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

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 response of chronic hepatitis B patients to IFNα treatment

technical field

The present invention relates to markers for predicting the response of chronic hepatitis B patients to IFNα treatment. The present invention also relates to a method for predicting the response of chronic hepatitis B patients to IFNα treatment, which comprises the step of measuring the expression level of such markers in PBMCs of chronic hepatitis B patients. The invention also relates to kits for use in the above methods.

Background technique

Hepatitis B virus infection, especially chronic hepatitis B virus infection, is one of the most important public health problems in the world. At present, there are more than 350 million people with chronic hepatitis B virus infection in the world. Chronic hepatitis B virus infection can cause liver diseases such as chronic hepatitis B (CHB), cirrhosis (Liver cirrhosis, LC) and primary hepatocellular carcinoma (Hepatocellular carcinoma, HCC). Hepatitis virus infection and the related diseases caused by it cause more than 1 million deaths every year in the world.

Most patients with chronic HBV infection show no obvious clinical symptoms, and about 10-30% of them develop liver cirrhosis or liver cancer. Chronic HBV infection is a dynamic process, and its natural history of infection can be divided into 4 different stages that are not necessarily necessarily related: 1) immune tolerance stage, characterized by HBeAg positive, active serum HBV DNA replication, ALT The level is normal or low, and fibrosis generally does not occur; 2) Active immune phase/immune clearance phase, characterized by HBeAg positive but its level begins to decline, HBV DNA replication level decreases, serum HBsAg level decreases, ALT continues to increase or fluctuation. In this stage, the CTL response of patients is activated, prone to moderate or severe inflammatory reactions and liver fibrosis of varying duration; 3) Inactive carrier stage, in this stage, the HBV DNA of patients is in the low replication stage, and the DNA level is extremely low Even if it cannot be detected, the HBsAg level is significantly reduced, the ALT level is normal, there is no or only mild inflammation in the liver tissue, HBeAg is negative, and anti-HBe is positive. This period means that the patient’s HBV infection has been under immune control, and it also indicates a Long-term and good prognosis, most patients have a very low chance of developing liver cirrhosis or even liver cancer; 4) HBeAg-negative hepatitis stage, which belongs to the late stage of chronic hepatitis B, and its main features are periodic fluctuations in HBV DNA and ALT levels and active hepatitis , and mainly HBV viruses with mutations in the former C region and/or basic core promoter (BCP), unable to express or express very low levels of HBeAg.

HBV virus has no direct pathogenic effect on host cells, and its main pathogenic mechanism is that HBV virus causes host immune response, which in turn causes immune liver tissue damage. The treatment of chronic HBV infection requires different antiviral treatments according to the different conditions of individual patients. The purpose of antiviral therapy is to continuously suppress HBV virus, prevent the development or transformation of hepatitis to fibrosis, liver cirrhosis and even liver cancer, prolong the survival period of patients and improve the quality of life.

There are currently 8 drugs available for antiviral treatment of CHB, including 6 nucleoside analogs: lamivudine (LAM), telbivudine (LdT), emtricitabine (FTC), entecavir (ETV), Defovir (ADV), tenofovir (TDF); 2 kinds of interferons: ordinary interferon (IFNα) and pegylated interferon (PEG-IFNα). The main function of nucleoside analogs is to inhibit the replication of HBV virus by inhibiting the activity of HBV polymerase. In 1976, Greenberg first reported the efficacy of IFNα in the treatment of chronic hepatitis B. IFNα was the first drug approved by the FDA for the treatment of chronic hepatitis B. PEG-IFNα was also used as the first choice drug for the treatment of chronic hepatitis B. It has dual functions of antiviral and immune regulation. effect. The main advantage of IFNα is that it has no drug resistance and has immune-mediated control of HBV infection, so that patients have a higher chance of HBeAg seroconversion, more durable virological response and HBsAg clearance at the end of treatment, making patients The level of HBV DNA was maintained at the lower limit of detection.

Antiviral therapy must ensure a certain degree of virological suppression and be able to produce biochemical remission, histological recovery and prevention of complications. The ideal end point of HBV treatment is the disappearance of HBsAg, but it is difficult for existing antiviral drugs to achieve this goal, so a more realistic treatment end point is to be able to induce a sustained state of virological suppression. The antiviral response can be divided into biochemical level, serum level, virological level and histological level. All responses can be assessed at certain time points during and after treatment. Clinical studies have shown that the response rate of CHB patients treated with conventional IFNα for 6 months is only 25-40%. Determining the specific immune response ability of CHB patients against HBV may be able to predict the expected therapeutic effect of CHB patients. For a long time, due to the lack of clear anti-HBV immune response indicators, serum ALT levels in CHB patients have been used as an indirect surrogate indicator to measure the host's anti-HBV immunity, but the prediction results are not ideal. If the response to treatment can be predicted before treatment, it is of great significance for optimizing drug selection, improving treatment compliance and ensuring curative effect.

Therefore, there is a need in the art for markers that can predict the response of CHB patients to IFNα treatment simply, conveniently, quickly, with high accuracy and specificity.

Contents of the invention

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps of cell culture, biochemistry, nucleic acid chemistry, and immunology used herein are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "IFNα" or "interferon-alpha" includes all natural or recombinant interferon-alpha, particularly preferably human interferon-alpha, such as recombinant human interferon-alpha, including but not limited to IFNα-1b (available for example from Schering Corporation, Kenilworth, NJ A interferon), IFNα-2a (such as available from Hoffmann-La Roche, Nutley, NJ -A interferon) or IFNα-2b (such as available from Schering Corporation, Kenilworth, NJ -Interferon A); for example a mixture of natural alpha-type interferons including but not limited to IFNα-n1 (for example available from Sumitomo, Japan or Glaxo-Wellcome Ltd., London, Great Britain Interferon α-n1) or IFN α-n3 (such as Alferon® available from Interferon Sciences interferon). In the present invention, the term "IFNα" or "interferon-α" also includes any substance having biological activity of IFNα, such as mutated or modified IFNα, such as PEG derivative of IFNα (PEG-IFNα). In the present invention, the term "IFNα" or "α-type interferon" is not limited by any specific source, and can be obtained from commercially available sources or produced by conventional techniques known to those skilled in the art. The production methods include But not limited to biological source extraction method and genetic engineering extraction method, which are described in detail in eg "Pestka S. Arch Biochem Biophys. 1983 Feb 15; 221(1): 1-37" (which is incorporated herein by reference).

As used herein, the term "HBV antigen" refers to proteins present in hepatitis B virus (HBV) that can induce an immune response in the body, including hepatitis B virus core antigen (HBcAg), hepatitis B virus surface antigen (HBsAg) and hepatitis B virus E antigen (HBeAg).

As used herein, the term "HBcAg" refers to the core antigen protein of hepatitis B virus (HBV), which is well known to those skilled in the art (see, eg, GENBANK accession number: CAM31905.1).

In the present invention, when referring to the amino acid sequence of HBcAg, it is described with reference to the sequence shown in SEQ ID NO:38. However, those skilled in the art understand that in the amino acid sequence of HBcAg, mutations or variations (including but not limited to, substitutions, deletions and/or additions, such as different genotypes, genotypes or different serotypes) can be naturally produced or artificially introduced , serum subtype of HBcAg), without affecting its biological function. Therefore, in the present invention, the term "HBcAg" shall include all such sequences, including for example the sequence shown in SEQ ID NO: 38 and its natural or artificial variants. And, when describing the sequence fragment of HBcAg, it not only includes the sequence fragment of SEQ ID NO: 38, but also includes the corresponding sequence fragment in its natural or artificial variant. For example, the expression "amino acid residues 1-183 of HBcAg" includes amino acid residues 1-183 of SEQ ID NO: 38, and corresponding fragments in its variants (natural or artificial). In the present invention, the expression "corresponding fragments" refers to fragments in the sequences being compared that are at equivalent positions when the sequences are optimally aligned, ie when the sequences are aligned for the highest percentage identity.

In the present invention, the term "HBcAg" is not limited by any specific protein synthesis method, and can be produced by conventional techniques known to those skilled in the art, such as DNA recombinant technology or chemical synthesis technology.

As used herein, the expression "antigenic fragment of HBcAg" refers to a truncated amino acid sequence fragment (ie, a polypeptide) of the HBcAg protein, which fragment has the same biological activity as the corresponding full-length protein, That is, PBMCs from chronic hepatitis B patients can be stimulated and activated. For example, the amino acid sequence fragment shown in SEQ ID NO: 3-37 in the present invention is the antigenic fragment of HBcAg. In the present invention, the antigenic fragment is not limited by any specific method of synthesizing the polypeptide, and can be produced by conventional techniques known to those skilled in the art, such as DNA recombination technique or chemical synthesis technique.

In the present invention, HBcAg or antigenic fragments thereof (for example, antigenic fragments respectively having the amino acid sequences shown in SEQ ID NO: 3-37) can be obtained by DNA recombinant technology, for example, by using a cell-free expression system from the Polynucleotides of these proteins or polypeptides are obtained (cell-free expression systems include, for example, expression systems based on reticulocyte lysates, expression systems based on wheat germ extracts, and expression systems based on E. coli extracts); or by using in vivo expression Systems (eg, E. coli prokaryotic expression system, yeast eukaryotic expression system) are obtained from polynucleotides encoding these proteins or polypeptides. Alternatively, HBcAg or antigenic fragments thereof (eg, antigenic fragments having the amino acid sequences shown in SEQ ID NO: 3-37, respectively) can be produced by chemical synthesis. Methods for chemical total synthesis of proteins or polypeptides are 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; and Wang P, et al., Tetrahedron Lett, 1998, 39(47):88711-14; which is incorporated herein by reference), and includes, but is not limited to: Solid Phase Peptide Synthesis (SPPS) or liquid phase fractional synthesis (e.g., Native Chemical Ligation (NCL), stacking Nitrogen method (Azide method), transfer activated ester method (Transfer Active Ester Condensation, TAEC)).

As used herein, the expression "antigenic fragments respectively having the amino acid sequences shown in SEQ ID NO:3-37" refers to the antigenic fragments having the amino acid sequences shown in SEQ ID NO:3, having the A combination of an antigenic fragment of the amino acid sequence shown in ID NO:4, ... an antigenic fragment of the amino acid sequence shown in SEQ ID NO:36, and an antigenic fragment of the amino acid sequence shown in SEQ ID NO:37.

As used herein, the term "immunological detection" refers to an assay that utilizes specific antigen-antibody interaction/binding affinity, which can generally be used to detect the presence or presence of a specific antigen or antibody in a sample. Level. Such immunological assays are well known to those skilled in the art and include, but are not limited to, ELISA assays, Elispot assays, Western blots, surface plasmon resonance methods, and the like. For a detailed description of immunological assays, see, eg, Fundamental Immunology, Ch. 7 Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989).

