CN109504737A - A kind of method of in-vitro screening PCSK9 inhibitor - Google Patents
A kind of method of in-vitro screening PCSK9 inhibitor Download PDFInfo
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- CN109504737A CN109504737A CN201811407344.9A CN201811407344A CN109504737A CN 109504737 A CN109504737 A CN 109504737A CN 201811407344 A CN201811407344 A CN 201811407344A CN 109504737 A CN109504737 A CN 109504737A
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Abstract
The invention belongs to PCSK9 inhibitor technical fields, and in particular to a kind of method of in-vitro screening PCSK9 inhibitor, comprising the following steps: the plasmid pGEX-EGFAB containing EGF-AB gene is imported Escherichia coli and expresses EGF-AB albumen；It extracts and purifies EGF-AB albumen；After purification EGF-AB albumen bed board coating, be placed in 4 DEG C overnight or 37 DEG C in 2h；Skimmed milk power is closed；The PCSK9 with His label and drug to be measured is added, while setting negative control；Then primary antibody is added and secondary antibody is incubated for；OD value is read after colour developing and calculates IC50.It is high-efficient the present invention is based on the method that molecular level devises primary screener PCSK9 inhibitor, it is at low cost.
Description
Technical field
The invention belongs to PCSK9 inhibitor technical fields, and in particular to a kind of method of in-vitro screening PCSK9 inhibitor.
Background technique
Atherosclerosis is the most common disease in disease of cardiovascular system, and endangers the common disease of human health.
The characteristics of atherosclerosis is the lesion of involvement artery since inner membrance, and successively there are many lesions to merge presence, including part
There are lipid and the accumulation of compound carbohydrate, proliferation of fibrous tissue and calcinosis to form patch, and has the gradually regression of arterial media, after
Hair venereal disease change still have patch internal haemorrhage, plaque rupture and local thrombus formed (referred to as atherosis-thrombosis,
atherosclerosis-thrombosis).Since the lipid appearance gathered in endarterium is athero- in yellow, because referred to herein as dynamic
Pulse atherosclerosis.
PCSK9 is the 9th member of invertase subtilopeptidase A family before kexin sample, by 692 amino acid residues
Composition, is mainly expressed in liver, kidney and small intestine, and be present in blood as a secreted protein.PCSK9 is main
It is synthesized in endoplasmic reticulum, is initially formed the precursor of a 75kDa, immature precursor includes a N-terminal signal peptide sequence, one
Predomain, a catalyst structure domain and one are rich in the C-terminal structural domain of cysteine.Subsequent PCSK9 in endoplasmic reticulum is at it
An autocatalysis is undergone to crack at 152nd residue, forming the predomain segment of a 14kDa and one includes catalysis
The 57kDa maturation segment of structural domain and C-terminal structural domain.Predomain after cracking is still combined with non-covalent bond in catalytic structure
On domain, a complex is formed with mature segment, and leave ER transport together with mature segment as a molecular chaperones
Into golgiosome, finally it is secreted into blood after a series of modifications such as acetylation in golgiosome.Predomain
It is that PCSK9 is correctly folded and left necessary to endoplasmic reticulum in conjunction with the effect that may play protease inhibition catalytic activity.Greatly
Most proprotein convertases can undergo secondary proteolysis elaboration after leaving endoplasmic reticulum, to discharge predomain, cruelly
Reveal enzymatic activity.And it leaves the PCSK9 after endoplasmic reticulum and does not undergo this secondary processing process, therefore PCSK9 is uniquely without egg
The subtilisin-like serine protease of white matter substrate.Liver is the main source of PCSK9 in blood.When due to detection
The difference of the specific antibody and standard value that use, the mean variation range of PCSK9 concentration is larger in human plasma.
Statins are fat-reducing medicaments most widely used at present, are to prevent and treat atherosclerotic cardiovascular disease
The foundation stone of disease, however the limitation of statins increasingly appears, research and development are directed to the novel lipid-lowering medicine of lipid metabolism difference target spot
Object becomes one of hot spot, and with the discovery of PCSK9, this kind of novel lipid-lowering medicine of PCSK9 inhibitor is greatly paid close attention to, and is lipid-loweringing
Treatment provides more choices, and brings good fortune can not to be much resistant to statins and the ineffective patient of statin treatment
Sound.
