CN101793898B - Method for screening antiviral medicament for degrading BST-2 activity by antagonistic Vpu - Google Patents
Method for screening antiviral medicament for degrading BST-2 activity by antagonistic Vpu Download PDFInfo
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Abstract
The invention discloses a method for screening an antiviral medicament for degrading BST-2 activity by antagonistic Vpu, which comprises the following steps: adopting a monoclonal Hela cell line which can stably express the Vpu protein and coating the monoclonal Hela cell line into a cell culture plate; incubating a BST-2 antibody and corresponding horse radish peroxidase HRP labeled secondary antibody and coating in cells of the cell culture plate; and finally, developing color through a TMB substrate, and determining BST-2 antigenic content on the cell surface according to an OD value at 450nm. The method for screening the antiviral medicament for degrading BST-2 activity by antagonistic Vpu can quickly and effectively screen the antiviral activity of the medicament to be tested with high flux, and is an achievable method for screening safe, reliable and effective anti-HIV virus medicaments.
Description
Technical field
The present invention relates to drug world, particularly relate to the screening model of anti-AIDS drug, be i.e. a kind of method of screening the antiviral drugs of antagonism Vpu degraded BST-2 activity.
Background technology
Acquired immune deficiency syndrome (AIDS) (acquired immunodeficiency syndrome, AIDS) be by human immunodeficiency virus (human immunodeficiency virus, HIV) infection causes human immunologic function's defective, and is easy to the clinical syndrome of generator opportunistic infections and tumour.Cause more than 2,000 ten thousand people dead in the past more than 20 year, whole world patients infected hiv has reached about 4,000 ten thousand at present.Acquired immune deficiency syndrome (AIDS) not only becomes the serious hygiene and health problem of China, simultaneously the annual direct economic loss that has caused more than one hundred billion.According to the ministry of Health of China circular, to the end of the year in 2009, will there be 740,000 patients infected hivs in China approximately; New HIV-positive reached 4.8 ten thousand in 2009.
Though traditional anti-AIDS drug of present clinical use has played very large effect aspect anti-HIV-1, but still there are some problems, as activity in human body is not high, oral administration biaavailability is low, it is high easily to produce drug resistance or cross resistance and production cost.The problem that faces in the Anti-AIDS Drugs treatment shows that research and development are still extremely urgent for the novel anti-AIDS drug of brand-new drug target.
The drug resistance of virus is to need the problem of solution in the present AIDS-treating medicine treatment most badly.Resistance is the result of the drug effect target position protein variant of virus.Because persister is often to one group of antiviral drugs resistance, and crossing drug resistant is very common between the same class medicine.Why HIV-1 can produce rapidly the drug resistance for the antiviral drugs of existing clinical practice, its main contributor be these medicines institute for target spot all be that virus is own.And the natural characteristics that just have high variation of pathogenic virus under the selection pressure of medicine, thereby are easy to cause that the rapid variation of virus produces resistance.Therefore, that one of the key that solves the drug resistance problem of HIV-1 is to break through is traditional " with pathogenic virus this as target " the drug development pattern.
In the very long process of biological evolution, host cell can form the defense system for Different Kinds of Pathogens virus, and virus also can form specific Antagonizing, to escape the inhibiting effect of host cell.Under the prerequisite that does not affect host's viability, raise specifically the natural Antiviral Mechanism of host and not only can effectively suppress copying of virus, simultaneously also can obviously reduce or even avoid medicine to the selection pressure of virus, thereby reduce the possibility that resistance mutation occurs.Therefore, the drug development pattern of " take the host as target " has become one of important research strategy that solves a present antiviral drugs drug resistance difficult problem.
Recently research is found, host's factor B ST-2 (also called after Tetherin/HM1.24/CD317) stops surface of cell membrane HIV-1 Virus release, the auxilin Vpu of HIV-1 then can come its activity of antagonism by the lysosomal pathway BST-2 that degrades, and improves viral burst size.Host's factor B ST-2 belongs to П type AQP-CHIP, whole albumen is homodimer, by the allos glycosylation, size is 30-36kDa, BST-2 is the constructive expression in Hela clone, in 293T, HOS, HT1080 clone, then do not express or expression very low, therefore discharge for HIV-1 virus, the Hela cell is that Vpu relies on, and 293T, HOS, HT1080 cell are the non-dependence of Vpu.But interferon abduction delivering BST-2 relies on so that 293T, HOS, HT1080 clone change Vpu into by the non-dependence of Vpu.If stop Vpu degraded BST-2, improve cell surface BST-2 content, then can suppress virus and discharge, thereby reach antiviral effect.
