CN105255995B - A kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method and its kit - Google Patents
A kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method and its kit Download PDFInfo
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Abstract
The present invention relates to pharmaceutical technology field, in particular to a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method and its kit.The detection method includes: that the nucleotide sequence for encoding Gln-Gly is inserted into luciferase plasmids, obtains the recombination luciferase containing Gln-Gly through external acellular transcription and translation；In-vitro recombination expression hand-foot-and-mouth disease toxalbumin enzyme, hand-foot-and-mouth disease toxalbumin enzyme are 2A or HRV 3CP；With the recombination luciferase containing Gln-Gly, tested material enzymatic reaction occurs for hand-foot-and-mouth disease toxalbumin enzyme, detects the bioluminescence intensity of fluorescein, obtains the anti-hand-foot-and-mouth-disease pharmaceutical activity of tested material.Detection method has the advantages that simple, micro, quick, and drug screening workload is small, and experiment is quickly accurate, and experimental result is reproducible, can be used for the screening and exploitation of anti-hand-foot-and-mouth-disease drug；Bioluminescent detection method of this law based on luciferase, high sensitivity, disturbing factor is few, strong by test product compatibility.
Description
Technical field
The present invention relates to pharmaceutical technology field, in particular to a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method and its reagent
Box.
Background technique
Hand-foot-and-mouth disease is pediatric disease caused by virus, belongs to the legal Class C infectious disease in China.0-6 years old infant be
The Susceptible population of the disease, wherein it is most commonly seen with 2-3 years old childhood infection, draw first at the positions such as four limbs and oral cavity after infant infection
Bleb or ulcer are played, most infants can the recovery from illness in 1-2 weeks after giving timely and effective treatment.However, a small number of infant disease progressions
Rapidly, central nervous system and cardiopulmonary infection, mortality risks with higher, case of surviving can occur within 1-5 days or so in morbidity
It still can there are sequelae.
According to WHO Report, about more than 20 kinds of the virus of hand-foot-and-mouth disease symptom can be caused, wherein with intestines
Road 71 types of virus (enterovirus 71, EV71) and coxsackievirus A16 (coxsachievirus A16, CoxA16)
Most commonly seen, other can cause the virus of hand-foot-and-mouth disease to specifically include that Coxsackie virus A group 2,4,5,7,9 and 10 types
(CoxA2, CoxA4, CoxA5, CoxA7, CoxA9 and CoxA10), coxsackie virus group B 1-5 type (CoxB1-5) and angstrom can disease
Malicious (ECHO) etc..Currently, it is not directly targeted the specific medicament or preventative vaccine of virus still, it is clinical mainly to take symptomatic treatment
Strategy.This status prompts medicament research and development personnel, and there is still a need for further investigate and sieve to anti-hand-foot-and-mouth-disease compound or composition
Choosing.
The 2A protease and HRV 3CP of brothers mouthful correlated virus are commonly used for doing screening target spot, and 2A and HRV 3CP belong to half
Cysteine proteases, the amido bond between specific recognition and scinderin matter glutamine (Gln) and glycine (Gly).In disease
It is primarily involved in the history of life of poison and interferes host protein to synthesize, virus protein original processing and mature, assistance viral nucleic acid is promoted to answer
System and starting host cell programmed death etc..Generally speaking, 2A and HRV 3CP have in the history of life of hand-foot-and-mouth disease poison
It plays an important role, while being also a good research target spot.But at present still do not have for the target spot drug official listing,
Which imply the drug screenings for the target spot to work it is still necessary to update, sensitiveer, more reliable approach.
Existing hand-foot-and-mouth disease toxalbumin inhibitor sifting method mainly has enzyme coupling method, yeast two-hybrid method and HPLC
Measure product method.Wherein, the accuracy of enzyme coupling method measurement is not high, the range of linearity is relatively narrow, and repeatability is bad；And yeast is double miscellaneous
Friendship method is long experimental period, and disturbing factor is more；The HPLC measurement product method period is long, flux is small, is directed to instrument and operator
The technical barrier of member is relatively high.Exactly because the bottleneck of screening technique is seriously constrained extensive, high based on this target drug
Flux screening and discovery.For example, Pfizer once used enzyme coupling method to screen to obtain HRV 3CP inhibitor rupintrivir simultaneously
Into development phase (Dragovich et al., J Med Chem, 1999,42:1213-1224), afterwards because not facing significantly
Bed drug effect is counted out.In short, up to now, being still not based on the drug formally granted listing of this target spot.Therefore, it is necessary to build
Vertical protease inhibitors screening technique quickly, accurate, disturbing factor is few, to push the anti-brothers mouthful of protease inhibitors class
Drug emerges in multitude.