PCSK9 has the function of powerful reduction LDL-C.Placebo-controlled study shows that PCSK9 inhibitor can make
LDL-C level reduces 50%-70% compared with baseline.Give every 2 weeks Evolocumab 140mg subcutaneous injection, can make LDL-C from
115mg/dl~124mg/dl is down to 39mg/dl~49mg/dl.In addition to the reduction of LDL-C, atherosclerotic plaque load and artery
Atherosis dissection is also related to Cardia cevent.The study found that compared with placebo, monthly evolocumab
420mg subcutaneous injection, atherosclerosis percent by volume decline 0.95%, difference is statistically significant, and 64.3% trouble
Person's atheromatous plaque subsides.And adverse reaction is few by evolocumab, and proportion is very low, there is not yet great adverse events.
The multinomial side effect of statins mentioned above, PCSK9 inhibitor have good safety and tolerance.In addition,
GAUSS-3 research discovery PCSK9 inhibitor can help to reduce the drug resistance of statins.Moreover, SLER and ODYSSEY two
Item test all shows that PCSK9 inhibitor can be substantially reduced the cardiocerebrovasculaevents events such as death, myocardial infarction, ishemic stroke.Cause
This, the PCSK9 of the cardiovascular and cerebrovascular diseases such as myocardial infarction, ishemic stroke caused by screening research and development treatment atherosclerosis inhibits
Agent is significant.
The primary screener of traditional PCSK9 inhibitor is by detection untested compound and the protein bound power of PCSK9
Reflect that inhibiting rate is strong and weak, needs using expensive Molecular interaction equipment, and need to prepare the recombination PCSK9 of very high-purity
Albumen, the interference by extraneous factor is larger, and error is also larger, and this method is directed to the path of PCSK9/LDLR, utilizes ELISA's
The in-vitro simulated mechanism of PCSK9 and LDLR of method, wherein EGF-AB albumen is the active region in LDLR with PCSK9 effect
Domain, then the influence by optical response signal sample to be tested to PCSK9 and EGF-AB interactions between protein is strong and weak.This method has
ELISA general advantage-is at low cost, high-efficient, and common microplate reader can be carried out detecting.Therefore, the application is based on this
A kind of method that principle devises in-vitro screening PCSK9 inhibitor.
Summary of the invention
It is high-efficient the present invention is based on the method that molecular level devises coarse sizing PCSK9 inhibitor, it is at low cost.
The object of the present invention is to provide a kind of methods of in-vitro screening PCSK9 inhibitor, comprising the following steps:
Plasmid pGEX-EGFAB containing EGF-AB gene is imported Escherichia coli and expresses EGF-AB albumen by S1；
S2 is extracted and is purified the EGF-AB albumen with GST label；
S3, after EGF-AB albumen after purification is diluted with DPBS bed board be coated with, be placed in 4 DEG C overnight or 37 DEG C in 2h, PBST
Cleaning；It is closed with 5g/100ml skimmed milk power, is placed in 2h in 37 DEG C, PBST cleaning；Be added PCSK9 with His label and
Drug to be measured, while negative control is set, it is incubated for 2h, PBST cleaning at room temperature；The primary antibody after being diluted with PBST is added, incubates at room temperature
Educate 2h, PBST cleaning；The secondary antibody after being diluted with PBST is added, is incubated for 1h, PBST cleaning at room temperature；TMB colour developing, color development stopping,
OD value is read under 450nm；Wherein the drug to be measured is PCSK9 inhibitor to be screened；
S4 calculates the OD mean value of drug to be measured, substitution formula: (1- drug OD mean value/negative control OD value to be measured) ×
100%, inhibiting rate is obtained, IC is calculated50, between inhibiting rate and drug concentration to be measured there is dosage effect to illustrate that drug to be measured has
Inhibit the effect of PCSK9 bioactivity.
Preferably, the process expressed in above-mentioned S1 are as follows: the Escherichia coli for having imported EGF-AB gene are cultivated to bacterium solution OD value
It is 0.7, IPTG is added and is allowed to final concentration of 1mmol/L, continues to collect thallus after cultivating 3h.
Preferably, the process of EGF-AB albumen is extracted and purified in above-mentioned S2 are as follows: cracking Escherichia coli are simultaneously prepared into suspension,
PBS is added and stablizes pH to 7.0-7.5, cross gst protein purification column and is eluted with reduced glutathione, under eluting
EGF-AB albumen is stored in the PBS solution containing 10% glycerol.
Preferably, EGF-AB albumen is diluted to 20 μ g/mL, PCSK9 concentration≤25 μ g/ with DPBS before above-mentioned bed board is coated with
mL。
Preferably, above-mentioned primary antibody is 1:2000 with the diluted ratio of PBST；Secondary antibody is 1:5000 with the diluted ratio of PBST.
