CN106399461A - Method of detection of transcription factor expression activity by luciferase reporter gene system - Google Patents
Method of detection of transcription factor expression activity by luciferase reporter gene system Download PDFInfo
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- CN106399461A CN106399461A CN201610825203.3A CN201610825203A CN106399461A CN 106399461 A CN106399461 A CN 106399461A CN 201610825203 A CN201610825203 A CN 201610825203A CN 106399461 A CN106399461 A CN 106399461A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
Abstract
The invention provides a method of detection of transcription factor expression activity by a luciferase reporter gene system; the method comprises the steps: based on the luciferase characteristics of being lasting, stable and easy to detect, co-transfecting cells with a luciferase reporter gene vector containing a target gene promoter and a transcription factor expression plasmid; after the cells are cultured, carrying out lysis of a certain number of cells with a cell lysis buffer liquid, collecting a lysate containing luciferase, and centrifuging to take a supernatant; and then real-timely detecting luciferase expression intensity data in a luciferase activity detection buffer liquid, at the same time, detecting the luciferase concentration, correcting, and then calculating to obtain the transcription factor expression quantity, namely the transcription factor expression activity needing to detection. The method is simpler and more feasible in whole operation process, shorter in time consuming, higher in flux, relatively high in repeatability and accuracy, and lower in operating costs, thereby having broad application prospects, and having excellent market potential.
Description
Technical field
The invention belongs to molecular biology and technical field of cell biology are and in particular to a kind of luciferase reporter gene system
The method of system detection transcription factor expression activity.
Background technology
The nucleotide sequence playing controlling gene expressional function in same nucleic acid chains is referred to as cis-acting elements, including startup
Son, enhancer, attenuator etc.；Transcription factor is also referred to as trans-acting factor, is a kind of specific effect in promoter region
Cis-acting elements so as to play the protein molecule of regulating and controlling effect to the gene expression in different nucleic acid chains.Transcription factor will
Play a role, it is necessary to have certain activity, this quantity depending on transcription factor and its parent being combined with cis-acting elements
And power.It can be seen that, the activity of detection transcription factor, for the rule of the expression regulation illustrating certain gene, significant, because
And be widely used in biology and medical research.
Particularly, the expression regulation of eukaryotic gene be current molecular field of biology forefront research direction it
One, and the regulation and control of transcriptional level are of paramount importance steps during gene expression.Due to protein-protein, protein with
Interaction between DNA, and the formation of some complicated macromolecular complexs, lead to the regulation and control of eucaryote transcriptional level more
For complexity；And, the detection of transcription factor and its expression regulation is the important prerequisite basis studying its function, efficient, special, clever
Quick, easy detection method has important effect for research eukaryotic gene expression.
It is presently used for detecting that the most commonly used method of transcription factor activity has fluorescence quantitative PCR detection, this detection method will
Transcription factor expression carrier is transfected in eukaryotic, and detects the expression of transcription factor by RT-PCR method, or logical
Cross protein expression level and indirectly reflect transcriptional activity；Wherein, RT-PCR detection method needs transcription factor transfecting eukaryotic cells,
By extracting total serum IgE in cell, enter performing PCR detection；However, RNA is temperature sensitive and degradable, need to design multipair during detection
Primer carries out purpose RNA amplification, chooses suitable internal reference and positive negative control simultaneously, and these defects above-mentioned lead to the result detecting not
The expression activity of transcription factor can truly be reflected, simultaneously repeatable poor, detection process takes longer.
Therefore, also provide in prior art and extract total protein in cell, and pass through Western blot (Western
Blotting) detect the expression of specified protein, thus reacting transcription factor expression amount from side；However, the behaviour of the method
Make more loaded down with trivial details, and, due to inevitably losing during protein extraction and degrading, measured result can not be true
The transcriptional expression amount of transcription factor in real reflection eukaryotic, additionally, the operating process uncontrollable factor of whole experiment is relatively more,
For therefore compared to popular RT-PCR detection method, more it is not suitable for detecting the expression activity of transcription factor.
