CN103127492A - Anti-hepatitis C virus protein and application thereof - Google Patents
Anti-hepatitis C virus protein and application thereof Download PDFInfo
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- CN103127492A CN103127492A CN2011103757908A CN201110375790A CN103127492A CN 103127492 A CN103127492 A CN 103127492A CN 2011103757908 A CN2011103757908 A CN 2011103757908A CN 201110375790 A CN201110375790 A CN 201110375790A CN 103127492 A CN103127492 A CN 103127492A
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Abstract
The invention relates to anti-hepatitis C virus protein and an application thereof. The inventor finds, for the first time, that the expression of TRIM 22 protein can inhibit the replication of HCV and expression of its gene and virus protein, which indicates that TRIM 22 plays an important role in the life cycle of HCV, and thus provides a new drug target for the development of anti-HCV drugs.
Description
Technical field
The invention belongs to biotechnology and field of pharmacology; More specifically, the present invention relates to a kind of albumen and uses thereof of anti-hepatitis c virus.
Background technology
Hepatitis C virus (HCV) is the single strand plus RNA virus of tool peplos, belongs to flaviviridae family.It is a kind of main global infectious disease that HCV infects the active chronic inflammation that causes.There are about 1.7 hundred million populations in the whole world, surpasses 3,800 ten thousand in China, namely accounts for the Chinese of 2-3% total population by hepatitis c virus infection.In the U.S., according to the report of 2005, there are every year 1-2 ten thousand people to die from HCV and infect; At home, the hepatitis C infection rate increases steadily in recent years.Show according to the Ministry of Public Health statistical data, the HCV number of the infected had risen to 130,000 people in 2009.Arranged after the hepatitis c virus infection human body long incubation period, and wherein 60～80% actute infection meetings transfer to chronicly, and 5-20% can transfer liver cirrhosis to, and final patient 1-7% can transfer hepatocarcinoma to.HCV has 6 kinds of different genotype at least, and it is distributed with region.The disease symptom of different genotype virus and all variant to replying for the treatment of.In chronic hepatitis C infection, the normal function that HCV not only can disturb immunocyte avoids being removed by immune system guaranteeing, and can induce the inflammatory reaction of liver, produces hepatic fibrosis and liver cirrhosis, finally causes hepatocarcinoma.
At present, the most effective therapy is long-acting interferon and ribavirin conjoint therapy clinically.After the course for the treatment of in week, in the part patient, obvious curative effects is arranged through 24-48, can effectively control or remove virus.The other patient without significantly replying, shows as virus load without obvious decline to interferon therapy.The other patient has certain replying to interferon therapy, but virus load in therapeutic process or treatment finish after bounce-back, cause treating unsuccessfully.In addition, there is no at present any vaccine and can effectively prevent hepatitis c virus infection.Because the combined therapy of Pegylation long-acting interferon and ribavirin is both expensive, again time-consuming (reaching 6-12 month), side effect is obvious, and in the territory, Great China region effective percentage only 50% common gene 1 type HCV the infected.
Therefore, for the whole world 100,017,000 HCV the infecteds, further research and develop more effective treatment means very urgent.
Summary of the invention
The object of the present invention is to provide albumen of a kind of anti-hepatitis c virus and uses thereof.
In a first aspect of the present invention, the purposes that TRIM22 albumen is provided or adjusts on it is for the preparation of the compositions (as medicine) of anti-hepatitis c virus (HCV).
In a preference, described anti-hepatitis c virus comprises: suppress copying of hepatitis C virus.
In another preference, described compositions also is used for:
Degraded TAB2 albumen and/or TAB3 albumen; Or
Suppress NF-κ B signal path.
In another preference, described TRIM22 albumen is:
(a) has the albumen of aminoacid sequence shown in SEQ ID NO:2;
(b) process of aminoacid sequence shown in SEQ ID NO:2 is one or more (as 1-30; Preferably 1-20; More preferably 1-10; 1-5 more preferably) replacement, disappearance or the interpolation of amino acid residue form, and have the albumen of the protein function that (a) limit;
(c) with the sequence homogeny of albumen of aminoacid sequence shown in SEQ ID NO:2 higher than 70% (preferably higher than 80%; More preferably higher than 90%; More preferably higher than 95%; More preferably higher than 98%; More preferably higher than 99%), and have the albumen of the protein function that (a) limit; Or
(d) active fragment that has the albumen of aminoacid sequence shown in the SEQ ID NO:2 of the protein function that (a) limit.
In another preference, the upper adjustment of described TRIM22 albumen is selected from:
Recombinant expression carrier contains an expression cassette in described expression vector, wherein comprise the gene of coding TRIM22 albumen; Or
Interferon; Or
p53。
In another preference, described recombinant expression carrier is: restructuring pcDNA carrier, its multiple clone site comprises the gene of the coding TRIM22 albumen of external source.
