CN105067574A

CN105067574A - EV71 3C protease action target point detection kit and EV71 3C protease action target point detection method

Info

Publication number: CN105067574A
Application number: CN201510342092.6A
Authority: CN
Inventors: 李冰清; 孟红; 秦立增; 李鹏; 李志会; 岳盈盈; 宋楠楠
Original assignee: INSTITUTE OF BASIC MEDICINE SAMS; Shandong University of Traditional Chinese Medicine
Current assignee: INSTITUTE OF BASIC MEDICINE SAMS; Shandong University of Traditional Chinese Medicine
Priority date: 2015-06-18
Filing date: 2015-06-18
Publication date: 2015-11-18
Anticipated expiration: 2035-06-18
Also published as: CN105067574B

Abstract

The present invention discloses an EV71 3C protease action target point detection kit, which comprises 3C protein, a polypeptide substrate and an enzymolysis reaction buffer solution, wherein the sequence of the 3C protein is represented by SEQ ID NO.1, and the polypeptide substrate is Dabcyl-EALFQGPPKFE-Edans. The present invention further discloses an EV71 3C protease action target point detection method, which comprises: adding a compound to be detected and 3C protein to an enzymolysis reaction buffer solution, adding a polypeptide substrate, uniformly mixing, and detecting the fluorescence intensity at a wavelength of 490 nm under 336 nm wavelength excitation, wherein it is indicated that the compound can inhibit the 3C protein if the fluorescence can not be detected, and it is indicated that the compound can not inhibit the 3C protein if the fluorescence can be detected. According to the present invention, with the detection kit and the detection method, the anti-EV71 ability of the compound to be detected can be effectively detected; and the method is simple and easy to perform, and the specificity and the sensitivity are high.

Description

EV71 HRV 3CP action target spot detection kit and detection method

Technical field

The present invention relates to a kind of EV713C proteinase action target spot detection kit, and detection method.

Background technology

Enterovirns type 71 (enterovirus71, EV71) is that the principal causative of hand-foot-and-mouth disease is one of former.Show as light symptoms clinically: heating, the bleb of brothers' oral area, herpangina more.Severe patient then causes neurological complication as AME, myelitis, neurogenic pulmonary edema etc.Severe patient is in peril of one's life.Clinical treatment EV71 infects mainly symptomatic treatment, there is no special antiviral drugs at present, finds and find that anti-EV71 virus drugs is the Chinese medicine problem faced at present.

Find anti-EV71 medicine and mainly contain 2 large approach at present: 1: on the basis of known viruse functional protein domain, carry out targeted design; 2: in occurring in nature extractions such as Chinese herbal medicine, sea life, land microorganisms, find.The important sources of the latter new construction, new mechanism antiviral drugs often, the research for antiviral drugs is significant, but the antivirus action target spot of the compound obtained in these natural resourcess but usually exists difficulty.Therefore, finding target spot and develop a set of suitable detection method is urgent problem.

Summary of the invention

For above-mentioned prior art, the present invention with 3C albumen for antivirus action target spot, develop a set of detection kit and detection method, its objective is whether detect compound to be detected can suppress 3C albumen, and then judge whether it can suppress EV71 virus, whether can carry out research application as the medicine of anti-EV71 virus.

3C albumen is the proteinase of hand-foot-and-mouth disease cause of disease EV71, its enzymatic activity directly affects viral replication efficiency and pathogenic virulence (WengK.F., LiM.L., HungC.T.etal.Enterovirus7l3CproteasecleavesanoveltargetC stF-64andihibitscellularpolyadenylation [J] .PLOSPathog, 2009,5 (9): elO00593.).This research, based on this proteinase measuring method for activity, establishes the reagent set of HRV 3CP activity, for detection of drugs target spot.If medicine can suppress the activity of HRV 3CP, then the action target spot of this medicine is pointed out to be activity center's (His41, Glu71 and Cys147 catalytic triads) of the HRV 3CP of EV71.

The present invention is achieved by the following technical solutions:

A kind of EV713C proteinase action target spot detection kit, comprises 3C albumen, peptide substrate, and enzyme digestion reaction damping fluid, the sequence following (as shown in SEQIDNO.1) of described 3C albumen:

GPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWVEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVVGIHIGGNGRQGFCAGLKRSYFASEQ。

Described peptide substrate is Dabcyl-EALFQGPPKFE-Edans, and its amino acid sequence is as shown in SEQIDNO.2, and N end connects Dabcyl group, and C end connects Edans group.