Summary of the invention
In view of this, the present invention provides a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method and its kits.The detection
Method has the advantages that simple, micro, quick, and drug screening workload is small, and experiment is quickly accurate, and experimental result is reproducible,
It can be used for the screening and exploitation of anti-hand-foot-and-mouth-disease drug；Bioluminescent detection method of this law based on luciferase, high sensitivity,
Disturbing factor is few, strong by test product compatibility.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection methods, include the following steps:
The nucleotide sequence for encoding Gln-Gly is inserted into luciferase plasmids, is contained through external acellular transcription and translation
The recombination luciferase of Gln-Gly；
In-vitro recombination expression hand-foot-and-mouth disease toxalbumin enzyme, hand-foot-and-mouth disease toxalbumin enzyme are 2A or HRV 3CP；
With the recombination luciferase containing Gln-Gly, tested material enzymatic reaction occurs for hand-foot-and-mouth disease toxalbumin enzyme, detects fluorescence
The bioluminescence intensity of element, obtains the anti-hand-foot-and-mouth-disease pharmaceutical activity of tested material.
The basic principle of detection method are as follows: after luciferase plasmids are loaded into the corresponding nucleotide sequence of substrate, lead to
Cross external acellular Transcription/Translation System synthesis recombination luciferase.In the present invention, the substrate of 2A and HRV 3CP is Gln-Gly
Dipeptides, corresponding nucleotide sequence can be CAAGGU.Figure 1A shows the corresponding nucleotide sequence of protein substrate (CAAGGU) in plasmid
Insertion point, plasmid synthesize recombination luciferase after external acellular transcription and translation.As shown in Figure 1B, luciferase is due to more
Gln-Gly two amino acid residues can not form correctly catalysis conformation, cause luciferase that cannot be catalyzed luciferin oxidation
It shines；Protease is added by after amino acid residue incision extra in luciferin, luciferase folds to form correctly catalysis structure
As being catalyzed luciferin oxyluminescence, therefore, luciferin luminous intensity characterizes prolease activity.It is raw to blank with tested material
The protease restraint of the inhibiting effect characterization tested material of object luminous intensity.
In some embodiments provided by the invention, hand-foot-and-mouth disease poison is selected from Coxsackie virus A 2 (CoxA2), A4
(CoxA4), A5 (CoxA5), A7 (CoxA7), A9 (CoxA9), A10 (CoxA10), A16 type (CoxA16), Coxsackie virus B 1
(CoxB1), B2 (CoxB2), B3 (CoxB3), B4 (CoxB4) and B5 (CoxB5) type, enterovirns type 71 (EV71), and angstrom
It can virus (ECHO).
In some embodiments provided by the invention, the nucleotides sequence for encoding Gln-Gly is classified as CAAGGU.According to codon
Degeneracy, other, which also can be used, can translate other nucleotide sequences of Gln-Gly, such as: CAAGGC, CAAGGA,
CAAGGG, CAGGGU, CAGGGC, CAGGGA, CAGGGG.
Preferably, luciferase plasmids are pGloSensorTM- 10F plasmid.
Preferably, the reaction system of enzymatic reaction are as follows:
Recombination luciferase containing Gln-Gly: 1~10 μ g
Hand-foot-and-mouth disease toxalbumin enzyme: 10~100IU
Tested material: 0 μm of ol/L of >
Hepes buffer: 10~200 μ L are complemented to.