As used herein, the term "antibody" refers to an immunoglobulin molecule, usually composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows the 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 ; Definition by Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

As used herein, an "antigen-binding fragment" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (e.g., OAS2 or USP18) to which the antibody binds, capable of interacting with Intact antibodies compete for specific binding to the antigen. See generally, Fundamental Immunology, Ch. 7 Paul, W., ed., 2nd Ed., Raven Press, N.Y. (1989), which is hereby incorporated by reference in its entirety for all purposes. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some instances, antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabody) and polypeptides comprising at least a portion of an antibody sufficient to confer on the polypeptide the ability to specifically bind an antigen.

As used herein, the term "nucleic acid aptamer (Aptamer)" refers to a single-stranded oligonucleotide capable of binding a target protein of interest or other biological target molecules with high affinity and high specificity, which can be folded to form For example, the thermodynamically stable three-dimensional space structure of stem-loop (Stem-Loop), hairpin (Hairpin), pseudoknot (Pseudoknot) or G-tetramer (G-tetramer), through such as structural complementarity, base stacking force, van der Waals Force, hydrogen bonding or electrostatic interaction to specifically bind to the target protein of interest or other biological target molecules. Nucleic acid aptamers can be DNA or RNA, and can also include nucleic acid analogs such as locked nucleic acid (LNA), peptide nucleic acid (PNA), diol nucleic acid (GNA) or threose nucleic acid (TNA)). Methods for obtaining nucleic acid aptamers that bind to specific target proteins are well known in the art, such as SELEX (Systematic evolution of ligands by exponential enrichment) screening technology.

As used herein, the term "targeting polypeptide" refers to a polypeptide molecule that can specifically bind to a target protein of interest. In the present invention, the targeting polypeptide may comprise natural amino acids, synthetic amino acids, or amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code as well as those amino acids that are subsequently modified, for example, hydroxyproline, gamma-hydroxyglutamate, O-phosphoserine, phosphothreonine or phosphotyrosine. In the present invention, the "specificity" between the targeting polypeptide and its target protein of interest can be determined based on the affinity, which can be determined by the dissociation equilibrium constant (i.e., _KD value) of the targeting polypeptide and the target protein of interest bound to it. to describe. The lower the K _D value, the stronger the binding strength between the targeting polypeptide and its bound target protein. It is generally known in the art that a _KD value greater than about 10 ⁻³ M is generally considered to indicate non-binding or non-specific binding. Depending on the specific target protein of interest, the targeted polypeptide specifically binding to the target protein can be obtained by methods known to those skilled in the art, such as screening by phage display technology or protein microarray technology.

As used herein, the expression "response to IFNα treatment" refers to the occurrence of virological response or serological response in chronic hepatitis B patients after receiving IFN/PEG-IFN treatment. Wherein, the virological response refers to HBVDNA<2000IU/ml 6 months after treatment, and it is maintained for 6 to 12 months after stopping the drug; the serological response refers to the negative conversion of HBeAg and the appearance of anti-HBe (see, EASL Chronic hepatitis B clinical practice guidelines (2012 edition)). Correspondingly, the expression "non-response to IFNα treatment" refers to the condition that the above-described conditions are not met in patients with chronic hepatitis B who receive IFN/PEG-IFN treatment.

As used herein, the term "statistical analysis value" refers to a value obtained after performing statistical analysis on detection results obtained by various detection methods. Various statistical analysis methods are well known in the art (see, for example, PCT International Application WO2009064901, which is incorporated herein by reference), and include, but are not limited to, linear combinations of test results, linear regression models, Logistic regression models, linear discriminant analysis ( LDA) model, nearest neighbor model or predictive analysis of microarray (PAM). Generally speaking, it is particularly preferred that the statistical analysis value is a value obtained by statistical analysis using a Logistic regression model. The Logistic regression model is specifically described in, for example, "Hu Chunyan. Combined detection of four tumor markers in serum of ovarian cancer [D]. Guangzhou: Sun Yat-sen University, 2008: 1-39", which is incorporated herein by reference in its entirety.

As used herein, the term "reference value" (also referred to as optimal diagnostic cut-off value) refers to a value that can reflect the status of a CHB patient population that does not respond to IFNα treatment. In the present invention, the reference value includes, for example, according to the level of markers in the samples of the CHB patient population who do not respond to IFNα treatment or according to the markers in the samples of the CHB patient population that do not respond to IFNα treatment before and after IFNα and/or HBV antigen stimulation The fold change of the level (relative expression level), a normal value or value range determined; and, the detection value (for example, the above-mentioned marker level of The value obtained by statistical analysis (statistical analysis value). Methods for determining optimal diagnostic cutoffs are well known in the art, including but not limited to Receiver Operating Characteristic (ROC) curve analysis, which is described in detail, for example, in "Habibzadeh F, et al., Biochem Med ( Zagreb). 2016; 26(3): 297-307" and "Chen Weizhong et al. Selection of the optimal operating point in the ROC curve [J]. China Health Statistics, 2006, 23: 157-158", all of which are by reference Incorporated into this article.

As used herein, the term "Ct value (Cycle threshold, cycle threshold)" refers to the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold in the fluorescent quantitative PCR detection. The Ct value of each template has a linear relationship with the logarithm of the initial copy number of the template, and the greater the initial copy number, the smaller the Ct value. A standard curve can be made by using the standard with known initial copy number, where the logarithm of the initial copy number is taken as the abscissa, and the Ct value is taken as the ordinate. Therefore, as long as the Ct value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.

As used herein, the term "comparative Ct method" is a method of relative quantitative analysis of mRNA well known in the art, which is described in detail, for example, in Livak KJ, et al. Methods. 2001 Dec; 25(4):402-8 (which is incorporated herein by reference). In the process of real-time PCR (quantitative PCR) reaction, it can be assumed that each PCR cycle doubles the amplification product, and the Ct value obtained in the exponential phase of the PCR reaction can reflect the copy number of the starting template, so if A difference of one cycle in the Ct values of different samples to be tested means that their initial template copy numbers have a 2-fold difference. Based on this, by comparing the Ct values of different samples, the relative expression level of the target gene in the sample to be tested can be judged.

As used herein, the term "normalization" or "standardization" refers to the elimination of variability that may exist in each step of the detection method used by comparing the expression level of the same gene in different samples. Yield-induced expression differences. Normalization methods are known in the art, including but not limited to methods described in detail in PCT International Application WO2013068422A1 or Chinese Patent Application 201280035399.6, such as the comparative Ct method based on internal reference genes.

As used herein, the term "expression level" refers to a measurable amount of a gene product produced by a gene of interest in a sample from a subject, wherein the gene product may be a transcription product or a translation product. Therefore, the expression "determining the expression level of the gene of interest" may refer to determining the level of the mRNA of the gene or a fragment of the mRNA, or the level of the cDNA of the gene or a fragment of the cDNA, or the expression level of the gene encoded by the gene. The level of the protein or its polypeptide fragments.

As used herein, the term "peripheral blood mononuclear cell (PBMC)" refers to a general term for cells with a single nucleus in peripheral blood, including but not limited to lymphocytes (T cells, B cells, NK cells), monocytes, or dendritic cells. Methods for obtaining PBMCs from peripheral blood are well known in the art, including but not limited to Ficoll's or Percoll's method. In the present invention, the term "reagents for separating PBMCs" refers to the reagents that need to be used in the method for obtaining PBMCs described above, such as polysucrose-diatrizoate for the Ficoll layering liquid method Amine solution. In the present invention, the term "PBMC isolation device" refers to a device capable of isolating and obtaining PBMC from a sample (for example, peripheral blood) from a subject, such devices are known in the art, including but not limited to detailed Cell separation devices described in Chinese patent applications CN1958776A, CN102286360A and CN105132278A, all of which are incorporated herein by reference.

As used herein, the term "peripheral blood buffy coat" refers to a component formed after natural sedimentation, centrifugation or density gradient centrifugation of peripheral anticoagulated blood, mainly composed of leukocytes (including peripheral blood mononuclear cells) and Platelet composition. After anticoagulant blood is centrifuged, it will form the upper layer of plasma, the lower layer of red blood cells, and a thin layer of white film between the two, accounting for about 1% of the total blood volume, known as the buffy coat.

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

In the present invention, the term "diluent" is preferably an electrolyte solution capable of maintaining cell osmotic pressure, and the solution also has the function of maintaining physiological pH value if necessary. Such solutions are well known in the art and include, but are not limited to, Alsever's solution, Earle's Balanced Salt Solution (EBSS), Gey's Balanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS), Phosphate Buffered Saline (PBS), Duchenne's Phosphate Buffered Saline (DPBS), Puck's Balanced Salt Solution, Ringer's Balanced Salt Solution (RBSS), Simm's Balanced Salt Solution (SBSS), TRIS Buffer (TBS), Tyrode's Balanced Salt Solution (TBSS), Physiological Saline or Ringer's Solution. In certain embodiments, the diluent is phosphate buffered saline or saline.

As used herein, the term "anticoagulant" refers to an agent or substance capable of preventing blood clotting, such substances are well known in the art, including but not limited to heparin, EDTA, oxalates (e.g., sodium oxalate , potassium oxalate, ammonium oxalate), sodium citrate (sodium citrate).

As used herein, the terms "culture solution" or "medium" refer to nutrients capable of maintaining cell activity. Usually the nutrients contain amino acids, vitamins, carbohydrates, inorganic salts and the like. Such nutrients are well known in the art and include, but are not limited to, RPMI-1640 medium or DMEM medium. In the present invention, the purpose of adding culture solution or culture medium to the sample from the subject is to maintain the activity of cells in the sample, especially PBMC. Methods for maintaining the activity of cells in blood components are well known in the art, and those skilled in the art can choose according to actual needs. In some embodiments, when the sample is whole blood, a culture solution can be added, such as adding an appropriate amount of glucose, sodium chloride, and potassium chloride to phosphate buffer or saline; In some embodiments, the culture medium is phosphate buffered saline with appropriate amount of glucose and potassium chloride added. In certain embodiments, when the sample is peripheral blood mononuclear cells (PBMC), peripheral blood buffy coat, or other blood components containing PBMC, a culture medium, such as a cell culture medium, such as is suitable for maintaining blood cells, particularly PBMC Active cell culture medium, such as RPMI-1640 medium or DMEM medium.

