CN107151697A

CN107151697A - Method and kit for predicting the response that Chronic Hepatitis B is treated to IFN α

Info

Publication number: CN107151697A
Application number: CN201710121980.4A
Authority: CN
Inventors: 葛胜祥; 王邵娟; 熊丽霞; 刘永亮; 夏宁邵
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2016-03-04
Filing date: 2017-03-03
Publication date: 2017-09-12
Anticipated expiration: 2037-03-03
Also published as: WO2017148432A1; CN107151697B

Abstract

The present invention relates to the mark for predicting the response that Chronic Hepatitis B is treated to IFN α.The invention further relates to the method for predicting the response that Chronic Hepatitis B is treated to IFN α, it includes determining the step of this kind of marker expression level in Chronic Hepatitis B PBMC.The invention further relates to the kit for the above method.

Description

Method and kit for predicting the response that Chronic Hepatitis B is treated to IFN α

Technical field

The present invention relates to the mark for predicting the response that Chronic Hepatitis B is treated to IFN α.The invention further relates to The method for the response treated in prediction Chronic Hepatitis B to IFN α, it includes determining this kind of mark in Chronic Hepatitis B PBMC The step of thing expression.The invention further relates to the kit for the above method.

Background technology

Hepatitis b virus infected, especially chronic HBV infection is that the mostly important public health in the whole world is asked One of topic, the current whole world about has more than 3.5 hundred million chronic hepatitis B virus infecton.Chronic HBV infection can be made Into chronic viral hepatitis type B (Chronic hepatitis B, CHB), hepatic sclerosis (Liver cirrhosis, LC) and original Hair property hepatocellular carcinoma (Hepatocellular carcinoma, HCC) etc. liver diseases, by chronic HBV infection and The death caused by relevant disease caused by it, the whole world is per year over 1000000 people.

Most chronic HBV infection patient is shown as without obvious clinical symptoms, wherein about 10-30% patient evolution into Hepatic sclerosis or liver cancer.Chronic HBV infection is a dynamic process, its natural history infected can be divided into 4 it is different and not Certainly exist the stage of positive connection：1) immune tolerant phase, feature is that HBeAg is positive, and serum HBV DNA replication dna is enlivened, ALT levels are normal or relatively low, and fibrosis does not occur typically；2) in active immunity stage/immune clearance solution stage, feature is HBeAg It is positive but its level is begun to decline, HBV DNA replication dnas level reduction, serum HBsAg level declines, and ALT is persistently raised or ripple It is dynamic.Ctl response in this phase patient is activated, and moderate or serious inflammatory reaction and liver that the duration is not waited easily occurs Fibrosis；3) inactive carrying stage, this Phase patient HBV DNA is in low replicative phase, and DNA level is extremely low or even detects not Out, HBsAg levels are significantly reduced, and ALT levels are shown normally, and hepatic tissue has no or only mild inflammation, HBeAg is negative, Anti-HBe is positive, and this period means that the HBV infection of patient has obtained immune control, also imply that one long-term and preferable Prognosis, the probability that hepatic sclerosis even liver cancer occurs for most of patient is very low；4) the HBeAg negative hepatitis phases, chronic hepatitis B is belonged to Late stage, its be mainly characterized in that HBV DNA and ALT horizontal periodicity fluctuation and active hepatitis, and with pre-core region and/ Or based on the HBV viruses of basic core promoter (BCP) variation, it is impossible to the HBeAg of expression or expression extremely low level.

HBV viruses do not have direct pathogenic effects to host cell, and its main pathogenesis is that HBV viruses cause place Main immune response, immune response and then causes immune liver tissue injury.The therapeutic modality of chronic HBV infection is needed according to patient The different situations of body give different antiviral therapies.The purpose of antiviral therapy is to be suppressed HBV viral persistences, is prevented Hepatitis extends the life cycle of patient and improved the quality of living to the development or conversion of fibrosis, hepatic sclerosis even liver cancer.

There are 8 kinds of medicines to can be used for CHB antiviral therapies, including 6 kinds of nucleoside analogs at present：Lamivudine (LAM), for than Husband fixed (LdT), emtricitabine (FTC), Entecavir (ETV), adefovirdipivoxil (ADV), tenofovir (TDF)；2 kinds of interferon： Plain interferon (IFN α) and Peg-IFN alpha-2b (PEG-IFN α).The main function of nucleoside analog is by suppressing HBV The activity of polymerase come suppress HBV virus duplication.Greenberg in 1976 reports IFN α treatment chronic hepatitis B first Curative effect, IFN α is to ratify the medicine for treating chronic hepatitis B by FDA earliest, and PEG-IFN α are also used as treatment chronic hepatitis B Choice drug, with antiviral and immunoregulatory double action.The major advantage of IFN α is to have no drug resistance and have to exempt from Epidemic disease mediation control HBV infection effect so that during treatment end patient have higher chance occur HBeAg Virus mutations, More longlasting virological response and HBsAg are removed, and the HBV DNA levels in patient is maintained Monitoring lower-cut.

Antiviral therapy must assure that virology to a certain extent suppresses and can produce biochemical alleviation, and histology is recovered And prevent the generation of complication.Preferable HBV treatments terminal is that HBsAg disappears, but existing antiviral drugs is difficult to reach This target, so more real treatment terminal is can to induce a lasting virology holddown.Antiviral response can To be divided into biochemistry level, serum levels, virology level and histological level.All responses can over the course for the treatment of and Some time points after treatment are estimated.Clinical studies show, CHB patient uses the response of 6 months of IFN α conventional therapy Rate is only 25-40%.CHB treatment patients may can be predicted for HBV specific immune responses ability by determining CHB patient Expected effect.For a long time, due to lacking univocal Anti-HBV activity immune response index, CHB patients serum's ALT level quilts As the indirect Substitute Indexes for weighing host's Anti-HBV activity immunocompetence, but it is unsatisfactory to predict the outcome.If can be controlled before the treatment Response prediction is treated, for optimization medicament selection, curative compliance is improved and ensures that curative effect tool is of great significance.

Therefore, this area be required to simply, easily and fast and with high accuracy and specifically predict CHB patient The mark for the response treated to IFN α.

The content of the invention

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The implication that personnel are generally understood that.Also, cell culture used herein, biochemistry, nucleic acid chemistry, Immunology Lab Operating procedure is widely used conventional steps in corresponding field.Meanwhile, for a better understanding of the present invention, phase is provided below Close the definition and explanation of term.

As used herein, term " IFN α " or " alpha-interferon " include all natural or restructuring α types interference Element, particularly preferably be human interferon-alpha, such as recombination human alpha type interferon, including but not limited to IFN α -1b (can for example be obtained From Schering Corporation, Kenilworth, N.J.'sA interferon), IFN α -2a (can for example obtain From Hoffmann-La Roche, Nutley, N.J.'s- A interferon) or IFN α -2b (be for example available from Schering Corporation, Kenilworth, N.J.'s- A interferon)；Such as natural α types interference The mixture of element, including but not limited to IFN α-n1 (Sumitomo is for example available from, Japan'sOr Glaxo-Wellcome Ltd., London, Great Britain'sInterferon alfa-n1) or IFN α-n3 (for example it is available from Interferon Sciences AlferonInterferon).In the present invention, term " IFN α " or " alpha-interferon " also includes the IFN α of any material with IFN α biological activity, such as mutation or modified, such as IFN α PEG derivatives (PEG-IFN α).In the present invention, term " IFN α " or " alpha-interferon " not by it is any it is specific come The limitation in source, can be obtained by commercial source or be produced by routine techniques well known by persons skilled in the art, the producer Method includes but is not limited to biological source extraction method and genetic engineering extraction method, and it is described in detail in such as " Pestka S.Arch Biochem Biophys.1983 Feb 15；221(1):1-37 " (it is incorporated herein by reference).

As used herein, term " HBV antigens " refers to that being present in hepatitis type B virus (HBV) to induce Body produces the albumen of immune response, including hepatitis B virus core antigen (HBcAg), hepatitis b virus s antigen And hepatitis B virus E antigen (HBeAg) (HBsAg).

As used herein, term " HBcAg " refers to, the core antigen protein of hepatitis type B virus (HBV), and it is Well known to a person skilled in the art (see, e.g. GENBANK accession number：CAM31905.1).

In the present invention, when referring to HBcAg amino acid sequence, with reference to SEQ ID NO：Sequence shown in 38 is carried out Description.However, it will be appreciated by those skilled in the art that in HBcAg amino acid sequence, can it is naturally-produced or be artificially introduced mutation or Variation (includes but is not limited to, displacement, missing and/or addition, such as different genotype, gene hypotype or different serotypes, serum The HBcAg of hypotype), without influenceing its biological function.Therefore, in the present invention, term " HBcAg " should include all such sequences Row, including such as SEQ ID NO：Sequence and its natural or artificial variant shown in 38.Also, when description HBcAg sequence During column-slice section, it not only includes SEQ ID NO：Corresponding sequence in 38 sequence fragment, in addition to its natural or artificial variants Fragment.For example, statement " HBcAg 1-183 amino acids residue " includes, SEQ ID NO：38 1-183 amino acids Respective segments in residue, and its variant (natural or artificial).In the present invention, statement " respective segments " refers to, when to sequence When row carry out optimal comparison, i.e., when sequence is compared to obtain highest percentage homogeneity, the sequence middle position being compared Fragment in equivalent site.

In the present invention, term " HBcAg " is not limited by the method for any specific synthetic proteins, can pass through this area skill Routine techniques known to art personnel is produced, such as DNA recombinant techniques or chemical synthesising technology.

As used herein, statement " HBcAg anti-genic fragment " refers to that HBcAg albumen is obtained after truncation Amino acid sequence segments (that is, polypeptide), the fragment have and corresponding full-length proteins identical biological activity, i.e. can pierce Swash and activate the PBMC from Chronic Hepatitis B.Such as SEQ ID NO in the present invention:Amino acid sequence segments shown in 3-37 As HBcAg anti-genic fragment.In the present invention, the anti-genic fragment is not limited by the method for any specific synthesis polypeptide System, can be produced, for example DNA recombinant techniques or chemical synthesising technology by routine techniques well known by persons skilled in the art.

In the present invention, HBcAg or its anti-genic fragment (for example, have such as SEQ ID NO respectively:Ammonia shown in 3-37 The anti-genic fragment of base acid sequence) it can be obtained by DNA recombinant techniques, such as by using Cell free expression system from coding (Cell free expression system includes such as expression based on reticulocyte lysate for the polynucleotides acquisition of these albumen or polypeptide System, the expression system based on Wheat Germ Extracts and the expression system based on E. coli extract)；Or by using internal Expression system (for example, escherichia coli prokaryotic expression system, yeast eukaryotic expression system) is from encoding many of these albumen or polypeptide Nucleotides is obtained.Alternatively, HBcAg or its anti-genic fragment (for example, have such as SEQ ID NO respectively:3-37 The anti-genic fragment of shown amino acid sequence) it can be produced by chemical synthesis.Albumen or the fully synthetic method of chemiluminescent polypeptide It is well known in the art (see, e.g., Raibaut L, et al., Top Curr Chem.2015；363:103-54； Thapa P,et al.Molecules.2014；19(9):14461-83；Dawson PE,et al.,Science,1994；266 (5186):776-9；With Wang P, et al., Tetrahedron Lett, 1998,39 (47):88711-14；It is by quoting It is incorporated herein), and include but is not limited to：Solid phase peptide synthesis technology (Solid Phase Peptide Synthesis, SPPS) Or liquid phase subsection synthesis technology is (for example, native chemical connection method (Native Chemical Ligation, NCL), azide method (Azide method), transfer active ester method (Transfer Active Ester Condensation, TAEC)).

As used herein, statement is " respectively with such as SEQ ID NO:The antigen of amino acid sequence shown in 3-37 Property fragment " refers to, with SEQ ID NO:The anti-genic fragment of amino acid sequence shown in 3, with SEQ ID NO:Shown in 4 The anti-genic fragment of amino acid sequence ... with SEQ ID NO:The anti-genic fragment of amino acid sequence shown in 36 and have SEQ ID NO:The combination of the anti-genic fragment of amino acid sequence shown in 37.

As used herein, term " immunology detection " refers to, utilizes the specific phase interaction between Ag-Ab The measure carried out with/binding affinity, it is typically used for detecting the presence in the sample of specific antigen or antibody or water It is flat.Such immunologic assay be well known to a person skilled in the art, include but is not limited to, ELISA detection, Elispot detection, Western blot, Surface Plasmon Resonance etc..On the detailed description of immunologic assay, reference can be made to for example, Fundamental Immunology, Ch.7 Paul, W., ed., second edition, Raven Press, N.Y. (1989).

As used herein, term " antibody " refers to, generally by two pairs of polypeptide chains, (each pair has one " light " (L) Chain and " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be categorized as κ and lambda light chain.Heavy chain can be categorized as μ, δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.In light chain and heavy chain, Variable region and constant region are connected by " J " area of about 12 or more amino acid, and heavy chain is also comprising about 3 or more ammonia " D " area of base acid.Each heavy chain is made up of weight chain variable district (VH) and heavy chain constant region (CH).Heavy chain constant region is by 3 domains (CH1, CH2 and CH3) is constituted.Each light chain is made up of light chain variable district (VL) and constant region of light chain (CL).Constant region of light chain is by one Individual domain C L compositions.The constant region of antibody can mediated immunity globulin and host tissue or the factor, including immune system is each Plant the combination of the first component (C1q) of cell (for example, effector cell) and classical complement system.VH and VL areas can be also subdivided into With denatured region (being referred to as complementary determining region (CDR)), the more conservative region for being referred to as framework region (FR) is interspersed with. Each VH and VL are by the following order：What FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 were arranged from amino terminal to carboxyl terminal 3 CDR and 4 FR compositions.The variable region (VH and VL) of each heavy chain/light chain pair forms paratope respectively.Amino acid is extremely The distribution of each region or domain follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia＆Lesk (1987)J.Mol.Biol.196:901-917；Chothia et al. (1989) Nature 342:878-883 definition.Term " antibody " is not limited by any specific method for producing antibody.For example, it includes, and especially, recombinant antibodies, monoclonal antibody And polyclonal antibody.Antibody can be the antibody of different isotypes, for example, IgG is (for example, IgG1, IgG2, IgG3 or IgG4 are sub- Type), IgA1, IgA2, IgD, IgE or IgM antibody.