Preferably, above-mentioned TMB colour developing 5-10min；Select 2mol/L sulfuric acid color development stopping.
Compared with prior art, the method for in-vitro screening PCSK9 inhibitor provided by the invention has the advantages that
The present invention is based on the method that molecular level devises coarse sizing PCSK9 inhibitor, for the path of PCSK9/LDLR,
Using the in-vitro simulated mechanism of PCSK9 and LDLR of the method for ELISA, wherein EGF-AB albumen be in LDLR with PCSK9
The active region of effect, then the influence by optical response signal sample to be tested to PCSK9 and EGF-AB interactions between protein is strong and weak, tool
Have the advantages that ELISA is general, at low cost, high-efficient, common microplate reader can be carried out detecting.
Detailed description of the invention
Fig. 1 is the inhibiting rate curve graph of positive inhibitor pep2-8 in embodiment 1.
Specific embodiment
The present invention is described in detail combined with specific embodiments below, but should not be construed as limitation of the invention.It is following
The test method of actual conditions is not specified in embodiment, operates usually according to normal condition, due to not being related to inventive point, thus it is not right
Its step is described in detail.
A kind of method of in-vitro screening PCSK9 inhibitor provided by the invention, comprising the following steps:
Plasmid pGEX-EGFAB containing EGF-AB gene is imported e. coli bl21 and expresses EGF-AB albumen by S1；
S2 is extracted and is purified the EGF-AB albumen with GST label；
S3, after EGF-AB albumen after purification is diluted with DPBS bed board be coated with, be placed in 4 DEG C overnight or 37 DEG C in 2h, PBST
Cleaning；It is closed with 5g/100ml skimmed milk power, is placed in 2h in 37 DEG C, PBST cleaning；Be added PCSK9 with His label and
Drug to be measured, while negative control is set, it is incubated for 2h, PBST cleaning at room temperature；The primary antibody after being diluted with PBST is added, incubates at room temperature
Educate 2h, PBST cleaning；The secondary antibody after being diluted with PBST is added, is incubated for 1h, PBST cleaning at room temperature；TMB colour developing, color development stopping,
OD value is read under 450nm；Wherein the drug to be measured is PCSK9 inhibitor to be screened；
S4 calculates the OD mean value of drug to be measured, substitution formula: (1- drug OD mean value/negative control OD value to be measured) ×
100%, inhibiting rate is obtained, IC is calculated50, between inhibiting rate and drug concentration to be measured there is dosage effect to illustrate that drug to be measured has
Inhibit the effect of PCSK9 bioactivity.
Preferably, the method for a kind of in-vitro screening PCSK9 inhibitor provided by the invention, specifically includes following embodiment.
In following embodiments, experiment reagent used is as follows: (1) (preparation method: the PBS of 20x takes 50mL to add to PBS buffer solution
Enter ddH2O is settled to 1L)；(2) (5% skimmed milk power, preparation method are as follows: weighing 2.5g skimmed milk power and be placed in 50mL for confining liquid
In test tube, adds TBST appropriate, shake up, 50mL graduated cylinder constant volume)；(3) Anti-6X His tag primary antibody (abcam, ab27025)；
(4) HRP secondary antibody (Vectorlabs, SA-5014)；(5) the efficient color developing agent of TMB (Suo Laibao biology, PR1200)；(6) terminate liquid
(2mol/L sulfuric acid, preparation method are as follows: taking concentrated sulfuric acid 109mL, use ddH2O is settled to 1L)；(7) DPBS (HyClone,
SH30028.02)；(8) (preparation method is as follows: the PBS of 750mL is added in the tween20 of 500 μ L to PBST, stirs evenly, NaOH tune
Whole pH is settled to 1L in 7.5 or so, PBS)；(9) Tween 20 (Aladdin, T104863-500mL)；(10) tryptone
(OXOID, LP0042)；(11) yeast extract (OXOID, LP0021)；(12) sodium chloride (raw work biology, A100241-
0500)；(13) agar (Lan Bolide, QZ01), ampicillin (INALCO, 1758-9314)；(14) IPTG (INALCO,
1758-1400)；(15) Bacterial Protein Extraction kit (health is century, CW0888M)；(16) (raw work is raw for reduced glutathione
Object, A100399-0050)；(17)ddH2O (Milli-Q preparation)；18) (preparation method is as follows: 10g pancreas egg for LB liquid medium
The ddH of 750mL is added in white peptone, 5g yeast extract, 10g sodium chloride2In O, adjustment pH7.2-7.4 or so, ddH2O is settled to 1L.
