CN108265078A - It is a kind of based on the anti-hepatitis B model foundation of host cell target spot and application - Google Patents
It is a kind of based on the anti-hepatitis B model foundation of host cell target spot and application Download PDFInfo
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Abstract
The present invention relates to using Heme oxygenases-1 as target spot, people's Heme oxygenases-1 gene and hepatitis B protein gene are transferred to liver cell expression by carrier, it realizes and targets Heme oxygenases-1 and its construction procedures and evaluation method with the anti-hepatic-B virus medicine evaluation model of hepatitis B interactions between protein simultaneously, particularly one kind generates specificity is high, correlation is strong anti-hepatitis B external model and its application in the research and development of incidence of hepatitis Mechanism Study, drug therapy and original new drug using host cell contents as target spot.
Description
Technical field
The present invention relates to a kind of method for building up of hepatitis B virus resisting medicine evaluation model, particularly a kind of to be directed to host
The external model and its operation sequence of cell target spot generate the molecular model of the anti-hepatitis B virus of specificity and its B-mode
Application in incidence of hepatitis Mechanism Study, drug therapy and original new drug research and development.
Background technology
Hepatitis B is a kind of infectious disease of very harmful worldwide prevalence as caused by hepatitis type B virus (HBV),
The whole world is there are about 3.5 hundred million HBV carrier at present, and China is HBV high infected zones, and HBV carrier accounts for total population ratio and is up to
10%, wherein Chronic Hepatitis B about 30,000,000, there are about 350,000 people every year to die of chronic hepatitis B relevant disease.Although both at home and abroad
Some anti-hbv drugs developed can suppressing virus replication, control progression of the disease, but its curative effect distance removes virus to a certain degree
The target of infection, the most hepatitis B patients of healing and virus carrier still has very over long distances.Hepatitis B and its relevant disease are still
The important public hygiene problem of China.Therefore, further investigation anti-hepatitis B original new drug is still the task of top priority.HBV is a kind of stringent
Cytozoon, in its entire life cycle, it is necessary to complete its duplication by host cell proteins.Therefore host is thin
Born of the same parents provide potentially large number of target spot for Anti-HBV drugs research.In antiviral drugs research, using host cell contents as disease-resistant
Toxic action target spot is it has been reported that some drugs have been enter into clinic, as Ribavirin has the work for inhibiting inosine monophosphate dehydrogenase
With approved is used for rsv infection.Obviously, find that Anti-HBV activity target spot is the weight of solution Anti-HBV drugs development bottleneck from host cell
Want one of approach.
Heme oxygenase (Heme oxygenase, HO) is the rate-limiting enzyme of internal catalysis ferroheme catabolism, there is three
Kind isoenzymes：HO-1, HO-2 and HO-3.HO-1 belongs to induction type, is mainly distributed on a variety of internal organs such as mammal liver, spleen, lung, has
There are adjusting and defencive function.HO-1 is external except cell particles are present in, and exists in nucleus, can cause cell oxygen by various
Change stress impairment factor induced, prompting HO-1 may also play a part of signal representation molecule.Research in recent years shows, HO-1
Effect degrade far beyond it ferroheme this function, HO-1 and its catabolite take part in that maintain cell self is flat
Weighing apparatus, and pass through anti-oxidative damage, the reaction that reduces inflammation, inhibit Apoptosis and adjust cell Proliferation, protection Various Tissues are risen
Important function.HO-1 overexpressions or increased activity to the protective effect of tissue damage and the improvement result of various chronic diseases,
Show that HO systems are of great significance in relevant disease clinical treatment and medicament research and development.
HO-1 is induced or is overexpressed other than showing the hepatoprotective effects such as its significant anti-apoptotic, anti-inflammatory and anti-fibrosis, most
Exciting is the discovery that its significant antivirus action, especially has significant directly Anti-HBV activity print effect, chronic
Application prospect in virus hepatitis has historically been a concern.HO-1 is entirely possible to cause liver as Anti-HBV activity and mitigation HBV
The new selection of damage.In view of HBV infection there is no the drug and method of radical cure so far, and lack specific target spot and reliable molecule
Model.Therefore, ideal anti-hepatitis B original new drug is developed, new Anti-HBV activity target spot need be explored, establish new appraisement system.