After the inventors of the present application tracked the response of chronic hepatitis B patients to IFNα treatment, they unexpectedly found that the level of OAS2 in PBMC samples of patients responding to IFNα treatment showed a significant Differences, or OAS2 and/or UPS18 levels in pre-treatment PBMC samples of patients responding to IFNα treatment showed significant differences compared with patients who did not respond to IFNα treatment after in vitro induction, so OAS2 and/or UPS18 can be used to predict CHB A marker of patient response to IFNα therapy. However, prior to this application, there has long been a lack of clear anti-HBV immune response indicators in the art to predict the response of CHB patients to IFNα treatment. Based on this finding, the present inventors developed a new method for predicting the therapeutic effect of IFNα on CHB patients or the response of CHB patients to IFNα treatment.

Therefore, in one aspect, the present invention provides a kit comprising a first reagent capable of detecting the expression level of a marker, optionally including IFNα and/or a stimulator; wherein the marker is selected from OAS2 or USP18, the stimulus is selected from HBV antigens, antigenic fragments of HBV antigens or any combination thereof.

In certain preferred embodiments, the HBV antigen is HBcAg. In some preferred embodiments, the HBcAg has the amino acid sequence shown in SEQ ID NO:38. In certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NO: 3-37.

In a particularly preferred embodiment, the stimuli comprise antigenic fragments having the amino acid sequences shown in SEQ ID NO: 3-37, respectively.

In certain preferred embodiments, the first reagent is a reagent capable of detecting the mRNA level of the marker. Such reagents are well known in the art and include, but are not limited to, nucleic acid probes that specifically bind to target sequences, primers that amplify target sequences, non-specific fluorescent dyes (eg, SYBR Green I), or combinations thereof. In some embodiments, the nucleic acid probe can be a single-labeled nucleic acid probe, such as a radionuclide (such as ³² P, ³ H, ³⁵ S, etc.) labeled probe, biotin-labeled probe, horseradish-treated Oxidase-labeled probes, digoxigenin-labeled probes or fluorescent group (such as FITC, FAM, TET, HEX, TAMRA, Cy3, Cy5, etc.) labeled probes; the nucleic acid probes can also be double-labeled nucleic acids Probes, such as Taqman probes, molecular beacons, displacement probes, scorpion primer probes, QUAL probes, FRET probes, etc. In certain embodiments, the first reagent comprises a Taqman probe. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence shown in SEQ ID NO:1. In certain embodiments, the cDNA of USP18 has a nucleotide sequence as shown in SEQ ID NO:2.

In certain preferred embodiments, the first reagent is a reagent capable of detecting the protein level of the marker. Such reagents are well known in the art, including but not limited to antibodies, targeting polypeptides or nucleic acid aptamers capable of specifically binding to OAS2 or USP18 proteins. In certain embodiments, such reagents are detectably labeled, such as enzymes (such as horseradish peroxidase, alkaline phosphatase, etc.), radionuclides (such as ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P, etc.), fluorescent dyes (such as FITC, TRITC, PE, Texas Red, quantum dots, Cy7, Alexa 750, etc.), acridinium ester compounds, magnetic beads (such as, ), colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (eg, streptavidin) modified with the above markers.

In certain preferred embodiments, the first reagent is used to determine the protein level of OAS2 or USP18 in the sample by immunological detection. In certain preferred embodiments, the immunological detection is selected from ELISA detection, Elispot detection, Western blot or surface plasmon resonance method. In certain embodiments, the first reagent comprises an antibody or antigen-binding fragment thereof against an OAS2 or USP18 protein.

In certain preferred embodiments, the kit of the present invention comprises a first reagent capable of detecting the expression level of OAS2, optionally including IFNα and/or a stimulator.

In certain preferred embodiments, the kit of the present invention comprises a first reagent capable of detecting the expression level of USP18 and IFNα, optionally further comprising a stimulator.

In certain preferred embodiments, the kit of the present invention further comprises a second reagent for pretreatment of a sample containing PBMCs, wherein the second reagent includes one or more reagents selected from the following: for diluting A diluent for the sample (e.g., phosphate buffered saline or saline); an anticoagulant (e.g., heparin) to prevent blood clotting; a reagent (e.g., culture medium or culture fluid) to maintain PBMC viability; and, Reagents for isolating PBMCs (eg, lymphocyte separation medium such as Ficoll-diatrizoate solution). In some preferred embodiments, the second reagent includes a reagent (for example, a culture medium or a culture solution) for maintaining the activity of PBMCs, and/or a reagent for isolating PBMCs (for example, a lymphocyte separation solution, such as Ficoll-diatrizoate solution).

In some preferred embodiments, the kit of the present invention further comprises a blood collection device (such as a pyrogen-free vacuum blood collection tube) and/or a PBMC separation device (such as a PBMC cell separation tube).

In some preferred embodiments, the kit of the present invention also includes a reagent capable of detecting the expression level of an internal reference gene. The expression of the internal reference gene in each tissue and cell is relatively constant, and will not Changes in expression occur, such as housekeeping genes; generally, the housekeeping genes encode proteins necessary to maintain basic cell life activities, including but not limited to β-actin, GAPDH, 18S rRNA, β2-MG, UBC or α-tubulin. In some preferred embodiments, the reagent capable of detecting the expression level of an internal reference gene is a reagent capable of detecting the mRNA level of the internal reference gene. In some preferred embodiments, the reagent capable of detecting the expression level of an internal reference gene is a reagent capable of detecting the protein level of the internal reference gene.

In another aspect, the present invention relates to the use of a reagent capable of detecting the expression level of a marker in the preparation of a kit for predicting the therapeutic effect of IFNα on a subject suffering from chronic hepatitis B or the subject Response to IFNα treatment; wherein the marker is selected from OAS2 or USP18.

In certain preferred embodiments, the kit further comprises IFNα and/or a stimulator selected from HBV antigens, antigenic fragments of HBV antigens, or any combination thereof.

In certain preferred embodiments, the HBV antigen is HBcAg. In some preferred embodiments, the HBcAg has the amino acid sequence shown in SEQ ID NO:38. In certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NO: 3-37.

In a particularly preferred embodiment, the stimuli comprise antigenic fragments having the amino acid sequences shown in SEQ ID NO: 3-37, respectively.

In certain preferred embodiments, the reagent capable of detecting the expression level of a marker is a reagent capable of detecting the mRNA level of the marker. Such reagents are well known in the art and include, but are not limited to, nucleic acid probes that specifically bind to target sequences, primers that amplify target sequences, non-specific fluorescent dyes (eg, SYBR Green I), or combinations thereof. In some embodiments, the nucleic acid probe can be a single-labeled nucleic acid probe, such as a radionuclide (such as ³² P, ³ H, ³⁵ S, etc.) labeled probe, biotin-labeled probe, horseradish-treated Oxidase-labeled probes, digoxigenin-labeled probes or fluorescent group (such as FITC, FAM, TET, HEX, TAMRA, Cy3, Cy5, etc.) labeled probes; the nucleic acid probes can also be double-labeled nucleic acids Probes, such as Taqman probes, molecular beacons, displacement probes, scorpion primer probes, QUAL probes, FRET probes, etc. In certain embodiments, the reagent capable of detecting the expression level of a marker comprises a Taqman probe. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence shown in SEQ ID NO:1. In certain embodiments, the cDNA of USP18 has a nucleotide sequence as shown in SEQ ID NO:2.

The mRNA level of the marker can be determined by an mRNA detection method known in the art, such as fluorescent quantitative PCR, Northern blotting, in situ hybridization, or including mRNA amplification (such as reverse transcription PCR) and the mRNA amplification. Methods for quantification of products such as electrophoresis and staining. In certain preferred embodiments, the reagent capable of detecting the expression level of the marker is used to quantitatively measure the mRNA level of the marker by PCR. In a specific embodiment, the reagent capable of detecting the expression level of the marker is used to measure the mRNA level of the marker by fluorescent quantitative PCR.

In certain preferred embodiments, the reagent capable of detecting the expression level of a marker is a reagent capable of detecting the protein level of the marker. Such reagents are well known in the art, including but not limited to antibodies, targeting polypeptides or nucleic acid aptamers capable of specifically binding to OAS2 or USP18 proteins. In certain embodiments, such reagents are detectably labeled, such as enzymes (such as horseradish peroxidase, alkaline phosphatase, etc.), radionuclides (such as ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P, etc.), fluorescent dyes (such as FITC, TRITC, PE, Texas Red, quantum dots, Cy7, Alexa 750, etc.), acridinium ester compounds, magnetic beads (such as, ), colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (eg, streptavidin) modified with the above markers.

In some preferred embodiments, the reagent capable of detecting the expression level of the marker is used to determine the protein level of OAS2 or USP18 in the sample by immunological detection. In certain preferred embodiments, the immunological detection is selected from ELISA detection, Elispot detection, Western blot or surface plasmon resonance method. In certain embodiments, the reagent capable of detecting the expression level of the marker comprises an antibody against OAS2 or USP18 protein or an antigen-binding fragment thereof.

In some preferred embodiments, the kit also includes a reagent capable of detecting the expression level of an internal reference gene. The expression of the internal reference gene in each tissue and cell is relatively constant, and will not occur under the condition of treatment factors. Altered expression, such as housekeeping genes; generally, the housekeeping genes encode proteins necessary to maintain basic cell life activities, including but not limited to β-actin, GAPDH, 18S rRNA, β2-MG, UBC or α-tubulin. In some preferred embodiments, the reagent capable of detecting the expression level of an internal reference gene is a reagent capable of detecting the mRNA level of the internal reference gene. In some preferred embodiments, the reagent capable of detecting the expression level of an internal reference gene is a reagent capable of detecting the protein level of the internal reference gene.

In some preferred embodiments, the kit further comprises one or more reagents or devices selected from 1)-6):

1) The diluent used to dilute the sample, such as phosphate buffer or saline;

2) Anticoagulants used to prevent blood clotting, such as heparin;

3) Reagents for maintaining PBMC activity, such as culture medium or culture fluid;

4) Reagents for separating PBMCs, such as lymphocyte separation fluid, such as polysucrose-diatrizoate solution;

5) blood collection devices, such as pyrogen-free vacuum blood collection tubes; and

6) PBMC separation device, such as a PBMC cell separation tube.

In certain preferred embodiments, the kit includes reagents (for example, culture medium or culture fluid) for maintaining the activity of PBMCs, and/or reagents for isolating PBMCs (for example, lymphocyte separation fluid, such as poly Sucrose-diatrizoate solution).

In certain preferred embodiments, the kit predicts the therapeutic effect of IFNα on a subject suffering from chronic hepatitis B or the response of the subject to IFNα treatment by a method comprising the following steps:

(1) determining the expression level of the marker in a sample from the subject using a reagent capable of detecting the expression level of the marker, wherein the marker is OAS2; and

(2) Comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, and comparing the statistical analysis value with the reference value, and judging whether the subject will respond to the Response to IFNα treatment;

Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat.