Preferably, the reaction system of enzymatic reaction are as follows:
Recombination luciferase containing Gln-Gly: 1~10 μ g
Hand-foot-and-mouth disease toxalbumin enzyme: 10~100IU
Tested material: 0 μm of ol/L of >
Hepes buffer: 100 μ L are complemented to
In some embodiments provided by the invention, the reaction system of enzymatic reaction are as follows:
Recombination luciferase containing Gln-Gly: 1 μ g
Hand-foot-and-mouth disease toxalbumin enzyme: 20IU
Tested material: 0 μm of ol/L of >
Hepes buffer: 10 μ L are complemented to
In other embodiments provided by the invention, the reaction system of enzymatic reaction are as follows:
Recombination luciferase containing Gln-Gly: 10 μ g
Hand-foot-and-mouth disease toxalbumin enzyme: 20IU
Tested material: 0 μm of ol/L of >
Hepes buffer: 25 μ L are complemented to
In other embodiments provided by the invention, the reaction system of enzymatic reaction are as follows:
Recombination luciferase containing Gln-Gly: 2 μ g
Hand-foot-and-mouth disease toxalbumin enzyme: 50IU
Tested material: 0 μm of ol/L of >
Hepes buffer: 50 μ L are complemented to
In other embodiments provided by the invention, the reaction system of enzymatic reaction are as follows:
Recombination luciferase containing Gln-Gly: 5 μ g
Hand-foot-and-mouth disease toxalbumin enzyme: 10IU
Tested material: 0 μm of ol/L of >
Hepes buffer: 150 μ L are complemented to
In other embodiments provided by the invention, the reaction system of enzymatic reaction are as follows:
Recombination luciferase containing Gln-Gly: 10 μ g
Hand-foot-and-mouth disease toxalbumin enzyme: 100IU
Tested material: 0 μm of ol/L of >
Hepes buffer: 200 μ L are complemented to
Preferably, the concentration of Hepes buffer is 10~100mM, pH 7.0~8.0.
Preferably, the concentration of Hepes buffer is 20mM, pH 7.5.
Preferably, the temperature of enzymatic reaction is 30 DEG C.
Preferably, the time of enzymatic reaction is 1~4h.
In some embodiments provided by the invention, the preparation method of the anti-hand-foot-and-mouth-disease pharmaceutical activity of tested material are as follows: root
According to bioluminescence intensity (RLU), the inhibiting rate of tested material is obtained.
In the present invention, inhibiting rate=100% of tested material × (1-RLUSample/RLUBlank)。
In other embodiments provided by the invention, after the inhibiting rate for obtaining tested material further include: according to tested material
Inhibiting rate obtain tested material to the EC of brothers' Aphthovirus protease50。
In the present invention, the EC of hand-foot-and-mouth disease toxalbumin enzyme50Calculation method is as follows:
A. the logarithm of tested material concentration and inhibiting rate, lg (tested material concentration) and lg (inhibiting rate) are taken respectively；
B. x=lg (tested material concentration) and y=lg (inhibiting rate), and y=ax+b are set respectively, according to different tested material concentration
Under corresponding inhibiting rate, substitute into y=ax+b equation in, find out a and b value；
C. y=lg (0.5) is substituted into equation, obtains x value, i.e. lg (EC50)；
D. it asks after the truth of a matter up to EC50Value.
In some embodiments provided by the invention, tested material is that antiviral drugs, compound, Chinese materia medica preparation or plant mention
Take object.
The present invention also provides a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection kits, comprising:
Recombination luciferase containing Gln-Gly；
Hand-foot-and-mouth disease toxalbumin enzyme；Hand-foot-and-mouth disease toxalbumin enzyme is 2A or HRV 3CP；
Hepes buffer；
Luciferin detection reagent.
In some embodiments provided by the invention, hand-foot-and-mouth disease poison be selected from Coxsackie virus A 2, A4, A5, A7, A9,
A10, A16 type, Coxsackie virus B 1, B2, B3, B4 and B5 type, enterovirns type 71 and echovirus.
In some embodiments provided by the invention, the recombination luciferase containing Gln-Gly are as follows: will encode Gln-Gly's
Nucleotide sequence is inserted into luciferase plasmids, obtains the recombination luciferase containing Gln-Gly through external acellular transcription and translation.
In some embodiments provided by the invention, the nucleotides sequence for encoding Gln-Gly is classified as CAAGGU.According to codon
Degeneracy, other, which also can be used, can translate other nucleotide sequences of Gln-Gly, such as: CAAGGC, CAAGGA,
CAAGGG, CAGGGU, CAGGGC, CAGGGA, CAGGGG.
Preferably, luciferase plasmids are pGloSensorTM- 10F plasmid.
Preferably, the concentration of Hepes buffer is 10~100mM, pH 7.0~8.0.
Preferably, the concentration of Hepes buffer is 20mM, pH 7.5.
In some embodiments provided by the invention, luciferin detection reagent is Bright-GloTMLuciferin detection reagent.
