CN107557430A - The method of high flux screening ALKBH5 micromolecular inhibitors - Google Patents
The method of high flux screening ALKBH5 micromolecular inhibitors Download PDFInfo
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Abstract
The present invention relates to medicinal chemistry art, specifically discloses the method for screening ALKBH5 micromolecular inhibitors respectively using the enzyme-linked fluorescence principles of FDH and AlphaScreen technologies.First successfully structure and Prokaryotic expression, purification obtain the higher ALKBH5 recombinant proteins of purity to this method, and the ssRNA of synthesis m6A modifications is as substrate.With the enzyme-linked fluorescence principles of FDH and AlphaScreen technology for detection ALKBH5 activity and establish its micromolecular inhibitor Screening Platform.The enzyme-linked fluorescence reaction stable systems of FDH, agents useful for same is cheap, is easy to get, and background is low；AlphaScreen reaction systems are stable, and the interference by sample is considerably less, and false positive false negative is low, can remove the interference of natural products autofluorescence, high sensitivity；Both approaches are easy to realize miniaturization, and flux is high and is complementary to one another, and the screening of ALKBH5 micromolecular inhibitors can be achieved.
Description
Technical field
The present invention relates to medicinal chemistry art, and in particular to is utilized respectively the enzyme-linked fluorescence principles of FDH and AlphaScreen skills
The method of art high flux screening ALKBH5 micromolecular inhibitors.
Background technology
Up to now, the eucaryote RNA being found, as mRNA, tRNA, rRNA and snRNA posttranscriptional modification have
Hundreds of, these modifications all have a great impact to RNA structure and function.6 N of the RNA adenines modification (m that methylates6A)
It is that most common and content highest methylates modification in eucaryote, it is primarily present in mRNA sequential coding area
(CDS), tanscription termination end (translation termination sites) and 3 ' non-translational regions (3 ' UTR), influence RNA's
Montage, core output, translation efficiency and stability, played in sperm development, stem cell differentiation, metabolism and tumor development
Key effect.
In mRNA m6During A modification regulations, transmethylase METTL3, METTL14 and demethylase FTO,
ALKBH5 is reported respectively.Demonstrate mRNA m6Dynamic regulation process, transcription and function to mRNA be present in A modifications
With certain effect.ALKBH5 is the RNA demethylases that another is found after FTO, can specific removals RNA with
6 N of the single stranded DNA adenine modification that methylates, itself and RNA transmethylase regulate and control the RNA balance that methylates jointly.
ALKBH5 genes are located at the 17p11 of chromosome, belong to the albumen of ALKB families, are to rely on Fe2+Double with ɑ -one glutaric acids add
Oxygenase.
Downstream target genes of the ALKBH5 as hypoxia inducible factor HIF-1 ɑ in the case where DNA damage and anoxic stimulate,
Its expression is influenceed by arginine methyltransferase 7 (PRMT7).MRNA conjugated protein groups credit analysis based on photo-crosslinking
Display ALKBH5 belongs to mRNA associated proteins.And the rat tubulose mRNA of ALKBH5 defects m6The horizontal obvious increase of A modifications,
The rat reproductive function is damaged, and declines sperm number, degradation.It has also been found that the transgenosis knocked out in ALKBH5 in the research
In mouse in p53 apoptosis pathway related gene expression change.It recent studies have shown that, RNA m6A is modified in human embryo stem cell certainly
Played a significant role in the acquisition of my updating ability, m is also observed in stem cell breaks up6Dynamic change horizontal A.In the recent period
Research report ALKBH5 high expression in neuroglial cytoma, prognosis and life cycle with patient have obvious correlation.On
These researchs stated all indicate RNA m6A modifications affect many biological processes, but its specific molecular mechanism and life
Thing function is also very unclear.ALKBH5 crystal structure is resolved out, further studies ALKBH5 on this basis
Micromolecular inhibitor will promote to m on mRNA6The research of the biological function of A modifications, while can also be relevant disease
Drug design provide important evidence.
The content of the invention
It is an object of the invention to provide one kind to be miniaturized, and flux is high, the screening ALKBH5 little molecules in inhibiting of high sensitivity
The method of agent.