In some preferred embodiments, when the expression level is greater than the reference value, or when the statistical analysis value of the expression level is greater than the reference value, it is indicated that the subject will respond to IFNα treatment , suitable for receiving IFNα treatment; when the expression level is not greater than the reference value, or when the statistical analysis value of the expression level is not greater than the reference value, it indicates that the subject will not respond to IFNα treatment , not suitable for IFNα treatment.

In some preferred embodiments, in step (2), the expression level is statistically analyzed using a Logistic regression model.

In some preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between samples and to make the samples comparable, the expression level values of the marker to be tested in different samples can usually be compared Perform normalization processing, such as comparing the expression level value of the marker to be tested in the sample with the expression level value of the internal reference gene. Therefore, in step (1), the expression level of the marker is the normalized expression level. The normalized expression level can be obtained by the following exemplary method: use a reagent capable of detecting the expression level of an internal reference gene to measure the expression level of the internal reference gene in a sample from the subject, and convert the expression of the marker to The level is compared with the expression level of the internal reference gene to obtain the normalized expression level of the marker. In a specific embodiment, in step (1), the expression level of the marker is the normalized mRNA level, which can be obtained by the following exemplary method: using the comparative Ct method to divide the marker in the sample to be tested The mRNA level of the substance is compared with the mRNA level of the internal reference gene to obtain a 2 ^-ΔCt value (ΔCt=Ct _{marker to be tested-} Ct _{internal reference gene} ), which is the normalized mRNA level of the marker, and also That is, the change factor of the expression level of the marker compared with the expression level of the internal reference gene.

In some preferred embodiments, before step (1), the method further includes one or more of the following steps: (a) obtaining a sample from the subject using a blood collection device; (b) using Anticoagulant treatment of a blood collection device or a sample from said subject; (c) treatment of a sample from said subject with a reagent for maintaining PBMC viability; (d) dilution of a sample from said subject with a diluent and (e) isolating PBMCs from the sample from said subject using a reagent for isolating PBMCs or a PBMC isolation device.

In certain preferred embodiments, the kit predicts the therapeutic effect of IFNα on a subject suffering from chronic hepatitis B or the response of the subject to IFNα treatment by a method comprising the following steps:

(1) Use IFNα to stimulate at least one sample from the subject as a test sample, and use a sample that has not been stimulated by IFNα as a control sample; or use IFNα and a stimulator to jointly stimulate the sample from the subject At least one sample is used as a test sample, and the sample stimulated by the stimulant but not stimulated by IFNα is used as a control sample; wherein the stimulator is selected from HBV antigen, antigenic fragments of HBV antigen or any combination thereof;

(2) Use a reagent capable of detecting the expression level of the marker to measure the expression level of the marker in each sample in step (1), and obtain the expression level of the marker in the sample to be tested compared with the control sample The fold change of the expression level of the marker in the test sample, the fold change is used as the relative expression level of the marker in the test sample, wherein the marker is selected from OAS2 or USP18; and

(3) Compare the relative expression level with a reference value, or perform statistical analysis on the relative expression level to obtain a statistical analysis value, and compare the statistical analysis value with the reference value, and determine whether the subject Will respond to IFNα treatment;

Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat.

In some preferred embodiments, when the relative expression level is less than the reference value, or when the statistical analysis value of the relative expression level is less than the reference value, it indicates that the subject will be treated with IFNα produce a response, suitable for receiving IFNα treatment; when the relative expression level is not less than the reference value, or when the statistical analysis value of the relative expression level is not less than the reference value, it indicates that the subject will not respond to Response to IFNα treatment is not suitable for IFNα treatment.

In some preferred embodiments, in step (2), the multiple of the mRNA level of the marker in the test sample compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method , that is, the relative mRNA expression level of the marker in the test sample.

In some preferred embodiments, in step (3), the relative expression level is statistically analyzed using a Logistic regression model.

In some preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between samples and to make the samples comparable, the expression level values of the marker to be tested in different samples can usually be compared Perform normalization processing, such as comparing the expression level value of the marker to be tested in the sample with the expression level value of the internal reference gene. Therefore, in step (2), the expression level of the marker is the normalized expression level. The normalized expression level can be obtained by the following exemplary method: use a reagent capable of detecting the expression level of an internal reference gene to measure the expression level of the internal reference gene in a sample from the subject, and convert the expression of the marker to The level is compared with the expression level of the internal reference gene to obtain the normalized expression level of the marker. In a specific embodiment, in step (2), the expression level of the marker is the normalized mRNA level, which can be obtained by the following exemplary method: using the comparative Ct method to divide the marker in the sample to be tested The mRNA level of the marker is compared with the mRNA level of the internal reference gene to obtain the normalized mRNA level of the marker. Further, the fold change of the normalized mRNA level of the marker in the sample to be tested compared to the normalized mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the factor described in the sample to be tested Relative mRNA expression levels of markers.

In some preferred embodiments, before step (1), the method further includes one or more of the following steps: (a) obtaining a sample from the subject using a blood collection device; (b) using Anticoagulant treatment of a blood collection device or a sample from said subject; (c) treatment of a sample from said subject with a reagent for maintaining PBMC viability; (d) dilution of a sample from said subject with a diluent and (e) isolating PBMCs from the sample from said subject using a reagent for isolating PBMCs or a PBMC isolation device.

In another aspect, the present invention provides a method for predicting the therapeutic effect of IFNα on a subject with chronic hepatitis B or the response of said subject to IFNα treatment, or a method of obtaining the result of a diagnostic assay, wherein the Described diagnostic analysis is used for predicting the therapeutic effect of IFNα to the experimenter suffering from chronic hepatitis B or described experimenter's response to IFNα treatment, it comprises the following steps:

(1) providing a sample from the subject;

(2) determining the expression level of a marker in the sample, wherein the marker is OAS2; and

(3) comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, and comparing the statistical analysis value with the reference value, and judging whether the subject will Response to IFNα treatment;

Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat.

In some preferred embodiments, after step (3), the method further includes the step of administering IFNα to the subject to treat chronic hepatitis B, wherein the subject is judged to have IFNα treatment answer.

In some preferred embodiments, when the expression level is greater than the reference value, or when the statistical analysis value of the expression level is greater than the reference value, it is indicated that the subject will respond to IFNα treatment , suitable for receiving IFNα treatment; when the expression level is not greater than the reference value, or when the statistical analysis value of the expression level is not greater than the reference value, it indicates that the subject will not respond to IFNα treatment , not suitable for IFNα treatment.

In some preferred embodiments, in step (2), the mRNA level or protein level of the marker is determined.

In some preferred embodiments, in step (2), the mRNA level of the marker is determined. The mRNA level of the marker can be determined by an mRNA detection method known in the art, such as fluorescent quantitative PCR, Northern blotting, in situ hybridization, or including mRNA amplification (such as reverse transcription PCR) and the mRNA amplification. Methods for quantification of products such as electrophoresis and staining. In certain preferred embodiments, the mRNA levels of the markers are quantified by PCR. In a specific embodiment, the mRNA level of the marker is determined by fluorescent quantitative PCR. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence shown in SEQ ID NO:1. In certain embodiments, the cDNA of USP18 has a nucleotide sequence as shown in SEQ ID NO:2.

In certain preferred embodiments, in step (2), the protein level of the marker is determined. In some preferred embodiments, in step (2), the protein level of the marker is determined by immunological detection. Further, in some preferred embodiments, the immunological detection is selected from ELISA detection, Elispot detection, Western blot or surface plasmon resonance method. In some embodiments, in step (2), an anti-OAS2 antibody or an antigen-binding fragment thereof is used to detect the protein level of OAS2.

In some preferred embodiments, in step (3), the expression level is statistically analyzed using a Logistic regression model.

In some preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between samples and to make the samples comparable, the expression level values of the marker to be tested in different samples can usually be compared Perform normalization processing, such as comparing the expression level value of the marker to be tested in the sample with the expression level value of the internal reference gene. Therefore, in step (2), the expression level of the marker is the normalized expression level. The normalized expression level can be obtained by the following exemplary method: use a reagent capable of detecting the expression level of an internal reference gene to measure the expression level of the internal reference gene in a sample from the subject, and convert the expression of the marker to The level is compared with the expression level of the internal reference gene to obtain the normalized expression level of the marker. In a specific embodiment, in step (2), the expression level of the marker is the normalized mRNA level, which can be obtained by the following exemplary method: using the comparative Ct method to divide the marker in the sample to be tested The mRNA level of the substance is compared with the mRNA level of the internal reference gene to obtain a 2 ^-ΔCt value (ΔCt=Ct _{marker to be tested-} Ct _{internal reference gene} ), which is the normalized mRNA level of the marker, and also That is, the change factor of the expression level of the marker compared with the expression level of the internal reference gene.

In some preferred embodiments, before step (1), one or more of the following steps are also included: (a) obtaining a sample from the subject; (c) obtain PBMCs or a blood component containing PBMCs (e.g., peripheral blood buffy coat) from a sample from said subject; (d) obtain a sample from said subject adding culture fluid or culture medium to the subject's sample; and, (e) diluting the sample from said subject.

In another aspect, the present invention provides a method for predicting the therapeutic effect of IFNα on a subject with chronic hepatitis B or the response of said subject to IFNα treatment, or a method of obtaining the result of a diagnostic assay, wherein the Described diagnostic analysis is used for predicting the therapeutic effect of IFNα to the experimenter suffering from chronic hepatitis B or described experimenter's response to IFNα treatment, it comprises the following steps:

(1) Provide at least two samples from the subject;

(2) Use IFNα to stimulate at least one sample as a test sample, and use a sample without IFNα stimulation as a control sample; or use IFNα and a stimulator to jointly stimulate at least one sample from the subject as a test sample Sample, and the sample stimulated by the stimulus but not stimulated by IFNα is used as a control sample; wherein the stimulus is selected from HBV antigen, antigenic fragments of HBV antigen or any combination thereof;

(3) Determining the expression level of the marker in each sample in step (2), and obtaining the change factor of the expression level of the marker in the sample to be tested compared to the expression level of the marker in the control sample, Taking the change factor as the relative expression level of the marker in the sample to be tested, wherein the marker is selected from OAS2 or USP18; and

(4) Compare the relative expression level with a reference value, or perform statistical analysis on the relative expression level to obtain a statistical analysis value, and compare the statistical analysis value with the reference value, and determine whether the subject Will respond to IFNα treatment;

Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat.

In some preferred embodiments, after step (4), the method further includes the step of administering IFNα to the subject to treat chronic hepatitis B, wherein the subject is judged to have IFNα treatment answer.