As used herein, " antigen-binding fragment " of antibody refers to, one or more parts of full length antibody, institute The ability for the same antigen (for example, OAS2 or USP18) that part keeps binding antibody to be combined is stated, can be competed with complete antibody Specific binding to antigen.Generally referring to, Fundamental Immunology, Ch.7Paul, W., ed., second edition, Raven Press, N.Y. (1989), it is merged into herein, for all purposes with its full text by quoting.Restructuring can be passed through DNA technique or enzymatic or chemical disruption generation antigen-binding fragment by complete antibody.In some cases, antigen binding fragment Section includes Fab, Fab', F (ab') 2, Fd, Fv, dAb and complementary determining region (CDR) fragment, single-chain antibody (for example, scFv), embedding Antibody, double antibody (diabody) and such polypeptide are closed, it includes and is enough to assign the antibody of polypeptid specificity combination antigenic capacity At least a portion.

As used herein, term " aptamer (Aptamer) " refers to, being capable of high-affinity and with high specificity The single stranded oligonucleotide of binding purpose target protein or other bio-target molecules, its foldable formation such as stem ring (Stem-Loop), The thermodynamically stable three dimensions knot of hair clip (Hairpin), false knot (Pseudoknot) or the G- tetramers (G-tetramer) Structure, for example, by complementary structure, base stacking force, Van der Waals force, hydrogen bond or electrostatic interaction and purpose target protein or other biologies Target molecule is specifically bound.Aptamer can be DNA or RNA, can also comprising nucleic acid analog (such as lock nucleic acid (LNA), Peptide nucleic acid (PNA), glycol nucleic acid (GNA) or threose nucleic acid (TNA)).The method for obtaining the aptamer for combining specific target protein It is it is known in the art that such as SELEX (Systematic evolution of ligands by exponential Enrichment) triage techniques.

As used herein, term " target polypeptide " refers to, can specifically bind the polypeptide point of purpose target protein Son.In the present invention, the target polypeptide can include natural amino acid, the amino acid of synthesis or use and naturally occurring amino acid The amino acid simulant (mimetics) that similar mode works.Naturally occurring amino acid is to be encoded by genetic code Those and those amino acid for modifying later, for example, hydroxy-proline, γ-Hydroxy GIutaminic Acid salt, O- phosphoserines, Phosphothreonine or phosphotyrosine.In the present invention, can be determined based on affinity target polypeptide and its purpose target protein it Between " specificity ", the affinity can use Dissociation equilibrium constant (that is, the K of target polypeptide and its purpose target protein combined_D Value) it is described.K_DValue is lower, and the bond strength between target polypeptide and its purpose target protein combined is stronger.In ability It is generally known that greater than about 10 in domain^-3M K_DValue is typically considered to represent non-binding or non-specific binding.Depending on specific Purpose target protein, can obtain the target polypeptide for specifically binding the target protein by method known to those skilled in the art, For example screened by display technique of bacteriophage or protein microarray technology.

As used herein, statement refers to that Chronic Hepatitis B receives IFN/PEG-IFN " to IFN α therapeutic response " After treatment, there is the situation of virological response or serology response.Wherein, the virological response refers to 6 months HBV after treatment DNA<Maintained 6 to 12 months after 2000IU/ml, and drug withdrawal；The serology response refers to that HBeAg the moon turns and anti-occurs HBe (referring to EASL chronic hepatitis Bs clinical practice guideline (2012 editions)).Correspondingly, statement " unresponsive is treated to IFN α " Refer to, Chronic Hepatitis B receives after IFN/PEG-IFN treatments, and the situation of condition as described above is not met.

As used herein, term " statistical analysis value " refers to, to the detection knot obtained by various detection methods Fruit carries out the value obtained after statistical analysis.Various statistical analysis techniques are well known in the art (see, e.g. PCT international applications WO2009064901, it is incorporated herein by reference), and including but not limited to linear combination, the linear regression of testing result Model, Logistic regression models, linear discriminant analysis (LDA) model, arest neighbors model or predictive analysis of microarrays (PAM). Typically, it is therefore particularly preferred that the statistical analysis value carries out statistical analysis by Logistic regression models and obtained Value.Logistic regression models are specifically described in such as " joint inspection of the tetra- kinds of tumor markerses of Hu Chunyan in ovarian cancer serum Survey [D] Guangzhou：Zhongshan University, 2008：1-39 ", its whole is incorporated herein by reference.

As used herein, term " reference value " (also referred to as excellent diagnostics dividing value) is to refer to reflection to control IFN α Treat the value of the situation of unresponsive CHB PATIENT POPULATIONs.In the present invention, reference value is included for example, being answered according to IFN α treatment nothing Marker levels in the CHB PATIENT POPULATION's samples answered or according to before and after IFN α and/or HBV antigenic stimulus to IFN α treatment without should The change multiple (relative expression levels) of marker levels in the CHB PATIENT POPULATION's samples answered, an identified normal number Value or number range；And, to the detected value that is obtained from the sample that unresponsive CHB PATIENT POPULATIONs are treated to IFN α (for example, preceding The change multiple of marker levels described in text) carry out being worth (statistical analysis value) obtained from statistical analysis.Determine excellent diagnostics The method of dividing value is well known in the art, including but not limited to Receiver operating curve's analysis (Receiver Operating Characteristic (ROC) curve analysis), it is described in detail in such as " Habibzadeh F, et al.,Biochem Med(Zagreb).2016；26(3):297-307 " and " best operating point in the medium .ROC curves of Chen Wei Select [J] China Healths statistics, 2006,23:157-158 ", its whole is incorporated herein by reference.

As used herein, term " Ct values (Cycle threshold, cycle threshold) " refers to, in fluorescent quantitation In PCR detections, the fluorescence signal in each reaction tube reaches the period undergone during given threshold.The Ct values of each template and There is linear relationship in the logarithm of the starting copy number of the template, starting copy number is more, and Ct values are smaller.Utilize known starting copies Several standard items can make standard curve, wherein using the logarithm of starting copy number as abscissa, ordinate is used as using Ct values.Cause This, as long as obtaining the Ct values of unknown sample, you can calculate the starting copy number of the sample from standard curve.

As used herein, term " comparing Ct methods " is a kind of mRNA relative quantitative assay sides well known in the art Method, it is described in detail in such as Livak KJ, et al. .Methods.2001 Dec；25(4):(it is incorporated by reference into 402-8 Herein).In real-time PCR (quantitative PCR) course of reaction is carried out, it can be assumed that the amplified production that each PCR cycle is doubled, And the Ct values drawn in the PCR exponential phases reacted can reflect the copy number of starting template, so if different testing samples Ct values differ a circulation, then mean that their initial template copy number has 2 times of difference.Based on this, by comparing The Ct values of different samples, you can the relative expression levels of target gene in testing sample are judged.

As used herein, term " normalization (normalization) " or " standardization " refer to, by than less It is that may be present variable in detection method each step used to eliminate with the expression of same gene in sample Differential expression caused by yield.Normalized method is known in the art, includes but is not limited to be described in detail in the PCT worlds Apply for the method in WO2013068422A1 or Chinese patent application 201280035399.6, such as comparison based on reference gene Ct methods.

As used herein, term " expression " refers to, is produced in the sample from subject by target gene Gene outcome measurable amount, wherein gene outcome can be transcription product or translation product.Therefore, statement " determines mesh Gene expression " can refer to, determine the level or the gene of the mRNA or described mRNA of gene fragment The level of cDNA or described cDNA fragment or the albumen by the gene code or the level of its polypeptide fragment.

As used herein, term " PMNC (Peripheral blood mononuclear Cell, PBMC) " refer to, the general name of the cell with individual cells core in peripheral blood, including but not limited to (T is thin for lymphocyte Born of the same parents, B cell, NK cells), monocyte or BMDC.The method that PBMC is obtained from peripheral blood be it is well known in the art, Including but not limited to Ficoll is layered liquid method or Percoll layering liquid methods.In the present invention, term " is used for the examination for separating PBMC Agent " refers to that the reagent used required in acquisition PBMC as described above method is for example layered liquid method for Ficoll Ficoll-Hypaque solution.In the present invention, term " PBMC separators " refers to, can be from the sample from subject Separation obtains PBMC device in product (for example, peripheral blood), and this kind of device is known in the art, includes but is not limited to retouch in detail The cell separation apparatus in Chinese patent application CN1958776A, CN102286360A and CN105132278A is set forth in, its is complete Portion is incorporated herein by reference.

As used herein, term " peripheral blood tunica albuginea layer " refers to, periphery anticoagulation by natural subsidence, centrifugation or The component formed after density gradient centrifugation, is mainly made up of leucocyte (including PMNC) and blood platelet. It is membranaceous that anticoagulation can form the blood plasma on upper strata, the red blood cell of lower floor and one layer of very thin therebetween white after centrifugation Thing, accounts for the 1% of total blood volume, is referred to as tunica albuginea layer.

As used herein, term " subject " includes but is not limited to various animals, particularly preferably people.

In the present invention, term " diluent " is preferably capable maintaining the electrolyte solution of Premeabilisation of cells pressure, if necessary should Solution also possesses the effect for keeping physiological ph.This kind of solution is well known in the art, including but not limited to Alsever's Solution (Alsever's solution), Earle's balanced salt solutions (EBSS), Gey's balanced salt solutions (GBSS), Hanks' balances Salting liquid (HBSS), phosphate buffer (PBS), Du Shi phosphate buffers (DPBS), Puck's balanced salt solutions, Ringer's balanced salt solutions (RBSS), Simm's balanced salt solutions (SBSS), TRIS buffer solutions (TBS), Tyrode's balances Salting liquid (TBSS), physiological saline or ringer's solution (Ringer's Solution).In some embodiments, the dilution Agent is phosphate buffer or physiological saline.

As used herein, term " anti-coagulants " refers to, can prevent the reagent or material of blood clotting, this kind of thing Matter is well known in the art, including but not limited to heparin, EDTA, oxalates (for example, sodium oxalate, potassium oxalate, ammonium oxalate), citron Sour sodium (sodium citrate).

As used herein, term " nutrient solution " or " culture medium " refer to, are able to maintain that the nutriment of cytoactive.It is logical Chang Suoshu nutriment contains amino acid, vitamin, carbohydrate, inorganic salts etc..This kind of nutriment is well known in the art, including but It is not limited to RPMI-1640 culture mediums or DMEM culture mediums.In the present invention, culture is added in the sample from the subject The purpose of liquid or culture medium is to maintain the particularly PBMC activity of the cell in sample.Maintain the work of cell in blood constituent The method of property is well known in the art, and those skilled in the art can be selected according to actual needs.In some embodiments In, when sample is whole blood, nutrient solution can be added, the nutrient solution is, for example, to add in phosphate buffer or physiological saline Plus appropriate glucose, sodium chloride and potassium chloride etc.；In certain embodiments, the nutrient solution is in phosphate buffer Addition appropriate glucose and potassium chloride.In certain embodiments, when sample is PMNC (PBMC), periphery When blood tunica albuginea layer or other blood constituents containing PBMC, culture medium can be added, such as cell culture medium is for example adapted to maintain The cell culture medium of haemocyte particularly PBMC activity, such as RPMI-1640 culture mediums or DMEM culture mediums.

Present inventor goes out people after research is tracked to the response situation that IFN α is treated to Chronic Hepatitis B Expect ground to find, to OAS2 levels in PBMC samples before the treatment of IFN α therapeutic response patient with treating unresponsive patient to IFN α Compared to showing notable difference, or to PBMC samples wherein OAS2 after external evoked before the treatment of IFN α therapeutic response patient And/or UPS18 levels show notable difference compared with treating unresponsive patient to IFN α, thus OAS2 and/or UPS18 can be with It is used as the mark for predicting CHB patient's IFN α therapeutic response.And before the application, this area lacks meaning for a long time Clear and definite Anti-HBV activity immune response index is to predict response situation that CHB patient treats to IFN α.Based on this discovery, the present invention People develops the new method for predicting the response that IFN α is treated to the therapeutic effect of CHB patient or CHB patient to IFN α.

Therefore, in one aspect, the invention provides a kind of kit, it includes that marker expression level can be detected First reagent, optionally also includes IFN α and/or stimulant；Wherein, the mark is selected from OAS2 or USP18, the stimulation Thing is selected from HBV antigens, the anti-genic fragment of HBV antigens or its any combinations.

In certain preferred aspects, the HBV antigens are HBcAg.In certain preferred aspects, it is described HBcAg has such as SEQ ID NO:Amino acid sequence shown in 38.In certain preferred aspects, the anti-genic fragment With selected from following amino acid sequence：SEQ ID NO:3-37.

In an especially preferred embodiment, the stimulant is comprising respectively with such as SEQ ID NO:Shown in 3-37 Amino acid sequence anti-genic fragment.