To realize the object of the invention, the present invention is utilized respectively the enzyme-linked fluorescence principles of FDH and AlphaScreen technologies are established
ALKBH5 micromolecular inhibitor Screening Platforms, screen ALKBH5 micromolecular inhibitor.
The enzyme-linked fluorescent method principles of FDH：
ALKBH5 is used as and depends on Fe2+With the dioxygenase of ɑ -one glutaric acids, it is removing the process of substrate methyl modification
In can produce formaldehyde, during formaldehyde dehydrogenase FDH is oxidized the formaldehyde into as formic acid, FDH coenzyme NAD can be reduced to
NADH, NADH can produce 460nm transmitting light in the case where 360nm excites light action, and the intensity of fluorescence can be detected by ELIASA.Profit
Reflect that ALKBH5 removes the power of m6A effects with the power of this fluorescence, so as to evaluate ALKBH5 activity and little molecules in inhibiting
Inhibitory activity of the agent to ALKBH5.
AlphaScreen technical principles：
AlphaScreen technologies are the technologies based on two microballons, and donor microballon and Acceptor beads are respectively comprising different
Chemical substance, when two microballons are close to each other, with 680nm light deexcitation donor microballon, its internal sensitising agent occurs one
The chemical reaction of series, oxygen molecule in reaction system around can be converted into the list of excitation state by caused energy during this
Line state oxygen, free oxygen spread in reaction system, transfer energy to Acceptor beads, it is produced 520-620nm transmitting light,
It is detected by ELIASA and launches the intensity of light to reflect that ALKBH5 removes m6The power of A effects, so as to evaluate ALKBH5 work
The inhibitory activity of property and micromolecular inhibitor to ALKBH5.
First successfully simultaneously Prokaryotic expression, purification obtains the higher FDH and ALKBH5 recombinant proteins of purity to structure to the present invention, synthesizes
M6A modification single-stranded DNA sequence be used as substrate, buy specific m6A antibody, using the enzyme-linked fluorescence principles of FDH with
AlphaScreen technologies establish ALKBH5 determination of activity platforms respectively, and optimize its reaction condition.Comprise the following steps that：
First, the expression and purification of FDH recombinant proteins
1st, the structure of FDH expression vectors：Select suitable expression vector and target gene active fragment.The expression of selection carries
Body will can give expression to the destination protein of solubility, to have clear and definite resistance and label, facilitate the expression and purifying of destination protein.Choosing
The objective gene sequence selected will include its activated centre, it is ensured that the feasibility of subsequent experimental；
Therefore, the expression vector that the present invention filters out is pET-28b；Objective gene sequence active fragment is FDH total lengths, will
It is cloned on expression vector.
2nd, the structure of ALKBH5 expression vectors：Select suitable expression vector and target gene active fragment.The table of selection
The destination protein of solubility can be given expression to up to carrier, to there is clear and definite resistance and label, facilitates the expression of destination protein and pure
Change.The objective gene sequence of selection will include its activated centre, it is ensured that the feasibility of subsequent experimental；
Therefore, the expression vector that the present invention filters out is pMCSG19；Objective gene sequence active fragment is that ALKBH5 is complete
It is long, it is cloned on expression vector.
3rd, the expression of FDH recombinant proteins：The purpose carrier successfully constructed conversion is entered into suitable expression bacterial strain, by bacterium solution
Even spread chooses monoclonal and filters out the bacterial strain containing purposeful plasmid and cultivated to being mixed with the solid medium of antibiotic.