HO-1 is as HBV infection host cell --- and a kind of inducible protein of liver cell, induced expression is in addition to showing that it is specific
Outside hepatoprotective effect, also there is significant directly Anti-HBV activity effect, prompt the potential target spot that HO-1 may be anti-hbv drug evaluation.
Therefore, establish targeting HO-1 drug evaluation model, to from host cell angle innovation drug, establish anti-hepatitis B prevention plan
It is somewhat significant.
Invention content
The technical problem to be solved in the present invention is to provide anti-hepatitis B of the one kind with DELTA rHO-1 (HO-1) for target spot
Viral evaluation model, the model-specific is high, correlation is strong, and one kind is provided for hepatitis B study of incident mechanism and original new drug evaluation
External reliable model.For this purpose, the present invention takes following technical scheme：
The main function target spot of Anti-HBV drugs research at present is conceived to virus, such as nucleoside analog (Lamivudine)
Main function target spot be HBV archaeal dna polymerases.Since HBV gene group easily morphs, lead to the Anti-HBV activity medicine using virus as target
Object is also easy to produce tolerance.Such as using HBV archaeal dna polymerases for target spot research and development nucleoside medicine there is drug resistance, be discontinued knock-on,
The limitations such as HBV cannot be removed, this is the bottleneck problem of urgent need to resolve in Anti-HBV drugs development.In addition, due to HBV gene group
Smaller, the potential target limited amount of coding also limits the development of Anti-HBV drugs.HBV is a kind of stringent cytozoicus
Object, in its entire life cycle, it is necessary to complete its duplication by host cell proteins.It is considered that host cell is anti-
HBV drugs provide potentially large number of action target spot.Obviously, find that Anti-HBV activity target spot is to solve Anti-HBV drugs hair from host cell
Open up the important channel of bottleneck.
One species specificity is high, correlation is strong, for from host cell --- and the Anti-HBV drugs of liver cell HO-1 are external
The method for building up of evaluation model.Specific preparation method is as follows：
Using bimolecular fluorescence complementary technology, target HO-1 and its with HBV interactions between protein, utilize the 2A polypeptides of viral source
Translation characteristic with self cutting, it is non-fluorescence by two of people HO-1 genes, HBV protein gene and fluorescin Venus
Complementary fragment VN155 and VC155, build on single carrier.The HL-7702 liver cells for waiting to cultivate in 1640 culture mediums are adherent
Afterwards, the wild type of structure and mutant vector are then first transfected into HL-7702 respectively with trial drug pretreatment cell 24 hours
Liver cell, while set up empty vector control, solvent control and untreated cell control.After transfection after 24,48 and 72 hours, analysis
The fluorescence intensity of each group cell.It is strong by detecting fluorescence since drug can influence HO-1 and HBV interactions between protein to the processing of cell
Degree, it will be appreciated that trial drug is to the intervention situation of HO-1 and HBV interactions between protein.After completing above-mentioned Fluorescence Intensity Assays, collect
Transfectional cell detects HO-1 expressions, the influence that transfectional cell HO-1 is expressed in analysis trial drug intervention.With reference to HO-1 with
The fluorescence intensity of HBV interactions between protein, using oxymatrine as positive control, the Anti-HBV effect of overall merit testing drug.
The present invention has the following advantages that compared with prior art：(1) using the host cell contents HO-1 of HBV infection as anti-second
Liver target spot；(2) at the same target HO-1 and its with HBV interactions between protein；(3) model-specific is high, correlation is strong, reliable and stable；
(4) the equal operational virus target spot of existing Anti-HBV drugs is overcome, there are drug resistance, knock-on of being discontinued, the limitations such as virus cannot be removed；
(5) suitable for incidence of hepatitis Mechanism Study and the screening of anti-hepatitis original new drug and early stage evaluation study.
Specific embodiment
Embodiment 1：The structure of anti-hepatitis virus model based on host cell target spot and evaluation
1 prepares following material：
1.1 people source HL-7702 liver cells：Purchased from the American Type Culture Collection committee of Chinese Academy of Sciences cell bank.