The ampicillin of 50mg/mL is added in mixed liquor after sterilizing with 1:1000 ratio, and is allowed to final concentration of 50 μ g/mL)；(19)LB
(preparation method is as follows: the agar of 1.5g is added in the LB liquid medium of 100mL solid medium, addition and liquid after sterilizing
The ammonia benzyl of same volume, paves plate)；(20) pGEX-EGFAB is given in Southwestern Medical Center, Texas ,Usa university
Johann Deisenhofer。
In following embodiments, experiment equipment used is as follows: 10KDa super filter tube (Millipore, UFC901096), gluathione
Peptide protein purification column (people from world and biology, S1001025), ELISA 96 orifice plates (Corning, 42592), shaking table, constant temperature training
Feeding case, 4 DEG C of refrigerators, -80 DEG C of refrigerators, centrifuge, super-clean bench, high-pressure sterilizing pot, spectrophotometer (Thermofisher),
PlateReader(PerkinElmer)。
Embodiment 1
A kind of method of in-vitro screening PCSK9 inhibitor, comprising the following steps:
Plasmid pGEX-EGFAB containing EGF-AB gene is imported e. coli bl21 and expresses EGF-AB albumen by S1；
37 DEG C are deposited in after the completion of inoculation under super-clean bench with the e. coli bl21 that conventional method imports EGF-AB gene
Incubator is stayed overnight；Picking single bacterium group is inoculated in the LB liquid medium of 5mL within second day, is vibrated at 37 DEG C with 240rpm
Night；Third day pours into bacterium solution in the LB culture medium of 500mL, 240rpm shaken cultivation at 37 DEG C, as spectrophotometric determination OD
IPTG is added at 0.7 in value, so that final concentration of 1mmol/L of the IPTG in culture solution, is further continued for the 240rpm at 37 DEG C and shakes
Swing culture 3 hours.5000g is centrifuged 10min and collects thallus after the completion of culture, and after abandoning supernatant, PBS is cleaned twice, is stored in -80 DEG C of ice
Case is spare.
S2 is extracted and is purified the EGF-AB albumen with GST label；
It extracts: albumen being extracted using kit, the Bacterial of the 20mL matched in kit is added in every gram of thallus
Protein Extraction Reagent, every milliliter of bacterium solution are added the DNase I's, the Lysozyme of 2 μ L and 10 μ L of 1 μ L
Protease Inhibitor cocktail cracks Escherichia coli and is prepared into suspension, and stands 20min at room temperature, thereafter
It is centrifuged 5 minutes under 15000g, abandons substrate and retain supernatant.
Purifying: it is centrifuged 10min with the super filter tube 4000g of 10KD, EGF-AB protein liquid is concentrated, PBS is added and stablizes pH extremely
7.0-7.5；Gst protein purification column 1h is balanced with PBS, EGF-AB albumen is added with 10 times of column volumes and stirs in the filter
Mix 1h；It is rinsed pillar 3-4 times with PBS, each 5min.With reduced glutathione elution pillar elution 5 times, 10mL/ times,
10min/ times；The PBS that 10% glycerol containing mass concentration is added after EGF-AB albumen 10KD super filter tube ultrafiltration under elution is protected
It is stored in -80 DEG C of refrigerators.
S3 includes the following steps:
EGF-AB albumen after purification dilutes 20 μ g/mL with DPBS, is coated in 96 orifice plates (reserved 3 holes with 100 holes μ L/
It is not coated with as blank control), it is placed in 2h in 4 DEG C overnight (12-16h) or 37 DEG C, is abandoned after liquid pats dry with 375 holes μ L/ use
PBST cleaning oscillation 3min.
It abandons liquid to pat dry, 5g/100ml skimmed milk power is added with 375 holes μ L/ and is closed, 2h is incubated in 37 DEG C, with 375 μ
The hole L/ is added PBST and cleans oscillation 3min, (if the same day does not continue to test, it is small should to clean 37 degree of lower dryings 1 after patting dry twice altogether
When, rear sealing plate is stored in 4 degree of refrigerators).
