 albumen pMx1 genetic fragment the step of include：
According to the gene order for the pig Mx1 albumen announced in Genebank, 5&apos is selected;Hold the startup containing 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 pig 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 porcine interferon alpha biological activity detection method according to claim 1, feature
It is, the genetic fragment of the pMx1 for the pig Mx1 albumen that PCR amplification is obtained is inserted into the 5&apos of pGL3-basic carrier luc genes;End,
The step of obtaining pMx1-luc fusion segments using PCR amplification again include：
The genetic fragment of pMx1 is connected into the 5&apos of pGL3-Basic plasmid luc genes by T4DNA ligases;End；
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 pig Mx1 albumen PCR amplification obtained is inserted into pGL3-basic carrier luc genes
5'End, then PCR amplification is used to obtain the enzyme selected in the step of product is carried out double digestion by pMx1-luc fusions segment
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 porcine interferon alpha biological activity detection method 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 porcine interferon alpha biological activity detection method 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 porcine interferon alpha biological activity detection method 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 porcine interferon alpha biological activity detection method according to claim 1 or 5,
It is characterized in that：The cell is pk-15 cells.
9. chemical-activated luciferase gene expression porcine interferon alpha biological activity detection method according to claim 1, feature
It is：The porcine interferon alpha standard items are the revealed a kind of recombinant porcine alpha interference of patent that notification number is CN101736062B
The recombinant porcine 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 porcine interferon alpha biological activity detection methods exist
Application in biological activity detection is carried out to natural or recombination porcine interferon alpha.
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| CN113621648A (en) * | 2021-08-30 | 2021-11-09 | 浙江大学 | Construction method and application of pBD1 gene fluorescence report intestinal epithelial cell line |
| 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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| CN113278592A (en) * | 2021-05-19 | 2021-08-20 | 中国农业大学 | High-throughput screening cell model for nutrient substances for regulating development of early-stage embryos of pigs as well as construction and application of cell model |
| CN113278592B (en) * | 2021-05-19 | 2022-05-10 | 中国农业大学 | High-throughput screening cell model for nutrient substances for regulating development of early-stage embryos of pigs as well as construction and application of cell model |
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