Summary of the invention
On the basis of above-mentioned research, the purpose of this invention is to provide a kind of method of screening the antiviral drugs of antagonism Vpu degraded BST-2 activity.Utilize the method can be fast, effectively, the antiviral activity of high flux screening medicine to be measured, provide practicable method for filtering out safe and reliable and effective anti HIV-1 virus medicine.
Cell ELISA (cell-ELISA) is to replace soluble antigen to be coated in a kind of effective ways that elisa plate (or Tissue Culture Plate) carries out immunology detection with cell.We use this technology, have set up the high flux screening model of screening antagonism Vpu degraded BST-2 activity.
Technical scheme of the present invention provides a kind of method of screening the antiviral drugs of antagonism Vpu degraded BST-2 activity, but it adopts the monoclonal Hela clone of stably express Vpu albumen to be coated in the Tissue Culture Plate, use the cell-ELISA technology, the two anti-cells that are coated in Tissue Culture Plate of hatching successively with BST-2 antibody and corresponding horseradish peroxidase HRP mark, by the colour developing of TMB nitrite ion, determine cell surface BST-2 antigenic content according to the size of the light absorption value OD of 450nm place at last.Cell surface BST-2 content is higher, and the antibody of then hatching combination is more, and the HRP enzymatic activity is higher, and the catalytic substrate reaction is faster, and is larger in the OD of 450nm place value.Can stop Vpu degraded BST-2 such as the compound that adds, then the OD of 450nm place value raises.
Method of the present invention, but wherein need the monoclonal Hela clone of model stably express Vpu albumen, and a kind of of construction method of described monoclonal Hela clone may further comprise the steps:
(1) the vpu fragment with amplification is inserted into eukaryotic expression vector pcDNA3.1 (+) multiple clone site (Invitrogen), obtains plasmid pcDNA3.1-vpu;
(2) inoculated and cultured Hela cell, transfection plasmid pcDNA3.1-vpu; Behind the cellar culture, but obtain the polyclone Hela clone of stably express Vpu;
(3) with the multi-clone cell line monoclonal, but obtain the monoclonal Hela clone of stably express Vpu.
Method of the present invention, build above-mentioned monoclonal Hela clone after, get the monoclonal Hela cell of stably express Vpu and cultivate, the inoculating cell culture plate, cultivate 16-24h (preferred 24h) after, add drug treating to be screened.Behind the drug treating 14-16h, abandon nutrient culture media, cell is after washing, fix, sealing, add 1: 5000～1: 50000 (preferred 1: 20000) incubated at room 1～2h of BST-2 antibody, clean cell, add HRP mark two anti-1: 1000～1: 10000 (preferred 1: 1500) incubated at room 0.5～2h, clean cell, after adding TMB nitrite ion lucifuge placement 20～40min, add 0.5M H
2SO
4Color development stopping is measured the OD value immediately in the 450nm place.
Technique scheme has following advantage: 1) utilize screening technique of the present invention can be fast, economy, high flux ground determines the activity of medicine to be measured; 2) as the screening model of cellular level, can avoid because the false positive that the non-specific factors such as cytotoxicity cause improves the accuracy rate of screening; 3) can get rid of the poor reactive compound of permeability cell, improve the one-tenth property of medicine of lead compound; 4) with endogenous BST-2 as target molecule, its expression, regulation and control, cell traffic and distribution are the normal physiological state.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
The action principle of screening technique of the present invention (screening model):
The monoclonal Hela clone of model stably express Vpu albumen, 96 hole ELISA Plate or Tissue Culture Plate inoculating cell are cultivated 16-24h, adding drug sample to be measured processes, cultivate again 14-16h, use the cell-ELISA technology, determine cell surface BST-2 content according to the size of the OD of 450nm place value.Can stop Vpu degraded BST-2 such as the compound that adds, then the OD of 450nm place value raises.
Embodiment 1: the structure of the monoclonal Hela clone of stably express Vpu
(1) makes up the Vpu expression vector
Use pcr amplification BH10 plasmid (NIH AIDS Research﹠amp; Reference Reagent Program) the vpu genetic fragment in.The primer: upstream primer 5 '-TCT GCA
GAA TTCGGG AAA GAC GCA AG-3 ' (shown in SEQ ID NO.1), downstream primer 5 ' AAG CCG
CTC GAGCCA TAA TAG ACT GTG AC-3 ' (shown in SEQ ID NO.2).
Reaction system:
Reaction conditions: 94 ℃ of 45s, 68 ℃ of 60s, totally 30 circulations.