The present invention provides a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method and its kits.The detection method includes:
The nucleotide sequence for encoding Gln-Gly is inserted into luciferase plasmids, is obtained through external acellular transcription and translation containing Gln-Gly's
Recombinate luciferase；In-vitro recombination expression hand-foot-and-mouth disease toxalbumin enzyme, hand-foot-and-mouth disease toxalbumin enzyme are 2A or HRV 3CP；Brothers
With the recombination luciferase containing Gln-Gly, tested material enzymatic reaction occurs for Aphthovirus protease, detects the bioluminescence of fluorescein
Intensity obtains the anti-hand-foot-and-mouth-disease pharmaceutical activity of tested material.The present invention at least has one of following advantage:
Detection method has the advantages that simple, micro, quick, and drug screening workload is small, and experiment is quickly accurate,
Experimental result is reproducible, can be used for the screening and exploitation of anti-hand-foot-and-mouth-disease drug；
The present invention is the bioluminescent detection method based on luciferase, high sensitivity, and disturbing factor is few；
It is strong by test product compatibility in the present invention.
Detailed description of the invention
Fig. 1 shows the basic schematic diagram of the method for the present invention；Wherein Figure 1A shows pGloSensorTMProtein substrate in -10F plasmid
(Gln-Gly) corresponding nucleotide sequence (CAAGGU) insertion point；Figure 1B shows recombination luciferase in 2A or HRV 3CP
Under effect, after recombinating Gln-Gly amino acid residue incision extra in luciferase, luciferase is folded to be formed and correctly be urged
Change conformation, is catalyzed luciferin oxyluminescence.
Specific embodiment
The invention discloses a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection methods and its kit, those skilled in the art can
To use for reference present disclosure, it is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this
It is it will be apparent that they are considered as being included in the present invention for the technical staff of field.Method and application of the invention is
Be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to herein
The methods and applications are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Agents useful for same, instrument can be by anti-hand-foot-and-mouth-disease pharmaceutical activity detection method provided by the invention and its kit
Market is bought.
Substrate: in pGloSensorTMIt is inserted into CAAGGU segment in -10F plasmid, is synthesized after external acellular transcription and translation
The luciferase of the amino acid residue containing Gln-Gly, is provided by Promege company.
Protease: EV71, CoxA16, CoxA2, CoxA4, CoxA5, CoxA7, CoxA9, CoxA10, CoxB1-5 and
The 2A and HRV 3CP of echo, enteric cytopathogenic human orphan virus are purified, purity > 85% by Promega company in-vitro recombination expression.
Reagent: trans- epoxy succinyl base-L- leucyl amido (4- guanidine radicals) butane (E-64) is purchased from the wide sharp biological section in Shanghai
Skill Co., Ltd, luciferin detection kit Bright-GloTMPurchased from Promega company, remaining reagent is that domestic analysis is pure.
Instrument: FLUOstar OPTIMA FL multi-function microplate reader is purchased from BMG LABTECH company.
Below with reference to embodiment, the present invention is further explained:
1 anti-hand-foot-and-mouth-disease pharmaceutical activity screening technique of embodiment
1, the corresponding nucleotide sequence of the substrate Gln-Gly of 2A and HRV 3CP (CAAGGU) is loaded into pGloSensorTM-
10F plasmid (Promege company) is transcribed in vitro translation system synthesis substrate using acellular and recombinates luciferase；
2, the 2A and HRV 3CP of each hand-foot-and-mouth disease poison of in-vitro recombination expression, including EV71, CoxA16, CoxA2, CoxA4,
The 2A and HRV 3CP of CoxA5, CoxA7, CoxA9, CoxA10, CoxB1-5 and echo, enteric cytopathogenic human orphan virus；
3, the recombination luciferase and 2A and HRV 3CP of acquisition, drug are incubated for 1~4h under the conditions of 30 DEG C jointly, examined
Survey the bioluminescence intensity (RLU) of fluorescein.
Mmp reaction system are as follows:
Substrate: 1~10 μ g
Protease: 10~100IU
Tested material: 0 μm of ol/L of >
Hepes buffer (10~100mM,
PH 7.0~8.0): complement to 100 μ L
Tested material is not added as blank, inhibiting rate of the drug to protease, calculation method are calculated are as follows:
Inhibiting rate=100% × (1-RLUSample/RLUBlank)
Drug is calculated to the half-maximal effect concentration (EC of each protease according to inhibiting rate50), calculation method is as follows:
A. the logarithm of tested material concentration and inhibiting rate, lg (tested material concentration) and lg (inhibiting rate) are taken respectively；
B. x=lg (tested material concentration) and y=lg (inhibiting rate), and y=ax+b are set respectively, according to different tested material concentration
Under corresponding inhibiting rate, substitute into y=ax+b equation in, find out a and b value；
C. y=lg (0.5) is substituted into equation, obtains x value, i.e. lg (EC50)；
D. it asks after the truth of a matter up to EC50Value.
Trans- epoxy succinyl base-L- leucyl amido (4- guanidine radicals) butane (E-64) of embodiment 2 inhibits test
Utilize the generally acknowledged trans- epoxy succinyl base-L- leucyl amido of cystatin (4- guanidine radicals) fourth
Alkane studies it to a variety of hand-foot-and-mouth disease poison 2A and HRV 3CP activity inhibition, verifies the reliability and universality of this method.