Additionally, Chinese patent CN1637417A is additionally provided with a kind of transcription factor activity detection method, the method is base
In the improvement of ELISA detection method, its special feature is to be built using a kind of new Double stranded oligonucleotide acid probe, to overcome
The deficiency that the design construction of ELISA detection method Double stranded oligonucleotide acid probe is brought.However, this new E LISA detection method
Yet suffer from complex operation and time-consuming longer technical problem.
Luciferase detection (luciferase assay) implemented by means of luciferase reporter gene system is current
Be widely used in eukaryotic gene expression and stechiology research, its application include receptor active, intracellular signal transduction,
MRNA processing and protein folding.Set up luciferase reporter gene detecting system simple, convenient, and luciferase is persistently steady
Fixed, thus can be used for detecting the research that transcription factor is combined with the special cis-acting elements in its promoter.
Content of the invention
In order to solve the above-mentioned problems in the prior art, that is, in order to provide a kind of short, high pass simple to operate, time-consuming
Amount, the detection method of the transcription factor expression activity that repeatable high and the degree of accuracy is high, inventor intends making full use of luciferase
Detection, that is, by means of luciferase reporter gene system, provides a kind of method of new detection transcription factor expression activity.
Specifically, the technical scheme that the present invention records provides a kind of luciferase reporter gene system detection transcription factor
The method of expression activity, comprises the following steps：
Step (1)：The protein composition of the transcription factor according to expression activity to be detected, determination can be with described transcription factor
In conjunction with target gene promoters sequence, the specific fragment of the target gene promoters screening insertion luciferase reporter gene is carried
The specific fragment of the described target gene promoters after body, and, insertion is located at the upstream of the expressed sequence of luciferase Luc, from
And the luciferase reporter gene carrier that comprise target gene promoters is obtained；
Step (2)：Carry out culture and the bed board of target cell, obtain cell to be transfected；
Step (3)：Prepare liposome, the described luciferase reporter gene carrier comprising target gene promoters and transcription because
The mixture of sub- expression plasmid, using the cell to be transfected obtained by this mixture transfection procedure (2), obtains transfected thin
Born of the same parents；
Step (4)：Cell lysis buffer solution is added to described transfected cell, is cracked, collect lysate, connect
, the collected lysate of centrifugation, take supernatant, add and carry out luciferase expression to luciferase assays buffer solution
Intensity detection, and calculate the expression of luciferase, thus detecting the expression activity of described transcription factor.
Wherein, described cracking refers to the cracking to cell membrane for cell lysis buffer solution A.
In the step (1) of said method, can be soft with known ncbi database and TargetScan biology
Part etc. is predicted and determines the target gene promoters sequence being combined with described transcription factor.
Preferably, detect in the method for transcription factor expression activity in above-mentioned luciferase reporter gene system, described glimmering
Light element enzyme Reporter gene vector is promoter enhancer report carrier pGL3/4-basic；This promoter enhancer report carrier
PGL3/4-basic is preferably purchased from Promega company of the U.S..
Preferably, detect in the method for transcription factor expression activity in above-mentioned luciferase reporter gene system, described step
Suddenly (2) include：Target cell is incubated at containing in hyclone, dual anti-complete medium, and maintains monolayer adherence to grow,
At 37 DEG C, in 5%CO2Incubator in incubated overnight, after cell confluency degree reaches 80%, then can come into effect step
(3).
Preferably, detect in the method for transcription factor expression activity in above-mentioned luciferase reporter gene system, described step
Suddenly in (3), the operation of transfection is：Described cell to be transfected is inoculated in 24 well culture plates for 100000 with every hole, will be described mixed
Compound and culture medium add this culture plate, are placed in the 5%CO at 37 DEG C2After incubation 6h in incubator, inhale and abandon described mixture, then
Plus fresh culture transfection culture 18～24h.
Present invention also offers a kind of cell lysis buffer solution, detect transcription for above-mentioned luciferase reporter gene system
In the method for factor expression activity.
Preferably, detect in the method for transcription factor expression activity in above-mentioned luciferase reporter gene system, described thin
Cellular lysate buffer solution has following components and proportioning：
0.2M KH2PO44-4.5ml, such as 4.1-4.45ml, 4.15-4.4ml, 4.2-4.35ml, 4.25-4.3ml；
0.2M K2HPO444-48ml, such as 44.5-47ml, 45-46.5ml, 45.75-46.25ml；
MQ H2O 48-52ml, such as 48.5-51.6ml, 48.8-50.4ml, 49.2-49.8ml；
Triton X-100 200μl.