Purpose inoculation is first enlarged culture for 37 DEG C into fluid nutrient medium during expressing protein, treats that bacterium is in growth period (OD
The expression of IPTG (isopropyl-β-D-thiogalactoside) induction destination proteins is added when 0.8-1.0)；
The expression bacterial strain selects BL21 (DE3), and the antibiotic selects kanamycins, the μ g/ml of final concentration 30；
4th, the expression of ALKBH5 recombinant proteins：The purpose carrier successfully constructed conversion is entered into suitable expression bacterial strain, will
Bacterium solution even spread chooses monoclonal and filters out the bacterial strain containing purposeful plasmid and trained to being mixed with the solid medium of antibiotic
Support.Purpose inoculation is first enlarged culture for 37 DEG C into fluid nutrient medium during expressing protein, treats that bacterium is in growth period (OD
The expression of IPTG (isopropyl-β-D-thiogalactoside) induction destination proteins is added when 0.8-1.0)；
The expression bacterial strain selects BL21 (DE3), and the antibiotic selects ammonia benzyl mycin, the μ g/ml of final concentration 10；
5th, the purifying of FDH and ALKBH5 recombinant proteins：The thalline in above-mentioned bacterium solution is collected by centrifugation, then ultrasonication discharges
Destination protein, high speed centrifugation discard bacterial chip, obtain destination protein solution.The carrier selected during according to construction of expression vector is special
Sign selects the purified pool protein samples such as suitable purification column, purification condition, buffer solution；
6th, expression and purification result：The protein sample collected during above-mentioned protein purification is taken, adds albumen loading buffer
Denaturation, then SDS-PAGE gel electrophoresis, contaminate glue, it is seen that have protein band at destination protein size with Coomassie brilliant blue dye liquor.
2nd, the foundation of the enzyme-linked fluorescence principle screening ALKBH5 micromolecular inhibitor platforms of FDH
According to the suitable detection reagent of feature selecting of the enzyme-linked fluorescence principles of FDH, in cushioning liquid, detection ALKBH5 and
The interaction of the single stranded RNA of m6A modifications.
It is of the invention preferred：The high FDH and ALKBH5 recombinant proteins of purity, coenzyme of the NAD as FDH, α-ketoglutaric acid,
Fe2+As ALKBH5 coenzyme, FDH activity is first detected using the formaldehyde of various concentrations as substrate, then makes mark song, it is determined that
Concentration of substrate, then using ssRNA as ALKBH5 substrate, detect ALKBH5 activity.FDH and first are obtained in this reaction system
The linear r2 of aldehyde reaction>0.995, when ssRNA concentration is 10 μM, detectable ALKBH5 activity, ssRNA concentration reach 40 μ
Process window value reaches 2 during M, can be according to NADH in 360nm, and 460nm fluorescence intensity screens ALKBH5 inhibitor.
3rd, the foundation of AlphaScreen technology screenings ALKBH5 micromolecular inhibitor platforms
According to the suitable detection reagent of the feature selecting of AlphaScreen technologies, in cushioning liquid, detection ALKBH5 and
The interaction of the single stranded DNA of m6A modifications.
It is of the invention preferred：The high ALKBH5 recombinant proteins of purity, α-ketoglutaric acid, Fe2+As ALKBH5 coenzyme,
Streptavidin Donor beads are raw as Acceptor beads as donor microballon, Protein A Accepter beads
Substrates of the ssDNA of thing element mark as ALKBH5, first with specific m6A ssDNA antibody and Protein A Accepter
Beads combination 1.5h, while make ALKBH5 and its substrate and coenzyme react 1h at 37 DEG C, then by Accepter after binding antibody
Beads is added in enzyme reaction system and is reacted at room temperature 1h, is eventually adding Streptavidin Donor beads room temperature reaction 0.5h,
Finally with Alpha detection methods detection luminous intensity, ALKBH5 inhibitor is screened according to luminous intensity.In this reaction system really
The optimum concentration for determining antibody and substrate is 1nM and 2.5 μM, and enzyme concentration is 3.5 μM.
The advantage of the invention is that：
1st, it is simple to operate：Board-washing is not required in experimentation, is directly detected after incubation.
2nd, miniaturization is easily achieved, flux is high.
3rd, material is easy to get, economical, is easy to high flux screening.
4th, reaction system is stable：The tolerable wider pH scopes of Alpha microballons, between 5.5-8.5.
5th, data reappearance is good：Background is low, and the special principle of luminosity of Alpha and detection method make it non-by the interference of sample
Often few, false positive false negative is low, can remove the interference of natural products autofluorescence, high sensitivity (detectable nM level substrates.