In another aspect of this invention, provide a kind of purposes of inhibitor of NF-κ B signal path, for the preparation of the compositions (as medicine) of anti-hepatitis c virus.
In a preference, the inhibitor of described NF-κ B signal path is TPCK.
In another aspect of this invention, provide the purposes of the inhibitor of a kind of TAB2 albumen or TAB3 albumen, for the preparation of the compositions (as medicine) of anti-hepatitis c virus.
In a preference, the inhibitor of described TAB2 albumen or TAB3 albumen is TRIM22 albumen.
In another aspect of this invention, provide the purposes of TRIM22 albumen, be used for the potential material of screening anti-hepatitis c virus.
In another aspect of this invention, provide a kind of method of screening the potential material of anti-hepatitis c virus, described method comprises:
(1) candidate substances is contacted with the system that comprises (as expressing) TRIM22 albumen;
(2) detect candidate substances to genetic transcription, protein expression or the active impact of TRIM22;
If described candidate substances promotes genetic transcription, protein expression or the activity of TRIM22, show that this candidate substances is the potential material of anti-hepatitis c virus.
In a preference, step (1) comprising: in the test group, candidate substances is joined in the system that comprises TRIM22 albumen; And/or
Step (2) comprising: genetic transcription, protein expression or the activity of TRIM22 in the system of detection test group, and compare with matched group, wherein said matched group is the system that comprises TRIM22 albumen of not adding described candidate substances;
If in the test group, the genetic transcription of TRIM22, protein expression or activity are statistically higher than (preferably be significantly higher than, more than 20%, better is high more than 50% as high; Better is high more than 80%) matched group, just show that this candidate substances is the potential material of anti-hepatitis c virus.
In another preference, in step (1), also comprise TAB2 albumen and/or TAB3 albumen in described system, and described TRIM22 albumen and TAB2 albumen and/or TAB3 protein-interacting (mutually combining); And
Step (2) comprising: detect candidate substances to the impact of TRIM22 albumen and TAB2 albumen and/or TAB3 protein-interacting;
If described candidate substances promotes TRIM22 albumen and TAB2 albumen and/or TAB3 protein-interacting (more preferably promoting the degraded of TAB2 albumen and/or TAB3 albumen), show that this candidate substances is the potential material of anti-hepatitis c virus.
In another preference, step (1) comprising: in the test group, candidate substances is joined in the system that comprises TRIM22 albumen and TAB2 albumen and/or TAB3 albumen; And/or
Step (2) comprising: the interaction of TRIM22 albumen and TAB2 albumen and/or TAB3 albumen in the system of detection test group, and compare with matched group, wherein said matched group is the system that comprises TRIM22 albumen and TAB2 albumen and/or TAB3 albumen of not adding described candidate substances;
(preferably significantly be better than, more than 20%, better is strong more than 50% as strong if in the test group, the interaction of TRIM22 albumen and TAB2 albumen and/or TAB3 albumen is better than statistically; Better is strong more than 80%) matched group, just show that this candidate substances is the potential material of anti-hepatitis c virus.
In another preference, described system is selected from: cell system, subcellular fraction system, solution system, organizational framework, organ systems or animal system.
In another preference, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, further to select from candidate substances and to determine for the useful compositions of anti-hepatitis c virus.
In another preference, described candidate substances is selected from (but being not limited to): for the micromolecular compound of TRIM22 albumen, and protein stabiliser, protein active promoter.
In another aspect of this invention, provide a kind of method of anti-hepatitis c virus, comprise the TRIM22 albumen or the adjustment on it that give experimenter's effective dose.
In a preference, the method right and wrong of described anti-hepatitis c virus are curative.
Other side of the present invention due to the disclosure of this paper, is apparent to those skilled in the art.
Description of drawings
Fig. 1, TRIM22 can be by IFN α inducible expressions.Wherein, A is the level of TRIM22mRNA that detects by PCR; B is for detecting by Western Blot the result that protein level changes.
In Fig. 2, HCV course of infection, TRIM22 is raised.Wherein, 1,2,3,4 represent respectively rear 0,8,24,48 hour of infection.
Fig. 3, mistake are expressed TRIM22 and are suppressed copying of HCV.Wherein vector represents the blank pcDNA3.1 carrier of transfection.
Fig. 4, the effect of NF-κ B path in HCV copies.Wherein, A is the variation of intracellular virus RNA, and B is the variation of viral RNA in supernatant.
Fig. 5, TRIM22 cause its degraded by the TAB2/3 combination.Wherein, A represents that TRIM22 is dose dependent to the Degradation of TAB2 and TAB3, B represents the exogenous interaction of TRIM22 and TAB2/3, and C represents that the endogenous of TRIM22 and TAB2/3 interacts, and D represents that the lysosome inhibitor is on the impact of TRIM22 degraded TAB2 effect.