1. materials and methods
1.1 substrate
In pGloSensorTMIt is inserted into CAAGGU segment in -10F plasmid, synthesizes and contains after external acellular transcription and translation
The luciferase of Gln-Gly amino acid residue is provided by Promege company.
1.2 protease
EV71, CoxA16, CoxA2, CoxA4, CoxA5, CoxA7, CoxA9, CoxA10, CoxB1-5 and echo, enteric cytopathogenic human orphan virus
2A and HRV 3CP, by Promega company in-vitro recombination expression purify, purity > 85%.
1.3 reagent
Purchased from Shanghai, extensively sharp biotechnology has trans- epoxy succinyl base-L- leucyl amido (4- guanidine radicals) butane (E-64)
Limit company, luciferin detection kit Bright-GloTMPurchased from Promega company, remaining reagent is that domestic analysis is pure.
1.4 instrument
FLUOstar OPTIMA FL multi-function microplate reader is purchased from BMG LABTECH company.
1.5 protease activity determination
Mmp reaction system (10 μ L) is Hepes buffer (20mM, pH 7.5), includes 1 μ g substrate, 20IU albumen
Enzyme sets a series of E-64 concentration gradients: 0, after 10,20,50,100,200,400 and 500 μm of ol/L, 30 DEG C of incubation 1h, waiting bodies
Bright-Glo is added in productTMLuciferin detection reagent after being incubated at room temperature 5min, measures bioluminescence intensity (RLU).Be not added by
Examination object is blank, calculates inhibiting rate=100% × (1-RLU of each concentration E-64Sample/RLUBlank), E-64 is calculated according to inhibiting rate
For the half-maximal effect concentration (EC of each protease50)。
2. result
This example has investigated the reliability of the method for the invention by taking the generally acknowledged inhibitor E-64 of 2A and HRV 3CP as an example
With universality, the method for the invention measures E-64 to each hand-foot-and-mouth disease poison 2A and HRV 3CP EC50For 214.37~357.48 μ
Mol/L etc., consistent with forefathers' report, it is reliable and pervasive for showing that this method is used for hand-foot-and-mouth disease toxalbumin inhibitor sifting
's.
EC of the table 1E-64 to each protease50(μm ol/L, mean value ± standard error, n=3)
3. conclusion
Using detection method of the present invention, screens hand-foot-and-mouth disease toxalbumin enzyme inhibitor and have reliability and pervasive
Property.
Embodiment 3 investigates different antiviral drugs to the inhibiting effect of EV71 virus HRV 3CP
Using the method for the invention, the sieve for inhibiting EV71 virus HRV 3CP active for existing antiviral compound
Choosing verifying
1. materials and methods
1.1 substrate
In pGloSensorTMIt is inserted into CAAGGU segment in -10F plasmid, synthesizes and contains after external acellular transcription and translation
The luciferase of Gln-Gly amino acid residue is provided by Promege company.
1.2 protease
EV71 virus HRV 3CP is purified, purity > 85% by Promega company in-vitro recombination expression.
1.3 reagent
Antiviral compound Ribavirin, Oseltamivir, Peramivir, Favipiravir and rupintrivir, are purchased from lark
Prestige Science and Technology Ltd., luciferin detection kit Bright-GloTMPurchased from Promega company, remaining reagent is domestic point
It analyses pure.
1.4 instrument
FLUOstar OPTIMA FL multi-function microplate reader is purchased from BMG LABTECH company.
1.5 protease activity determination
Mmp reaction system (25 μ L) is Hepes buffer (20mM, pH 7.5), includes 10 μ g substrates, 20IU albumen
Enzyme, each reagent set a series of concentration gradients: 0, after 10,20,50,100,200,400 and 500 μm of ol/L, 30 DEG C of incubation 1h, etc.