1.2 oxymatrine：Suzhou Baozetang Medicine Science ＆ Technology Co., Ltd.'s product.
2 construction methods are as follows：
2.1 people source HL-7702 liver cells, using containing 100 μ g/mL of 10% fetal calf serum, penicillin 100IU/m and streptomysin
1640 culture mediums, the routine culture under the conditions of 37 DEG C, 5%CO2.
2.2 use bimolecular fluorescence complementary technology, have the translation characteristic of self cutting using the 2A polypeptides of viral source,
By two of people HO-1 genes, HBV protein gene and fluorescin Venus non-fluorescence complementary fragment VN155 and VC155, structure
It build on single carrier.
2.3 after HL-7702 liver cells are adherent, first with trial drug pretreatment cell 24 hours, then by the open country of structure
Raw type and mutant vector transfect HL-7702 liver cells respectively, at the same set up empty vector control, solvent control and it is untreated (not
Transfection) cell controls.
3 evaluation methods are as follows：
After 3.1 transfections after 24,48 and 72 hours, the glimmering of each group cell is analyzed with fluorescence microscope and its related software respectively
Luminous intensity understands intervention situation of the trial drug to HO-1 and HBV interactions between protein.
3.2 collect transfectional cell, and HO-1mRNA expressions are detected using RT-PCR technology；Using Western
Blotting technologies detect HO-1 protein expression levels, and analysis trial drug intervention is to transfectional cell HO-1mRNA and protein expression
Horizontal influence.
3.3 combine the fluorescence intensity of HO-1 and HBV interactions between protein and HO-1mRNA and protein expression level information, and synthesis is commented
Valency tests the Anti-HBV effect of compound.
Claims (10)
1. a kind of method for building up of novel hepatitis B (HBV) drug evaluation model, including model construction program and evaluation side
Method, it is characterised in that using host cell contents as target spot, build specificity is high, correlation is strong anti-hepatitis B external model and its
Application in hepatopathy Mechanism Study and Antihepatitis medicament evaluation.
2. the method for building up of novel hepatitis B drug evaluation model according to claim 1, it is characterised in that described
Target spot be from liver cell DELTA rHO-1 (HO-1).
3. the method for building up of the novel hepatitis B drug evaluation model according to claim 1 and 2, it is characterised in that institute
The action target spot stated includes the interaction of HO-1 albumen and HBV albumen.
4. the method for building up of novel hepatitis B drug evaluation model according to claim 1, it is characterised in that described
Transfectional cell be Normal human tissue source liver cell such as HL-7702 cells.
5. the method for building up of the novel hepatitis B drug evaluation model according to claim 1 and 3, it is characterised in that institute
Carrier construction method is bimolecular fluorescence complementary (BiFC) technology.
6. the method for building up of the novel hepatitis B drug evaluation model according to claim 1 and 5, it is characterised in that institute
BiFC technologies are in list by people HO-1 genes, HBV protein gene and the non-fluorescence complementary fragment structure of fluorescin two
Transfectional cell on one carrier or different carriers.
7. the method for building up of novel hepatitis B drug evaluation model according to claim 1, it is characterised in that described
Fluorescence detection method analyzed for fluorescence microscope and its related software.
8. the method for building up of novel hepatitis B drug evaluation model according to claim 1, it is characterised in that described
HO-1 detection of expression method be RT-PCR and Western Blotting technologies.
9. the method for building up of novel hepatitis B drug evaluation model according to claim 1, it is characterised in that described
Model evaluation standard change for fluorescence intensity, HO-1 expression quantity raising.
10. the method for building up of novel hepatitis B drug evaluation model according to claim 1, it is characterised in that institute's structure
The model built has high specific and good correlation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113549651A (en) * | 2021-07-19 | 2021-10-26 | 中国计量大学 | Method for establishing anti-novel coronavirus drug screening and evaluating model and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113549651A (en) * | 2021-07-19 | 2021-10-26 | 中国计量大学 | Method for establishing anti-novel coronavirus drug screening and evaluating model and application |
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Application publication date: 20180710 |