In certain preferred aspects, first reagent is the examination for the mRNA level in-site that can detect the mark Agent.This kind of reagent is it is known in the art that including but is not limited to the nucleic acid probe of specific binding target sequence, amplification target sequence Primer, non-specific fluorescence dyestuff (for example, SYBR Green I) or its combination of row.In some embodiments, the core Acid probe can be for single nucleic acid probe marked, such as radionuclide (such as³²P、³H、³⁵S etc.) label probe, biotin labeling Probe, horseradish peroxidase-labeled probe, digoxin labelled probe or fluorophor (such as FITC, FAM, TET, HEX, TAMRA, Cy3, Cy5 etc.) label probe；The nucleic acid probe can also be double labelling nucleic acid probe, such as Taqman probes, Molecular beacon, displacement probe, scorpions probe, QUAL probes, FRET probes etc..In some embodiments, described first Reagent includes Taqman probes.In some embodiments, the cDNA of the OAS2 has such as SEQ ID NO:Nucleosides shown in 1 Acid sequence.In some embodiments, the cDNA of the USP18 has such as SEQ ID NO:Nucleotide sequence shown in 2.

In certain preferred aspects, first reagent is the examination for the protein level that can detect the mark Agent.This kind of reagent be it is well known in the art, include but is not limited to can with OAS2 or USP18 protein-specifics combine antibody, Target polypeptide or aptamer.In some embodiments, this kind of reagent carries detectable mark, such as enzyme (such as horseradish mistake Oxide enzyme, alkaline phosphatase etc.), radionuclide (such as³H、¹²⁵I、³⁵S、¹⁴C、³²P etc.), fluorescent dye (such as FITC, TRITC, PE, Texas Red, quantum dot, Cy7, Alexa 750 etc.), acridine ester type compound, magnetic bead (for example,), collaurum or coloured glass or plastics (for example, polystyrene, polypropylene, latex, etc.) pearl, Yi Jiyong In the biotin for combining the Avidin (for example, Streptavidin) that above-mentioned label is modified.

In certain preferred aspects, first reagent determines OAS2 in the sample by immunology detection Or USP18 protein level.In certain preferred aspects, the immunology detection is selected from ELISA detections, Elispot Detection, western blot or Surface Plasmon Resonance.In some embodiments, first reagent include anti-OAS2 or The antibody of USP18 albumen or its antigen-binding fragment.

In certain preferred aspects, kit of the invention, which is included, can detect the first examination of OAS2 expressions Agent, optionally also includes IFN α and/or stimulant.

In certain preferred aspects, kit of the invention, which is included, can detect the first of USP18 expressions Reagent and IFN α, optionally also include stimulant.

In certain preferred aspects, kit of the invention, which is also included, is used to pre-process the sample containing PBMC Second reagent, wherein second reagent includes being selected from following one or more reagents：Diluent (example for dilute sample Such as, phosphate buffer or physiological saline)；Anti-coagulants (for example, heparin) for preventing blood clotting；For maintaining PBMC to live The reagent (for example, culture medium or nutrient solution) of property；With for separating PBMC reagent (for example, lymphocyte separation medium, for example Ficoll-Hypaque solution).In certain preferred aspects, second reagent includes being used to maintain PBMC activity Reagent (for example, culture medium or nutrient solution), and/or for separating PBMC reagent (for example, lymphocyte separation medium, for example Ficoll-Hypaque solution).

In certain preferred aspects, kit of the invention (is adopted also comprising blood-taking device by such as apyrogeneity vacuum Blood vessel) and/or PBMC separators (such as PBMC cell Separating tubes).

In certain preferred aspects, kit of the invention, which is also included, can detect reference gene expression Reagent, expression relative constancy of the reference gene in each tissue and cell, and will not under conditions of processing factor effect Generation expression changes, such as house-keeping gene；Generally, the house-keeping gene coding is to necessary to maintenance radical cellular activities Protein, it includes but is not limited to β-actin, GAPDH, 18S rRNA, β 2-MG, UBC or α-tubulin.Some preferred In embodiment, the reagent that can detect reference gene expression is that can detect the mRNA level in-site of the reference gene Reagent.In certain preferred aspects, the reagent that can detect reference gene expression is can detect institute State the reagent of the protein level of reference gene.

In another aspect, the present invention relates to can detect use of the reagent of marker expression level in reagent preparation box On the way, the kit is used to predict that IFN α controls IFN α the therapeutic effect of the subject with chronic hepatitis B or the subject The response for the treatment of；Wherein, the mark is selected from OAS2 or USP18.

In certain preferred aspects, the kit also includes IFN α and/or stimulant, and the stimulant is selected from HBV antigens, the anti-genic fragment of HBV antigens or its any combinations.

In certain preferred aspects, the reagent that can detect marker expression level is described for that can detect The reagent of the mRNA level in-site of mark.This kind of reagent is it is known in the art that including but is not limited to specific binding target sequence Nucleic acid probe, expand the primer of target sequence, non-specific fluorescence dyestuff (for example, SYBR Green I) or its combination. In some embodiments, the nucleic acid probe can be for single nucleic acid probe marked, such as radionuclide (such as³²P、³H、³⁵S Deng) label probe, biotinylated probe, horseradish peroxidase-labeled probe, digoxin labelled probe or fluorophor are (such as FITC, FAM, TET, HEX, TAMRA, Cy3, Cy5 etc.) label probe；The nucleic acid probe can also be visited for the nucleic acid of double labelling Pin, such as Taqman probes, molecular beacon, displacement probe, scorpions probe, QUAL probes, FRET probes.In some realities Apply in mode, the reagent that can detect marker expression level includes Taqman probes.In some embodiments, it is described OAS2 cDNA has such as SEQ ID NO:Nucleotide sequence shown in 1.In some embodiments, the cDNA of the USP18 With such as SEQ ID NO:Nucleotide sequence shown in 2.

The mRNA level in-site of the mark, such as fluorescent quantitation can be determined using mRNA detection methods known in the art PCR, Northern blotting, hybridization in situ, or including mRNA amplifications (such as reverse transcription PCR) and the mRNA amplified productions Quantitative (such as electrophoresis and dyeing) method.In certain preferred aspects, it is described to detect marker expression water Flat reagent quantitative determines the mRNA level in-site of the mark by PCR.It is described to examine in a specific embodiment The reagent for surveying mark expression determines the mRNA level in-site of the mark by quantitative fluorescent PCR.

In certain preferred aspects, the reagent that can detect marker expression level is described for that can detect The reagent of the protein level of mark.This kind of reagent is well known in the art, and including but is not limited to can be with OAS2 or USP18 egg Antibody, target polypeptide or the aptamer specifically bound in vain.In some embodiments, this kind of reagent carries detectable mark Note, such as enzyme (as horseradish peroxidase, alkaline phosphatase), radionuclide (such as³H、¹²⁵I、³⁵S、¹⁴C、³²P etc.), it is glimmering Photoinitiator dye (such as FITC, TRITC, PE, Texas Red, quantum dot, Cy7, Alexa 750), acridine ester type compound, magnetic bead (for example,), collaurum or coloured glass or plastics (for example, polystyrene, polypropylene, latex, etc.) pearl, And for combining the biotin for the Avidin (for example, Streptavidin) that above-mentioned label is modified.

In certain preferred aspects, the reagent that can detect marker expression level passes through immunology detection To determine the protein level of OAS2 or USP18 in the sample.In certain preferred aspects, the immunology detection choosing From ELISA detections, Elispot detections, western blot or Surface Plasmon Resonance.In some embodiments, the energy The reagent of enough detection marker expression levels includes the antibody or its antigen-binding fragment of anti-OAS2 or USP18 albumen.

In certain preferred aspects, the kit, which is also included, can detect the examination of reference gene expression Agent, expression relative constancy of the reference gene in each tissue and cell, and will not be sent out under conditions of the effect of processing factor Raw expression changes, such as house-keeping gene；Generally, house-keeping gene coding egg necessary to maintaining radical cellular activities White matter, it includes but is not limited to β-actin, GAPDH, 18S rRNA, β 2-MG, UBC or α-tubulin.In some preferred realities Apply in scheme, the reagent that can detect reference gene expression is that can detect the mRNA level in-site of the reference gene Reagent.In certain preferred aspects, the reagent that can detect reference gene expression is described for that can detect The reagent of the protein level of reference gene.

In certain preferred aspects, the kit also comprising one or more selected from 1) -6) reagent or dress Put：

1) it is used for the diluent of dilute sample, such as phosphate buffer or physiological saline；

2) it is used for the anti-coagulants for preventing blood clotting, such as heparin；

3) it is used for the reagent for maintaining PBMC activity, such as culture medium or nutrient solution；

4) it is used for the reagent for separating PBMC, such as such as lymphocyte separation medium, Ficoll-Hypaque solution；

5) blood-taking device, such as apyrogeneity vacuum blood collection tube；With

6) PBMC separators, such as PBMC cell Separating tubes.

In certain preferred aspects, the kit includes being used for the reagent for maintaining PBMC activity (for example, culture Base or nutrient solution), and/or for separating PBMC reagent (for example, lymphocyte separation medium, such as Ficoll-Hypaque are molten Liquid).

In certain preferred aspects, the kit predicts IFN α to trouble by the method comprised the steps The response that the therapeutic effect or the subject for having the subject of chronic hepatitis B are treated to IFN α：

(1) use can detect the reagent of marker expression level to determine described in the sample from the subject and indicate The expression of thing, wherein the mark is OAS2；With

(2) expression and reference value are compared, or the expression is carried out statistical analysis to be united Assay value is counted, and the statistical analysis value is compared with reference value, and judges whether the subject can treat to IFN α and is produced Raw response；

Wherein, the sample includes PMNC (PBMC), such as whole blood (such as anticoagulated whole blood), peripheral blood Mononuclearcell (PBMC) or peripheral blood tunica albuginea layer.

In certain preferred aspects, when the expression is more than the reference value, or when the expression water When flat statistical analysis value is more than the reference value, shows that the subject can produce response to IFN α treatment, suitably receive IFN α is treated；When the expression is not more than the reference value, or when the statistical analysis value of the expression be not more than it is described During reference value, show that the subject will not produce response to IFN α treatment, be not suitable for receiving IFN α treatment.

In certain preferred aspects, in step (2), using Logist ic regression models to the expression water It is flat to carry out statistical analysis.

In certain preferred aspects, in order to eliminate the difference of the mark initial value to be measured between sample and sample It is different, so as to have comparativity between sample, can generally normalizing be carried out to the expression value of mark to be measured in different samples Change is handled, for example, be compared the expression value of the expression value of mark to be measured in sample and reference gene.Therefore, In step (1), the expression of the mark is normalization expression.The normalization expression can be by following Illustrative methods are obtained：Institute in the sample from the subject is determined using the reagent that can detect reference gene expression The expression of reference gene is stated, and the expression of the expression of the mark and the reference gene is compared Compared with to obtain the normalization expression of the mark.In a specific embodiment, in step (1), the mark The expression of will thing is normalization mRNA level in-site, and it can be obtained by following exemplary method：Will be to be measured using Ct methods are compared The mRNA level in-site of mark described in sample and the mRNA level in-site of the reference gene are compared, and obtain 2^-ΔCtIt is worth (Δ Ct= Ct_{Mark to be measured}-Ct_{Reference gene}), the value is the normalization mRNA level in-site of the mark, namely the expression quantity of the mark is compared In the change multiple of the expression quantity of reference gene.

In certain preferred aspects, before step (1), methods described also comprise the following steps in one or It is multinomial：(a) sample is obtained from the subject using blood-taking device；(b) blood-taking device is handled or from described using anti-coagulants The sample of subject；(c) using sample of the agent treatment from the subject for being used to maintain PBMC activity；(d) using dilute Release sample of the dilution agent from the subject；(e) using be used to separating PBMC reagent or PBMC separators from from Separation obtains PBMC in the sample of the subject.

(1) IFN α is used to stimulate at least a sample from the subject as testing sample, and will be without IFN α The sample of stimulation is used as control sample；Or, use at least a sample of IFN α and stimulant Co stituation from the subject Product will stimulate through the stimulant as testing sample but be used as control sample without the sample that IFN α is stimulated；Wherein, it is described Stimulant is selected from HBV antigens, the anti-genic fragment of HBV antigens or its any combinations；

(2) use can detect mark described in each sample in the reagent determination step (1) of marker expression level Expression, and obtain the expression of the mark in the testing sample compared to the mark in the control sample Expression change multiple, using the change multiple as mark described in the testing sample relative expression levels, Wherein described mark is selected from OAS2 or USP18；With

(3) relative expression levels are compared with reference value, or statistical analysis is carried out to the relative expression levels To obtain statistical analysis value, and the statistical analysis value is compared with reference value, and judges whether the subject can be to IFN α treatments produce response；

In certain preferred aspects, when the relative expression levels are less than the reference value, or when the phase When being less than the reference value to the statistical analysis value of expression, show that the subject can produce response to IFN α treatment, fit Preferably receive IFN α treatment；When the relative expression levels are not less than the reference value, or when the system of the relative expression levels When counting assay value not less than the reference value, show that the subject will not produce response to IFN α treatment, be not suitable for receiving IFN α is treated.

In certain preferred aspects, in step (2), the mark in the testing sample is obtained by comparing Ct methods The mRNA level in-site of will thing is compared to the change multiple of the mRNA level in-site of the mark in the control sample, i.e., described testing sample Described in mark mRNA relative expression levels.

In certain preferred aspects, in step (3), using Logist ic regression models to the relative table Statistical analysis is carried out up to level.