Brief description of the drawings
Fig. 1 is pET-28b-FDH and pMCSG19-ALKBH5 purification results, wherein, a pET-28b-FDH, 1 is in figure
Albumen Marker, 2 be 200mMl imidazole elutions, and 3 be 50mM imidazole elutions, and 4-5 is 5mM imidazole elutions.
B is pMCSG19-ALKBH5, and 1-3 is the foreign protein flowed through in figure, and 4-7 is eluent, and 8 be albumen Marker.Fig. 2
To utilize the schematic diagram of the enzyme-linked fluorescence of FDHFDH.
Fig. 3 is FDH determination of activity curve maps.
Fig. 4 is ALKBH5 determination of activity curve maps in the enzyme-linked fluorescent methods of FDH.
Fig. 5 is the schematic diagram of AlphaScreen technologies.
Fig. 6 is the optimal conditions curve map of antibody concentration and concentration of substrate in AlphaScreen methods.
Fig. 7 is the optimal conditions curve map in ALKBH5 concentration and reaction time in AlphaScreen methods.
Fig. 8 is reaction system z value measurement results in AlphaScreen methods.
Embodiment
It is as follows for embodiment for the present invention is better described：In addition to without adding explanation, percentage as described below contains
Amount is weight/mass percentage composition.
Embodiment 1
First, the expression and purification of pET-28b-FDH recombinant proteins
Reagent and method：
Block the preparation of that penicillin：Weigh 1g ampicillin ultra-pure waters to dissolve and be settled to 10ml, in super-clean bench
Filtered with the 0.22um of sterilizing filter, -20 DEG C of preservations；
100mM IPTG (isopropyl-β-D-thiogalactoside) preparation：2.383g IPTG are weighed to be dissolved with ultra-pure water
And 100ml is settled to, filtered in super-clean bench with the 0.22um of sterilizing filter, -20 DEG C of preservations；
The preparation of competent cell：It will express in fluid nutrient mediums of the bacterial strain BL21 (DE3) by 1 ‰ access sterilizings, 37 DEG C
Shaking table culture is taken in the EP pipes that 1ml bacterium solutions sterilize to 1.5ml, 3000rpm centrifugations 5min collects thalline, often to exponential phase
The CaCl of 100ul 0.1M sterilizing precoolings is added in pipe2Solution, piping and druming is uniform, and 25ul 50% is then often added in pipe and is sterilized
Glycerine, mix, -80 DEG C preservation, it is standby.
The preparation of fluid nutrient medium：Weigh respectively in 6g peptones, 6g sodium chloride, 3g yeast extracts to 1L conical flasks,
Add ultra-pure water 600ml, 121 DEG C of autoclaving 20min；
The preparation of solid medium：Weigh 2g peptones, 2g sodium chloride, 1g yeast extracts, 3g agar powders extremely respectively
In 500ml conical flasks, ultra-pure water 200ml, 121 DEG C of autoclaving 20min are added.Sterilizing puts solid medium room temperature after terminating
Put to it is cool and it is not solidifying when add 200 μ l ampicillins (0.5mg/ml), shake up, pour into sterile culture dish, 4 DEG C after solidification
Preserve, it is standby；
Binding buffer component：50mM sodium dihydrogen phosphates, 300mM sodium chloride, 5mM imidazoles, pH 7.4；
Washing buffer component：50mM sodium dihydrogen phosphates, 300mM sodium chloride, 50mM imidazoles, pH 7.4；
Elution buffer component：50mM sodium dihydrogen phosphates, 300mM sodium chloride, 200mM imidazoles, pH 7.4.
1st, the structure of pET-28b-FDH expression vectors：By the full-length clone of FDH base sequences to expression vector pET-28b
On, connected through sequence verification correct；
2nd, the expression of pET-28b-FDH recombinant proteins：The recombinant vector pET-28b-FDH successfully constructed conversions are entered into table
Up to bacterial strain BL21 (DE3), by bacterium solution even spread to being mixed with the solid medium of that penicillin of card, 37 DEG C of overnight incubations, choose
Monoclonal is cultivated into fluid nutrient medium.