Drug pep2-8 to be measured is prepared into 50 μm of ol/L, 12.5 μm of ol/L, 3.13 μm of ol/L, 0.78 μ respectively with DMSO
Mol/L, 0.2 μm of ol/L, the gradient concentration of 0.05 μm of ol/L separately set equivalent is only added in three blank controls DMSO, Mei Geti
Degree concentration is separately added into the PCSK9 solution of equivalent, and an addition equivalent PCSK9 solution is as negative right in three blank controls
According to the ddH of equivalent is only added in other two blank control2O is incubated for 1h, Mei Geti under room temperature (23-27 DEG C) as blank control
It spends strength solution to be added in above-mentioned 96 orifice plate with the amount in 100 holes μ L/, 3 holes are one group, should be added not for one group in two groups of blank controls
Albumen containing EGF-AB is coated with hole, and negative control group is normally added EGF-AB albumen coating hole, at room temperature (23-27 DEG C) oscillation incubation
2h is abandoned after liquid pats dry, with the PBST in 375 holes μ L/, 3min/ progress oscillation cleaning, and totally 3 times.
After abandoning liquid pats dry, the primary antibody after being diluted with PBST with 1:2000, room temperature (23-27 are added with the amount in 100 holes μ L/
DEG C) oscillation incubation 2h, with the PBST in 375 holes μ L/, 3min/ progress oscillation cleaning, totally 4 times.
After abandoning liquid pats dry, the secondary antibody after being diluted with PBST with 1:5000, room temperature (23-27 are added with the amount in 100 holes μ L/
DEG C) oscillation incubation 1h, with the PBST in 375 holes μ L/, 3min/ progress oscillation cleaning totally 4 times, is abandoned liquid and is patted dry.
TMB is added with the amount in 100 holes μ L/ and is protected from light oscillation colour developing 5-10min, then the sulfuric acid of 2mol/L is added with 50 holes μ L/
After mixing, OD value is read with microplate reader at 450 nm for solution color development stopping.
S4 calculates the OD mean value of drug to be measured, substitution formula: (1- drug OD mean value/negative control OD value to be measured) ×
100%, inhibiting rate is obtained, draws inhibiting rate curve, then calculate IC50, there is dosage effect between inhibiting rate and drug concentration to be measured
Illustrate that drug to be measured has the effect of inhibiting PCSK9 bioactivity.OD value test result is as follows table 1, pep2-8 difference are dense
Inhibiting rate curve such as Fig. 1 of degree.
The test result of OD value in 1 embodiment 1 of table
Embodiment 2
The exploration of EGF-AB albumen and PCSK9 albumen optimization concentration parameter:
Since there may be unstable differences between EGF-AB albumen and PCSK9 albumen batch, determined by preliminary experiment
Stable bond concentration of the EGF-AB albumen to appropriate PCSK9 albumen: EGF-AB protein concentration is respectively configured by dilution of DPBS
For 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, three groups of elisa plates are coated with, separately reserved three holes are not coated with as blank control；PCSK9
Albumen is through ddH2O dilution is respectively set 400 μ g/mL, 200 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, and 0
The concentration gradient of μ g/mL is then respectively adding three groups of coating plates, and every group of each three Kong Chongfu of concentration, 100 holes μ L/ are reserved before
Blank control wells with 100 holes μ L/ be added ddH2O；Primary antibody, secondary antibody concentration temporarily use the minimum extension rate suggested in specification
1:1000.By testing EGF-AB albumen difference between batch can be excluded in 20 μ g/mL and stablize to 25 μ g/mL and following concentration
PCSK9 albumen obtain more suitable test data (i.e. developing time 5-10 minutes, under 450nm between OD value 0.9-1.2),
Therefore the optium concentration of EGF-AB albumen is 20 μ g/mL, and PCSK9 concentration≤25 μ g/mL.
The re-optimization of PCSK9 protein concentration: due to PCSK9 protein concentration can advanced optimize and different batches between
The stability difference of PCSK9 can carry out gradient survey to PCSK9 protein concentration dosage using before new batch PCSK9 albumen test
Examination.EGF-AB albumen is coated with bed board (staying three holes as blank control to use) using the concentration of 20 μ g/mL, and the setting of PCSK9 albumen is dense
Spending gradient is 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.13 μ g/mL, 1.63 μ g/mL, 0.82 μ g/mL, 0 μ g/mL, primary antibody
Diluted concentration is 1:2000, and secondary antibody diluted concentration is 1:5000.As a result i.e. developing time 5-10min is taken, OD value 0.9- under 450nm
PCSK9 protein concentration between 1.2 is as test concentrations.