In certain preferred aspects, in order to eliminate the difference of the mark initial value to be measured between sample and sample It is different, so as to have comparativity between sample, can generally normalizing be carried out to the expression value of mark to be measured in different samples Change is handled, for example, be compared the expression value of the expression value of mark to be measured in sample and reference gene.Therefore, In step (2), the expression of the mark is normalization expression.The normalization expression can be by following Illustrative methods are obtained：Institute in the sample from the subject is determined using the reagent that can detect reference gene expression The expression of reference gene is stated, and the expression of the expression of the mark and the reference gene is compared Compared with to obtain the normalization expression of the mark.In a specific embodiment, in step (2), the mark The expression of will thing is normalization mRNA level in-site, and it can be obtained by following exemplary method：Will be to be measured using Ct methods are compared The mRNA level in-site of mark described in sample and the mRNA level in-site of the reference gene are compared, and obtain returning for the mark One changes mRNA level in-site.Further, the homogenization mRNA level in-site phase of the mark in the testing sample is obtained by comparing Ct methods Than the change multiple of the homogenization mRNA level in-site of the mark in the control sample, i.e., indicate described in described testing sample The mRNA relative expression levels of thing.

In another aspect, the invention provides for predicting therapeutic effect of the IFN α to the subject with chronic hepatitis B Or the method for response that the subject treats to IFN α, or the method for obtaining the result of diagnostic analysis, wherein the diagnosis point Analyse for predicting the response that IFN α is treated to the therapeutic effect of the subject with chronic hepatitis B or the subject to IFN α, its Comprise the steps：

(1) sample from the subject is provided；

(2) expression of mark in the sample is determined, wherein the mark is OAS2；With

(3) expression and reference value are compared, or the expression is carried out statistical analysis to be united Assay value is counted, and the statistical analysis value is compared with reference value, and judges whether the subject can treat to IFN α and is produced Raw response；

It is some preferred embodiment in, after step (3), methods described also include give the subject apply The step of IFN α is to treat chronic hepatitis B, wherein the subject is judged as that response can be produced to IFN α treatment.

In certain preferred aspects, in step (2), the mRNA level in-site or albumen water of the mark are determined It is flat.

In certain preferred aspects, in step (2), the mRNA level in-site of the mark is determined.It can use MRNA detection methods known in the art determine the mRNA level in-site of the mark, such as quantitative fluorescent PCR, Northern traces Method, hybridization in situ, or quantitative (such as electrophoresis including mRNA amplifications (such as reverse transcription PCR) and the mRNA amplified productions And dyeing) method.In certain preferred aspects, the mRNA level in-site of the mark is quantitative determined by PCR.One In individual specific embodiment, the mRNA level in-site of the mark is determined by quantitative fluorescent PCR.In some embodiments, The cDNA of the OAS2 has such as SEQ ID NO:Nucleotide sequence shown in 1.In some embodiments, the USP18 CDNA has such as SEQ ID NO:Nucleotide sequence shown in 2.

In certain preferred aspects, in step (2), the protein level of the mark is determined.Some excellent In the embodiment of choosing, in step (2), the protein level of the mark is determined by immunology detection.Further, exist In some preferred embodiments, the immunology detection is selected from ELISA detections, El ispot detections, western blot or table Face Plasmon Resonance.In some embodiments, in step (2), anti-OAS2 antibody or its antigen-binding fragment are used To detect OAS2 protein level.

In certain preferred aspects, in step (3), using Logistic regression models to the expression Carry out statistical analysis.

In certain preferred aspects, in order to eliminate the difference of the mark initial value to be measured between sample and sample It is different, so as to have comparativity between sample, can generally normalizing be carried out to the expression value of mark to be measured in different samples Change is handled, for example, be compared the expression value of the expression value of mark to be measured in sample and reference gene.Therefore, In step (2), the expression of the mark is normalization expression.The normalization expression can be by following Illustrative methods are obtained：Institute in the sample from the subject is determined using the reagent that can detect reference gene expression The expression of reference gene is stated, and the expression of the expression of the mark and the reference gene is compared Compared with to obtain the normalization expression of the mark.In a specific embodiment, in step (2), the mark The expression of will thing is normalization mRNA level in-site, and it can be obtained by following exemplary method：Will be to be measured using Ct methods are compared The mRNA level in-site of mark described in sample and the mRNA level in-site of the reference gene are compared, and obtain 2^-ΔCtIt is worth (Δ Ct= Ct_{Mark to be measured}-Ct_{Reference gene}), the value is the normalization mRNA level in-site of the mark, namely the expression quantity of the mark is compared In the change multiple of the expression quantity of reference gene.

In certain preferred aspects, before step (1), one or more in also comprising the following steps：(a) Sample is obtained from the subject；(b) anti-coagulants, such as heparin are added into the sample from the subject；(c) from from PBMC or the blood constituent (for example, peripheral blood tunica albuginea layer) containing PBMC are obtained in the sample of the subject；(d) to from institute State and nutrient solution or culture medium are added in the sample of subject；With sample of (e) dilution from the subject.

(1) at least two parts samples from the subject are provided；

(2) IFN α is used to stimulate at least a sample as testing sample, and using the sample stimulated without IFN α as right Product in the same old way；Or, at least a sample of IFN α and stimulant Co stituation from the subject is used as testing sample, and It will be stimulated through the stimulant but be used as control sample without the sample that IFN α is stimulated；Wherein, the stimulant is anti-selected from HBV The former, anti-genic fragment of HBV antigens or its any combinations；

(3) in determination step (2) in each sample mark expression, and obtain the mark in the testing sample The expression of thing compared to the expression of the mark in the control sample change multiple, using the change multiple as The relative expression levels of mark described in the testing sample, wherein the mark is selected from OAS2 or USP18；With

(4) relative expression levels are compared with reference value, or statistical analysis is carried out to the relative expression levels To obtain statistical analysis value, and the statistical analysis value is compared with reference value, and judges whether the subject can be to IFN α treatments produce response；

It is some preferred embodiment in, after step (4), methods described also include give the subject apply The step of IFN α is to treat chronic hepatitis B, wherein the subject is judged as that response can be produced to IFN α treatment.

In certain preferred aspects, in step (2), the HBV antigens are HBVcAg.In some preferred realities Apply in scheme, the HBVcAg has such as SEQ ID NO:Amino acid sequence shown in 38.In certain preferred aspects, The anti-genic fragment, which has, is selected from following amino acid sequence：SEQ ID NO:3-37.In a particularly preferred embodiment party In case, the stimulant is comprising respectively with such as SEQ ID NO:The anti-genic fragment of amino acid sequence shown in 3-37.

In certain preferred aspects, in step (3), the mRNA level in-site of the mark is determined.It can use MRNA detection methods known in the art determine the mRNA level in-site of the mark, such as quantitative fluorescent PCR, Northern traces Method, hybridization in situ, or quantitative (such as electrophoresis including mRNA amplifications (such as reverse transcription PCR) and the mRNA amplified productions And dyeing) method.In certain preferred aspects, the mRNA level in-site of the mark is quantitative determined by PCR.One In individual specific embodiment, the mRNA level in-site of the mark is determined by quantitative fluorescent PCR.In some embodiments, The cDNA of the OAS2 has such as SEQ ID NO:Nucleotide sequence shown in 1.In some embodiments, the USP18 CDNA has such as SEQ ID NO:Nucleotide sequence shown in 2.

In certain preferred aspects, in step (3), the protein level of the mark is determined.Some excellent In the embodiment of choosing, in step (3), the protein level of the mark is determined by immunology detection.Further, exist In some preferred embodiments, the immunology detection is selected from ELISA detections, Elispot detections, western blot or table Face Plasmon Resonance.In some embodiments, in step (3), using the antibody of anti-OAS2 or USP18 albumen or it is anti- Former binding fragment detects the protein level of the mark.

In certain preferred aspects, in step (3), the mark in the testing sample is obtained by comparing Ct methods The mRNA level in-site of will thing is compared to the change multiple of the mRNA level in-site of the mark in the control sample, i.e., described testing sample Described in mark mRNA relative expression levels.

In certain preferred aspects, in step (4), using Logistic regression models to the relative expression Level carries out statistical analysis.

In certain preferred aspects, in order to eliminate the difference of the mark initial value to be measured between sample and sample It is different, so as to have comparativity between sample, can generally normalizing be carried out to the expression value of mark to be measured in different samples Change is handled, for example, be compared the expression value of the expression value of mark to be measured in sample and reference gene.Therefore, In step (3), the expression of the mark is normalization expression.The normalization expression can be by following Illustrative methods are obtained：Institute in the sample from the subject is determined using the reagent that can detect reference gene expression The expression of reference gene is stated, and the expression of the expression of the mark and the reference gene is compared Compared with to obtain the normalization expression of the mark.In a specific embodiment, in step (3), the mark The expression of will thing is normalization mRNA level in-site, and it can be obtained by following exemplary method：Will be to be measured using Ct methods are compared The mRNA level in-site of mark described in sample and the mRNA level in-site of the reference gene are compared, and obtain returning for the mark One changes mRNA level in-site.Further, the homogenization mRNA level in-site phase of the mark in the testing sample is obtained by comparing Ct methods Than the change multiple of the homogenization mRNA level in-site of the mark in the control sample, i.e., indicate described in described testing sample The mRNA relative expression levels of thing.

The present invention also includes following exemplary embodiment：

1. the stimulation for external evoked interferon-induced gene (Interferon-stimulated genes, ISGs) Combination and/or the IFN α of thing, its total length comprising HbcAg or Partial Fragment.

2. the mark for predicting Chronic Hepatitis B interferon alfa effect, wherein the mark is interferon Induced gene (Interferon-stimulated genes, ISGs).

3. the mark of project 2, wherein the interferon-induced gene is selected from OAS2 and USP18.

4. screening the method for the mark for predicting Chronic Hepatitis B interferon alfa effect, methods described includes Following step：

1) whole blood sample before Chronic Hepatitis B medication is collected, PBMC is separated；

2) with the combination induction PBMC samples of HbcAg polypeptide segment or IFN α or HbcAg polypeptide segment and IFN α Product；

3) the PBMC samples after induction are collected, RNA is extracted, ISGs mRNA level in-sites are detected after reverse transcription；

4) make a collection of specimens clinical IFN α therapeutic response result, screens ISGs；

Wherein, difference ISGs mRNA level in-sites, IFN α under IFN α therapeutic response group inductive condition identical with unresponsive group are compared Therapeutic response group and unresponsive group of ISGs mRNA level in-site height it is different can as therapeutic effect foundation.

5. the method for project 4, wherein the HbcAg polypeptide segment is the 1-183 amino acids of HbcAg Polypeptide segment.

6. the kit for predicting Chronic Hepatitis B interferon alfa effect, it includes HbcAg polypeptide segment With the reagent of IFN α inducer, and detection mark OAS2 and USP18 level.

7. the kit of project 6, wherein the HbcAg polypeptide segment is the 1-183 bit aminos of HbcAg The polypeptide segment of acid.

8. detect that the reagent of mark OAS2 and/or USP18 level is being prepared for predicting Chronic Hepatitis B alpha interferon Purposes in the diagnosticum of therapeutic effect.

9. detect that the reagent of mark OAS2 and/or USP18 level is being prepared for predicting Chronic Hepatitis B alpha interferon Purposes in the kit of therapeutic effect.

10. mark OAS2 and/or USP18 are used for the purposes for predicting Chronic Hepatitis B interferon alfa effect.

11. the purposes in project any one of 8-10, wherein the detection of the mark OAS2 and/or USP18 includes quantifying Detect mark OAS2 and/or USP18 mRNA level in-site.

12. the purposes in project any one of 8-10, wherein quantitatively detecting mark OAS2 and/or USP18 by fluorescent PCR MRNA level in-site.

13. the purposes in project any one of 8-10, wherein the mRNA level in-site for detecting mark OAS2 and/or USP18 The sequence of primer and probe be：

The beneficial effect of invention

The present invention gropes to be found that OAS2 and/or USP18 expression are controlled to IFN α by many experiments and repeatedly Treat response and IFN α is treated in unresponsive the two colonies of CHB patient, there is significant difference, thereby establish a kind of letter It is single, easily and fast and with the therapeutic effect of high accuracy and specific prediction IFN α to the subject with chronic hepatitis B Or the Forecasting Methodology of response that the subject treats to IFN α.

Compared with prior art, technical scheme has the advantages that：

(1) find that Chronic Hepatitis B PBMC can be used in without or through OAS2 expressions after stimulated in vitro first pre- Survey the result of CHB patient's IFN α treatment；

(2) find that Chronic Hepatitis B PBMC USP18 expressions after stimulated in vitro can be used in predicting CHB first The result of patient's IFN α treatment；

(3) it is simple, convenient involved by：For example, by gathering whole blood, separation PBMC, being dispensed to cell training Support plate and add after IFN α, be subsequently placed in constant incubator and cultivate；Cultivate and mRNA or protein level are carried out after certain time Detection；

(4) to the less demanding of experiment condition, the technical capability of personnel, equipment and environment；

(5) it is high to IFN α therapeutic response prediction of result accuracy rate；

(6) cost is not high, it is easy to promote, wide using scope.

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but people in the art Member will be understood that drawings below and embodiment are merely to illustrate the present invention, rather than the restriction to the scope of the present invention.With reference to the accompanying drawings With the following detailed description of preferred embodiment, various purposes of the invention and favourable aspect are to those skilled in the art It will be apparent.