In some preferred embodiments, when the relative expression level is less than the reference value, or when the statistical analysis value of the relative expression level is less than the reference value, it indicates that the subject will be treated with IFNα produce a response, suitable for receiving IFNα treatment; when the relative expression level is not less than the reference value, or when the statistical analysis value of the relative expression level is not less than the reference value, it indicates that the subject will not respond to Response to IFNα treatment is not suitable for IFNα treatment.

In some preferred embodiments, in step (2), the HBV antigen is HBVcAg. In some preferred embodiments, the HBVcAg has the amino acid sequence shown in SEQ ID NO:38. In certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NO: 3-37. In a particularly preferred embodiment, the stimuli comprise antigenic fragments having the amino acid sequences shown in SEQ ID NO: 3-37, respectively.

In some preferred embodiments, in step (3), the mRNA level of the marker is determined. The mRNA level of the marker can be determined by an mRNA detection method known in the art, such as fluorescent quantitative PCR, Northern blotting, in situ hybridization, or including mRNA amplification (such as reverse transcription PCR) and the mRNA amplification. Methods for quantification of products such as electrophoresis and staining. In certain preferred embodiments, the mRNA levels of the markers are quantified by PCR. In a specific embodiment, the mRNA level of the marker is determined by fluorescent quantitative PCR. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence shown in SEQ ID NO:1. In certain embodiments, the cDNA of USP18 has a nucleotide sequence as shown in SEQ ID NO:2.

In some preferred embodiments, in step (3), the protein level of the marker is determined. In some preferred embodiments, in step (3), the protein level of the marker is determined by immunological detection. Further, in some preferred embodiments, the immunological detection is selected from ELISA detection, Elispot detection, Western blot or surface plasmon resonance method. In some embodiments, in step (3), an antibody against OAS2 or USP18 protein or an antigen-binding fragment thereof is used to detect the protein level of the marker.

In some preferred embodiments, in step (3), the multiple of the mRNA level of the marker in the test sample compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method , that is, the relative mRNA expression level of the marker in the test sample.

In some preferred embodiments, in step (4), the relative expression level is statistically analyzed using a Logistic regression model.

In some preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between samples and to make the samples comparable, the expression level values of the marker to be tested in different samples can usually be compared Perform normalization processing, such as comparing the expression level value of the marker to be tested in the sample with the expression level value of the internal reference gene. Therefore, in step (3), the expression level of the marker is the normalized expression level. The normalized expression level can be obtained by the following exemplary method: use a reagent capable of detecting the expression level of an internal reference gene to measure the expression level of the internal reference gene in a sample from the subject, and convert the expression of the marker to The level is compared with the expression level of the internal reference gene to obtain the normalized expression level of the marker. In a specific embodiment, in step (3), the expression level of the marker is the normalized mRNA level, which can be obtained by the following exemplary method: using the comparative Ct method to divide the marker in the sample to be tested The mRNA level of the marker is compared with the mRNA level of the internal reference gene to obtain the normalized mRNA level of the marker. Further, the fold change of the normalized mRNA level of the marker in the sample to be tested compared to the normalized mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the factor described in the sample to be tested Relative mRNA expression levels of markers.

In some preferred embodiments, before step (1), one or more of the following steps are also included: (a) obtaining a sample from the subject; (c) obtain PBMCs or a blood component containing PBMCs (e.g., peripheral blood buffy coat) from a sample from said subject; (d) obtain a sample from said subject adding culture fluid or culture medium to the subject's sample; and, (e) diluting the sample from said subject.

The present invention also includes the following exemplary embodiments:

1. A stimulator for inducing interferon-stimulated genes (ISGs) in vitro, which comprises a combination of full-length or partial fragments of HBV core antigen and/or IFNα.

2. A marker for predicting the therapeutic effect of alpha interferon in patients with chronic hepatitis B, wherein the marker is an interferon-stimulated gene (Interferon-stimulated genes, ISGs).

3. The marker according to item 2, wherein the interferon-inducible gene is selected from OAS2 and USP18.

4. Screening is used to predict the method for the marker of alpha interferon therapeutic effect of chronic hepatitis B patient, described method comprises the steps:

1) Collect whole blood samples from patients with chronic hepatitis B before medication, and separate PBMC;

2) Inducing PBMC samples with HBV core antigen polypeptide fragments or IFNα or a combination of HBV core antigen polypeptide fragments and IFNα;

3) Collect induced PBMC samples, extract RNA, and detect ISGs mRNA levels after reverse transcription;

4) Collect the clinical IFNα treatment response results of specimens and screen ISGs;

Among them, comparing the IFNα treatment response group and the non-response group under the same induction conditions and different ISGs mRNA levels, the difference in the level of ISGs mRNA between the IFNα treatment response group and the non-response group can be used as the basis for the treatment effect.

5. The method according to item 4, wherein the HBV core antigen polypeptide fragment is a polypeptide fragment of amino acids 1-183 of the HBV core antigen.

6. A kit for predicting the therapeutic effect of alpha interferon in patients with chronic hepatitis B, which comprises HBV core antigen polypeptide fragments and IFNα inducers, and reagents for detecting the levels of markers OAS2 and USP18.

7. The kit according to item 6, wherein the HBV core antigen polypeptide fragment is a polypeptide fragment of amino acids 1-183 of HBV core antigen.

8. The use of the reagent for detecting the level of markers OAS2 and/or USP18 in the preparation of a diagnostic agent for predicting the therapeutic effect of alpha interferon in patients with chronic hepatitis B.

9. Use of the reagent for detecting the level of markers OAS2 and/or USP18 in the preparation of a kit for predicting the therapeutic effect of alpha interferon in patients with chronic hepatitis B.

10. The use of markers OAS2 and/or USP18 for predicting the therapeutic effect of alpha interferon in patients with chronic hepatitis B.

11. The use according to any one of items 8-10, wherein the detection of the marker OAS2 and/or USP18 comprises quantitative detection of the mRNA level of the marker OAS2 and/or USP18.

12. The use according to any one of items 8-10, wherein the mRNA levels of the markers OAS2 and/or USP18 are quantitatively detected by fluorescent PCR.

13. The use in any one of items 8-10, wherein the sequences of the primers and probes for detecting the mRNA levels of markers OAS2 and/or USP18 are:

Beneficial Effects of the Invention

Through a large number of experiments and repeated explorations, the present invention has found that there are significant differences in the expression levels of OAS2 and/or USP18 in the two groups of CHB patients who respond to IFNα treatment and those who do not respond to IFNα treatment, thus establishing a simple, A method for predicting the therapeutic effect of IFNα on a subject suffering from chronic hepatitis B or the response of the subject to IFNα treatment, which is convenient, rapid, highly accurate and specific.

Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

(1) For the first time, it was found that the expression level of OAS2 in PBMC of chronic hepatitis B patients without or after in vitro stimulation can be used to predict the outcome of IFNα treatment in CHB patients;

(2) For the first time, it was found that the expression level of USP18 in PBMCs of patients with chronic hepatitis B after in vitro stimulation can be used to predict the outcome of IFNα treatment in CHB patients;

(3) The operations involved are simple and convenient: for example, by collecting whole blood, separating PBMCs, distributing them to cell culture plates and adding IFNα, and then placing them in a constant temperature incubator for culture; after culturing for a certain period of time, mRNA or Detection of protein level;

(4) The requirements for experimental conditions, technical ability of personnel, equipment and environment are not high;

(5) The prediction accuracy of IFNα treatment response is high;

(6) The cost is not high, easy to popularize, and widely used.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the present invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiment.

Description of drawings

Figure 1 shows that under different treatment conditions, there is a significant difference in OAS2 mRNA levels in PBMCs of CHB patients between responders (Rs) and non-responders (NRs) to IFNα treatment. Among them, Figure 1a shows that PBMCs were not induced in vitro, and the mRNA level of OAS2 in the response group was significantly higher than that in the non-response group. Figure 1b shows that after PBMCs were induced by IFNα in vitro, the relative expression level of OAS2 mRNA in the response group was significantly lower than that in the non-response group. (Note: * means p<0.05, **** means p<0.0005)

Figure 2 shows that under different treatment conditions, there is a significant difference in USP18 mRNA levels in PBMCs of CHB patients between responders (Rs) and non-responders (NRs) to IFNα treatment. Among them, Figure 2a shows that after PBMCs are induced by IFNα in vitro, the relative expression level of USP18 mRNA in the response group is significantly lower than that of the non-response group; Figure 2b shows that after PBMCs are co-induced by IFNα and HBcAg polypeptide library, the relative expression level of USP18 mRNA in the response group significantly lower than that of the non-response group. (Note: *** means p<0.001, **** means p<0.0005)

Figure 3 shows the ROC curves of the OAS2N assay and the OAS2(IFNa+N-N) assay. The results showed that both OAS2N detection method and OAS2(IFNa+N-N) detection method had good sensitivity and specificity for predicting the response of CHB patients to IFNα treatment.

Figure 4 shows the ROC curves of the OAS2 (IFNa+N-N) detection method, the USP18 (IFNa+N-N) detection method and their combined detection. The results showed that OAS2(IFNa+N-N) detection method, USP18(IFNa+N-N) detection method and their combined detection had good sensitivity and specificity for predicting the response of CHB patients to IFNα treatment.

sequence information

Sequence information related to the present invention is provided in Table 1 below.