Described enzyme digestion reaction damping fluid is reaction buffer liquid, and it comprises following component: 25mMTris-HCl (pH8.0), 200mMNaCl.

A kind of EV713C proteinase action target spot detection method is as follows: compound to be detected, 3C albumen to be joined in enzyme digestion reaction damping fluid, then add peptide substrate, mixing, after reaction 30min, under 336nm wavelength excites, detect its fluorescence intensity at 490nm place; If can't detect fluorescence, then show that this compound to be detected can suppress 3C albumen, have anti-EV71 virus capable, the medicine that can be used as anti-EV71 virus further studies application; If fluorescence can be detected, then show that this compound to be detected can not suppress 3C albumen.

Further, detection method is as follows: peptide substrate, under 336nm wavelength excites, obtains its fluorescence intensity level A at 490nm place (representing fluorescence intensity when not degrading).After 3C polypeptide substrate being joined reaction buffer liquid, after mixing reaction 30min, pour in glass cuvette, under 336nm wavelength excites, obtain its fluorescence intensity level B at 490nm place (fluorescence intensity after degraded).Certain density compound to be detected is added in above-mentioned reaction system, and repeat the operation of above step, obtain fluorescence intensity C when compound exists, by comparing A, B, C tri-is worth size, (, between A, B, the numerical value of C is less for the numerical value of C, illustrates that the suppression of compound to be detected to the enzymatic activity of HRV 3CP is more obvious on the impact of the enzymatic activity of HRV 3CP for deterministic compound, the numerical value of C is larger, illustrates that the suppression of compound to be detected to the enzymatic activity of HRV 3CP is more weak).

Principle of the present invention is: this complete detection reagent is primarily of 3C albumen and fluorescent peptide substrates composition.After 3C albumen carries out enzymolysis to substrate, because the group at substrate two ends is separated, fluorescent quenching disappears, and reaction product inspires fluorescence signal.By wilder group of fluorescence emission curves difference with control group, judge whether compound have impact on the degrading activity of HRV 3CP to substrate, and then the target compound of screening antagonism EV713C proteinase, follow-up also by calculating milosevic constant km value and the catalytic efficiency Kcat value of 3C, quantitatively judge the rejection ability of target compound.

Detection kit of the present invention and method, the anti-EV71 ability (whether with 3C albumen for target spot) effectively can treating detection compound detects, and method is easy, easy, specificity and sensitivity high.

Accompanying drawing explanation

Fig. 1: inhibitor is on the impact of 3C protein degradation 2C-3A polypeptide.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated.

Instrument, reagent, material etc. involved in following embodiment, unless otherwise noted, be existing conventional instrument, reagent, material etc. in prior art, obtain by regular commercial sources.Experimental technique involved in following embodiment, detection method etc., unless otherwise noted, are existing normal experiment method in prior art, detection method etc.

The research of experiment EV713C proteinase action target spot detection kit and detection method

1: material:

1.1:3C albumen

Adopt molecular cloning method to obtain the prokaryotic expression plasmid of 3C gene, use Escherichia coli Bl21 bacterial strain to carry out the expression and purification (known technology) of albumen.Peptide sequence following (as shown in SEQIDNO.1):

The C of 3C albumen the 147th halfcystine activity center is the key amino acid of activity center.

1.2: fluorescent peptide substrates

Inventor have detected HRV 3CP early stage and lives to the enzyme of 6 10 peptide substrates, searches out a good stability by the polypeptide (table 1) of 3C albumen efficient degradation in aqueous.This substrate is based on 2C-3A protein sequence design on EV71.Enzymolysis site is the peptide bond between glutamine and glycocoll.The N end of polypeptide connects Dabcyl group, and C end connects Edans group.

Dabcyl and Edans two group near time there is fluorescence and urge and going out, do not show fluorescence.After polypeptide is degraded, two groups are separated, and under 336nm wavelength light excites, fluorescence can be detected at 490nm place.

Table 1 fluorescent peptide substrates

2 method steps: prolease activity determination experiment

2.1: Enzyme assay

2.1.1 enzyme reaction system alive is as follows:

3C albumen 2ul (0.5uM);

Peptide substrate 5ul (10uM);

Reaction buffer liquid (25mMTris-HClpH8.0,200mMNaCl) 13ul.