Present invention also offers a kind of luciferase assays buffer solution, for above-mentioned luciferase reporter gene system
In the method for detection transcription factor expression activity.
Preferably, detect in the method for transcription factor expression activity in above-mentioned luciferase reporter gene system, described glimmering
Light element Enzyme assay buffer solution has following components and proportioning：
0.25M Tris pH7.8 700μl
1M MgSO4100-115 μ l, such as 102-112 μ l, 103-110 μ l, 104-108 μ l, 105-107 μ l；
dH2O 5870-5880 μ l, such as 5872-5879 μ l, 5875-5878 μ l；
0.25M ATP 250-300 μ l, such as 255-290 μ l, 260-285 μ l, 270-280 μ l,
Luc 43-50 μ l, such as 44-48.5 μ l, 45-48 μ l, 46-47 μ l.
What deserves to be explained is, in described cell lysis buffer solution and described luciferase assays buffer solution, each component
Although proportioning illustrate in the form of numerical value is combined with unit, however it is not limited to the shape that above numerical value is combined with unit
Formula, in other words, the actual each group timesharing prepared in described cell lysis buffer solution and described luciferase assays buffer solution,
As long as ensureing that each ratio of components meets said ratio.
Preferably, detect in the method for transcription factor expression activity in above-mentioned luciferase reporter gene system, described step
Suddenly (4) include：
(4.1) inhale the culture medium abandoning in described culture plate, add the PBS of 400 μ l/ hole precoolings, rinse described transfected
Cell 1-2 time, then inhales and abandons PBS；
(4.2) described cell lysis buffer solution A preparing in advance to each in the hole addition 50 μ l of described culture plate, connects
, described culture plate is placed in 4 DEG C of shaking table and rocks 5-15 minute, during rocking, check whether cell comes off；
(4.3) collect lysate, put in the 1.5ml EP pipe of a set of mark, with centrifuge at 4 DEG C with 12000rpm
Rotating speed centrifugation 10min, obtain supernatant；
(4.4) now join described luciferase assays buffer B, therefrom take 146 μ l to add to another set of mark
In 1.5ml EP pipe, and this EP pipe is inserted in holding low temperature on ice；
(4.5) supernatant taking gained in 20 μ l (4.3) adds to the EP pipe described in (4.4), detects luciferase
Expression intensity data；
(4.6) luciferase concentration, correction are detected；And calculate the expression of luciferase, thus detect described turning
The expression activity of the record factor.
Above-mentioned luciferase reporter gene system detect transcription factor expression activity method in, due to described transcription because
Son expression activity show as can activation target gene promoter, therefore, if described transcription factor can open activation target gene
Mover, then luciferase gene will express, and as shown in figure 1, luciferase can produces chemiluminescence Enzyme assay buffering
Fluorescein (Luc) in liquid B reacts and produces fluorescence, and the expression of luciferase is become with the expression activity of described transcription factor
Direct ratio.Just because of the presence of this Gene Expression Mechanism and corresponding proportional relation, above-mentioned luciferase reporter gene system inspection
The method surveying transcription factor expression activity just can be carried out.
As can be seen here, the present invention builds target gene promoters region luciferase reporter gene by the method for molecular cloning
Plasmid, for detecting the expression of the Intracellular transcription factor, and then is applied to grinding of eukaryotic transcription factor expression mechanism
Study carefully.
In sum, a kind of luciferase reporter gene system of inventor successful implementation detection transcription factor expression activity
Method, the method is ingenious to be make use of luciferase itself lasting stability and is easy to the characteristic detecting, will comprise target base first
Luciferase reporter gene carrier because of promoter is transfected to cell jointly with transcription factor expression plasmid, continues culture transfected
Cell after, by a number of cell with cell lysis buffer solution crack, collect the lysate containing luciferase, then from
The heart takes supernatant, and then in luciferase assays buffer solution, real-time detection goes out luciferase expression intensity data, and with
When record luciferase concentration, after correction, you can be calculated the expression of described transcription factor, i.e. the transcription of required detection
The expression activity of the factor；As can be seen here, compared to prior art, the whole operation process of the method is simpler easy, takes
Shorter, flux is higher, and has higher repeatability and the degree of accuracy.