Fig. 6, TRIM22 come negative regulation NF-κ B path by acting on TAB2 (A) or TAB3 (B).
The specific embodiment
The present invention has disclosed the albumen of a kind of anti-hepatitis c virus (HCV) and relevant drug target.The inventor finds that first the TRIM22 protein expression can suppress the expression of copying of HCV and gene and virus protein.The present invention points out TRIM22 to bring into play important function in the biocycle of HCV, and provides new drug target for the research and development of anti-HCV medicament.
TRIM22 albumen
TRIM albumen also is called RBCC albumen, comprises a RING domain, one or two B-box domain and a coiled-co domain, and 60% TRIM albumen also has a SPRY domain at C-terminal.TRIM family protein structure is conservative, and is relevant with the different physiological roles of body, as cell proliferation, differentiation, growth and apoptosis etc.
TRIM22 has another name called Staf50 (Stimulating transacting factor, 50kDa), is interferon-induced expressing gene.In the normal physiological situation, mainly high expressed in PERIPHERAL BLOOD MONONUCLEAR CELL, lymphoid tissue and ovary, there are some researches show that TRIM22 is relevant with the differentiation of lymphocytes activation and erythrocyte.(comprise liver) in other histoorgans, TRIM22 only has basal expression, studies confirm that interferon and p53 can obviously raise the expression of TRIM22 gene in some cell lines but have.
In the present invention, described TRIM22 albumen (polypeptide) can be naturally occurring, such as its can be separated or purification from animal.In addition, described TRIM22 albumen can be also artificial preparation, such as producing according to the genetic engineering recombinant technique of routine restructuring TRIM22 albumen.
Any suitable TRIM22 albumen all can be used for the present invention.Described TRIM22 albumen comprises TRIM22 albumen or its bioactive fragment (or being called active fragment) of total length.For example, the aminoacid sequence of described TRIM22 albumen can be substantially the same with the sequence of (SEQ ID NO:2) shown in GenBank accession number AAH22281.1; Its nucleotide sequence can be substantially the same with the sequence shown in SEQ ID NO:1.
The TRIM22 albumen that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form or the aminoacid sequence of its bioactive fragment are also included within the present invention.TRIM22 albumen or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and described sequence through amino acid substitution does not affect its activity or kept the activity of its part.Suitably replacing aminoacid is technology well known in the art, and described technology can be implemented at an easy rate, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, basically can not change biological activity at the inessential area change single amino acids of a peptide species.See the Molecular Biology ofThe Gene such as Watson, the 4th edition, 1987, The Benjamin/Cummings Pub.Co.P224.
The bioactive fragment of any TRIM22 albumen can be applied in the present invention.Here, the implication of the bioactive fragment of TRIM22 albumen refers to as a peptide species, and it still can keep all or part of function of the TRIM22 albumen of total length.Generally, described bioactive fragment keeps the activity of 50% total length TRIM22 albumen at least.Under preferred condition, described active fragment can keep 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length TRIM22 albumen.
TRIM22 albumen has been known in many animals and has existed with conservative highly.Therefore, be appreciated that deriving from different vegeto-animal TRIM22 all is contained in the present invention.Preferably, they are to compare with aminoacid sequence shown in SEQ ID NO:2, the sequence homogeny is higher than 60%, being more preferably higher than 70%, is more preferably higher than 80%, is more preferably higher than 85%, more preferably higher than 88%, being more preferably higher than 90%, is more preferably higher than 95%, is more preferably higher than 98%.
The present invention also can adopt TRIM22 albumen modified or improvement, such as, can adopt the TRIM22 albumen of being modified or improveing for the effect that promotes its half-life, effectiveness, metabolism and/or albumen.Described can be a kind of conjugate of TRIM22 albumen through the TRIM22 albumen of modifying or improve, or it can comprise substituted or artificial aminoacid.Described can be to have less common ground with naturally occurring TRIM22 albumen through the TRIM22 albumen of modifying or improve, but also can bring into play antiviral effect, and can not bring other harmful effect or toxicity.That is to say, any bioactive version that does not affect TRIM22 albumen all can be used in the present invention.
Agonist of TRIM22 and uses thereof
Based on the inventor's above-mentioned new discovery, the invention provides the purposes of the upper adjustment of a kind of TRIM22, for the preparation of the compositions of anti-hepatitis c virus.
As used herein, the upper adjustment of described TRIM22 has comprised promoter, agonist etc.The material of transcribing and translating of the activity of any TRIM22 of raising albumen, the stability of keeping TRIM22 albumen, promotion TRIM22 protein expression, the secretion that promotes TRIM22 albumen, prolongation TRIM22 albumen effective acting time or promotion TRIM22 all can be used for the present invention, as the active substance that can be used for anti-hepatitis c virus.