Table 1: Sequence information

Nucleotide sequence of OAS2 cDNA (SEQ ID NO: 1)

ATGGGAAATGGGGAGTCCCAGCTGTCCTCGGTGCCTGCTCAGAAGCTGGGTTGGTTTATCCAGGAATACCTGAAGCCCTACGAAGAATGTCAGACACTGATCGACGAGATGGTGAACACCATCTGTGACGTCCTGCAGGAACCCGAACAGTTCCCCCTGGTGCAGGGAGTGGCCATAGGTGGCTCCTATGGACGGAAAACAGTCTTAAGAGGCAACTCCGATGGTACCCTTGTCCTCTTCTTCAGTGACTTAAAACAATTCCAGGATCAGAAGAGAAGCCAACGTGACATCCTCGATAAAACTGGGGATAAGCTGAAGTTCTGTCTGTTCACGAAGTGGTTGAAAAACAATTTCGAGATCCAGAAGTCCCTTGATGGGTTCACCATCCAGGTGTTCACAAAAAATCAGAGAATCTCTTTCGAGGTGCTGGCCGCCTTCAACGCTCTGAGCTTAAATGATAATCCCAGCCCCTGGATCTATCGAGAGCTCAAAAGATCCTTGGATAAGACAAATGCCAGTCCTGGTGAGTTTGCAGTCTGCTTCACTGAACTCCAGCAGAAGTTTTTTGACAACCGTCCTGGAAAACTAAAGGATTTGATCCTCTTGATAAAGCACTGGCATCAACAGTGCCAGAAAAAAATCAAGGATTTACCCTCGCTGTCTCCGTATGCCCTGGAGCTGCTTACGGTGTATGCCTGGGAACAGGGGTGCAGAAAAGACAACTTTGACATTGCTGAAGGCGTCAGAACCGTTCTGGAGCTGATCAAATGCCAGGAGAAGCTGTGTATCTATTGGATGGTCAACTACAACTTTGAAGATGAGACCATCAGGAACATCCTGCTGCACCAGCTCCAATCAGCGAGGCCAGTAATCTTGGATCCAGTTGACCCAACCAATAATGTGAGTGGAGATAAAATATGCTGGCAATGGCTGAAAAAAGAAGCTCAAACCTGGTTGACTTCTCCCAACCTGGATAATGAGTTACCTGCACCATCTTGGA ATGTTCTGCCTGCACCACTCTTCACGACCCCAGGCCACCTTCTGGATAAGTTCATCAAGGAGTTTCTCCAGCCCAACAAATGCTTCCTAGAGCAGATTGACAGTGCTGTTAACATCATCCGTACATTCCTTAAAGAAAACTGCTTCCGACAATCAACAGCCAAGATCCAGATTGTCCGGGGAGGATCAACCGCCAAAGGCACAGCTCTGAAGACTGGCTCTGATGCCGATCTCGTCGTGTTCCATAACTCACTTAAAAGCTACACCTCCCAAAAAAACGAGCGGCACAAAATCGTCAAGGAAATCCATGAACAGCTGAAAGCCTTTTGGAGGGAGAAGGAGGAGGAGCTTGAAGTCAGCTTTGAGCCTCCCAAGTGGAAGGCTCCCAGGGTGCTGAGCTTCTCTCTGAAATCCAAAGTCCTCAACGAAAGTGTCAGCTTTGATGTGCTTCCTGCCTTTAATGCACTGGGTCAGCTGAGTTCTGGCTCCACACCCAGCCCCGAGGTTTATGCAGGGCTCATTGATCTGTATAAATCCTCGGACCTCCCGGGAGGAGAGTTTTCTACCTGTTTCACAGTCCTGCAGCGAAACTTCATTCGCTCCCGGCCCACCAAACTAAAGGATTTAATTCGCCTGGTGAAGCACTGGTACAAAGAGTGTGAAAGGAAACTGAAGCCAAAGGGGTCTTTGCCCCCAAAGTATGCCTTGGAGCTGCTCACCATCTATGCCTGGGAGCAGGGGAGTGGAGTGCCGGATTTTGACACTGCAGAAGGTTTCCGGACAGTCCTGGAGCTGGTCACACAATATCAGCAGCTCTGCATCTTCTGGAAGGTCAATTACAACTTTGAAGATGAGACCGTGAGGAAGTTTCTACTGAGCCAGTTGCAGAAAACCAGGCCTGTGATCTTGGACCCAGCCGAACCCACAGGTGACGTGGGTGGAGGGGACCGTTGGTGTTGGCATCTTCTGGCAAAAGAAGCAAAGGAATGGTTATCCTCTCC CTGCTTCAAGGATGGGACTGGAAACCCAATACCACCTTGGAAAGTGCCGGTAAAAGTCATCTAA

Nucleotide sequence of USP18 cDNA (SEQ ID NO: 2)

ATGAGCAAGGCGTTTGGGCTCCTGAGGCAAATCTGTCAGTCCATCCTGGCTGAGTCCTCGCAGTCCCCGGCAGATCTTGAAGAAAAGAAGGAAGAAGACAGCAACATGAAGAGAGAGCAGCCCAGAGAGCGTCCCAGGGCCTGGGACTACCCTCATGGCCTGGTTGGTTTACACAACATTGGACAGACCTGCTGCCTTAACTCCTTGATTCAGGTGTTCGTAATGAATGTGGACTTCACCAGGATATTGAAGAGGATCACGGTGCCCAGGGGAGCTGACGAGCAGAGGAGAAGCGTCCCTTTCCAGATGCTTCTGCTGCTGGAGAAGATGCAGGACAGCCGGCAGAAAGCAGTGCGGCCCCTGGAGCTGGCCTACTGCCTGCAGAAGTGCAACGTGCCCTTGTTTGTCCAACATGATGCTGCCCAACTGTACCTCAAACTCTGGAACCTGATTAAGGACCAGATCACTGATGTGCACTTGGTGGAGAGACTGCAGGCCCTGTATACGATCCGGGTGAAGGACTCCTTGATTTGCGTTGACTGTGCCATGGAGAGTAGCAGAAACAGCAGCATGCTCACCCTCCCACTTTCTCTTTTTGATGTGGACTCAAAGCCCCTGAAGACACTGGAGGACGCCCTGCACTGCTTCTTCCAGCCCAGGGAGTTATCAAGCAAAAGCAAGTGCTTCTGTGAGAACTGTGGGAAGAAGACCCGTGGGAAACAGGTCTTGAAGCTGACCCATTTGCCCCAGACCCTGACAATCCACCTCATGCGATTCTCCATCAGGAATTCACAGACGAGAAAGATCTGCCACTCCCTGTACTTCCCCCAGAGCTTGGATTTCAGCCAGATCCTTCCAATGAAGCGAGAGTCTTGTGATGCTGAGGAGCAGTCTGGAGGGCAGTATGAGCTTTTTGCTGTGATTGCGCACGTGGGAATGGCAGACTCCGGTCATTACTGTGTCTACATCCGGAATGCTGTGGATGGAAAATGGTTCTGCT TCAATGACTCCAATATTTGCTTGGTGTCCTGGGAAGACATCCAGTGTACCTACGGAAATCCTAACTACCACTGGCAGGAAACTGCATATCTTCTGGTTTACATGAAGATGGAGTGCTAA

HBcAg polypeptide library peptide sequence (P1-P35) (SEQ ID NO: 3-37)

P1: MDIDPYKEFGASVEL (SEQ ID NO: 3)

P2: YKEFGASVELLSSFLP (SEQ ID NO: 4)

P3: ASVELLSFLPSDFFP (SEQ ID NO: 5)

P4: LSFLPSDFPSVRDL (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: PRRRTPSPPRRRRSQS (SEQ ID NO: 34)

P33:PSPRRRRSQSPRRRR (SEQ ID NO: 35)

P34: RRSQSPRRRRSQSRE (SEQ ID NO: 36)

P35: QSPRRRRSQSRESQC (SEQ ID NO: 37)

Amino acid sequence of HBcAg (SEQ ID NO: 38)

MDIDPYKEFGATVELLSFLPSDFFPSVRGLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

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: TTGGATTTTCCCTTTTTGTGC (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)

detailed description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it. Unless otherwise indicated, the experiments and methods described in the examples (eg, molecular biology assays and immunoassays) were performed essentially according to conventional methods well known in the art and described in various references. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), It is hereby incorporated by reference in its entirety. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1. Peripheral blood PBMC isolation and in vitro induction culture

1.1 Peripheral blood PBMC isolation

(1) The whole blood was centrifuged at 4000rpm for 10min.

(2) Aspirate the serum and add an equal amount of PBS to dilute the remaining whole blood.

(3) A 15 mL centrifuge tube was prepared, and an equal amount of whole blood lymphocyte separation medium (Ficoll-page TmPLUS, GE Helathcare Life Sciences) was added.

(4) Slowly add the diluted whole blood on top of the lymphocyte separation solution along the tube wall to keep the layered state.

(5) The above liquid was centrifuged at 1000 g for 22 min, and the rate of speed up and speed down was adjusted to the minimum.

(6) Carefully draw the white cloudy lymphocyte layer into another centrifuge tube, add PBS to dilute and centrifuge at 600g×7min, discard the supernatant. Then add PBS to dilute, centrifuge at 400g×7min, and discard the supernatant. Resuspend the cells with 1ml of culture medium.

(7) Add 10 μl of the prepared sample cell suspension and 40 μl of 0.4% trypan blue into a 1.5 ml centrifuge tube to prepare a 1:5 cell dilution. 10 μl of trypan blue/cell mixed suspension was added to a hemocytometer and counted viable cells, wherein the number of cells per ml=cell count×dilution factor×10 ⁴ .

1.2 Induction and culture of PBMC by IFNα and/or stimulators in vitro

The above PBMC cells were seeded in a 24-well plate, 5×10 ⁵ cells were added to each well, and 1 ml of RPM1640 medium containing 10% fetal bovine serum was added. A total of four wells were set up for each sample, wherein the first well was marked N without adding inducing substances (the inducing substances included IFNα and HBcAg polypeptide library), and the second well was marked with IFNα (Andafin, Anhui Anke Bioengineering (Group) Co., Ltd.) (1×10 ⁴ IU/mL), the third well was added with a peptide library containing 35 HBcAg antigenic fragments (SEQ ID NO:3-37) (synthesized by Shanghai Bioengineering Co., Ltd.) (1 μg/ strips/mL, 35 strips in total), and the above-mentioned IFNα and HBcAg polypeptide libraries were added to the fourth well. After the plate was placed in a carbon dioxide incubator at 37°C for 24 hours, the cells were collected.

Embodiment 2. Fluorescence quantitative PCR method detects the mRNA level of gene

The present invention establishes the fluorescence of 17 genes including USP18, OAS2, LGP1, LAP3, GIP3, CEB1, ATF5, GIP2, OAS3, RPLP2, STXBP5, Viperin, RPS28, PI3KAP1, MX1, DUSP1, ETEF1 and internal reference β-actin Quantitative PCR detection system.

2.1 Extraction of RNA from PBMC

(1) Collect the PBMC cells obtained in Example 1 by centrifugation, discard the supernatant; add 250 μl RPMI1640 medium and 750 μl Tripure to each well, and mix well; add 200 μl chloroform to each 1 ml of cells, mix up and down, and statically Set for 10min.

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

(3) After the above treatment, the sample in each tube is divided into three layers, absorb the top layer (be careful not to absorb the protein layer) and transfer it to a new EP tube treated with DEPC, add an equal volume of isopropanol , mix evenly by inverting up and down, and let stand at -20°C for 15 minutes.

(4) Centrifuge at 12,000 rpm at 4°C for 10 min, discard the supernatant.

(5) Add 1 mL of 75% ethanol to wash twice, centrifuge at 12,000 rpm, 4°C for 5 min.

(6) Discard the ethanol, air-dry for 5-10 minutes, dissolve the RNA with 35 μL of DEPC water preheated at 55-60°C, and freeze the extracted RNA in a -80°C refrigerator for storage.