Additionally, present invention also offers a kind of cell lysis buffer solution and luciferase assays buffer solution, for institute
The method that the luciferase reporter gene system stated detects transcription factor expression activity, compared to the corresponding fluoroscopic examination work of purchase
For making liquid, cost is cheaper；Currently, the Promega company monopolizing luciferase expression Activity determination industry overwhelming majority
Market, the price of its 1000 fluorescence detection reagent kit (working solution containing fluoroscopic examination) producing is about 12000 yuan；Average each
The price of sample detection reaches 120 yuan；About 100 detection at least 160-200 unit/samples；Fluoroscopic examination work provided by the present invention
The cost price making liquid is about 3000 yuan, it is possible to implement about 3300 times, under identical price, can the number of times of examinations be state foreign minister
Answer product 12-15 times, thus there is splendid price advantage, economical and practical.Therefore, luciferase report provided by the present invention
Accuse method and cell lysis buffer solution and the luciferase assays buffering that genic system detects transcription factor expression activity
Liquid, has the above-mentioned comprehensive advantage not available for existing transcription factor expression activity test method, the method is in expression activity
Context of detection has broad application prospects, and has excellent market potential.
Brief description
Fig. 1 is that luciferase reporter gene system of the present invention detects fluorescein in the active method of transcription factor expression
Enzymatic mechanism schematic diagram, illustrated therein is fluorescein luminescence reaction equation it is seen then that fluorescein is under luciferase catalysis, and
Mg2+And have issued fluorescence under ATP effect；
Fig. 2 is that luciferase reporter gene system of the present invention detects in the active method of transcription factor expression preferably
The collection of illustrative plates of pGL3/4-basic carrier.
Specific embodiment
With reference to specific embodiment, the present invention is further elaborated, but the present invention is not limited to following embodiment party
Formula.
The invention provides a kind of method that luciferase reporter gene system detects transcription factor expression activity, including with
Lower step：
Step (1)：The protein composition of the transcription factor according to expression activity to be detected, determination can be with described transcription factor
In conjunction with target gene promoters sequence, with genomic DNA as template, using PCR reaction expanded, obtain target gene promoters
Control region fragment；Then, described target gene promoters control region fragment is carried out digestion purifying, screen recon；Finally, will sieve
The specific fragment insertion luciferase reporter gene carrier of the target gene promoters chosen, and, the described target gene after insertion
The specific fragment of promoter is located at the upstream of the expressed sequence of luciferase Luc, thus be obtained comprising the glimmering of target gene promoters
Light element enzyme Reporter gene vector；
Step (2)：Carry out culture and the bed board of target cell, obtain cell to be transfected；
Step (3)：Prepare liposome, the described luciferase reporter gene carrier comprising target gene promoters and transcription because
The mixture of sub- expression plasmid, using the cell to be transfected obtained by this mixture transfection procedure (2), obtains transfected thin
Born of the same parents；
Step (4)：Cell lysis buffer solution A is added to described transfected cell, is cracked, collect lysate, connect
, the collected lysate of centrifugation, take supernatant, add and carry out luciferase table to luciferase assays buffer B
Reach intensity detection, and calculate the expression of luciferase, thus detecting the expression activity of described transcription factor.
In a preferred embodiment, described luciferase reporter gene carrier is promoter enhancer report carrier
pGL3/4-basic.Wherein, pGL3/4-basic Vector map is as shown in Figure 2.
In a preferred embodiment, described step (2) includes：Target cell is incubated at containing hyclone, dual anti-
Complete medium in, and maintain monolayer adherence grow, at 37 DEG C, in 5%CO2Incubator in incubated overnight, treat cell
After degree of converging reaches 80%, then can come into effect step (3).
In a preferred embodiment, in described step (3), the operation of transfection is：By described cell to be transfected with every hole
100000 are inoculated in 24 well culture plates, described mixture and culture medium are added this culture plate, is placed in 5% at 37 DEG C
CO2After incubation 6h in incubator, inhale and abandon described mixture, then plus fresh culture transfection culture 18-24h.