As optimal way of the present invention, the upper adjustment of described TRIM22 includes, but is not limited to: interferon or p53.
As optimal way of the present invention, the upper adjustment of described TRIM22 albumen includes, but is not limited to: expression vector or the expression constructs that can express (the preferred mistake expressed) TRIM22 after changing cell over to.Usually, described expression vector comprises a box gene, and the gene that described box gene contains the TRIM22 that encodes reaches the connected expression regulation sequence of operability with it.Described " operability is connected " or " operationally being connected in " refer to a kind of like this situation, and namely the activity of same linear DNA sequence other parts can be regulated or control to some part of linear DNA sequence.For example, if the transcribing of promoter control sequence, it is exactly operationally to be connected in coded sequence so.
In the present invention, the TRIM22 polynucleotide sequence can be inserted in recombinant expression carrier.As long as can copy in host and stablize, any plasmid and carrier may be used to the present invention.A key character of expression vector is usually to contain origin of replication, promoter, marker gene and translation control element.
Method well-known to those having ordinary skill in the art can be used for build the DNA sequence that contains the TRIM22 that encodes and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc.Described DNA sequence can be effectively connected on suitable promoter in expression vector, and is synthetic to instruct mRNA.Conversion carrier also comprises ribosome binding site and the transcription terminator that translation initiation is used.
Compositions
The present invention also provides a kind of compositions, and it contains effective dose (as 0.000001-50wt%; Better 0.00001-20wt%; Better, described TRIM22 albumen 0.0001-10wt%) or adjustment on it, and pharmaceutically acceptable carrier.
Compositions of the present invention can be directly used in anti-hepatitis c virus.In addition, also can unite use with other therapeutic agent or adjuvant simultaneously.
Usually, these materials can be formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, preferably, pH is about 6-8.
As used herein, term " contains " the various compositions of expression and can be applied to together in mixture of the present invention or compositions.Therefore, term " mainly by ... form " and " by ... composition " be included in during term " contains ".As used herein, term " effective dose " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.
Compositions of the present invention contains TRIM22 albumen and the pharmaceutically acceptable carrier of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usually pharmaceutical preparation should be complementary with administering mode, and pharmaceutical composition of the present invention can be made into the injection form, for example with normal saline or contain glucose and the aqueous solution of other adjuvant is prepared by conventional method.Described pharmaceutical composition should be made under aseptic condition.The dosage of active component is the treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.
TRIM22 albumen of the present invention or the effective dose adjusted on it can change with the order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (for example by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: described TRIM22 albumen or the pharmacokinetic parameter biological example utilization rate of adjusting on it, metabolism, half-life etc.; The order of severity of the disease that the patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Usually, adjust giving with the about dosage of 0.00001mg-50mg/kg the weight of animals (better 0.0001mg-10mg/kg the weight of animals) every day when TRIM22 albumen of the present invention or on it, can obtain gratifying effect.For example, by an urgent demand for the treatment of situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
The present invention also provides a kind of method of anti-hepatitis c virus, comprises the TRIM22 albumen or the adjustment on it that give experimenter's effective dose.When being used for anti-hepatitis c virus, the preferred TRIM22 albumen that adopts restructuring.
TRIM22 albumen of the present invention or the administering mode of adjusting on it have no particular limits, can be whole body or local.For example, TRIM22 albumen of the present invention or on it adjustment can give animal by the mode of spinal cord intrathecal injection, lumbar injection, intravenous injection, oral, subcutaneous injection, intradermal injection etc., spinal cord intrathecal injection comparatively preferably.
After the purposes of the described TRIM22 albumen of cicada, can adopt several different methods well known in the art that described TRIM22 albumen or its encoding gene or its pharmaceutical composition are delivered medicine to mammal.Preferably, can adopt the means of gene therapy to carry out, such as can be directly with TRIM22 albumen by delivering medicine to the experimenter such as methods such as injections; Perhaps, can be delivered on target spot by the ceneme (such as expression vector or virus etc.) that certain approach will carry the TRIM22 gene, and make it the TRIM22 albumen of expression activity.
As one embodiment of the present invention, described TRIM22 albumen directly can be delivered medicine to mammal (such as the people), perhaps, the gene of the coding TRIM22 albumen method by routine can be cloned in suitable carrier (as conventional protokaryon or carrier for expression of eukaryon or viral vector such as herpesvirus vector or adenovirus vector), described carrier is imported in the cell that can express described TRIM22 albumen, make described cellular expression TRIM22 albumen.Can by appropriate described cell being incorporated into the suitable position of body of mammals, realize the TRIM22 protein expression.
The administering mode of the upper adjustment of TRIM22 albumen depends primarily on type and the characteristic of described upper adjustment, and this is that those skilled in the art can assess.