2.2 RNA reverse transcription

The concentration and purity of RNA samples were determined using a spectrophotometer. , Take 2 μL RNA sample and measure the absorbance value of A260 and A280 in a 2 μL RNase-freewater calibration instrument, and detect the ratio of A260 and A280 to evaluate the purity of the RNA sample. After testing, the RNA purity: A260/280≥1.80.

Reverse transcription system:

Reverse transcription was carried out at 42° C. for 30 min by the above-mentioned reverse transcription system. The obtained cDNA is the template in the fluorescent quantitative PCR detection system, which is stored at -20°C.

2.3 Establishment of fluorescent quantitative PCR system

(1) RT-PCR system:

Amplification reaction process:

95°C for 3min, then 95°C for 10sec, 55°C for 45sec, a total of 39 cycles.

Primer software was used to design the detection primers and probes for RT-PCR, which were synthesized by Yingjun Biotechnology Co., Ltd. The specific sequences are shown in Table 2 below:

Table 2. Primers and probes used for real-time quantitative PCR reactions

(2) Primer pre-experiment

Preliminary experiments were performed on 18 sets of primers (Table 2 above) designed with the primer software, and two templates from CHB patients (marked as CHB 1 and CHB 2 respectively) were randomly selected, and the genes to be tested and internal reference primers were detected by fluorescent quantitative PCR Ct value. The identification results are shown in Table 3. The Ct values of the primers of 17 sets of genes to be tested and the internal reference β-actin primer are all between 15-35, indicating that they can be effectively amplified; while the Neg group (without template) was not detected To effectively amplify, it further shows that the 18 sets of primers listed in Table 2 above can be used for the fluorescent quantitative PCR detection of the corresponding gene to be tested.

Table 3. Preliminary experiments of primers in the fluorescent quantitative PCR detection system

Example 3. Differences in the expression of OAS2 and USP18 in CHB patients who respond to and do not respond to IFNα treatment

The present invention has clinically tracked 150 cases of chronic hepatitis B specimens, of which 66 cases received IFNα treatment and had 24-week treatment results; among them, 47 cases did not respond to IFNα treatment, and 19 cases responded to IFNα treatment. Table 4 shows the feature data of the selected research samples in this embodiment, and the results show that there is no significant difference in age and sex between the response group (Rs) and the non-response group (NRs), before treatment ALT, AST and HBsAg, HBV DNA, HBeAg results were also not significantly different.

Table 4. Characteristics of selected research specimens

Peripheral blood is collected from chronic hepatitis B patients who have not received IFNα treatment, PBMCs are extracted according to the method of the above-mentioned embodiment 1, and the PBMCs are processed and the method of embodiment 2 is extracted RNA, and fluorescent quantitative PCR is carried out to detect and calculate 17 genes to be tested ( mRNA levels of USP18, OAS2, LGP1, LAP3, GIP3, CEB1, ATF5, GIP2, OAS3, RPLP2, STXBP5, Viperin, RPS28, PI3KAP1, MX1, DUSP1, ETEF1), which were determined by relative quantification (compared to Ct method ) is calculated, and the calculation method is: subtract the Ct value of each internal reference β-actin from the measured Ct value of each target gene to obtain the ΔCt value of each target gene (ΔCt=Ct _{target gene} -Ct _{internal reference gene} ) , calculate the 2 ^-ΔCt value, and what it represents is the change factor (that is, the normalized mRNA level of this gene relative to the internal reference gene) of the expression amount of the gene in the sample to be tested relative to the expression amount of the internal reference gene; then The ΔCt value of the gene in the sample to be tested is subtracted from the ΔCt value of the gene in the control sample to obtain the ΔΔCt value (ΔΔCt=ΔCt sample _{to be tested} -ΔCt _{control sample} ), and finally calculate the 2- ^ΔΔCt value, which represents the value of the gene to be tested The fold change of the expression level of the gene in the test sample relative to the expression level of the gene in the control sample (that is, the relative mRNA expression level of the gene in the test sample relative to the gene in the control sample).

By comparing the differences in the mRNA levels of the above genes in the response group and the non-response group, it was found that the mRNA levels of OAS2 and USP18 were significantly different between the response group and the non-response group. The results are shown in Figure 1 and Figure 2, wherein the PBMC cells of the OAS2N group were not induced in vitro, and what this group represented was the normalized mRNA level of OAS2 in the sample to be tested relative to the internal reference gene; OAS2(IFNa+N-N) Group, USP18 (IFNa+N-N) group and the PBMC in vitro induction conditions of USP18 (IFNa+HBcAg-HBcAg) group are as shown in Table 5, and the specific steps of its in vitro induction culture are carried out with reference to embodiment 1.2, wherein, OAS2 (IFNa+N-N ) group represents the relative mRNA expression level of OAS2 in the test sample relative to the control sample; Relative mRNA expression levels of USP18 relative to the respective control samples.

Table 5. In vitro induction conditions of test samples and control samples in different groups

Figure 1a shows the detection results of the OAS2N group, the results showed that in the PBMC samples of CHB patients without any in vitro induction before IFNα treatment, the normalized mRNA level of OAS2 in the response group was significantly higher than that in the non-response group. Figure 1b shows the detection results of the OAS2 (IFNa+N-N) group, the results show that in the PBMC samples of CHB patients induced by IFNα in vitro before receiving IFNα treatment, the relative expression level of OAS2 mRNA in the responder group was significantly lower than that in the non-responder group of patients.

Figure 2a shows the detection results of the USP18(IFNa+N-N) group, the results show that in the PBMC samples of CHB patients induced by IFNα in vitro before receiving IFNα treatment, the relative expression level of USP18 mRNA in the responder group was significantly lower than that of the non-responder group of patients. Figure 2b shows the detection results of the USP18 (IFNa+HBcAg-HBcAg) group, and the results show that the CHB patients induced and cultured in vitro by IFNα and HBcAg polypeptide library (containing the HBcAg antigenic fragment shown in SEQ ID NO:3-37) received In the PBMC samples before IFNα treatment, the relative expression level of USP18 mRNA in the response group was significantly lower than that in the non-response group.

The above results indicated that OAS2 levels in pre-treatment PBMC samples of patients responding to IFNα treatment were significantly higher than those of patients not responding to IFNα treatment; at the same time, pre-treatment PBMC samples of patients responding to IFNα treatment were induced by IFNα and/or HBcAg in vitro The levels of OAS2 and/or UPS18 were significantly lower in patients who did not respond to IFNα treatment. Therefore, OAS2 and/or UPS18 can be used to predict the response of CHB patients to IFNα therapy.

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

According to the experimental conditions of the OAS2 N group and the OAS2 (IFNa+N-N) group in Example 3, the corresponding OAS2 N detection method and the OAS2 (IFNa+N-N) detection method were established respectively, and Table 6 records the main steps of the above two methods . In this example, the above two methods were used to detect the samples of CHB patients recorded in Example 3, and two diagnostic indicators of the samples were obtained respectively: normalized mRNA level of OAS2 and relative mRNA expression level of OAS2 ; Then use SPSS 17.0 software to take the normalized mRNA level of OAS2 and the relative expression level of OAS2 mRNA as diagnostic indicators, and draw the ROC curve to test whether the OAS2 N detection method or OAS2 (IFNa+N-N) detection method alone can predict the effect of IFNα on CHB patients. The therapeutic effect or performance of CHB patients in response to IFNα treatment. The results of the ROC curve analysis are shown in Figure 3 and Table 7. The results show that the areas under the ROC curve (AUC) of the OAS2 N detection method and the OAS2 (IFNa+N-N) detection method are 0.774 and 0.814 respectively; the sensitivities are 72%, 71%, respectively. %; specificities were 80%, 83%.

The above results show that both detection methods have high sensitivity and specificity, and OAS2N detection method or OAS2(IFNa+N-N) detection method alone can be used to predict the therapeutic effect of IFNα on CHB patients or the response of CHB patients to IFNα treatment. response with high sensitivity and specificity.

Table 6. Main steps of OAS2 N assay and OAS2(IFNa+N-N) assay

Table 7. ROC analysis of OAS2 N detection method and OAS2 (IFNa+N-N) detection method

Example 5. OAS2 (IFNa+N-N) detection method, USP18 (IFNa+N-N) detection method and the ROC curve analysis of the combined detection of the two

According to the experimental conditions of the OAS2 (IFNa+N-N) group and the USP18 (IFNa+N-N) group in Example 3, the corresponding OAS2 (IFNa+N-N) detection method and USP18 (IFNa+N-N) detection method were respectively established, and Table 8 records The main steps of the above two methods are described. In this example, the above two methods were used to detect the CHB patient samples recorded in Example 3, and two diagnostic indicators were obtained respectively: the relative mRNA expression level of OAS2 and the relative mRNA expression level of USP18; and then Using SPSS 17.0 software, the relative expression level of OAS2 mRNA or the relative expression level of USP18 mRNA was used as a diagnostic index, and the ROC curve was drawn to test whether the OAS2 (IFNa+N-N) detection method or USP18 (IFNa+N-N) detection method alone was used in predicting IFNα. The therapeutic effect of CHB patients or the performance of CHB patients in response to IFNα treatment; and the above indicators are used as joint detection indicators, and the statistical analysis value is obtained through the Logistic regression model, and the ROC curve is drawn again with this value to evaluate the joint detection of the two. predictive performance. The results of ROC curve analysis are shown in Figure 4 and Table 9. The results showed that the areas under the ROC curve (AUC) of the two methods were 0.814 and 0.823 respectively; the sensitivities were 71% and 60% respectively; the specificities were 83% and 95% respectively. %. The AUC of the combined detection of the two methods can reach 0.861, and the sensitivity and specificity are 74% and 89%, respectively. The above results show that OAS2(IFNa+N-N) detection method and USP18(IFNa+N-N) detection method and combined detection all have good sensitivity and specificity, and further show that whether using OAS2N detection method alone or OAS2(IFNa+N-N) detection method , or the combined detection of the two have good sensitivity and specificity for predicting the therapeutic effect of IFNα on CHB patients or the response of CHB patients to IFNα treatment.