The vpu fragment of amplification is inserted into eukaryotic expression vector pcDNA3.1 (+) multiple clone site (EcoRI and XhoI) (Invitrogen), obtains plasmid pcDNA3.1-vpu, express HIV-1 auxilin Vpu.
(2) foundation of the monoclonal Hela clone of stably express Vpu
6 orifice plates inoculation Hela cell 2 * 10
5Individual, 37 ℃, 5% CO
2After cultivating 24h, according to FuGENE HD Transfection Reagent (Roche) instructions transfection plasmid pcDNA3.1-vpu, the transfection ratio is 4: 1.Concrete operations are as follows: with 100 μ l DMEM in high glucose nutrient culture media dilution plasmid pcDNA3.1-vpu 1.0 μ g and 4 μ l FuGENE HD TransfectionReagent, incubated at room 15min is added in the cells and supernatant of 6 orifice plates.
Behind the transfection 24h, abandon nutrient culture media, with the digestive juice vitellophag that contains 0.25% pancreatin and 0.02%EDTA, with dilution inoculation in 1: 20.After cultivating 24h, adding final concentration is that 400 μ g/mlG418 (aminoglycoside antibiotics, the great Bioisystech Co., Ltd in Shanghai) carry out the selectivity cultivation, and every 3d changes liquid once, behind the cultivation 15d, obtains the polyclone Hela clone of stably express Vpu.Use limiting dilution assay with the multi-clone cell line monoclonal, finally obtain the monoclonal Hela clone of a strain stably express Vpu, called after Hela-Vpu.
Embodiment 2: the screening process of medicine
(1) cell is cultivated
Get the Hela-Vpu cell and cultivate, after cell covers with culture flask, abandon old nutrient culture media, with the digestive juice digestion that contains 0.25% pancreatin and 0.02%EDTA.Treat cell rounding, abandon digestive juice, add immediately the DMEM in high glucose nutrient culture media (HyClone) contain 10%FBS (hyclone), at the bottom of blowing and beating gently bottle with suction pipe, cell is broken away from bottle fully at the bottom of and make it to be separated into single cell suspension.Behind the blood counting chamber counting, the every hole of 96 porocyte culture plates inoculating cell 1 * 10
4Individual, behind the cultivation 24h, add drug treating.
(2) sample preparation
Compound sample: 10mg sterling compound is molten in 1mlDMSO, and 50%DMSO is diluted to 1mg/ml, gets 1.0 μ l and acts on 100 μ l cell systems, and making its final concentration is 10 μ g/ml.
Fermentation broth sample: strain fermentation, picking one fritter cultivation species enter to fill the 250ml triangular flask of 50ml fermentation medium from the inclined-plane, 28 ℃, 190rpm rotary shaker cultivation 4d.Get the 10ml fermentation liquor with the acetone extracting of 10ml, after volatilizing, use the DMSO dissolving of 1ml.Get 10 μ l and add 10 μ l water doubling dilutions, get 1.0 μ l and act on 100 μ l cell systems.
ConcanamycinA is available from Sigma company, and DMSO dissolves, and concentration is 10 μ M, gets 0.5 μ l and acts on 100 μ l cell systems.Concanamycin A is lysosome degradation pathway inhibitor, in this screening model as positive compound.
(3) sample is active detects
Experiment component is three groups, negative control group: add 1.0 μ l 50%DMSO in cells and supernatant, this group reflection Vpu is to the palliating degradation degree of BST-2.
Positive drug control group: add the 10 μ M Concanamycin A of 0.5 μ l in the cell system of 100 μ l, the final concentration that makes ConcanamycinA is 50nM.This group reflection lysosome degradation pathway inhibitor C oncanamycinA is to the inhibition degree of Vpu degraded BST-2.
Sample experimental group: add the sample 1.0 μ l of screening in the cell system of 100 μ l.This group reflection screening sample is to the inhibition degree of Vpu degraded BST-2.
Drug treating 14-16h, abandon nutrient culture media, PBS washes cell 2 times, 4% paraformaldehyde room temperature is 20min fixedly, PBS washes cell 3 times, 5% skimmed milk power room temperature sealing 1h, 1: 20000 incubated at room 2h of BST-2 antibody (NIH provides), PBS washes cell 3 times, 1: 1500 incubated at room 1h of HRP mark donkey anti-rabbit IgG (available from Santa Cruz), PBS washes cell 3 times, and every hole adds 100 μ lTMB nitrite ions (be century available from health), after lucifuge was placed 30min, every hole added 50 μ l 0.5M H
2SO
4Color development stopping is measured the OD value immediately in the 450nm place.