The method of the potential material of screening anti-hepatitis c virus
Get the described TRIM22 albumen of cicada after the purposes aspect anti-hepatitis c virus, can screen expression or the active material that promotes TRIM22 based on this feature.
Therefore, the invention provides a kind of method that screening can be used for the potential material of anti-hepatitis c virus, described method comprises: candidate substances is contacted with the system that comprises (as expressing) TRIM22; With the impact of detection candidate substances on TRIM22; If described candidate substances can improve the transcribing of TRIM22, expresses, active or secretion, show that this candidate substances is the potential material that can be used for anti-hepatitis c virus.
In optimal way of the present invention, when screening, in order to be easier to observe transcribing, express, secrete or active change of TRIM22, also matched group can be set, described matched group can be the system of not adding the expression TRIM22 of described candidate substances.
The system of described expression TRIM22 can be for example cell (or cell culture) system, and described cell can be the cell of endogenous expression TRIM22; It can be maybe the cell of recombinant expressed TRIM22.The system of described expression TRIM22 can also be (but being not limited to) subcellular fraction system, solution system, organizational framework, organ systems or animal system (as animal model) etc.
As a kind of optimal way of the present invention, described system is the cell system of expressing TRIM22, for example U937 cell.
As optimal way of the present invention, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, with further selection and definite for the real useful material of anti-hepatitis c virus.
The present invention has no particular limits for the detection method of TRIM22 protein expression, activity, amount or secretion situation.Can adopt conventional protein quantification or half-quantitative detection technology, for example (but being not limited to): SDS-PAGE method, Western-Blot method, ELISA etc.
On the other hand, the present invention also provides the potential material that can be used for anti-hepatitis c virus that adopts described screening technique to obtain.The material that these Preliminary screening go out can consist of a screening storehouse so that people finally can therefrom filter out can be for the real useful material of anti-hepatitis c virus, thereby be used for clinical.
The inhibitor of NF-κ B signal path
The inventor also finds, it is requisite in time multiplexed cell system institute that NF-κ B signal path is HCV virus, by blocking-up NF-κ B signal path, can to HCV copy and inhibitory action is played in the expression of gene and virus protein.
Based on the inventor's above-mentioned new discovery, provide a kind of purposes of inhibitor of NF-κ B signal path, for the preparation of the compositions of anti-hepatitis c virus.The inhibitor of any existing NF-κ B signal path all can be applied in the present invention.For example, the inhibitor of described NF-κ B signal path is TPCK.
The inventor also finds, TRIM22 albumen can be by being combined with TAB2/3 and causing its degraded; And the signal path of NF-κ B has finally been blocked in the degraded of TAB2/3 (expression TAB2 or TAB3).
Based on above-mentioned new discovery of the present invention, also provide the purposes of the inhibitor of a kind of TAB2 albumen or TAB3 albumen, for the preparation of the compositions of anti-hepatitis c virus.The inhibitor of described TAB2 albumen or TAB3 albumen is for example TRIM22 albumen.
As used herein, described inhibitor has comprised time adjustment, antagonist, blocker, blocker etc.
The inhibitor of described TAB2/3 gene or albumen refers to the activity of any TAB2/3 of reduction albumen, the stability that reduces TAB2/3 gene or albumen, downward TAB2/3 protein expression, reduces the material of transcribing and translating of TAB2/3 albumen effective acting time or inhibition TAB2/3 gene, these materials all can be used for the present invention, as for suppressing the useful material of TAB2/3.
The inhibitor of described NF-κ B signal path refers to the activity of any NF-of reduction κ B signaling pathway protein, the stability that reduces NF-κ B signal path gene or albumen, the expression of lowering NF-κ B signaling pathway protein, minimizing NF-κ B signaling pathway protein effective acting time or suppresses the material of transcribing and translating of NF-κ B signal path gene, these materials all can be used for the present invention, as for suppressing the useful material of NF-κ B signal path.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is write according to normal condition such as J. Pehanorm Brooker etc. usually, molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Unless otherwise defined, the same meaning that all specialties and scientific words and the one skilled in the art who uses in literary composition is familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
The expression that embodiment 1, IFN α induce lower TRIM22
The human interferon IFN α that adds 500 units/ml in U937 cell (available from ATCC) received sample every 6 hours, extracted mRNA and carried out RT-PCR.
The results are shown in Figure 1A, Trim22 is raised by IFN α, and arrives peaking at the 18th hour.Further whether research TRIM22 is raised by IFN α on protein level, (Trim22 Mus source multi-resistance is available from Abnova company with the endogenous antibody of anti-TRIM22, Cat.#H00010346-B01) carry out Western Blot, discovery Trim22 on protein level is raised by IFN α equally, and arrived peaking at the 24th hour, as Figure 1B.
Therefore, TRIM22 can be significantly by IFN α inducible expression.