Brief description of the drawings

Fig. 1 shows that under the conditions of different disposal the PBMC of CHB patient OAS2mRNA levels are to IFN α therapeutic response There is significant difference between group (Rs) and unresponsive group (NRs).Wherein, Fig. 1 a show PBMC without external evoked, response group OAS2 mRNA level in-site is significantly higher than unresponsive group.Fig. 1 b show PBMC through IFN α it is external evoked after, response group OAS2 mRNA Relative expression levels are substantially less than unresponsive group.(note：* p is represented<0.05, * * * * represent p<0.0005)

Fig. 2 shows that under the conditions of different disposal the PBMC of CHB patient USP18mRNA levels are answered to IFN α treatment Answer between group (Rs) and unresponsive group (NRs) and there is significant difference.Wherein, Fig. 2 a show PBMC through IFN α it is external evoked after, should The mRNA relative expression levels for answering group USP18 are substantially less than unresponsive group；；Fig. 2 b show PBMC through IFN α and HBcAg peptide libraries After common induction, response group USP18 mRNA relative expression levels are substantially less than unresponsive group.(note：* * represent p< 0.001, * * * * represent p<0.0005)

Fig. 3 shows the ROC curve of OAS2N detection methods and OAS2 (IFNa+N-N) detection method.As a result show, OAS2N inspections The response that survey method and OAS2 (IFNa+N-N) detection methods treats for prediction CHB patient to IFN α be respectively provided with good sensitivity with Specificity.

Fig. 4 shows OAS2 (IFNa+N-N) detection method, USP18 (IFNa+N-N) detection methods and the combined detection ROC curve.As a result show, OAS2 (IFNa+N-N) detection method, USP18 (IFNa+N-N) detection methods and both joint-detections pair The response treated in prediction CHB patient to IFN α has good sensitivity and specificity.

Sequence information

In the table 1 that sequence information of the present invention is provided below.

Table 1：Sequence information

OAS2 cDNA nucleotide sequence (SEQ ID NO：1)

ATGGGAAATGGGGAGTCCCAGCTGTCCTCGGTGCCTGCTCAGAAGCTGGGTTGGTTTATCCAGGAATAC CTGAAGCCCTACGAAGAATGTCAGACACTGATCGACGAGATGGTGAACACCATCTGTGACGTCCTGCAGGAACCCGA ACAGTTCCCCCTGGTGCAGGGAGTGGCCATAGGTGGCTCCTATGGACGGAAAACAGTCTTAAGAGGCAACTCCGATG GTACCCTTGTCCTCTTCTTCAGTGACTTAAAACAATTCCAGGATCAGAAGAGAAGCCAACGTGACATCCTCGATAAA ACTGGGGATAAGCTGAAGTTCTGTCTGTTCACGAAGTGGTTGAAAAACAATTTCGAGATCCAGAAGTCCCTTGATGG GTTCACCATCCAGGTGTTCACAAAAAATCAGAGAATCTCTTTCGAGGTGCTGGCCGCCTTCAACGCTCTGAGCTTAA ATGATAATCCCAGCCCCTGGATCTATCGAGAGCTCAAAAGATCCTTGGATAAGACAAATGCCAGTCCTGGTGAGTTT GCAGTCTGCTTCACTGAACTCCAGCAGAAGTTTTTTGACAACCGTCCTGGAAAACTAAAGGATTTGATCCTCTTGAT AAAGCACTGGCATCAACAGTGCCAGAAAAAAATCAAGGATTTACCCTCGCTGTCTCCGTATGCCCTGGAGCTGCTTA CGGTGTATGCCTGGGAACAGGGGTGCAGAAAAGACAACTTTGACATTGCTGAAGGCGTCAGAACCGTTCTGGAGCTG ATCAAATGCCAGGAGAAGCTGTGTATCTATTGGATGGTCAACTACAACTTTGAAGATGAGACCATCAGGAACATCCT GCTGCACCAGCTCCAATCAGCGAGGCCAGTAATCTTGGATCCAGTTGACCCAACCAATAATGTGAGTGGAGATAAAA TATGCTGGCAATGGCTGAAAAAAGAAGCTCAAACCTGGTTGACTTCTCCCAACCTGGATAATGAGTTACCTGCACCA TCTTGGAATGTTCTGCCTGCACCACTCTTCACGACCCCAGGCCACCTTCTGGATAAGTTCATCAAGGAGTTTCTCCA GCCCAACAAATGCTTCCTAGAGCAGATTGACAGTGCTGTTAACATCATCCGTACATTCCTTAAAGAAAACTGCTTCC GACAATCAACAGCCAAGATCCAGATTGTCCGGGGAGGATCAACCGCCAAAGGCACAGCTCTGAAGACTGGCTCTGAT GCCGATCTCGTCGTGTTCCATAACTCACTTAAAAGCTACACCTCCCAAAAAAACGAGCGGCACAAAATCGTCAAGGA AATCCATGAACAGCTGAAAGCCTTTTGGAGGGAGAAGGAGGAGGAGCTTGAAGTCAGCTTTGAGCCTCCCAAGTGGA AGGCTCCCAGGGTGCTGAGCTTCTCTCTGAAATCCAAAGTCCTCAACGAAAGTGTCAGCTTTGATGTGCTTCCTGCC TTTAATGCACTGGGTCAGCTGAGTTCTGGCTCCACACCCAGCCCCGAGGTTTATGCAGGGCTCATTGATCTGTATAA ATCCTCGGACCTCCCGGGAGGAGAGTTTTCTACCTGTTTCACAGTCCTGCAGCGAAACTTCATTCGCTCCCGGCCCA CCAAACTAAAGGATTTAATTCGCCTGGTGAAGCACTGGTACAAAGAGTGTGAAAGGAAACTGAAGCCAAAGGGGTCT TTGCCCCCAAAGTATGCCTTGGAGCTGCTCACCATCTATGCCTGGGAGCAGGGGAGTGGAGTGCCGGATTTTGACAC TGCAGAAGGTTTCCGGACAGTCCTGGAGCTGGTCACACAATATCAGCAGCTCTGCATCTTCTGGAAGGTCAATTACA ACTTTGAAGATGAGACCGTGAGGAAGTTTCTACTGAGCCAGTTGCAGAAAACCAGGCCTGTGATCTTGGACCCAGCC GAACCCACAGGTGACGTGGGTGGAGGGGACCGTTGGTGTTGGCATCTTCTGGCAAAAGAAGCAAAGGAATGGTTATC CTCTCCCTGCTTCAAGGATGGGACTGGAAACCCAATACCACCTTGGAAAGTGCCGGTAAAAGTCATCTAA

USP18 cDNA nucleotide sequence (SEQ ID NO：2)

ATGAGCAAGGCGTTTGGGCTCCTGAGGCAAATCTGTCAGTCCATCCTGGCTGAGTCCTCGCAGTCCCCG GCAGATCTTGAAGAAAAGAAGGAAGAAGACAGCAACATGAAGAGAGAGCAGCCCAGAGAGCGTCCCAGGGCCTGGGA CTACCCTCATGGCCTGGTTGGTTTACACAACATTGGACAGACCTGCTGCCTTAACTCCTTGATTCAGGTGTTCGTAA TGAATGTGGACTTCACCAGGATATTGAAGAGGATCACGGTGCCCAGGGGAGCTGACGAGCAGAGGAGAAGCGTCCCT TTCCAGATGCTTCTGCTGCTGGAGAAGATGCAGGACAGCCGGCAGAAAGCAGTGCGGCCCCTGGAGCTGGCCTACTG CCTGCAGAAGTGCAACGTGCCCTTGTTTGTCCAACATGATGCTGCCCAACTGTACCTCAAACTCTGGAACCTGATTA AGGACCAGATCACTGATGTGCACTTGGTGGAGAGACTGCAGGCCCTGTATACGATCCGGGTGAAGGACTCCTTGATT TGCGTTGACTGTGCCATGGAGAGTAGCAGAAACAGCAGCATGCTCACCCTCCCACTTTCTCTTTTTGATGTGGACTC AAAGCCCCTGAAGACACTGGAGGACGCCCTGCACTGCTTCTTCCAGCCCAGGGAGTTATCAAGCAAAAGCAAGTGCT TCTGTGAGAACTGTGGGAAGAAGACCCGTGGGAAACAGGTCTTGAAGCTGACCCATTTGCCCCAGACCCTGACAATC CACCTCATGCGATTCTCCATCAGGAATTCACAGACGAGAAAGATCTGCCACTCCCTGTACTTCCCCCAGAGCTTGGA TTTCAGCCAGATCCTTCCAATGAAGCGAGAGTCTTGTGATGCTGAGGAGCAGTCTGGAGGGCAGTATGAGCTTTTTG CTGTGATTGCGCACGTGGGAATGGCAGACTCCGGTCATTACTGTGTCTACATCCGGAATGCTGTGGATGGAAAATGG TTCTGCTTCAATGACTCCAATATTTGCTTGGTGTCCTGGGAAGACATCCAGTGTACCTACGGAAATCCTAACTACCA CTGGCAGGAAACTGCATATCTTCTGGTTTACATGAAGATGGAGTGCTAA

HBcAg peptide libraries peptide section sequence (P1-P35) (SEQ ID NO：3~37)

P1:MDIDPYKEFGASVEL(SEQ ID NO：3)

P2:YKEFGASVELLSFLP(SEQ ID NO：4)

P3:ASVELLSFLPSDFFP(SEQ ID NO：5)

P4:LSFLPSDFFPSVRDL(SEQ ID NO：6)

P5:SDFFPSVRDLLDTAS(SEQ ID NO：7)

P6:SVRDLLDTASALYRE(SEQ ID NO：8)

P7:LDTASALYREALESP(SEQ ID NO：9)

P8:ALYREALESPEHCSP(SEQ ID NO：10)

P9:ALESPEHCSPHHTAL(SEQ ID NO：11)

P10:EHCSPHHTALRQAIL(SEQ ID NO：12)

P11:HHTALRQAILCWGEL(SEQ ID NO：13)

P12:RQAILCWGELMNLAT(SEQ ID NO：14)

P13:CWGELMNLATWVGSN(SEQ ID NO：15)

P14:MNLATWVGSNLEDPA(SEQ ID NO：16)

P15:WVGSNLEDPASRELV(SEQ ID NO：17)

P16:LEDPASRELVVSYVN(SEQ ID NO：18)

P17:SRELVVSYVNVNMGL(SEQ ID NO：19)

P18:VSYVNVNMGLKIRQL(SEQ ID NO：20)

P19:VNMGLKIRQLLWFHI(SEQ ID NO：21)

P20:KIRQLLWFHISCLTF(SEQ ID NO：22)

P21:LWFHISCLTFGRETV(SEQ ID NO：23)

P22:SCLTFGRETVLEYLV(SEQ ID NO：24)

P23:GRETVLEYLVSFGVW(SEQ ID NO：25)

P24:LEYLVSFGVWIRTPP(SEQ ID NO：26)

P25:SFGVWIRTPPAYRPP(SEQ ID NO：27)

P26:IRTPPAYRPPNAPIL(SEQ ID NO：28)

P27:AYRPPNAPILSTLPE(SEQ ID NO：29)

P28:NAPILSTLPETTVVR(SEQ ID NO：30)

P29:STLPETTVVRRRGRS(SEQ ID NO：31)

P30:TTVVRRRGRSPRRRT(SEQ ID NO：32)

P31:RRGRSPRRRTPSPRR(SEQ ID NO：33)

P32:PRRRTPSPRRRRSQS(SEQ ID NO：34)

P33:PSPRRRRSQSPRRRR(SEQ ID NO：35)

P34:RRSQSPRRRRSQSRE(SEQ ID NO：36)

P35:QSPRRRRSQSRESQC(SEQ ID NO：37)

HBcAg amino acid sequence (SEQ ID NO：38)

MDIDPYKEFGATVELLSFLPSDFFPSVRGLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLA TWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPET TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

Primer (5 ' -3 ') (SEQ ID NO：39~74)

USP18-Forward:CAGACCCTGACAATCCACCT(SEQ ID NO：39)

USP18-Reverse:AGCTCATACTGCCCTCCAGA(SEQ ID NO：40)

OAS2-Forward:TCAGCGAGGCCAGTAATCTT(SEQ ID NO：41)

OAS2-Reverse:GCAGAACATTCCAAGATGGT(SEQ ID NO：42)

LGP1-Forward:GCAGGAAGACAGTGGAGAGC(SEQ ID NO：43)

LGP1-Reverse:GAGCCAGCACTTCTGGGTAG(SEQ ID NO：44)

LAP3-Forward:GGTGCCATGGATGTAGCTTT(SEQ ID NO：45)

LAP3-Reverse:AGAGAGGCATCCTCCAGACA(SEQ ID NO：46)

GIP3-Forward:CTCGCTGATGAGCTGGTCT(SEQ ID NO：47)

GIP3-Reverse:ATACTTGTGGGTGGCGTAGC(SEQ ID NO：48)

CEB1-Forward:GATTGCTGGAGGGAATCAAA(SEQ ID NO：49)

CEB1-Reverse:TTGGATTTCCCTTTTTGTGC(SEQ ID NO：50)

ATF5-Forward:AGCCCCCTGTCTTGGATACT(SEQ ID NO：51)

ATF5-Reverse:CGAGAAGGTTGAGGTGGAGA(SEQ ID NO：52)

GIP2-Forward:CGCAGATCACCCAGAAGATC(SEQ ID NO：53)

GIP2-Reverse:GCCCTTGTTATTCCTCACCA(SEQ ID NO：54)

OAS3-Forward:GTCAAACCCAAGCCACAAGT(SEQ ID NO：55)

OAS3-Reverse:GGGCGAATGTTCACAAAGTT(SEQ ID NO：56)

RPLP2-Forward:GCTGTAGCCGTCTCTGCTG(SEQ ID NO：57)

RPLP2-Reverse:AAAAAGGCCAAATCCCATGT(SEQ ID NO：58)