Table 8. Main steps of OAS2(IFNa+N-N) assay and USP18(IFNa+N-N) assay

Table 9. ROC analysis of OAS2(IFNa+N-N) detection method and USP18(IFNa+N-N) detection method and their combined detection

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the claims appended hereto and any equivalents thereof.

sequence listing

<110> Xiamen University

<120> Method and kit for predicting response of chronic hepatitis B patients to IFNα treatment

<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

<400> 1

atgggaaatg gggagtccca gctgtcctcg gtgcctgctc agaagctggg ttggtttatc 60

caggaatacc tgaagccccta 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 accccagcccc 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

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

1 5 10 15

<210> 38

<211> 183

<212> PRT

<213> 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 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 Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

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

aaaatgct ggcaatggct gaa 23

<210> 77

<211> 21

<212>DNA

<213> Artificial sequence

<220>

<223> probe

<400> 77

cttgggggagg 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

tggtcccgctgttctgctgg 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

aggagaggagat ggatgaatgt gagca 25

<210> 92

<211> 25

<212>DNA

<213> Artificial sequence

<220>

<223> probe

<400> 92

ccgacaggat gcagaaggag atcac 25

Claims (9)

1. A test kit comprising the first reagent capable of detecting marker expression levels, optionally also including IFNα and/or stimulators; wherein the markers are selected from OAS2 or USP18, and the stimulators are selected from HBV antigens, antigenic fragments of HBV antigens, or any combination thereof; Preferably, the HBV antigen is HBcAg; more preferably, the HBcAg has an amino acid sequence as shown in SEQ ID NO: 38; preferably, the antigenic fragment has an amino acid sequence selected from the following: SEQ ID NO: 3-37; Preferably, the stimulator comprises antigenic fragments respectively having the amino acid sequences shown in SEQ ID NO: 3-37; Preferably, the first reagent is a reagent capable of detecting the mRNA level or protein level of the marker, for example, a reagent capable of detecting the mRNA level of the marker. 2. The kit of claim 1, comprising a first reagent capable of detecting the expression level of OAS2, optionally further comprising IFN[alpha] and/or a stimulator. 3. The kit of claim 1, comprising a first reagent capable of detecting the expression level of USP18 and IFN[alpha], optionally further comprising a stimulator. 4. The kit of any one of claims 1-3, further comprising a second reagent for pretreatment of a sample containing PBMCs, wherein the second reagent comprises one or more reagents selected from the group consisting of: Diluents to dilute samples (eg, phosphate buffered saline or saline); anticoagulants to prevent blood clotting (eg, heparin); reagents to maintain PBMC viability (eg, culture medium or broth) and, a reagent for separating PBMCs (for example, a lymphocyte separation fluid, such as Ficoll-diatrizoate solution); more preferably, the second reagent includes a reagent for maintaining PBMC activity (for example, a culture medium or culture medium), and/or reagents for isolating PBMCs (for example, lymphocyte separation medium, such as Ficoll-diatrizoate solution); Preferably, the kit further comprises a blood collection device (such as a pyrogen-free vacuum blood collection tube) and/or a PBMC separation device (such as a PBMC cell separation tube). 5. The use of a reagent capable of detecting the expression level of a marker in the preparation of a kit for predicting the therapeutic effect of IFNα on a subject suffering from chronic hepatitis B or the response of the subject to IFNα treatment; Wherein, the marker is selected from OAS2 or USP18; Preferably, the kit further comprises IFNα and/or a stimulator selected from HBV antigens, antigenic fragments of HBV antigens, or any combination thereof; Preferably, the HBV antigen is HBcAg; more preferably, the HBcAg has an amino acid sequence as shown in SEQ ID NO: 38; preferably, the antigenic fragment has an amino acid sequence selected from the following: SEQ ID NO: 3-37; Preferably, the stimulator comprises antigenic fragments respectively having the amino acid sequences shown in SEQ ID NO: 3-37; Preferably, the reagent capable of detecting the expression level of the marker is a reagent capable of measuring the mRNA level or protein level of the marker, for example, a reagent capable of measuring the mRNA level of the marker; more preferably, the reagent capable of The reagent for detecting the expression level of the marker is quantitatively determined by PCR to measure the mRNA level of the marker; Preferably, the kit further comprises one or more reagents or devices selected from 1)-6): 1) The diluent used to dilute the sample, such as phosphate buffer or saline; 2) Anticoagulants used to prevent blood clotting, such as heparin; 3) Reagents for maintaining PBMC activity, such as culture medium or culture fluid; 4) Reagents for separating PBMCs, such as lymphocyte separation fluid, such as polysucrose-diatrizoate solution; 5) blood collection devices, such as pyrogen-free vacuum blood collection tubes; and 6) PBMC separation device, such as PBMC cell separation tube; Preferably, the kit includes reagents (for example, medium or culture fluid) for maintaining the activity of PBMCs, and/or reagents for isolating PBMCs (for example, lymphocyte separation fluid, such as polysucrose-diatrizoate solution). 6. The use of claim 5, wherein the test kit predicts the therapeutic effect of IFNα on a subject suffering from chronic hepatitis B or the response of the subject to IFNα treatment by a method comprising the following steps: (1) determining the expression level of the marker in a sample from the subject using a reagent capable of detecting the expression level of the marker, wherein the marker is OAS2; and (2) Comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, and comparing the statistical analysis value with the reference value, and judging whether the subject will respond to the Response to IFNα treatment; Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat; Preferably, in step (2), the expression level is statistically analyzed using a Logistic regression model; Preferably, before step (1), the method further comprises one or more of the following steps: (a) using a blood collection device to obtain a sample from the subject; (b) treating the blood collection device with an anticoagulant or a sample from the subject; (c) treating the sample from the subject with a reagent for maintaining PBMC activity; (d) diluting the sample from the subject with a diluent; and (e ) isolating PBMCs from a sample from said subject using a reagent for isolating PBMCs or a PBMC isolation device. 7. The use of claim 5, wherein the test kit predicts the therapeutic effect of IFNα on a subject suffering from chronic hepatitis B or the response of the subject to IFNα treatment by a method comprising the following steps: (1) Use IFNα to stimulate at least one sample from the subject as a test sample, and use a sample that has not been stimulated by IFNα as a control sample; or use IFNα and a stimulator to jointly stimulate the sample from the subject At least one sample is used as a test sample, and the sample stimulated by the stimulant but not stimulated by IFNα is used as a control sample; wherein the stimulator is selected from HBV antigen, antigenic fragments of HBV antigen or any combination thereof; (2) Use a reagent capable of detecting the expression level of the marker to measure the expression level of the marker in each sample in step (1), and obtain the expression level of the marker in the sample to be tested compared with the control sample The fold change of the expression level of the marker in the test sample, the fold change is used as the relative expression level of the marker in the test sample, wherein the marker is selected from OAS2 or USP18; and (3) Compare the relative expression level with a reference value, or perform statistical analysis on the relative expression level to obtain a statistical analysis value, and compare the statistical analysis value with the reference value, and determine whether the subject Will respond to IFNα treatment; Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat; Preferably, in step (2), the multiple of change of the mRNA level of the marker in the test sample compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the relative mRNA expression levels of the markers in the sample; Preferably, in step (3), statistical analysis is performed on the relative expression level using a Logistic regression model; Preferably, before step (1), the method further comprises one or more of the following steps: (a) using a blood collection device to obtain a sample from the subject; (b) treating the blood collection device with an anticoagulant or a sample from the subject; (c) treating the sample from the subject with a reagent for maintaining PBMC activity; (d) diluting the sample from the subject with a diluent; and (e ) isolating PBMCs from a sample from said subject using a reagent for isolating PBMCs or a PBMC isolation device. 8. A method for predicting the therapeutic effect of IFNα on a subject with chronic hepatitis B or the response of said subject to IFNα treatment, or a method of obtaining the result of a diagnostic assay, wherein said diagnostic assay is used to predict IFNα A therapeutic effect on a subject suffering from chronic hepatitis B or a response of said subject to IFNα treatment comprising the steps of: (1) providing a sample from the subject; (2) determining the expression level of a marker in the sample, wherein the marker is OAS2; and (3) comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, and comparing the statistical analysis value with the reference value, and judging whether the subject will Response to IFNα treatment; Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat; Preferably, in step (2), the mRNA level or protein level of the marker is determined, for example, the mRNA level of the marker is determined; more preferably, in step (2), the marker is quantitatively determined by PCR The mRNA level of the animal; Preferably, in step (3), the expression level is statistically analyzed using a Logistic regression model; Preferably, before step (1), one or more of the following steps are further included: (a) obtaining a sample from the subject; (b) adding anticoagulation to the sample from the subject (c) obtain PBMC or blood components containing PBMC (for example, peripheral blood buffy coat) from the sample from the subject; (d) add to the sample from the subject a culture fluid or culture medium; and, (e) diluting a sample from said subject. 9. A method for predicting the therapeutic effect of IFNα on a subject with chronic hepatitis B or the response of said subject to IFNα treatment, or a method of obtaining the result of a diagnostic assay, wherein said diagnostic assay is used to predict IFNα A therapeutic effect on a subject suffering from chronic hepatitis B or a response of said subject to IFNα treatment comprising the steps of: (1) Provide at least two samples from the subject; (2) Use IFNα to stimulate at least one sample as a test sample, and use a sample without IFNα stimulation as a control sample; or use IFNα and a stimulator to jointly stimulate at least one sample from the subject as a test sample Sample, and the sample stimulated by the stimulus but not stimulated by IFNα is used as a control sample; wherein the stimulus is selected from HBV antigen, antigenic fragments of HBV antigen or any combination thereof; (3) Determining the expression level of the marker in each sample in step (2), and calculating the change factor of the expression level of the marker in the sample to be tested compared to the expression level of the marker in the control sample, Taking the change factor as the relative expression level of the marker in the sample to be tested, wherein the marker is selected from OAS2 or USP18; and (4) Compare the relative expression level with a reference value, or perform statistical analysis on the relative expression level to obtain a statistical analysis value, and compare the statistical analysis value with the reference value, and determine whether the subject Will respond to IFNα treatment; Wherein, the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or peripheral blood buffy coat; Preferably, in step (2), the HBV antigen is HBVcAg; more preferably, the HBVcAg has the amino acid sequence shown in SEQ ID NO: 38; preferably, the antigenic fragment has amino acids selected from the following Sequence: SEQ ID NO: 3-37; More preferably, the stimulus comprises antigenic fragments having the amino acid sequences shown in SEQ ID NO: 3-37; Preferably, in step (3), the mRNA level or protein level of the marker is determined; for example, the mRNA level of the marker is determined; more preferably, in step (3), the marker is quantitatively determined by PCR The mRNA level of the animal; Preferably, in step (3), the multiple of change of the mRNA level of the marker in the test sample compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the relative mRNA expression levels of the markers in the sample; Preferably, in step (4), the relative expression level is statistically analyzed using a Logistic regression model; Preferably, before step (1), one or more of the following steps are further included: (a) obtaining a sample from the subject; (b) adding anticoagulation to the sample from the subject (c) obtain PBMC or blood components containing PBMC (for example, peripheral blood buffy coat) from the sample from the subject; (d) add to the sample from the subject a culture fluid or culture medium; and, (e) diluting a sample from said subject.