3rd, above-mentioned BL21 (DE3)-pET-28b-FDH is inoculated into 37 DEG C of expansion trainings in fluid nutrient medium in 1 ‰ ratio
Support, 0.5mM IPTG (isopropyl-β-D-thiogalactoside) 30 are added when bacterium is in exponential phase (OD 0.6-0.9)
FDH expression is induced DEG C overnight.
4th, the purifying of pET-28b-FDH recombinant proteins：A. thalline is collected：8000rpm 5min are collected by centrifugation in above-mentioned bacterium solution
Thalline, with 5mM imidazole solutions be resuspended thalline, 8000rpm 5min centrifugation abandon supernatant；B. ultrasonication:Add 5mM imidazoles
Thalline, ultrasonication (ice bath is resuspended in solution；Ultrasonic 3s, it is spaced 15s), high speed centrifugation discards bacterial chip, obtains containing purposeful
The supernatant of albumen, 0.22um filter membranes filter；C. pillar is balanced:Protein purification device is connected, first with ultra-pure water emptying system
Bubble, then connect upper albumen purification column, it is first substantially steady to ultraviolet detection value with ultrapure water pillar, then change into
Binding buffer balance pillars are steady to ultraviolet detection value, and absorbance is zeroed；D. loading:Protein sample is flowed through into albumen
Purification column, destination protein and a small amount of foreign protein are combined with pillar, and other foreign proteins directly flow through；E. foreign protein is washed:With
Washing buffer flushing pillars are steady to ultraviolet detection value, and the foreign protein combined on pillar is eluted；F. purpose egg is eluted
In vain:With the destination protein combined on Elution buffer elution pillars；G. successively with Binding buffer, ultrapure water
Pillar is steady to ultraviolet detection value respectively；H. pillar is preserved with 20% ethanol.
5th, expression and purification result：50ul is taken out in the protein sample collected from protein purification procedures respectively, adds 10 μ l
6 × loading of albumen buffer, 100 DEG C of denaturation 10min, then SDS-PAGE gel electrophoresis, contaminate glue with Coomassie brilliant blue dye liquor,
It is destination protein pET-28b-FDH it was observed that there is protein band at 43KDa.
2nd, the expression and purification of pMCSG19-ALKBH5 recombinant proteins
Reagent and method：
The preparation of ampicillin：Weigh 1g ampicillin ultra-pure waters to dissolve and be settled to 10ml, in super-clean bench
Filtered with the 0.22um of sterilizing filter, -20 DEG C of preservations；
Binding buffer component：50mM sodium dihydrogen phosphates, 300mM sodium chloride, pH 8.0；Elution buffer
Component：50mM sodium dihydrogen phosphates, 300mM sodium chloride, 10mM maltose, pH 8.0.
1st, the structure of pMCSG19-ALKBH5 expression vectors：By the full-length clone of ALKBH5 base sequences to expression vector
On pMCSG19, connected through sequence verification correct；
2nd, the expression of pMCSG19-ALKBH5 recombinant proteins：The recombinant vector pMCSG19-ALKBH5 successfully constructed is converted
Into expression bacterial strain BL21 (DE3), by bacterium solution even spread to being mixed with the solid medium of ampicillin, 37 DEG C were incubated
At night, choose monoclonal and cultivated into fluid nutrient medium.
3rd, above-mentioned BL21 (DE3)-pMCSG19-ALKBH5 is inoculated into 37 DEG C of expansion in fluid nutrient medium in 1 ‰ ratio
Culture, 0.5mM IPTG (isopropyl-β-D-thiogalactoside) are added when bacterium is in exponential phase (OD 0.6-0.9)