Inhibition of degradation rate=(sample experimental group OD-negative control group OD)/(positive group OD-negative control group OD) * 100%
The selection result is, screens altogether 3000 samples from combinatorial chemical library, obtains 11 of positive compounds (Inhibition of degradation rate＞50%), and positive rate is 0.37%.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Sequence table
＜110〉Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
＜120〉method of the antiviral drugs of screening antagonism Vpu degraded BST-2 activity
<130>KHP10112228.7
<160>2
<170>PatentIn version 3.5
<210>1
<211>26
<212>DNA
＜213〉artificial sequence
<400>1
tctgcagaat tcgggaaaga cgcaag 26
<210>2
<211>29
<212>DNA
＜213〉artificial sequence
<400>2
aagccgctcg agccataata gactgtgac 29
Claims (2)
1. method of screening the antiviral drugs of antagonism Vpu degraded BST-2 activity, it is characterized in that, but adopt the monoclonal Hela clone of stably express Vpu albumen to be coated in the Tissue Culture Plate, then resist with two of BST-2 antibody and corresponding horseradish peroxidase HRP mark and hatch successively the cells that are coated in Tissue Culture Plate, by the colour developing of TMB nitrite ion, determine cell surface BST-2 antigenic content according to the size of the light absorption value OD of 450nm place at last;
But the monoclonal Hela clone of stably express Vpu albumen is coated in the Tissue Culture Plate, is specially: the monoclonal Hela cell of getting stably express Vpu is cultivated, and the inoculating cell culture plate behind the cultivation 16-24h, adds drug treating to be screened;
But the preparation method of the monoclonal Hela clone of stably express Vpu albumen may further comprise the steps:
(1) the vpu genetic fragment in the application pcr amplification BH10 plasmid, upstream primer is shown in SEQ ID NO.1, and downstream primer is shown in SEQ ID NO.2;
Reaction system:
Reaction conditions: 94 ℃ of 45s, 68 ℃ of 60s, totally 30 circulations;
The vpu fragment of amplification is inserted into eukaryotic expression vector pcDNA3.1 (+) multiple clone site (Invitrogen), obtains plasmid pcDNA3.1-vpu;
(2) inoculated and cultured Hela cell, transfection plasmid pcDNA3.1-vpu; Behind the cellar culture, but obtain the polyclone Hela clone of stably express Vpu;
(3) with the multi-clone cell line monoclonal, but obtain the monoclonal Hela clone of stably express Vpu;
Two of described BST-2 antibody and corresponding horseradish peroxidase HRP mark anti-ly hatched successively, be specially BST-2 antibody 1:20000 incubated at room 2h after, two anti-1:1500 incubated at room 1h of HRP mark.
2. the method for claim 1, it is characterized in that, behind the drug treating 14-16h, abandon nutrient culture media, cell is after washing, fix, sealing, add BST-2 antibody 1:20000 incubated at room 2h, clean cell, add HRP mark two anti-1:1500 incubated at room 1h, clean cell, after adding TMB nitrite ion lucifuge placement 20～40min, add 0.5M H
2SO
4Color development stopping is measured the OD value immediately in the 450nm place.
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Citations (4)
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|---|---|---|---|---|
| CN1210260A (en) * | 1997-09-02 | 1999-03-10 | 中国科学院生物物理研究所 | Optical spectrum method for determining features and effectness of medicine against AlDs virus |
| CN1451042A (en) * | 1999-01-25 | 2003-10-22 | Musc研究发展基金会 | Method of monitoring HIV drug resistance |
| US6770752B2 (en) * | 2001-01-09 | 2004-08-03 | Becton, Dickinson And Company | Sequences for detection of HIV-1 |
| CN101591645A (en) * | 2008-05-29 | 2009-12-02 | 中国医学科学院医药生物技术研究所 | A kind of screening method of inverase |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1210260A (en) * | 1997-09-02 | 1999-03-10 | 中国科学院生物物理研究所 | Optical spectrum method for determining features and effectness of medicine against AlDs virus |
| CN1451042A (en) * | 1999-01-25 | 2003-10-22 | Musc研究发展基金会 | Method of monitoring HIV drug resistance |
| US6770752B2 (en) * | 2001-01-09 | 2004-08-03 | Becton, Dickinson And Company | Sequences for detection of HIV-1 |
| CN101591645A (en) * | 2008-05-29 | 2009-12-02 | 中国医学科学院医药生物技术研究所 | A kind of screening method of inverase |
Non-Patent Citations (1)
| Title |
|---|
| Yukie Iwabu et al.HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes.《JOURNAL OF BIOLOGICAL CHEMISTRY》.2009,第284卷(第50期),35060-35072. * |
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