STXBP5-Forward:GTTCATCTGATGGGCTTCGT(SEQ ID NO：59)

STXBP5-Reverse:TTTGTTGTGGTGGTTCTCCA(SEQ ID NO：60)

Viperin-Forward:CTTTTGCTGGGAAGCTCTTG(SEQ ID NO：61)

Viperin-Reverse:CAGCTGCTGCTTTCTCCTCT(SEQ ID NO：62)

RPS28-Forward:CCGTGTGCAGCCTATCAAG(SEQ ID NO：63)

RPS28-Reverse:TTTACATTGCGGATGATGGA(SEQ ID NO：64)

PI3KAP1-Forward:CTGCAGAGAGCTTTCCATCC(SEQ ID NO：65)

PI3KAP1-Reverse:GTCTCTGGCTCATCGTCACA(SEQ ID NO：66)

MX1-Forward:GTGCATTGCAGAAGGTCAGA(SEQ ID NO：67)

MX1-Reverse:CTGGTGATAGGCCATCAGGT(SEQ ID NO：68)

DUSP1-Forward:CCAACCATTTTGAGGGTCAC(SEQ ID NO：69)

DUSP1-Reverse:ACCCTTCCTCCAGCATTCTT(SEQ ID NO：70)

ETEF1-Forward:AGCGGAAGGAGGAGAAAAAG(SEQ ID NO：71)

ETEF1-Reverse:GTACTCTTGGGCAGGTGAGC(SEQ ID NO：72)

β-actin-Forward:CAAAGACCTGTACGCCAACACA(SEQ ID NO：73)

β-actin-Reverse:GGAGTACTTGCGCTCAGGAGG(SEQ ID NO：74)

Probe (5 ' -3 ') (SEQ ID NO：75~92)

USP18-probe:TCTGCCACTCCCTGTACTTCCCC(SEQ ID NO：75)

OAS2-probe:AAAATATGCTGGCAATGGCTGAA(SEQ ID NO：76)

LGP1-probe:CTTGGGGAGGCCTCTCTGCAG(SEQ ID NO：77)

LAP3-probe:AATTCATCCTGGCTCTGGAACAA(SEQ ID NO：78)

GIP3-probe:TAGTGGCCACGCTGCAGAGC(SEQ ID NO：79)

CEB1-probe:TCCTACTCTGAATGAAGGGACTGTAA(SEQ ID NO：80)

ATF5-probe:AACGAGGCCGGGCAGGAGGA(SEQ ID NO：81)

GIP2-probe:TCTGGCTGTCCACCCGAGCG(SEQ ID NO：82)

OAS3-probe:TCAACAGTGGCTGCCAAGGG(SEQ ID NO：83)

RPLP2-probe:TGCTGCTGGTTCTGCCCCTG(SEQ ID NO：84)

STXBP5-probe:AACTCACCACTTAAACAGTCTCCAGG(SEQ ID NO：85)

Viperin-probe:TGGTCCCGCTGTTCTGCTGG(SEQ ID NO：86)

RPS28-probe:TTCTCAGGGACAGTGCACGCAG(SEQ ID NO：87)

PI3KAP1-probe:AGGCTGCTCTGCGGCGTGCG(SEQ ID NO：88)

MX1-probe:ATCCTGGGATTTTGGGGCTTTC(SEQ ID NO：89)

DUSP1-probe:AGCATCCCTGTGGAGGACAACC(SEQ ID NO：90)

ETEF1-probe:AGGAGGAGATGGATGAATGTGAGCA(SEQ ID NO：91)

β-actin-probe:CCGACAGGATGCAGAAGGAGATCAC(SEQ ID NO：92)

Embodiment

The present invention is described referring now to the following embodiment for being intended to illustration (and non-limiting present invention) of the invention.Unless Specialize, otherwise carried out basically according to known in the art and described in various bibliography conventional methods real Apply the experiment described in example and method (for example, experimental methods of molecular biology and immunodetection).See, e.g., Sambrook et al., Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.(1989)；With Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), its whole passes through Reference is merged into herein.Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market. Those skilled in the art know that embodiment describes the present invention by way of example, and it is claimed to be not intended to limit the present invention Scope.Entire disclosure case mentioned in this article and other references are merged into herein with its full text by quoting.

The peripheral blood PBMC of embodiment 1. is separated and external evoked culture

1.1 peripheral blood PBMC are separated

(1) whole blood is centrifuged into 10min under the conditions of 4000rpm.

(2) serum is suctioned out, plus the PBS of equivalent dilutes remaining whole blood.

(3) prepare 15mL centrifuge tube, add lymphocyte separation medium (the Ficoll-page Tm with whole blood equivalent PLUS,GE Helathcare Life Sciences)。

(4) whole blood after dilution is slowly added on lymphocyte separation medium along tube wall, keeps stratification state.

(5) aforesaid liquid is centrifuged into 22min under the conditions of 1000g, and the speed of raising speed and reduction of speed is transferred to minimum.

(6) it is careful to draw white cloud buffy coat, into another centrifuge tube, centrifuged after adding PBS dilutions, 600g × 7min, abandons supernatant.PBS dilutions are added, 400g × 7min is centrifuged, abandons supernatant.Cell is resuspended with 1ml nutrient solution.

(7) 0.4% trypan blue that 10 μ l have prepared sample cell suspension and 40 μ l is added in 1.5ml centrifuge tubes, is prepared 1：5 cell diluents.10 μ l trypan blues/mixing with cells suspension is taken to add blood cell counting plate and carry out viable count, wherein Every milliliter of cell quantity=cell count × extension rate × 10⁴。

The external evoked culture of 1.2 IFN αs and/or stimulant to PBMC

Above-mentioned PBMC cells are inoculated in 24 orifice plates, 5 × 10 are added per hole⁵Individual cell, and 1ml is added containing 10% tire ox The RPM1640 culture mediums of serum.Every part of sample sets four holes altogether, wherein the first hole is without the inducing substance (inducer Matter includes IFN α and HBcAg peptide libraries) N is labeled as, (Anda is fragrant, Anhui peace section bioengineering (group) for the second hole addition IFN α Limited company) (1 × 10⁴IU/mL), the 3rd hole addition contains 35 articles of HBcAg anti-genic fragments (SEQ ID NO:3-37) Peptide library (bioengineering limited company synthesizes by Shanghai) (1 μ g/ bars/mL, totally 35), above-mentioned IFN α is added in the 4th hole With HBcAg peptide libraries.Culture plate is placed in 37 DEG C of CO2gas incubators after Fiber differentiation 24h, cell is collected.

The fluorescence quantitative PCR method of embodiment 2. detects the mRNA level in-site of gene

The present invention to including USP18, OAS2, LGP1, LAP3, GIP3, CEB1, ATF5, GIP2, OAS3, RPLP2, It is glimmering that 17 kinds of genes such as STXBP5, Viperin, RPS28, PI3KAP1, MX1, DUSP1, ETEF1 and internal reference β-actin are established Fluorescent Quantitative PCR detection architecture.

RNA extraction in 2.1 PBMC

(1) PBMC cells, supernatant discarding obtained in embodiment 1 is collected by centrifugation；250 μ l RPMI1640 trainings are added per hole Base and 750 μ l Tripure are supported, and are well mixed；200 μ l chloroforms, mixing of turning upside down, 4 DEG C of standings are added in per 1ml cells 10min。

(2) 12,000rpm, 4 DEG C, centrifuge 15min.

(3) after above-mentioned processing, often manage interior sample and be divided into three layers, draw one layer of the top and (be careful not to be drawn onto albumen Layer) it is transferred in a new EP pipes treated with DEPC, plus isometric isopropanol, mixing of turning upside down, -20 DEG C of standings 15min。

(4) 12,000rpm, 4 DEG C, 10min is centrifuged, supernatant is abandoned.

(5) add the ethanol of 1mL 75% to wash twice, 12,000rpm, 4 DEG C, centrifuge 5min.

(6) ethanol is discarded, air air-dries 5-10min, dissolves RNA with the 55-60 DEG C of preheated μ L of DEPC water 35, extract RNA freeze and preserved in -80 DEG C of refrigerators.

2.2 RNA reverse transcriptions

Using the concentration and purity of spectrophotometric determination RNA sample., 2 μ L RNA samples are taken in 2 μ L RNase-free A260 and A280 light absorption values, detection A260 and A280 ratios are determined in water calibration instruments so as to evaluate the purity of RNA sample, After testing, RNA purity：A260/280≥1.80.

Reverse transcription system：

By above-mentioned reverse transcription system, in 42 DEG C of reverse transcription 30min.The cDNA of acquisition is fluorescence quantitative PCR detection body Template in system, is stored in -20 DEG C.

2.3 quantitative fluorescent PCR Establishings

(1) RT-PCR systems：

Amplified reaction process：

95 DEG C of 3min, then 95 DEG C of 10sec, 55 DEG C of 45sec, totally 39 circulations.

It is used for RT-PCR detection primer and probe using primer Software for Design, and by Ying Jun Bioisystech Co., Ltd Synthesis, particular sequence see the table below 2：

Table 2. is used for the primer and probe that quantitative fluorescent PCR reacts

(2) primer preliminary experiment

To carrying out preliminary experiment with 18 sets of primers (table 2 above) of primer Software for Design, randomly select two parts and suffer from from CHB The template (being respectively labeled as CHB 1, CHB 2) of person, passes through fluorescence quantitative PCR detection testing gene and the Ct values of internal control primer. As shown in table 3, the primer and internal reference β-actin primer Ct values of 17 sets of testing genes show it to qualification result between 15-35 Can effectively it be expanded；Neg groups (being free of template) are not detected by effective amplification simultaneously, further demonstrate that listed in table 2 above The 18 sets of primers lifted can be used for the fluorescence quantitative PCR detection of corresponding testing gene.

The preliminary experiment of primer in the fluorescence quantitative PCR detection system of table 3.

Differential expression of embodiment 3.OAS2, USP18 in IFN α therapeutic response and unresponsive CHB patient

Clinic has followed the trail of 150 chronic hepatitis B samples to the present invention altogether, wherein receiving that IFN α is treated and having 24 weeks treatment knots Totally 66 of fruit；Wherein to unresponsive totally 47 of IFN α treatment, to IFN α therapeutic response totally 19.Table 4 shows the present embodiment It is selected in the characteristic of research sample, as a result shows, response group (Rs) and unresponsive group (NRs) do not have in age, sex Significant difference, ALT, AST and HBsAg, HBV DNA, HBeAg results are also not significantly different before treatment.

The selected research sample feature of table 4.

Pair do not receive the Chronic Hepatitis B collection peripheral blood of IFN α treatment also, extracted according to the method for above-described embodiment 1 PBMC and PBMC is handled and embodiment 2 method extract RNA, carry out quantitative fluorescent PCR with detect calculate 17 it is to be measured Gene (USP18, OAS2, LGP1, LAP3, GIP3, CEB1, ATF5, GIP2, OAS3, RPLP2, STXBP5, Viperin, RPS28, PI3KAP1, MX1, DUSP1, ETEF1) mRNA level in-site, the mRNA level in-site passes through relative quantification method (comparing Ct methods) Calculating is obtained, and its calculation is：The Ct values of each target gene measured are subtracted to respective internal reference β-actin Ct values, obtained To Δ Ct values (the Δ Ct=Ct of each target gene_{Target gene}-Ct_{Reference gene}), calculate 2^-ΔCtValue, during represented by it is sample to be tested Change multiple (that is, gene normalizing relative to reference gene of the expression quantity of the gene relative to the expression quantity of reference gene Change mRNA level in-site)；Then the Δ Ct values of the gene of this in sample to be tested and the Δ Ct values of the gene of this in check sample are subtracted each other, obtained Δ Δ Ct values (Δ Δ Ct=Δs Ct_{Sample to be tested}-ΔCt_{Check sample}), finally calculate 2^-ΔΔCtValue, represented by it is the sample to be tested base (that is, the gene of this in sample to be tested is relative to right relative to the change multiple of the expression quantity of the check sample gene for the expression quantity of cause In the same old way in this gene mRNA relative expression levels).

By comparing the difference of the mRNA level in-site of each above-mentioned gene in response group and unresponsive group, find wherein OAS2 and There is notable difference between response group and unresponsive group in USP18 mRNA level in-site.As a result as depicted in figs. 1 and 2, wherein The PBMC cells of OAS2N groups without any external evoked, represented by the group be in sample to be tested OAS2 relative to reference gene Normalization mRNA level in-site；OAS2 (IFNa+N-N) group, USP18 (IFNa+N-N) groups and USP18 (IFNa+HBcAg-HBcAg) As shown in table 5, the specific steps of its external evoked culture are carried out the external evoked conditions of PBMC of group with reference to embodiment 1.2, wherein, Represented by OAS2 (IFNa+N-N) groups is mRNA relative expression water of the OAS2 relative to OAS2 in check sample in sample to be tested It is flat；That USP18 (IFNa+N-N) groups and USP18 (IFNa+HBcAg-HBcAg) groups are represented respectively is USP18 in sample to be tested Relative to the mRNA relative expression levels of USP18 in respective check sample.

The external evoked condition of sample to be tested and check sample in the different groups of table 5.

Fig. 1 a show the testing result of OAS2N groups, as a result show, IFN is received without any external evoked CHB patient In PBMC samples before α treatments, the OAS2 normalization mRNA level in-site of response group patient is significantly higher than unresponsive group of patient.Fig. 1 b The testing result of OAS2 (IFNa+N-N) groups is shown, is as a result shown, the CHB patient through the external evoked culture of IFN α receives IFN α In PBMC samples before treatment, the OAS2 of response group patient mRNA relative expression levels are substantially less than unresponsive group of patient.