16 DEG C induce ALKBH5 expression overnight.
4th, the purifying of pMCSG19-ALKBH5 recombinant proteins：A. thalline is collected：Above-mentioned bacterium is collected by centrifugation in 8000rpm 5min
Thalline in liquid, thalline is resuspended with Binding buffer, supernatant is abandoned in 8000rpm 5min centrifugations；B. ultrasonication:Add
Thalline, ultrasonication (ice bath is resuspended in Binding buffer；Ultrasonic 3s, it is spaced 15s), high speed centrifugation discards bacterial chip, obtains
To the supernatant containing destination protein, 0.22um filter membranes filter；C. pillar is balanced:Protein purification device is connected, first uses ultra-pure water
Bubble in emptying system, upper albumen purification column is then connected, it is first substantially steady to ultraviolet detection value with ultrapure water pillar,
It is steady to ultraviolet detection value to change Binding buffer balance pillars into again, absorbance is zeroed；D. loading:By protein sample stream
Through protein purification post, destination protein and a small amount of foreign protein are combined with pillar, and other foreign proteins directly flow through；E. foreign protein is washed:
It is steady to ultraviolet detection value with Binding buffer flushing pillars, the foreign protein combined on pillar is eluted；F. purpose is eluted
Albumen:With the destination protein combined on Elution buffe elution pillars；G. rushed successively with Binding buffer, ultra-pure water
It is steady to ultraviolet detection value respectively to wash pillar；H. pillar is preserved with 20% ethanol.
5th, expression and purification result：50ul is taken out in the protein sample collected from protein purification procedures respectively, adds 10 μ l
6 × loading of albumen buffer, 100 DEG C of denaturation 10min, then SDS-PAGE gel electrophoresis, contaminate glue with Coomassie brilliant blue dye liquor,
It is destination protein pMCSG19-ALKBH5 it was observed that there is protein band at 90KDa.3rd, the enzyme-linked fluorescence principle screenings of FDH
The foundation of ALKBH5 micromolecular inhibitor platforms
Reagent and method：
The preparation of HEPES buffer solution：20mM Hepes, pH8.0.
L-AA：PH8.0,20mM Hepes prepare 20mM L-AAs, final concentration of 2mM during use.
ɑ -one glutaric acids：PH8.0,20mM Hepes prepare 4mM ɑ -one glutaric acids, final concentration of 200 μM during use.
Iron ammonium sulfate：PH8.0,20mM Hepes prepare 200 μM of iron ammonium sulfates, final concentration of 20 μM during use.
NAD:PH8.0,20mM Hepes prepare 10mM NAD, final concentration of 1mM during use.
1st, FDH coenzyme, α-ketoglutaric acid, Fe are used as using NAD2+As ALKBH5 coenzyme, first with the first of various concentrations
For aldehyde as substrate in 384 microwell plates, 20 μ l reaction systems detect FDH activity, then make mark song.
2nd, using ssRNA as ALKBH5 substrate, in 384 microwell plates, 20 μ l reaction systems detect ALKBH5 activity.This
FDH and the linear r of formaldehyde reaction are obtained in reaction system2>0.995, can during reaction time 60min when ssRNA concentration is 10 μM
ALKBH5 activity is detected, Process window value reaches 2 when ssRNA concentration reaches 40 μM, can be according to NADH in 360nm, 460nm
Fluorescence intensity screen ALKBH5 inhibitor.
4th, the foundation of AlphaScreen principles screening ALKBH5 micromolecular inhibitor platforms
Reagent and method：
The preparation of HEPES buffer solution：20mM Hepes, pH8.0.
L-AA：PH8.0,20mM Hepes prepare 20mM L-AAs, final concentration of 2mM during use.
ɑ -one glutaric acids：PH8.0,20mM Hepes prepare 4mM ɑ -one glutaric acids, final concentration of 200 μM during use.
Iron ammonium sulfate：PH8.0,20mM Hepes prepare 200 μM of iron ammonium sulfates, final concentration of 20 μM during use.
1st, according to 23 DEG C of crosslinking Protein A Acceptor beads of kit requirement and a series of m of concentration6A resists
Body.
2nd, with α-ketoglutaric acid, Fe2+As ALKBH5 coenzyme, a series of bottoms of concentration ssRNA as ALKBH5 is prepared
Thing, in 384 microwell plates, 10 μ l reaction systems carry out enzyme reaction.
The 3rd, Protein A Acceptor beads that 5 μ l have been crosslinked to antibody add the reaction of microwell plate enzyme reaction system
1h, add 5 μ lDonor Beads reactions, ELIASA measure AlphaScreen signal intensities.Observation signal value determines most suitable
Antibody concentration and concentration of substrate.