The expression of TRIM22 in embodiment 2, HCV course of infection
With HCV virus (referring to Zhong, P. etc., Robust hepatitis C virus infection in vitro.Proc.Natl.Acad.Sci.U.S.A., 10226 (2005), pp.9294-9299), 3 * 10
5Moi 0.3) infect human liver cancer cell Huh-7 (referring to Zhong, P. etc., Robust hepatitis C virus infection in vitro.Proc.Natl.Acad.Sci.U.S.A., 10226 (2005), pp.9294-9299), time segment is received sample, extracts cell mRNA and carries out conventional RT-QPCR.
Result such as Fig. 2, in the HCV course of infection, the expression of TRIM22 in cell occurs significantly to raise as can be known.
Embodiment 3, mistake are expressed TRIM22 to the inhibitory action of HCV
Build the pcDNA-TRIM22-FLAG carrier, construction method is as follows: the pcDNA3.1 carrier is available from Invitrogen company, and the method that obtains 5 ' Flag-pcDNA3.1-TRIM22 carrier is as follows:
IFN α stimulated the Hela cell 16 hours, Trizol extracting cell total rna, and the cDNA that obtains take its reverse transcription reaction is as template.TRIM22 (containing ClaI/XhoI) design of primers is as follows:
Forward primer: 5 '-CCATCGATATGGATTTCTCAGTAAAGGTA-3 ';
Downstream primer: 5 '-CCGCTCGAGTCAGGAGCTCGGTGGGCACAC-3 ';
React by PCR, at TRIM22 two ends introducing ClaI and XhoI restriction enzyme site.Digest respectively pcr amplification product and the 5.-Flag-pcDNA3.1 carrier of TRIM22 with Cobra venom endonuclease, reclaim the purpose fragment, transformed competence colibacillus DH5 α bacterium after connecting with the T4 ligase.After identifying, bacterium colony PCR checking, enzyme action evaluation and DNA sequencing obtain 5 ' Flag-pcDNA3.1-TRIM22 expression vector.Sequence verification is correct.
Transfection the previous day with the Huh-7 cell with trypsinization, counting and in six orifice plates bed board, making transfection day cell fusion degree is 70-90%.Avoid using antibiotic during bed board and transfection.Transfection pcDNA-TRIM22-FLAG carrier after 24 hours, and take the blank pcDNA3.1 carrier of while transfection as contrast, incubated overnight in the CO2 gas incubator of 37 ℃.After about 18 hours, the Huh-7 cell after transfection is blown afloat gently with 5ml training liquid, and in 12 orifice plates bed board (every hole 0.5ml).After 24 hours, with HCV virus (3 * 10
5, Moi 0.3) and infection Huh-1, incubated overnight in the CO2 gas incubator of 37 ℃.Renew bright training liquid after 24 hours.Changed after liquid 48 hours, and collected cell sample, extract cell total rna, (cell total rna comprises cell RNA and viral RNA to carry out the Real-time pcr analysis; Cell RNA is essentially identical in control cells (vector) and transfection TRIM22 cell, and what that intracellular virus infects what of the RNA amount that therefore records can represent).Repeatedly.
Result such as Fig. 3 have observed in transfection during in the Huh-7 cell of TRIM22, total RNA amount significantly is less than matched group, and visible copying of HCV obviously suppressed.
NF-κ B path is considered to have antiviral effect usually, but some previous works show, it is requisite in time multiplexed cell system institute that it is HCV virus equally.The inventor is by selecting inhibitor TPCK (the N-Tosyl-L-Phenylalanine Chloromethyl Ketone of NF-κ B path; Available from Sigma company) verify this hypothesis.
HCV virus (3 * 10 with 0.3moi
5Moi 0.3) infection Huh-7 cell, adding final concentration in rear 24 hours in infection is the TPCK of 50nM, (Fig. 4 a) and the variation of the middle viral RNA of supernatant (Fig. 4 b) when utilizing Real-time PCR to detect respectively 36h and 48h in cell, can obtain, in the repressed situation of NF-κ B path, copying also of HCV significantly suppressed.
The above results proves, in the HCV virus replication, NF-κ B path has also played requisite effect.
The molecular mechanism of the anti-HCV virus of embodiment 5, TRIM22
According to early-stage Study as a result the inventor infer, TRIM22 suppresses copying of HCV by suppressing NF-κ B path, may be by the lysosomal pathway TAB2/3 (being TAB2 or TAB3) that degrades, thereby suppress the activation of NF-κ B.In order to verify this supposition, the inventor is in the impact of cell-based assay TRIM22 on NF-κ B path, and determines whether it is to act on the TAB2/TAB3 path.