In a preferred embodiment, described cell lysis buffer solution A has following components and proportioning：
In a preferred embodiment, described luciferase assays buffer B has following components and proportioning：
In a preferred embodiment, described step (4) includes：
(4.1) inhale the culture medium abandoning in described culture plate, add the PBS of 400 μ l/ hole precoolings, rinse described transfected
Cell 1-2 time, then inhales and abandons PBS；
(4.2) described cell lysis buffer solution A preparing in advance to each in the hole addition 50 μ l of described culture plate, connects
, described culture plate is placed in 4 DEG C of shaking table and rocks 5-15 minute, during rocking, check whether cell comes off；
(4.3) collect lysate, put in the 1.5ml EP pipe of a set of mark, with centrifuge at 4 DEG C with 12000rpm
Rotating speed centrifugation 10min, obtain supernatant；
(4.4) now join described luciferase assays buffer B, therefrom take 146 μ l to add to another set of mark
In 1.5ml EP pipe, and this EP pipe is inserted in holding low temperature on ice；
(4.5) supernatant taking gained in 20 μ l (4.3) adds to the EP pipe described in (4.4), detects luciferase
Expression intensity data；
(4.6) luciferase concentration, correction are detected；And calculate the expression of luciferase, thus detect described turning
The expression activity of the record factor.
Step in following embodiments is routine operation if no special instructions, and described reagent if no special instructions all can be from
Disclosure is either commercially available.
Embodiment 1
The main agents bought and instrument：
Promoter enhancer report carrier pGL3/4-basic (plasmid) is purchased from Promega company of the U.S.；PBS (phosphate
Buffer solution) it is purchased from HyClone company；LipofectaminTM2000 liposomes are purchased from Invitrogen company；Luciferase expression intensity
Detector is Promega Glomax 20/20 biology/chemiluminescence detector.Preparation of reagents：
100ml cell lysis buffer solution A (is prepared before experiment) in advance, wherein：
6988 μ l luciferase assays buffer B (taking 2*24 hole as a example, experiment is now joined after starting), wherein：
Start to test, operating procedure is as follows：
(1)：Specify the protein composition of transcription factor, determine the target gene promoters sequence being combined with described transcription factor
Row, with genomic DNA as template, are expanded using PCR reaction, are obtained target gene promoters control region fragment；Then, by institute
State target gene promoters control region fragment and carry out digestion purifying, screen recon；Finally, by the target gene promoters screening
Specific fragment inserts luciferase reporter gene carrier, and, the specific fragment of described target gene promoters after inserting is located at
The upstream of the expressed sequence of luciferase Luc, thus be obtained the luciferase reporter gene carrier comprising target gene promoters；
(2)：Target cell is incubated at containing 10% hyclone and contains in dual anti-complete medium, and remain single
Layer adherent growth, at 37 DEG C, in 5%CO2Incubator in stand overnight culture, object observing cell growth state, treat mesh
Mark cell growth is counted with trypan blue respectively to during logarithmic phase, is inoculated in 24 well culture plates with the cell concentration in 100000 every holes,
It is placed in the 5%CO at 37 DEG C2Cultivate in incubator, when cell confluency degree reaches 80%, obtain cell to be transfected, come into effect
Following steps (3)；
(3)：Prepare the promoter enhancer report carrier pGL3/4-basic/ transcription factor table comprising target gene promoters
Reach plasmid/LipofectaminTMThe mixture of 2000 liposomes, transfects described cell to be transfected using this mixture, obtains quilt
The cell of transfection, specifically includes following steps：
(3.1) by promoter enhancer report carrier pGL3/4-basic, 60ng of comprising target gene promoters of 300ng
Transcription factor expression plasmid, be dissolved in 50 μ l serum free mediums dilution, under room temperature, be incubated 5min；
(3.2) by the Lipofectamin of 2 μ lTM2000 liposomes are dissolved in 50 μ l serum free mediums in the same way
Dilution, is incubated 5min under room temperature；
(3.3) the soft reagent mixing (3.1) and preparation in (3.2), room temperature stands 20～30 minutes, obtains comprising target base
Promoter enhancer report carrier pGL3/4-basic/ transcription factor expression plasmid/Lipofectamin because of promoterTM2000
The mixture of liposome；
(3.4) inhale the former culture medium abandoning in 24 well culture plates, then cleaned 3 times with PBS (phosphate buffer), will be described
Cell to be transfected is inoculated in this 24 well culture plate for 100000 with every hole, and every hole adds 500 μ l serum free mediums, then by institute
State mixture to be added dropwise in this culture plate, and gently rocker uniformly, is placed in the 5%CO at 37 DEG C2After incubation 6h in incubator,
Described mixture is abandoned in suction, then plus fresh culture transfection culture 18-24h, obtain transfected cell；
(3.5) control group pGL3/4-basic empty carrier implements parallel transfection, and every group is all done 3 secondary orifices and be repeated 3 times to protect
The accuracy of card experimental result.