4th, immobilized substrate concentration and antibody concentration, the ALKBH5 of various concentrations is prepared, detects ALKBH5 activity and determine
ALKBH5 concentration.
5th, AlphaScreen signal (exciting lights are detected in different time：680nm, launch light：520-620nm) intensity is true
Determine the reaction time.
6th, it is 1nM finally to determine antibody concentration, and ssRNA concentration of substrate is 2.5 μM, ALKBH5 concentration
3.5 μM, reaction time 30min.
7th, multiple experiments are repeated, calculate z values, the stability of evaluation response system.
Under these conditions, with ketoglutaric acid on α, Fe2+As ALKBH5 coenzyme, Streptavidin Donor
Beads is as donor microballon, and Protein A Accepter beads are as Acceptor beads, the ssDNA conducts of biotin labeling
ALKBH5 substrate, first with specific m6A ssDNA antibody and Protein A Accepter beads combination 1.5h, make simultaneously
ALKBH5 reacts 1h at 37 DEG C with its substrate and coenzyme, then Accepter beads after binding antibody are added in enzyme reaction system
1h is reacted at room temperature, Streptavidin Donor beads room temperature reaction 0.5h is eventually adding, is finally examined with Alpha detection methods
Luminous intensity is surveyed, ALKBH5 inhibitor is screened according to luminous intensity.Due to Cleaning Principle special AlphaScreen and detection ripple
It is long, compound autofluorescence can be avoided to disturb, ambient interferences, 384 orifice plates 20μlReaction system realizes the high flux of reaction.
Claims (5)
1. one kind screening ALKBH5 little molecules in inhibiting agent methods, it is characterised in that small using enzyme-linked fluorescence principle screening ALKBH5
Molecule inhibitor, specific method are as follows：
（1）Using pET-28b as expression vector, FDH full-length gene orders are active fragment, are cloned on expression vector, structure
Build simultaneously Prokaryotic expression, purification FDH recombinant proteins；
（2）Using pMCSG19 as expression vector, ALKBH5 full-length gene orders are active fragment, are cloned on expression vector,
Build simultaneously Prokaryotic expression, purification ALKBH5 recombinant proteins；
（3）FDH activity is detected using the formaldehyde of various concentrations as substrate, makees standard curve, determines concentration of substrate；Then with
Substrates of the ssRNA as ALKBH5, detect ALKBH5 activity；
（4）In cushioning liquid, FDH and ALKBH5 recombinant proteins, coenzyme of the NAD as FDH, α-ketoglutaric acid, Fe are added2+As
ALKBH5 coenzyme, according to NADH in 360nm, 460nm fluorescence intensity screening ALKBH5 inhibitor；
Substrate ssRNA concentration selects 10 μM -40 μM.
2. screening ALKBH5 little molecules in inhibiting agent methods as claimed in claim 1, it is characterised in that
The expression of FDH recombinant proteins：The purpose carrier successfully constructed conversion is entered into BL21 (DE3) expression bacterial strains, bacterium solution is equal
Even be applied to is mixed with the solid medium of kanamycins antibiotic, is chosen monoclonal and is filtered out the bacterial strain progress containing purposeful plasmid
Culture；Purpose inoculation is first enlarged culture for 37 DEG C into fluid nutrient medium during expressing protein, treats that bacterium is in growth period
The expression of isopropyl-β-D-thiogalactoside induction destination protein is added during OD 0.8-1.0；
The expression of ALKBH5 recombinant proteins：The purpose carrier successfully constructed conversion is entered into BL21 (DE3) expression bacterial strains, by bacterium solution
Even spread chooses monoclonal and filters out the bacterial strain containing purposeful plasmid and to being mixed with the solid medium of ammonia benzyl mycin antibiotic
Row culture；Purpose inoculation is first enlarged culture for 37 DEG C into fluid nutrient medium during expressing protein, treats that bacterium is in growth
The expression of isopropyl-β-D-thiogalactoside induction destination protein is added during phase OD 0.8-1.0.