Bright-Glo is added in volumeTMLuciferin detection reagent after being incubated at room temperature 5min, measures bioluminescence intensity (RLU).To be not added
Tested material is blank, calculates inhibiting rate=100% × (1-RLU of each compoundSample/RLUBlank), chemical combination is calculated according to inhibiting rate
Object is directed to the half-maximal effect concentration (EC of EV71 virus HRV 3CP50)。
2. result
The present embodiment uses the method for the invention, and it is living to the inhibition of EV71 virus HRV 3CP to investigate antiviral compound
Property.As shown in table 2, Ribavirin, Oseltamivir, Peramivir and Favipiravir do not have EV71 virus HRV 3CP and inhibit
Activity, wherein the anti-viral targets of Ribavirin are induction viral nucleic acid copy errors, Oseltamivir and Peramivir it is disease-resistant
Malicious target spot is to inhibit neuraminidase, and the antiviral target spot of Favipiravir is to inhibit RNA synzyme, is lived with HRV 3CP is inhibited
Property is unrelated.And rupintrivir then has significant inhibiting effect, EC to HRV 3CP50=38.61 ± 4.83nmol/L, this implementation
Example result and forefathers report consistent (Dragovich et al., J Med Chem, 1999,42:1213-1224).
EC of 2 compound of table to EV7 virus HRV 3CP50(nmol/L, n=3)
| Compound | EC50(nmol/L) |
| Ribavirin | ND |
| Oseltamivir | ND |
| Peramivir | ND |
| Favipiravir | ND |
| Rupintrivir | 38.61±4.83 |
Note: ND expression is not detected.
3. brief summary
The present embodiment is consistent for the selection result and drug self character of antiviral chemical drug and forefathers' report, institute of the present invention
The method of stating can be used for chemical drug and inhibit the active anti-brothers mouthful screening compound of HRV 3CP.
Embodiment 4 investigates different compounds to the inhibiting effect of EV71 virus HRV 3CP
Anti- EV71 compound rutin, fisetin, luteolin and apiolin inhibit the active sieve of EV71 virus HRV 3CP
Choosing.
1. materials and methods
1.1 substrate
In pGloSensorTMIt is inserted into CAAGGU segment in -10F plasmid, synthesizes and contains after external acellular transcription and translation
The luciferase of Gln-Gly amino acid residue is provided by Promege company.
1.2 protease
EV71 virus HRV 3CP is purified, purity > 85% by Promega company in-vitro recombination expression.
1.3 reagent
Rutin, fisetin, luteolin and apiolin standard items are purchased from National Institute for Food and Drugs Control, luciferin inspection
Test agent box Bright-GloTMPurchased from Promega company, remaining reagent is that domestic analysis is pure.
1.4 instrument
FLUOstar OPTIMA FL multi-function microplate reader is purchased from BMG LABTECH company.
1.5 protease activity determination
Mmp reaction system (50 μ L) is Hepes buffer (10mM, pH 7.0), includes 2 μ g substrates, 50IU EV71
Viral HRV 3CP, test drug concentrations are 400 μm of ol/L, and after 30 DEG C of incubation 2h, Bright-Glo is added in equal volumeTMLuciferin
Detection reagent after being incubated at room temperature 5min, measures bioluminescence intensity (RLU).Sample is not added as blank, each compound is calculated
Inhibiting rate=100% × (1-RLUSample/RLUBlank)。
2. result
As shown in table 3,400 μm of ol/L rutins and fisetin significantly inhibit EV71 virus HRV 3CP,
Respectively 75.81 ± 4.37% and 68.17 ± 3.38%；And under same concentrations, luteolin and apiolin to HRV 3CP then
There is no apparent inhibiting effect.Lin etc. is research shows that rutin and fisetin have apparent inhibit to EV71 virus HRV 3CP
Act on (Lin et al, Journal of Virological Methods, 2012,182:93-98), and Lv etc. research shows that
Luteolin and apiolin only have weaker inhibition to HRV 3CP and make although having apparent inhibiting effect to EV71 virus
With, two compound effects target spots be not HRV 3CP (Lv et al, Antiviral Research, 2014,109:30-
41).It is consistent using protease inhibitors STUDY ON SCREENING acquired results of the present invention and forefathers' report, show that this method can
With the anti-brothers mouthful screening compound for being inhibited based on protease.
Inhibiting rate (%, mean value ± standard error, n=3) of 3 compound of table to EV7 virus HRV 3CP
| Compound | Inhibiting rate (%) |
| Rutin | 75.81±4.37 |
| Fisetin | 68.17±3.38 |
| Luteolin | 9.07±2.26 |
| Apiolin | 8.47±3.46 |
3. brief summary
This section embodiment shows the anti-brothers mouthful compound sieve that the method for the invention can be used for inhibiting based on protease
Choosing.