(4)：Cell lysis buffer solution A is added to described transfected cell, is cracked, collect lysate, then, from
Lysate collected by the heart, takes supernatant, adds and carries out luciferase expression intensity to luciferase assays buffer B
Detection, and calculate the expression of luciferase, thus detecting the expression activity of described transcription factor, concrete operations are as follows：
(4.1) inhale the culture medium abandoning in described transfected cell place culture plate, add the PBS of 400 μ l/ hole precoolings,
Rinse described transfected cell 1-2 time, then inhale and abandon PBS；
(4.2) described cell lysis buffer solution A preparing in advance to each in the hole addition 50 μ l of described culture plate, connects
, described culture plate is placed in 4 DEG C of shaking table and rocks 5-15 minute, during rocking, check whether cell comes off；
(4.3) collect lysate, put in the 1.5ml EP pipe of a set of mark, with centrifuge at 4 DEG C with 12000rpm
Rotating speed centrifugation 10min, obtain supernatant；
(4.4) now join described luciferase assays buffer B, therefrom take 146 μ l to add to another set of mark
In 1.5ml EP pipe, and this EP pipe is inserted in holding low temperature on ice；
(4.5) supernatant taking gained in 20 μ l (4.3) adds to the EP pipe described in (4.4), detects luciferase
Expression intensity data；
(4.6) luciferase concentration, correction are detected；And calculate the expression of luciferase, thus detect described turning
The expression activity of the record factor.
Above the specific embodiment of the present invention is described in detail, but it has been only used as example, the present invention has been not intended to limit
In particular embodiments described above.To those skilled in the art, any equivalent modifications that this practicality is carried out and replacing
In generation, is also all among scope of the invention.Therefore, the equalization made without departing from the spirit and scope of the invention converts and repaiies
Change, all should cover within the scope of the invention.
Claims (9)
1. a kind of luciferase reporter gene system detects the method for transcription factor expression activity it is characterised in that including following
Step：
Step（1）：The protein composition of the transcription factor according to expression activity to be detected, determination can be combined with described transcription factor
Target gene promoters sequence, the specific fragment of the target gene promoters screening is inserted luciferase reporter gene carrier,
And, the specific fragment of the described target gene promoters after inserting is located at the upstream of the expressed sequence of luciferase Luc, thus making
The luciferase reporter gene carrier of target gene promoters must be comprised；
Step（2）：Carry out culture and the bed board of target cell, obtain cell to be transfected；
Step（3）：Prepare liposome, the described luciferase reporter gene carrier comprising target gene promoters and transcription factor table
Reach the mixture of plasmid, using this mixture transfection procedure（2）Obtained cell to be transfected, obtains transfected cell；
Step（4）：Cell lysis buffer solution is added to described transfected cell, is cracked, collect lysate, then, from
Lysate collected by the heart, takes supernatant, adds and carries out luciferase expression intensity to luciferase assays buffer solution
Detection, and calculate the expression of luciferase, thus detecting the expression activity of described transcription factor.
2. the method that luciferase reporter gene system according to claim 1 detects transcription factor expression activity, it is special
Levy and be, described luciferase reporter gene carrier is promoter enhancer report carrier pGL3/4-basic.