3. one kind screening ALKBH5 little molecules in inhibiting agent methods, it is characterised in that utilize AlphaScreen technology screenings ALKBH5
Micromolecular inhibitor, specific method are as follows：
（1）Using pMCSG19 as expression vector, ALKBH5 full-length gene orders are active fragment, are cloned on expression vector,
Build simultaneously Prokaryotic expression, purification ALKBH5 recombinant proteins；
（2）Using ssRNA as ALKBH5 substrate, ALKBH5 activity is detected；
（3）In cushioning liquid, ALKBH5 recombinant proteins, α-ketoglutaric acid, Fe are added2+As ALKBH5 coenzyme,
Streptavidin Donor beads are raw as Acceptor beads as donor microballon, Protein A Accepter beads
Substrates of the ssDNA of thing element mark as ALKBH5, first with specific m6A ssDNA antibody and Protein A Accepter
Beads is combined, while ALKBH5 is added with its substrate and coenzyme in 37 DEG C of reactions, then by Accepter beads after binding antibody
Enter and reacted at room temperature in enzyme reaction system, be eventually adding Streptavidin Donor beads room temperature reactions, finally use
AlphaScreen detection methods detect luminous intensity, and ALKBH5 inhibitor is screened according to luminous intensity.
4. screening ALKBH5 little molecules in inhibiting agent methods as claimed in claim 3, it is characterised in that
The expression of ALKBH5 recombinant proteins：The purpose carrier successfully constructed conversion is entered into BL21 (DE3) expression bacterial strains, by bacterium solution
Even spread chooses monoclonal and filters out the bacterial strain containing purposeful plasmid and to being mixed with the solid medium of ammonia benzyl mycin antibiotic
Row culture；Purpose inoculation is first enlarged culture for 37 DEG C into fluid nutrient medium during expressing protein, treats that bacterium is in growth
The expression of isopropyl-β-D-thiogalactoside induction destination protein is added during phase OD 0.8-1.0.
5. the screening ALKBH5 little molecules in inhibiting agent methods as described in claim 3 or 4, it is characterised in that
Antibody concentration is 1nM, and ssRNA concentration of substrate is 2.5 μM, and ALKBH5 concentration is 3.5 μM, reaction time 30min.
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| CN108593615A (en) * | 2018-05-02 | 2018-09-28 | 浠思（上海）生物技术有限公司 | The method for screening PD1/PD-L1 blocking agents using HTRF one-step method |
| CN109517877A (en) * | 2018-10-15 | 2019-03-26 | 北京大学 | Screen m6A goes nucleotides substrate, kit and the method for modification enzyme inhibitor |
| CN110567906A (en) * | 2019-09-12 | 2019-12-13 | 深圳大学 | Method for representing RNA methylation modification and application |
| CN111330009A (en) * | 2020-04-02 | 2020-06-26 | 南通大学 | Application of m6A modified related gene ALKBH5 in promotion of nerve axon injury repair |
| CN112245421A (en) * | 2020-09-25 | 2021-01-22 | 北京大学 | New application of nemostat in preparation of demethylase inhibitor |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593615A (en) * | 2018-05-02 | 2018-09-28 | 浠思（上海）生物技术有限公司 | The method for screening PD1/PD-L1 blocking agents using HTRF one-step method |
| CN109517877A (en) * | 2018-10-15 | 2019-03-26 | 北京大学 | Screen m6A goes nucleotides substrate, kit and the method for modification enzyme inhibitor |
| CN109517877B (en) * | 2018-10-15 | 2021-07-20 | 北京大学 | Nucleotide substrates, kits and methods for screening m6A for de-modified enzyme inhibitors |
| CN110567906A (en) * | 2019-09-12 | 2019-12-13 | 深圳大学 | Method for representing RNA methylation modification and application |
| CN111330009A (en) * | 2020-04-02 | 2020-06-26 | 南通大学 | Application of m6A modified related gene ALKBH5 in promotion of nerve axon injury repair |
| CN112245421A (en) * | 2020-09-25 | 2021-01-22 | 北京大学 | New application of nemostat in preparation of demethylase inhibitor |
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