Fig. 2 a show the testing result of USP18 (IFNa+N-N) groups, as a result show, through the external evoked culture of IFN α CHB patient receives in the PBMC samples before IFN α treatment, and the USP18 of response group patient mRNA relative expression levels are significantly low In unresponsive group of patient.Fig. 2 b show the testing result of USP18 (IFNa+HBcAg-HBcAg) groups, as a result show, through IFN α (contain SEQ ID NO with HBcAg peptide libraries:HBcAg anti-genic fragments shown in 3-37) the CHB patient of external evoked culture connects In PBMC samples before being treated by IFN α, the USP18 of response group patient mRNA relative expression levels are substantially less than unresponsive Group patient.

The above results show, to OAS2 levels in PBMC samples before the treatment of IFN α therapeutic response patient compared to IFN α Unresponsive patient is treated significantly to raise；Meanwhile, to PBMC samples before the treatment of IFN α therapeutic response patient through IFN α and/or Wherein OAS2 and/or UPS18 levels are significantly reduced compared to the unresponsive patient of IFN α treatment after HBcAg is external evoked.Therefore OAS2 and/or UPS18 can be used for prediction CHB patient to IFN α therapeutic response.

The ROC curve analysis of embodiment 4.OAS2 N detection methods, OAS2 (IFNa+N-N) detection method

The experiment condition organized according to OAS2 N groups in embodiment 3 and OAS2 (IFNa+N-N), is established corresponding respectively OAS2 N detection methods and OAS2 (IFNa+N-N) detection method, table 6 describe the key step of above two method.The present embodiment is adopted CHB clinical samples described in embodiment 3 are detected respectively with two methods as described above, sample is obtained respectively This two diagnosis indexs：OAS2 normalizes mRNA level in-site and OAS2 mRNA relative expression levels；Then SPSS 17.0 is used Software draws ROC curve to examine using OAS2 normalization mRNA level in-sites and OAS2 mRNA relative expression levels as diagnosis index OAS2 N detection methods or OAS2 (IFNa+N-N) detection methods is used alone in therapeutic effect or CHB of the prediction IFN α to CHB patient Performance in terms of the response that patient treats to IFN α.As a result ROC curve analysis result show as shown in Fig. 3 and table 7, OAS2 N Area (AUC) is respectively 0.774,0.814 under the ROC curve of detection method and OAS2 (IFNa+N-N) detection method；Sensitivity is distinguished For 72%, 71%；Specificity is respectively 80%, 83%.

The above results show that 2 kinds of detection methods are respectively provided with higher sensitivity and specificity, and OAS2N detections are used alone Method or OAS2 (IFNa+N-N) detection method can be used for prediction IFN α and the therapeutic effect of CHB patient or CHB patient controlled IFN α The response for the treatment of, with higher sensitivity and specificity.

The key step of table 6.OAS2 N detection methods and OAS2 (IFNa+N-N) detection method

The ROC analyses of table 7.OAS2 N detection methods, OAS2 (IFNa+N-N) detection method

The ROC of embodiment 5.OAS2 (IFNa+N-N) detection method, USP18 (IFNa+N-N) detection methods and the combined detection Tracing analysis

According to the experiment condition that OAS2 (IFNa+N-N) groups and USP18 (IFNa+N-N) are organized in embodiment 3, establish respectively Corresponding OAS2 (IFNa+N-N) detection methods and USP18 (IFNa+N-N) detection method, table 8 describe the main of above two method Step.The present embodiment employs two methods as described above and CHB clinical samples described in embodiment 3 is carried out respectively Detection, obtains two diagnosis indexs respectively：OAS2 mRNA relative expression levels and USP18 mRNA relative expression levels；So Referred to afterwards using the softwares of SPSS 17.0 using the mRNA relative expression levels of OAS2 mRNA relative expression levels or USP18 as diagnosis Mark, draws ROC curve to examine and OAS2 (IFNa+N-N) detection methods or USP18 (IFNa+N-N) detection methods is used alone in prediction Performance in terms of the response that IFN α is treated to the therapeutic effect of CHB patient or CHB patient to IFN α；And using These parameters as Joint-detection index, by Logistic regression models, obtains statistical analysis value, ROC curve is drawn again with the value, evaluate two The estimated performance of person's joint-detection.As a result ROC curve analysis result show as shown in Fig. 4 and table 9, the ROC curve of 2 kinds of methods Lower area (AUC) is respectively 0.814,0.823；Sensitivity is respectively 71%, 60%；Specificity is respectively 83%, 95%.2 kinds The AUC of method joint-detection can reach 0.861, and sensitivity and specificity are respectively 74%, 89%.The above results show OAS2 (IFNa+N-N) detection method and USP18 (IFNa+N-N) detection methods and joint-detection are respectively provided with good sensitivity and specificity, Further either show that OAS2N detection methods or OAS2 (IFNa+N-N) detection method, or the combined detection is used alone, There is good sensitivity and special to the response that prediction IFN α is treated to the therapeutic effect of CHB patient or CHB patient to IFN α Property.

The key step of table 8.OAS2 (IFNa+N-N) detection methods and USP18 (IFNa+N-N) detection method

The ROC of table 9.OAS2 (IFNa+N-N) detection methods and USP18 (IFNa+N-N) detection methods and the combined detection Analysis

Although the embodiment of the present invention has obtained detailed description, it will be appreciated by those skilled in the art that：Root According to all teachings announced, various modifications and changes can be carried out to details, and these change the guarantor in the present invention Within the scope of shield.The whole of the present invention is divided into be provided by appended claims and its any equivalent.

Sequence table

<110>Xiamen University

<120>Method and kit for predicting the response that Chronic Hepatitis B is treated to IFN α

<130> IDC160055

<150> 201610121177.6

<151> 2016-03-04

<160> 92

<170> PatentIn version 3.5

<210> 1

<211> 2064

<212> DNA

<213>Homo sapiens（Homo sapiens）

<400> 1

atgggaaatg gggagtccca gctgtcctcg gtgcctgctc agaagctggg ttggtttatc 60

caggaatacc tgaagcccta cgaagaatgt cagacactga tcgacgagat ggtgaacacc 120

atctgtgacg tcctgcagga acccgaacag ttccccctgg tgcagggagt ggccataggt 180

ggctcctatg gacggaaaac agtcttaaga ggcaactccg atggtaccct tgtcctcttc 240

ttcagtgact taaaacaatt ccaggatcag aagagaagcc aacgtgacat cctcgataaa 300

actggggata agctgaagtt ctgtctgttc acgaagtggt tgaaaaacaa tttcgagatc 360

cagaagtccc ttgatgggtt caccatccag gtgttcacaa aaaatcagag aatctctttc 420

gaggtgctgg ccgccttcaa cgctctgagc ttaaatgata atcccagccc ctggatctat 480

cgagagctca aaagatcctt ggataagaca aatgccagtc ctggtgagtt tgcagtctgc 540

ttcactgaac tccagcagaa gttttttgac aaccgtcctg gaaaactaaa ggatttgatc 600

ctcttgataa agcactggca tcaacagtgc cagaaaaaaa tcaaggattt accctcgctg 660

tctccgtatg ccctggagct gcttacggtg tatgcctggg aacaggggtg cagaaaagac 720

aactttgaca ttgctgaagg cgtcagaacc gttctggagc tgatcaaatg ccaggagaag 780

ctgtgtatct attggatggt caactacaac tttgaagatg agaccatcag gaacatcctg 840

ctgcaccagc tccaatcagc gaggccagta atcttggatc cagttgaccc aaccaataat 900

gtgagtggag ataaaatatg ctggcaatgg ctgaaaaaag aagctcaaac ctggttgact 960

tctcccaacc tggataatga gttacctgca ccatcttgga atgttctgcc tgcaccactc 1020

ttcacgaccc caggccacct tctggataag ttcatcaagg agtttctcca gcccaacaaa 1080

tgcttcctag agcagattga cagtgctgtt aacatcatcc gtacattcct taaagaaaac 1140

tgcttccgac aatcaacagc caagatccag attgtccggg gaggatcaac cgccaaaggc 1200

acagctctga agactggctc tgatgccgat ctcgtcgtgt tccataactc acttaaaagc 1260

tacacctccc aaaaaaacga gcggcacaaa atcgtcaagg aaatccatga acagctgaaa 1320

gccttttgga gggagaagga ggaggagctt gaagtcagct ttgagcctcc caagtggaag 1380

gctcccaggg tgctgagctt ctctctgaaa tccaaagtcc tcaacgaaag tgtcagcttt 1440

gatgtgcttc ctgcctttaa tgcactgggt cagctgagtt ctggctccac acccagcccc 1500

gaggtttatg cagggctcat tgatctgtat aaatcctcgg acctcccggg aggagagttt 1560

tctacctgtt tcacagtcct gcagcgaaac ttcattcgct cccggcccac caaactaaag 1620

gatttaattc gcctggtgaa gcactggtac aaagagtgtg aaaggaaact gaagccaaag 1680

gggtctttgc ccccaaagta tgccttggag ctgctcacca tctatgcctg ggagcagggg 1740

agtggagtgc cggattttga cactgcagaa ggtttccgga cagtcctgga gctggtcaca 1800

caatatcagc agctctgcat cttctggaag gtcaattaca actttgaaga tgagaccgtg 1860

aggaagtttc tactgagcca gttgcagaaa accaggcctg tgatcttgga cccagccgaa 1920

cccacaggtg acgtgggtgg aggggaccgt tggtgttggc atcttctggc aaaagaagca 1980

aaggaatggt tatcctctcc ctgcttcaag gatgggactg gaaacccaat accaccttgg 2040

aaagtgccgg taaaagtcat ctaa 2064

<210> 2

<211> 1119

<212> DNA

<213>Homo sapiens（Homo sapiens）

<400> 2

atgagcaagg cgtttgggct cctgaggcaa atctgtcagt ccatcctggc tgagtcctcg 60

cagtccccgg cagatcttga agaaaagaag gaagaagaca gcaacatgaa gagagagcag 120

cccagagagc gtcccagggc ctgggactac cctcatggcc tggttggttt acacaacatt 180

ggacagacct gctgccttaa ctccttgatt caggtgttcg taatgaatgt ggacttcacc 240

aggatattga agaggatcac ggtgcccagg ggagctgacg agcagaggag aagcgtccct 300

ttccagatgc ttctgctgct ggagaagatg caggacagcc ggcagaaagc agtgcggccc 360

ctggagctgg cctactgcct gcagaagtgc aacgtgccct tgtttgtcca acatgatgct 420

gcccaactgt acctcaaact ctggaacctg attaaggacc agatcactga tgtgcacttg 480

gtggagagac tgcaggccct gtatacgatc cgggtgaagg actccttgat ttgcgttgac 540

tgtgccatgg agagtagcag aaacagcagc atgctcaccc tcccactttc tctttttgat 600

gtggactcaa agcccctgaa gacactggag gacgccctgc actgcttctt ccagcccagg 660

gagttatcaa gcaaaagcaa gtgcttctgt gagaactgtg ggaagaagac ccgtgggaaa 720

caggtcttga agctgaccca tttgccccag accctgacaa tccacctcat gcgattctcc 780

atcaggaatt cacagacgag aaagatctgc cactccctgt acttccccca gagcttggat 840

ttcagccaga tccttccaat gaagcgagag tcttgtgatg ctgaggagca gtctggaggg 900

cagtatgagc tttttgctgt gattgcgcac gtgggaatgg cagactccgg tcattactgt 960

gtctacatcc ggaatgctgt ggatggaaaa tggttctgct tcaatgactc caatatttgc 1020

ttggtgtcct gggaagacat ccagtgtacc tacggaaatc ctaactacca ctggcaggaa 1080

actgcatatc ttctggttta catgaagatg gagtgctaa 1119

<210> 3

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P1

<400> 3

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu

1 5 10 15

<210> 4

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P2

<400> 4

Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu Ser Phe Leu Pro

1 5 10 15

<210> 5

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P3

<400> 5

Ala Ser Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro

1 5 10 15

<210> 6

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P4

<400> 6

Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu

1 5 10 15

<210> 7

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P5

<400> 7

Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

1 5 10 15

<210> 8

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P6

<400> 8

Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu

1 5 10 15

<210> 9

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P7

<400> 9

Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro

1 5 10 15

<210> 10

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P8

<400> 10

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro

1 5 10 15

<210> 11

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P9

<400> 11

Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu

1 5 10 15

<210> 12

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P10

<400> 12

Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu

1 5 10 15

<210> 13

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P11

<400> 13

His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu

1 5 10 15

<210> 14

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P12

<400> 14

Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Asn Leu Ala Thr

1 5 10 15

<210> 15

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P13

<400> 15

Cys Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn

1 5 10 15

<210> 16

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P14

<400> 16

Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala

1 5 10 15

<210> 17

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P15

<400> 17

Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val

1 5 10 15

<210> 18

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P16

<400> 18

Leu Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn

1 5 10 15

<210> 19

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P17

<400> 19

Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu

1 5 10 15

<210> 20

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P18

<400> 20

Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu

1 5 10 15

<210> 21

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P19

<400> 21

Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile

1 5 10 15

<210> 22

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P20

<400> 22

Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe

1 5 10 15

<210> 23

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P21

<400> 23

Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

1 5 10 15

<210> 24

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P22

<400> 24

Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val

1 5 10 15

<210> 25

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P23

<400> 25

Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp

1 5 10 15

<210> 26

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P24

<400> 26

Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro

1 5 10 15

<210> 27

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P25

<400> 27

Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro

1 5 10 15

<210> 28

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P26

<400> 28

Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu

1 5 10 15

<210> 29

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P27

<400> 29

Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu

1 5 10 15

<210> 30

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P28

<400> 30

Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg

1 5 10 15

<210> 31

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P29

<400> 31

Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser

1 5 10 15

<210> 32

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P30

<400> 32

Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

1 5 10 15

<210> 33

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P31

<400> 33

Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg

1 5 10 15

<210> 34

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P32

<400> 34

Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser

1 5 10 15

<210> 35

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P33

<400> 35

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg

1 5 10 15

<210> 36

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P34

<400> 36

Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu

1 5 10 15

<210> 37

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> P35

<400> 37

Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln Cys

1 5 10 15

<210> 38

<211> 183

<212> PRT

<213>Hepatitis type B virus（Hepatitis B virus）

<400> 38

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Gly Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210> 39