2.1.2 enzyme activity determination method

Peptide substrate, under 336nm wavelength excites, obtains its fluorescence intensity level A at 490nm place (representing fluorescence intensity when not degrading).After 3C polypeptide substrate being joined reaction buffer liquid, after mixing reaction 30min, pour in glass cuvette, under 336nm wavelength excites, obtain its fluorescence intensity level B at 490nm place (fluorescence intensity after degraded).In above-mentioned reaction system, add certain density compound to be detected, and repeat the operation of above step, obtain fluorescence intensity C when compound exists, by comparing A, B, C tri-is worth size, and deterministic compound is on the impact of the enzymatic activity of HRV 3CP.

2.1.3 enzyme activity determination principle

The N end of polypeptide connects Dabcyl group, and C end connects Edans group.Dabcyl and Edans two group near time there is fluorescence and urge and going out, do not show fluorescence.After polypeptide is degraded by HRV 3CP gradually, two groups be separated, under 336nm wavelength light excites, fluorescence can be detected at 490nm place, and along with degraded acceleration fluorescence gradually grow until react completely.By the impact of comparative evaluation given the test agent on reaction product fluorescence intensity, and then determine that whether it is by suppressing the antiviral drug effect of HRV 3CP activation plays.

2.2: experimental result

2.2.1 inhibitor Lu Ping Qu Wei (Rupintrivir) lives to the enzyme of 3C protein degradation substrate 2C-3A and suppresses

Get in the reaction system that 0.5uM3C albumen joins containing peptide substrate respectively, measure 336nm wavelength after in mixing reaction 30min to excite down, in the fluorescence intensity at 490nm place, according to above experimental technique, detect fluorescence intensity when existing with or without the bent reed of 5uM inhibitor Lu Ping respectively, mapping.

As shown in Figure 1, as seen from the figure, when the bent reed of unrestraint agent Lu Ping exists, 3C albumen is not suppressed result, degradable peptide substrate, stronger fluorescence can be detected.When there being the bent reed of 5uM inhibitor Lu Ping to exist, 3C albumen is suppressed, and peptide substrate of not degrading, almost can't detect fluorescence.

Claims

1. an EV713C proteinase action target spot detection kit, is characterized in that: comprise 3C albumen, peptide substrate, and enzyme digestion reaction damping fluid, the sequence following (as shown in SEQIDNO.1) of described 3C albumen:

GPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWVEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVVGIHIGGNGRQGFCAGLKRSYFASEQ；

2. EV713C proteinase action target spot detection kit according to claim 1, is characterized in that: described enzyme digestion reaction damping fluid is reaction buffer liquid, and it comprises following component: 25mMTris-HCl (pH8.0), 200mMNaCl.

3. EV713C proteinase action target spot detection kit according to claim 1 and 2, is characterized in that: comprise following material: 0.5uM3C albumen 2ul; 10uM peptide substrate 5ul; Reaction buffer liquid 13ul.

4. the method utilizing the EV713C proteinase action target spot detection kit according to any one of claim 1 ~ 3 to carry out detecting, it is characterized in that: compound to be detected, 3C albumen are joined in enzyme digestion reaction damping fluid, then peptide substrate is added, mixing, after reaction 30min, under 336nm wavelength excites, detect its fluorescence intensity at 490nm place; If can't detect fluorescence, then show that this compound to be detected can suppress 3C albumen, there is anti-EV71 virus capable; If fluorescence can be detected, then show that this compound to be detected can not suppress 3C albumen.

5. detection method according to claim 4, is characterized in that: peptide substrate, under 336nm wavelength excites, obtains its fluorescence intensity level A at 490nm place, represents fluorescence intensity when not degrading; After 3C polypeptide substrate being joined reaction buffer liquid, after mixing reaction 30min, pour in glass cuvette, under 336nm wavelength excites, obtain its fluorescence intensity level B at 490nm place, represent the fluorescence intensity after degrading; In above-mentioned reaction system, add certain density compound to be detected, and repeat the operation of above step, obtain fluorescence intensity C when compound exists, by comparing A, B, C tri-is worth size, determines the impact of compound to be detected on the enzymatic activity of HRV 3CP.