Embodiment 5 investigates Chinese materia medica preparation to the inhibiting effect of EV71 virus HRV 3CP
It lists anti-hand-foot-and-mouth-disease Chinese medicine and inhibits EV71 virus HRV 3CP screening active ingredients.
1. materials and methods
1.1 substrate
In pGloSensorTMIt is inserted into CAAGGU segment in -10F plasmid, synthesizes and contains after external acellular transcription and translation
The luciferase of Gln-Gly amino acid residue is provided by Promege company.
1.2 protease
EV71 virus HRV 3CP is purified, purity > 85% by Promega company in-vitro recombination expression.
1.3 test medicine
Antiviral oral liquor (Jiangxi QingFeng Pharmacy Co., Ltd), gold vibration oral solution (the limited public affairs of Jiangsu Kang Yuan medicine company share
Department), Lanqin oral liquid (Yangzijiang Pharmaceutical Group Co., Ltd), Camptotheca acuminata leaves (Jiangxi QingFeng Pharmacy Co., Ltd), heat
Malicious injection for curing (Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov), tanreqin injection (Shanghai Kaibao Pharmaceutical Co., Ltd) are country
The clinical treatment hand-foot-and-mouth disease that State Administration of Traditional Chinese Medicines's " traditional Chinese medicine treatment hand-foot-and-mouth disease clinical technology guide (version in 2012) " includes
Chinese medicine is used in recommendation.
1.4 instrument
FLUOstar OPTIMA FL multi-function microplate reader is purchased from BMG LABTECH company.
1.5 protease activity determination
Mmp reaction system (150 μ L) is Hepes buffer (50mM, pH 7.5), includes 5 μ g substrates, 10IU EV71
Viral HRV 3CP, test medicine extension rate are 50,100,200,500 and 1000, isometric to be added after 30 DEG C of incubation 4h
Bright-GloTMLuciferin detection reagent after being incubated at room temperature 5min, measures bioluminescence intensity (RLU).Sample is not added as sky
It is white, calculate inhibiting rate=100% × (1-RLU of each preparationSample/RLUBlank)。
2. result
As shown in table 4, the hand-foot-and-mouth disease drug being recommended to use by State Administration of Traditional Chinese Medicine is to EV71 virus HRV 3CP
All have different degrees of inhibiting effect, under different dilutions, the inhibiting rate of drug is 8.63~72.41% to differ, and shows this
A little drugs all have certain inhibiting effect to EV71 virus HRV 3CP.Forefathers' studies have shown that antiviral oral liquor (Dong Qiaoli
Deng paediatrics pharmaceutical journal, 2012,18:27-29), gold vibration oral solution (Liu et al., PLoS One, 2014,9:e94466),
Lanqin oral liquid (Wen Zhiyuan and Gu Yiwen, Contemporary Chinese medicine, 2013,20:55-56), (Guan Chen is immunized Camptotheca acuminata leaves
Learn magazine, 2013,29:737-744), Reduning injection (Cao Ze Yu, Chinese herbal medicine, 2014,45:1450-1455) and Tanreqing
The Chinese materia medica preparations such as injection (fourth is red, practical drug and clinical, 2008,11:87-88) have specific curative effect to hand-foot-and-mouth disease,
The present embodiment the selection result is consistent with previous karyotype studies and clinical efficacy, and therefore, the method for the invention can be used for being directed to
The Chinese materia medica preparation protease inhibiting activity of hand-foot-and-mouth disease screens.
Inhibiting rate (%, mean value ± standard error, n=3) of the different Chinese materia medica preparations of table 4 to EV71 virus HRV 3CP
3. brief summary
This section embodiment shows the anti-brothers mouthful Chinese materia medica preparation that the method for the invention can be used for inhibiting based on protease
Screening.
Embodiment 6 investigates plant extracts to the inhibiting effect of EV71 virus HRV 3CP
The positions such as honeysuckle, cape jasmine, radix scutellariae, Radix Isatidis, Fructus Forsythiae and fritillary bulb water, n-butanol, ethyl acetate and petroleum ether
Inhibit EV71 virus HRV 3CP screening active ingredients.
1. materials and methods
1.1 substrate
In pGloSensorTMIt is inserted into CAAGGU segment in -10F plasmid, synthesizes and contains after external acellular transcription and translation
The luciferase of Gln-Gly amino acid residue is provided by Promege company.
1.2 protease
EV71 virus HRV 3CP is purified, purity > 85% by Promega company in-vitro recombination expression.