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 39

cagaccctga caatccacct 20

<210> 40

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 40

agctcatact gccctccaga 20

<210> 41

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 41

tcagcgaggc cagtaatctt 20

<210> 42

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 42

gcagaacatt ccaagatggt 20

<210> 43

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 43

gcaggaagac agtggagagc 20

<210> 44

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 44

gagccagcac ttctgggtag 20

<210> 45

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 45

ggtgccatgg atgtagcttt 20

<210> 46

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 46

agagaggcat cctccagaca 20

<210> 47

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 47

ctcgctgatg agctggtct 19

<210> 48

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 48

atacttgtgg gtggcgtagc 20

<210> 49

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 49

gattgctgga gggaatcaaa 20

<210> 50

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 50

ttggatttcc ctttttgtgc 20

<210> 51

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 51

agccccctgt cttggatact 20

<210> 52

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 52

cgagaaggtt gaggtggaga 20

<210> 53

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 53

cgcagatcac ccagaagatc 20

<210> 54

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 54

gcccttgtta ttcctcacca 20

<210> 55

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 55

gtcaaaccca agccacaagt 20

<210> 56

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 56

gggcgaatgt tcacaaagtt 20

<210> 57

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 57

gctgtagccg tctctgctg 19

<210> 58

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 58

aaaaaggcca aatcccatgt 20

<210> 59

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 59

gttcatctga tgggcttcgt 20

<210> 60

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 60

tttgttgtgg tggttctcca 20

<210> 61

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 61

cttttgctgg gaagctcttg 20

<210> 62

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 62

cagctgctgc tttctcctct 20

<210> 63

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 63

ccgtgtgcag cctatcaag 19

<210> 64

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 64

tttacattgc ggatgatgga 20

<210> 65

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 65

ctgcagagag ctttccatcc 20

<210> 66

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 66

gtctctggct catcgtcaca 20

<210> 67

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 67

gtgcattgca gaaggtcaga 20

<210> 68

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 68

ctggtgatag gccatcaggt 20

<210> 69

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 69

ccaaccattt tgagggtcac 20

<210> 70

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 70

acccttcctc cagcattctt 20

<210> 71

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 71

agcggaagga ggagaaaaag 20

<210> 72

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 72

gtactcttgg gcaggtgagc 20

<210> 73

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 73

caaagacctg tacgccaaca ca 22

<210> 74

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>Primer

<400> 74

ggagtacttg cgctcaggag g 21

<210> 75

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 75

tctgccactc cctgtacttc ccc 23

<210> 76

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 76

aaaatatgct ggcaatggct gaa 23

<210> 77

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 77

cttggggagg cctctctgca g 21

<210> 78

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 78

aattcatcct ggctctggaa caa 23

<210> 79

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 79

tagtggccac gctgcagagc 20

<210> 80

<211> 26

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 80

tcctactctg aatgaaggga ctgtaa 26

<210> 81

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 81

aacgaggccg ggcaggagga 20

<210> 82

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 82

tctggctgtc cacccgagcg 20

<210> 83

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 83

tcaacagtgg ctgccaaggg 20

<210> 84

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 84

tgctgctggt tctgcccctg 20

<210> 85

<211> 26

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 85

aactcaccac ttaaacagtc tccagg 26

<210> 86

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 86

tggtcccgct gttctgctgg 20

<210> 87

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 87

ttctcaggga cagtgcacgc ag 22

<210> 88

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 88

aggctgctct gcggcgtgcg 20

<210> 89

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 89

atcctgggat tttggggctt tc 22

<210> 90

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 90

agcatccctg tggaggacaa cc 22

<210> 91

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 91

aggaggagat ggatgaatgt gagca 25

<210> 92

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223>Probe

<400> 92

ccgacaggat gcagaaggag atcac 25

Claims

1. a kind of kit, it includes the first reagent that can detect marker expression level, optionally also include IFN α and/or Stimulant；Wherein, the mark is selected from OAS2 or USP18, and the stimulant is selected from HBV antigens, the antigenic piece of HBV antigens Section or its any combinations；

Preferably, the HBV antigens are HBcAg；It is highly preferred that the HBcAg has such as SEQ ID NO:Amino shown in 38 Acid sequence；Preferably, the anti-genic fragment, which has, is selected from following amino acid sequence：SEQ ID NO:3-37；

Preferably, the stimulant is comprising respectively with such as SEQ ID NO:The antigenic piece of amino acid sequence shown in 3-37 Section；

Preferably, first reagent is the reagent for the mRNA level in-site or protein level that can detect the mark, is, for example, The reagent of the mRNA level in-site of the mark can be detected.

2. the kit described in claim 1, it optionally also includes comprising that can detect the first reagent of OAS2 expressions IFN α and/or stimulant.

3. the kit described in claim 1, it is comprising that can detect the first reagent and IFN α of USP18 expressions, optionally Ground also includes stimulant.

4. the kit described in claim any one of 1-3, it, which is also included, is used for the second examination for pre-processing the sample containing PBMC Agent, wherein second reagent includes being selected from following one or more reagents：Diluent for dilute sample is (for example, phosphorus Phthalate buffer or physiological saline)；Anti-coagulants (for example, heparin) for preventing blood clotting；For maintaining PBMC activity Reagent (for example, culture medium or nutrient solution)；With for separating PBMC reagent (for example, lymphocyte separation medium, such as poly- sugarcane Sugar-cardiografin solution)；It is highly preferred that second reagent includes being used for the reagent for maintaining PBMC activity (for example, culture medium Or nutrient solution), and/or for separating PBMC reagent (for example, lymphocyte separation medium, such as Ficoll-Hypaque are molten Liquid)；

Preferably, the kit also includes blood-taking device (such as apyrogeneity vacuum blood collection tube) and/or PBMC separator (examples Such as PBMC cell Separating tubes).

5. purposes of the reagent of marker expression level in reagent preparation box can be detected, the kit is used to predict IFN α The response that therapeutic effect or the subject to the subject with chronic hepatitis B are treated to IFN α；Wherein, the mark Selected from OAS2 or USP18；

Preferably, the kit also includes IFN α and/or stimulant, the stimulant be selected from HBV antigens, HBV antigens it is anti- Immunogenic fragment or its any combinations；

Preferably, the reagent that can detect marker expression level is that can determine the mRNA level in-site or egg of the mark The reagent of white level, is, for example, the reagent for the mRNA level in-site that can determine the mark；It is highly preferred that described can detect mark The reagent of will thing expression quantitative determines the mRNA level in-site of the mark by PCR；

Preferably, the kit also comprising one or more selected from 1) -6) reagent or device：

6) PBMC separators, such as PBMC cell Separating tubes；

Preferably, the kit includes the reagent (for example, culture medium or nutrient solution) for being used to maintain PBMC activity, and/or uses In separation PBMC reagent (for example, lymphocyte separation medium, such as Ficoll-Hypaque solution).

6. the purposes described in claim 5, wherein the kit predicts IFN α to trouble by the method comprised the steps The response that the therapeutic effect or the subject for having the subject of chronic hepatitis B are treated to IFN α：

(1) use can detect that the reagent of marker expression level determines mark described in the sample from the subject Expression, wherein the mark is OAS2；With

(2) expression and reference value are compared, or the expression is carried out statistical analysis to obtain statistical Analysis value, and the statistical analysis value is compared with reference value, and judge whether the subject can be answered IFN α treatment generation Answer；

Wherein, the sample includes PMNC (PBMC), and such as whole blood (such as anticoagulated whole blood), peripheral blood are single Nucleus (PBMC) or peripheral blood tunica albuginea layer；

Preferably, in step (2), statistical analysis is carried out to the expression using Logistic regression models；

Preferably, before step (1), methods described also comprise the following steps in one or more：(a) blood-taking device is used Sample is obtained from the subject；(b) blood-taking device or the sample from the subject are handled using anti-coagulants；(c) use Sample of the agent treatment from the subject for maintaining PBMC activity；(d) using dilution dilution agent from described tested The sample of person；Using the reagent or PBMC separator that are used to separate PBMC from sample from the subject divided (e) From acquisition PBMC.

7. the purposes described in claim 5, wherein the kit predicts IFN α to trouble by the method comprised the steps The response that the therapeutic effect or the subject for having the subject of chronic hepatitis B are treated to IFN α：

(1) use IFN α to stimulate at least a sample from the subject as testing sample, and will be stimulated without IFN α Sample be used as control sample；Or, made using at least a sample of IFN α and stimulant Co stituation from the subject For testing sample, and it will be stimulated through the stimulant but be used as control sample without the sample that IFN α is stimulated；Wherein, it is described to stimulate Thing is selected from HBV antigens, the anti-genic fragment of HBV antigens or its any combinations；

(2) use can detect the table of mark described in each sample in the reagent determination step (1) of marker expression level Up to level, and obtain table of the expression compared to the mark in the control sample of the mark in the testing sample Up to horizontal change multiple, using the change multiple as mark described in the testing sample relative expression levels, wherein The mark is selected from OAS2 or USP18；With

(3) relative expression levels are compared with reference value, or the relative expression levels is carried out statistical analysis to obtain Statistical analysis value is obtained, and the statistical analysis value is compared with reference value, and judges whether the subject can be controlled IFN α Treat and produce response；

Preferably, in step (2), by compare Ct methods obtain the mRNA level in-site of the mark in the testing sample compared to The change multiple of the mRNA level in-site of the mark in the control sample, i.e., the mRNA phases of mark described in described testing sample To expression；

Preferably, in step (3), statistical analysis is carried out to the relative expression levels using Logistic regression models；

8. for predicting that IFN α is answered IFN α treatment the therapeutic effect of the subject with chronic hepatitis B or the subject The method answered, or the method for obtaining the result of diagnostic analysis, wherein the diagnostic analysis is used to predict IFN α to chronic The response that the therapeutic effect of the subject of liver or the subject treat to IFN α, it comprises the steps：

(1) sample from the subject is provided；

(3) expression and reference value are compared, or the expression is carried out statistical analysis to obtain statistical Analysis value, and the statistical analysis value is compared with reference value, and judge whether the subject can be answered IFN α treatment generation Answer；

Preferably, in step (2), the mRNA level in-site or protein level of the mark is determined, for example, determines the mark MRNA level in-site；It is highly preferred that in step (2), the mRNA level in-site of the mark is quantitative determined by PCR；

Preferably, in step (3), statistical analysis is carried out to the expression using Logistic regression models；

Preferably, before step (1), one or more in also comprising the following steps：(a) sample is obtained from the subject Product；(b) anti-coagulants, such as heparin are added into the sample from the subject；(c) from the sample from the subject Obtain PBMC or the blood constituent (for example, peripheral blood tunica albuginea layer) containing PBMC；(d) add into the sample from the subject Enter nutrient solution or culture medium；With sample of (e) dilution from the subject.

9. for predicting that IFN α is answered IFN α treatment the therapeutic effect of the subject with chronic hepatitis B or the subject The method answered, or the method for obtaining the result of diagnostic analysis, wherein the diagnostic analysis is used to predict IFN α to chronic The response that the therapeutic effect of the subject of liver or the subject treat to IFN α, it comprises the steps：

(1) at least two parts samples from the subject are provided；

(2) IFN α is used to stimulate at least a sample to be used as control sample as testing sample, and using the sample stimulated without IFN α Product；Or, at least a sample of IFN α and stimulant Co stituation from the subject is used as testing sample, and will be through The stimulant stimulates but is used as control sample without the sample that IFN α is stimulated；Wherein, the stimulant is selected from HBV antigens, HBV The anti-genic fragment of antigen or its any combinations；

(3) in determination step (2) in each sample mark expression, and calculate the mark in the testing sample Expression compared to the expression of the mark in the control sample change multiple, using the change multiple as described The relative expression levels of mark described in testing sample, wherein the mark is selected from OAS2 or USP18；With

(4) relative expression levels are compared with reference value, or the relative expression levels is carried out statistical analysis to obtain Statistical analysis value is obtained, and the statistical analysis value is compared with reference value, and judges whether the subject can be controlled IFN α Treat and produce response；

Preferably, in step (2), the HBV antigens are HBVcAg；It is highly preferred that the HBVcAg has such as SEQ ID NO:Amino acid sequence shown in 38；Preferably, the anti-genic fragment, which has, is selected from following amino acid sequence：SEQ ID NO:3-37；It is highly preferred that the stimulant is comprising respectively with such as SEQ ID NO:The antigen of amino acid sequence shown in 3-37 Property fragment；

Preferably, in step (3), the mRNA level in-site or protein level of the mark are determined；For example determine the mark MRNA level in-site；It is highly preferred that in step (3), the mRNA level in-site of the mark is quantitative determined by PCR；

Preferably, in step (3), by compare Ct methods obtain the mRNA level in-site of the mark in the testing sample compared to The change multiple of the mRNA level in-site of the mark in the control sample, i.e., the mRNA phases of mark described in described testing sample To expression；

Preferably, in step (4), statistical analysis is carried out to the relative expression levels using Logistic regression models；