3. the method that luciferase reporter gene system according to claim 1 detects transcription factor expression activity, it is special
Levy and be, described step（2）Including：Target cell is incubated at containing in hyclone, dual anti-complete medium, and maintains
Monolayer adherence grows, at 37 DEG C, in 5%CO2Incubator in incubated overnight, after cell confluency degree reaches 80%, then can open
Beginning implementation steps（3）.
4. the method that luciferase reporter gene system according to claim 1 detects transcription factor expression activity, it is special
Levy and be, described step（3）The operation of middle transfection is：Described cell to be transfected is inoculated in 24 holes cultures for 100000 with every hole
In plate, described mixture and culture medium are added this culture plate, is placed in the 5%CO at 37 DEG C2After incubation 6h in incubator, suction is abandoned
Described mixture, then plus fresh culture transfection culture 18-24h.
5. the method that luciferase reporter gene system according to claim 1 detects transcription factor expression activity, it is special
Levy and be, according to volume ratio, described cell lysis buffer solution has following components：
0.2M KH2PO44-4.5ml
0.2M K2HPO444-48ml
MQ H20 48-52ml
Triton X-100 200μl.
6. the method that luciferase reporter gene system according to claim 1 detects transcription factor expression activity, it is special
Levy and be, according to volume ratio, described luciferase assays buffer solution has following components：
0.25M Tris pH7.8 700μl
1M MgSO4100-115μl
dH2O 5870-5880μl
0.25M ATP 250-300μl
Luc 43-50μl.
7. the method that luciferase reporter gene system according to claim 1 detects transcription factor expression activity, it is special
Levy and be, described step（4）Including：
（4.1）Inhale the culture medium abandoning in described culture plate, add the PBS of 400 μ l/ hole precoolings, rinse described transfected cell
1-2 time, then inhale and abandon PBS；
（4.2）Described cell lysis buffer solution A preparing in advance to each in the hole addition 50 μ l of described culture plate, then,
Described culture plate is placed in 4 DEG C of shaking table and rocks 5-15 minute, during rocking, check whether cell comes off；
（4.3）Collect lysate, put in the 1.5ml EP pipe of a set of mark, with centrifuge at 4 DEG C with 12000 rpm turn
Speed centrifugation 10 min, obtain supernatant；
（4.4）Now join described luciferase assays buffer B, therefrom take 146 μ l to add to the 1.5ml of another set of mark
In EP pipe, and this EP pipe is inserted in holding low temperature on ice；
（4.5）Take 20 μ l（4.3）The supernatant of middle gained add to（4.4）Described in EP pipe in, detect luciferase expression
Intensity data；
（4.6）Detection luciferase concentration, correction；And calculate the expression of luciferase, thus detect described transcription because
The expression activity of son.
8. a kind of luciferase reporter gene system for described in claim 1 detects the method for transcription factor expression activity
Cell lysis buffer solution is it is characterised in that according to volume ratio, described cell lysis buffer solution has following components：
0.2M KH2PO44-4.5ml
0.2M K2HPO444-48ml
MQ H20 48-52ml
Triton X-100 200μl.
9. a kind of luciferase reporter gene system for described in claim 1 detects the method for transcription factor expression activity
Luciferase assays buffer solution is it is characterised in that according to volume ratio, described luciferase assays buffer solution has
Following components：
0.25M Tris pH7.8 700μl
1M MgSO4100-115μl
dH2O 5870-5880μl
0.25M ATP 250-300μl
Luc 43-50μl.
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| CN108004270B (en) * | 2017-11-24 | 2021-06-18 | 上海交通大学 | Method for detecting activity of duck IFN-beta promoter by using dual-luciferase reporter gene |
| CN109022546A (en) * | 2018-07-28 | 2018-12-18 | 扬州大学 | A kind of verification method of Nanos2 promoter nucleus key transcription factor |
| CN110029146A (en) * | 2018-12-20 | 2019-07-19 | 中国食品药品检定研究院 | The biological activity of reporter gene method measurement recombinant human horny cell growth factor-2 |
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| CN111944874B (en) * | 2020-07-20 | 2023-06-30 | 广东省微生物研究所（广东省微生物分析检测中心） | Method for screening and identifying stress response gene expression regulatory factor |
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