Embodiment 3
The optimization of the diluted concentration of primary antibody and secondary antibody: since primary antibody secondary antibody is all made of import reagent therefore optimize extension rate to drop
Low experimental cost.1. secondary antibody optimization experiment: being coated with using the EGF-AB albumen bed board of 20 μ g/mL concentration, still reserve one group of sky
As control, PCSK9 still takes the configuration of previous run, but repeats three groups in white hole.Primary antibody is still 1:1000 dilution.Secondary antibody
Diluted concentration is set as 1:1000,1:2000, and 1:5000 respectively corresponds three groups of PCSK9.Primary antibody 1:1000 after tested, secondary antibody 1:
It can still be obtained when 5000 and the previous run result that there was no significant difference.2. primary antibody optimization experiment: EGF-AB albumen and PCSK9 are equal
Using previous configuration, primary antibody concentration is set as 1:1000,1:2000, and 1:5000 respectively corresponds three groups of PCSK9, and secondary antibody concentration is adopted
With the 1:5000 of previous determination.After tested, primary antibody is before 1:2000, secondary antibody still can keep and optimize in 1:5000 without conspicuousness
The result of difference.
It should be noted that involved in the present invention when numberical range, it is thus understood that two endpoints of each numberical range with
And any one numerical value can be selected between two endpoints, since the step method of use is identical as embodiment, go to live in the household of one's in-laws on getting married in order to prevent
It states, the present invention describes preferred embodiment., but technology people in the art although preferred embodiments of the present invention have been described
Member is once know basic creative concept, then additional changes and modifications can be made to these embodiments.So appended right
It is required that being intended to be construed to includes preferred embodiment and all change and modification for falling into the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (6)
1. a kind of method of in-vitro screening PCSK9 inhibitor, which comprises the following steps:
Plasmid pGEX-EGFAB containing EGF-AB gene is imported Escherichia coli and expresses EGF-AB albumen by S1；
S2 is extracted and is purified the EGF-AB albumen with GST label；
S3, bed board is coated with after EGF-AB albumen after purification is diluted with DPBS, and being placed in 4 DEG C, 2h, PBST are cleaned overnight or in 37 DEG C；
It is closed with 5g/100ml skimmed milk power, is placed in 2h in 37 DEG C, PBST cleaning；The PCSK9 with His label and medicine to be measured is added
Object, while negative control is set, it is incubated for 2h, PBST cleaning at room temperature；The primary antibody after being diluted with PBST is added, is incubated for 2h at room temperature,
PBST cleaning；The secondary antibody after being diluted with PBST is added, is incubated for 1h, PBST cleaning at room temperature；TMB colour developing, color development stopping, 450nm
Lower reading OD value；Wherein the drug to be measured is PCSK9 inhibitor to be screened；
S4 calculates the OD mean value of drug to be measured, substitutes into formula: (1- drug OD mean value/negative control OD value to be measured) × 100%,
Inhibiting rate is obtained, IC is calculated50, illustrate that drug to be measured has with dosage effect between inhibiting rate and drug concentration to be measured and inhibit
The effect of PCSK9 bioactivity.
2. the method for in-vitro screening PCSK9 inhibitor according to claim 1, which is characterized in that the process expressed in S1
Are as follows: it is 0.7 that the Escherichia coli for having imported EGF-AB gene, which are cultivated to bacterium solution OD value, and IPTG is added and is allowed to final concentration of 1mmol/
L continues to collect thallus after cultivating 3h.
3. the method for in-vitro screening PCSK9 inhibitor according to claim 1, which is characterized in that extract and purify in S2
The process of EGF-AB albumen are as follows: cracking Escherichia coli are simultaneously prepared into suspension, and PBS is added and stablizes pH to 7.0-7.5, crosses glutathione
Protein purification column is simultaneously eluted with reduced glutathione, and the EGF-AB albumen under eluting is stored in the PBS solution containing 10% glycerol.
4. the method for in-vitro screening PCSK9 inhibitor according to claim 1, which is characterized in that EGF- before bed board is coated with
AB albumen is diluted to 20 μ g/mL, PCSK9 concentration≤25 μ g/mL with DPBS.
5. the method for in-vitro screening PCSK9 inhibitor according to claim 1, which is characterized in that primary antibody is diluted with PBST
Ratio be 1:2000；Secondary antibody is 1:5000 with the diluted ratio of PBST.
6. the method for in-vitro screening PCSK9 inhibitor according to claim 1, which is characterized in that TMB colour developing 5-10min；
Select 2mol/L sulfuric acid color development stopping.
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Application publication date: 20190322 |