(Trizol extracting Hela cell total rna, the cDNA that obtains take its reverse transcription reaction is as template to build the expression plasmid of pcDNA-TAB2-HA and pcDNA-TAB3-HA.Design of primers is as follows:
TAB2 (containing BamH I/EcoR I):
Forward primer: 5 '-CGGGATCCATGGCCCAAGGAAGCCACCAA (SEQ ID NO:3);
Downstream primer: 5 '-CGGAATTCTCAGAAATGCCTTGGCATCTC (SEQ ID NO:4);
TAB3 (containing BamH I/EcoR I):
Forward primer: 5 '-CGGGATCCATGGCGCAAAGCAGCCCACAG (SEQ ID NO:5);
Downstream primer: 5 '-CGGAATTCTCAGGTGTACCGTGGCATCTC (SEQ ID NO:6).
React by PCR, at TAB2 or TAB3 two ends introducing BamHI/EcoRI restriction enzyme site.Digest respectively pcr amplification product TAB2, TAB3 and 5 ' HA-pcDNA3.0 carrier (available from Invitrogen company) with Cobra venom endonuclease BamHI/EcoRI, reclaim the purpose fragment, transformed competence colibacillus DH5 α bacterium after connecting with the T4 ligase.After identifying, bacterium colony PCR checking, enzyme action evaluation and DNA sequencing obtain 5 ' HA-pcDNA3.0-TAB 2 and 5 ' HA-pcDNA3.0-TAB3 expression vector), and in HEK293T cell (ATCC), utilize Lipofectamine reagent with these two kinds of plasmids of variable concentrations gradient (with the pcDNA3.0 empty carrier as blank) respectively with the common transfection of pcDNA-TRIM22-FLAG, whether detect TRIM22 has Degradation for TAB2/3.Collected cell sample in 24 hours after transfection, carry out Western Blot and analyze.
Found that, when TRIM22 crossed expression, the expression of TAB2/3 on protein level obviously suppressed, and this inhibitory action be have dose dependent (Fig. 5 a).And then test by co-immunoprecipitation, find TRIM22 can interact with TAB2/3 (Fig. 5 b, c) on level in cell.In order to verify whether TRIM22 realizes by lysosomal pathway this Degradation of TAB2/3, the inhibitor that added lysosomal pathway in 24 hours after the HEK293T cell transfecting---ammonium chloride (10Mm, 20mM), collect cell sample after 6 hours and carry out Western Blot analysis, result such as Fig. 5 d, thus proved that this effect realizes by lysosomal pathway.
And then inventor's utilization verifies with the luciferase assay of NF-κ B promoter whether RIM22 exists inhibitory action for the NF-κ B path that TAB2/3 activates.When with pcDNA-TAB2-HA or pcDNA-TAB3-HA respectively with the luciferase reporting plasmid that contains NF-κ B promoter together the transfection meeting expression of luciferase fluorescence can be detected due to the activation of NF-κ B promoter.Dye pcDNA-TAB2-HA (200ng) at the HEK293T transit cell respectively, the pcDNA3.0 empty carrier of pcDNA-TAB3-HA (200ng) and equivalent is as blank, change simultaneously the pcDNA-TRIM22-FLAG (0 that increases in gradient over to, 200,400 and 600ng) plasmid, and add NF-κ B reporter gene (50ng), pRL-TK (10ng).Doing luciferase after transfection in 24 hours detects.
Result such as Fig. 6.Along with the increase of TRIM22 transfection amount, the expression of the luciferase fluorescence that NF-κ B promoter activates weakens gradually, thereby has proved that TRIM22 comes negative regulation NF-κ B path by acting on TAB2/3, and this negative regulation has dose-dependent effect.
In sum, TRIM22 albumen can be by being combined with TAB2/3 and causing its degraded; The signal path of NF-κ B has finally been blocked in the degraded of TAB2/3.
Embodiment 6, drug screening
Method 1:
The first step: the preparation of cell model: as described in Example 3 with pcDNA-TRIM22-FLAG expression vector transfection Huh-7 cell, obtain to stablize the reconstitution cell of expressing TRIM22.With the cell model of this kind cell as the medicine that is used for screening anti-HCV virus.
Second step: drug screening:
Test group: the culture of the above-mentioned cell of processing with candidate substances;
Matched group: the culture of the above-mentioned cell of processing without candidate substances.
Appropriate time after processing adopts Western Blot method to measure the TRIM22 protein expression of described cell.If compare with matched group, the TRIM22 protein expression in the test group significantly rises more than 30%, illustrates that this candidate substances is the material of potential anti-HCV virus.
Checking: utilize IFN α to process the culture of above-mentioned cell as candidate substances in the test group, found that, the TRIM22 protein expression significantly rises 30%, so IFN α is useful for antiviral.
Method 2:
The first step: the preparation of cell model: with the pcDNA-TAB2-HA expression plasmid corotation HEK293T cell of pcDNA-TRIM22-FLAG expression vector and embodiment 5 preparations, obtain to stablize the reconstitution cell of expressing TRIM22 and TAB2 as described in Example 3.With the cell model of this kind cell as the medicine that is used for screening anti-HCV virus.