The preparation at 1.3 medicinal material different solvents positions
Honeysuckle, cape jasmine, radix scutellariae, Radix Isatidis, Fructus Forsythiae and fritillary bulb drying and crushing simultaneously cross 45 meshes, and it is each to weigh medicinal material
200g, 95% alcohol reflux extract 3 times, and the time is respectively 3,3 and 2 hours, and solvent is respectively 10,8 and 6 times of amounts, close after filtering
And filtrate, it is concentrated under reduced pressure；100mL water dispersion medicinal extract is successively extracted 3 times with petroleum ether, ethyl acetate and each 100mL of n-butanol,
Each extract liquor and aqueous solution decompression freeze-drying, obtain each position dry cream.
1.4 instrument
FLUOstar OPTIMA FL multi-function microplate reader is purchased from BMG LABTECH company.
1.5 protease activity determination
Mmp reaction system (200 μ L) is Hepes buffer (100mM, pH 8.0), includes 10 μ g substrates, 100IU
EV71 virus HRV 3CP, each position dry cream concentration is 1mg/mL, and after 30 DEG C of incubation 1h, Bright-Glo is added in equal volumeTMFirefly
Light element detection reagent after being incubated at room temperature 5min, measures bioluminescence intensity (RLU).Sample is not added as blank, each combination is calculated
Inhibiting rate=100% of object × (1-RLUSample/RLUBlank)。
2. result
As shown in table 5, the water, n-butanol, ethyl acetate of honeysuckle, cape jasmine, radix scutellariae, Radix Isatidis, Fructus Forsythiae and fritillary bulb and
Petroleum ether 4 extraction positions have different degrees of inhibiting effect, generally speaking, water position pair to EV71 virus HRV 3CP
EV71 virus HRV 3CP inhibitory activity is weaker, and organic position has compared with strong inhibitory activity, shows that this method can be used for Chinese medicine
The screening study of material difference extraction position protease inhibitors.
Inhibiting rate (%, mean value ± standard error, n=3) of the 5 medicinal material different parts of table to EV71 virus HRV 3CP
3. brief summary
This section embodiment shows the anti-brothers mouthful plant extract that the method for the invention can be used for inhibiting based on protease
Position screening.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method, which comprises the steps of:
The nucleotide sequence for encoding Gln-Gly is inserted into luciferase plasmids, is obtained through external acellular transcription and translation and contains Gln-
The recombination luciferase of Gly；
In-vitro recombination expression hand-foot-and-mouth disease toxalbumin enzyme, the hand-foot-and-mouth disease toxalbumin enzyme are 2A or HRV 3CP；
In cell-free system, pH value under 7~8 detection architecture, the hand-foot-and-mouth disease toxalbumin enzyme contains Gln-Gly with described
Recombination luciferase, tested material enzymatic reaction occurs, detect the bioluminescence intensity of fluorescein, obtain the anti-of the tested material
Hand-foot-and-mouth disease pharmaceutical activity；
The hand-foot-and-mouth disease poison be selected from Coxsackie virus A 2, A4, A5, A7, A9, A10, A16 type, Coxsackie virus B 1, B2, B3,
B4 and B5 type, enterovirns type 71 and echovirus.
2. detection method according to claim 1, which is characterized in that the reaction system of the enzymatic reaction are as follows:
3. detection method according to claim 1, which is characterized in that the temperature of the enzymatic reaction is 30 DEG C, the time 1
~4h.
4. detection method according to claim 1, which is characterized in that the anti-hand-foot-and-mouth-disease pharmaceutical activity of the tested material
Preparation method are as follows: according to bioluminescence intensity, obtain the inhibiting rate of tested material.
5. a kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection kit characterized by comprising
Recombination luciferase containing Gln-Gly；
Hand-foot-and-mouth disease toxalbumin enzyme；The hand-foot-and-mouth disease toxalbumin enzyme is 2A or HRV 3CP；
Hepes buffer；
Luciferin detection reagent.
6. kit according to claim 5, which is characterized in that the recombination luciferase containing Gln-Gly are as follows: will compile
The nucleotide sequence of code Gln-Gly is inserted into luciferase plasmids, obtains the recombination containing Gln-Gly through external acellular transcription and translation
Luciferase.
7. kit according to claim 6, which is characterized in that the nucleotides sequence for encoding the Gln-Gly is classified as
CAAGGU。
8. kit according to claim 6, which is characterized in that the luciferase plasmids are pGloSensorTM-10F
Plasmid.
9. kit according to claim 5, which is characterized in that the concentration of the Hepes buffer is 10~100mM,
PH 7.0~8.0.
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