Second step: drug screening:
Test group: the culture of the above-mentioned cell of processing with candidate substances;
Matched group: the culture of the above-mentioned cell of processing without candidate substances.
Appropriate time after processing utilizes the method identical with embodiment 5, detects the degraded situation of TAB2, is the material of potential anti-HCV virus if the degraded of test group TAB2 than the remarkable rising 20% of matched group, illustrates this candidate substances.
Conclusion
Because interferon-ALPHA (IFN α) is the active drug that present unique treatment HCV infects, treatment lacks persistence and replys quite a few patient to IFN α.Wherein host factor has been brought into play important effect for the immunne response of IFN α.Research in recent years is found, increasing TRIM family molecule (as TRIM5 α, TRIM19, TRIM25 and TRIM28 etc.) plays an important role in the antiviral inherent immunity, and many TRIM family molecules also can be induced rise by disturbed element, and whether effect is arranged is but unknown but the TRIM family protein infects for treatment HCV.
The present invention finds that by a series of virusology experiments expressing TRIM22 albumen can suppress copying of HCV virus excessively in the Huh7 cell.And then proved that by a series of molecular biology experiments TRIM22 albumen can be by being combined with TAB2/3 and causing its degraded; And the signal path of NF-κ B has finally been blocked in the degraded of TAB2/3.And that NF-κ B signal path is HCV virus is requisite in time multiplexed cell system institute.This has pointed out TRIM22 by blocking-up NF-κ B signal path, so to HCV copy and inhibitory action is played in the expression of gene and virus protein.
Therefore, TRIM22 albumen has been brought into play important function in the biocycle of HCV, for the research and development of anti-HCV medicament provide new possible drug target.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1.TRIM22 albumen or the purposes of adjusting on it are for the preparation of the compositions of anti-hepatitis c virus.
2. purposes as claimed in claim 1, is characterized in that, described compositions also is used for:
Degraded TAB2 albumen and/or TAB3 albumen; Or
Suppress NF-κ B signal path.
3. purposes as claimed in claim 1, is characterized in that, the upper adjustment of described TRIM22 albumen is selected from:
Recombinant expression carrier contains an expression cassette in described expression vector, wherein comprise the gene of coding TRIM22 albumen; Or
Interferon; Or
p53。
4. the purposes of the inhibitor of a NF-κ B signal path is for the preparation of the compositions of anti-hepatitis c virus.
5. purposes as claimed in claim 4, is characterized in that, the inhibitor of described NF-κ B signal path is TPCK.
6. the purposes of the inhibitor of a TAB2 albumen or TAB3 albumen is for the preparation of the compositions of anti-hepatitis c virus.
7.TRIM22 the purposes of albumen is for the potential material of screening anti-hepatitis c virus.
8. method of screening the potential material of anti-hepatitis c virus, described method comprises:
(1) candidate substances is contacted with the system that comprises TRIM22 albumen;
(2) detect candidate substances to genetic transcription, protein expression or the active impact of TRIM22;
If described candidate substances promotes genetic transcription, protein expression or the activity of TRIM22, show that this candidate substances is the potential material of anti-hepatitis c virus.
9. method as claimed in claim 8, is characterized in that, step (1) comprising: in the test group, candidate substances is joined in the system that comprises TRIM22 albumen; And/or
Step (2) comprising: genetic transcription, protein expression or the activity of TRIM22 in the system of detection test group, and compare with matched group, wherein said matched group is the system that comprises TRIM22 albumen of not adding described candidate substances;
If in the test group, the genetic transcription of TRIM22, protein expression or activity are statistically higher than matched group, just show that this candidate substances is the potential material of anti-hepatitis c virus.
10. method as claimed in claim 9, is characterized in that, in step (1), also comprises TAB2 albumen and/or TAB3 albumen in described system, and described TRIM22 albumen and TAB2 albumen and/or TAB3 protein-interacting; And
Step (2) comprising: detect candidate substances to the impact of TRIM22 albumen and TAB2 albumen and/or TAB3 protein-interacting;
If described candidate substances promotes TRIM22 albumen and TAB2 albumen and/or TAB3 protein-interacting, show that this candidate substances is the potential material of anti-hepatitis c virus.
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| CN108271364A (en) * | 2015-05-29 | 2018-07-10 | 宾夕法尼亚大学理事会 | For the composition and method of the protein for false folding of degrading |
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| WO2006088438A1 (en) * | 2005-02-14 | 2006-08-24 | Applera Corporation | Biomarkers for interferon-alpha response in hepatitis c virus infected patients |
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| WO2006088438A1 (en) * | 2005-02-14 | 2006-08-24 | Applera Corporation | Biomarkers for interferon-alpha response in hepatitis c virus infected patients |
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