TWI330200B - Hbv pre-core pretein having particle-forming ability - Google Patents

Description

1330200 玖, invention description: [Minghujin Xuanyuanbei invention field] The present invention relates to a HBV pronuclear protein which can form nuclear particles such as HBV virus. I. Prior Art 3 Background of the Invention The diagnosis of viral infection is mainly carried out by a method of detecting a virus and a virus-associated component (protein and nucleic acid) and a method of measuring a specific antibody produced by a virus infection. In the case of hepatitis B virus (HBV) infection, a diagnostic test for HBs antigen/antibody, HBc antibody, HBe antigen/antibody, 113 yen-1, and the like has been introduced. In general, the presence or absence of HBV infection can be detected using HBs antigen and HBc antibodies. In addition, not only the infectivity of HBV, but also the measurement of the amount of HBe antigen/antibody and HBV-DNA is used as an indicator for determining the condition of the HBV-bearing person and the effect of the treatment. Among them, the HBe antigen/antibody immunoassay is simple and inexpensive to use. It is the most widely used, but in the prokaryotic mutant which cannot produce/secret the HBe antigen, since the HBe antigen is negative, the proliferation of HBV is still strong, so There are cases where the infectivity and immunogenicity are strong and the pathology is quite active. On the other hand, regardless of the wild strain or the mutant strain, the measurement of the amount of HBV-DNA reflecting the proliferation/replication state of HBV is important, but the pretreatment method and the measurement method are complicated, and the problems of reproducibility and stability are lacking.

Recently, HBV nuclear-associated antigen assays have been developed which are considered to reflect the proliferative/replicative state of HBV 5 1330200, either in the presence of HBV nuclear-related antibodies (HBc antibodies and HBe antibodies) or in the presence of variants. Kimura et al (Journal of Clinical Microbiology, 40, 439-445, 2002) use a monoclonal antibody specific for HBV-associated antigen (HBV pronuclear/nuclear genetic product containing HBc antigen and HBe antigen), which is simple before The treatment method has developed an immunoassay method for simultaneously detecting HBc antigen and HBe antigen in serum, and has been shown to have a relationship with the NTA test for detecting a viral genome. In the literature report so far, the HBV pronuclear/nuclear genetic code encodes a full-length 212 amino acid residue (amino acid number: -29 to 183 (the first amino acid of the HBc antigen is 1. The same is true below) )) The protein consists of a 19-residue message peptide (signa pepti (Je) at its N-terminus. The so-called message peptide is composed of a hydrophobic amino acid residue, and its function is used as a secreted protein. The sequence of the message bound to the membrane, herein refers to the sequence consisting of the _29--11 amino acid.

Once the nucleoprotein is decoded, it passes through the endoplasmic reticulum membrane, and after the 19-residue message peptide is cleaved, the end (four) binding region is cleaved into a plate antigen (amino acid number. 10-10-149) and secreted into the blood. In the past, the HBe antigen was thought to be present in the blood in combination with serum proteins such as albumin and γ-globulin, and there was no report of the formation of HBV particles. In addition, although the HBe antigen is also maintained with immune tolerance, Reports related to the suppression of viral replication, but only for the replication of HBV. The part of the biological role is still unknown. The W nuclear/nuclear genetic gene has a second start message. The product translated from the beginning of the message forms a plate antigen (amino acid number: i 6 1330200 ~ 183), and the HBV progenitor RNA is incorporated into one side to form a viral capsid, and the jacket formed by the HBs is placed on the jacket ( After envelope, it is released into the cell as a complete virus particle. Although the first _144 amino acid is present in the HBc antigen, nuclear particle formation occurs, but an infectious virus is formed. Nuclear particle The molecule is considered to be composed of a protein of the first 183 amino acid sequence. As described above, the HBc antigen and the HBe antigen are both proteins having most of the common sequences, and at the same time have very different properties. The main cause of the difference is the cysteine residue of the amino acid No. 7 of the HBe antigen. The cysteine acid forms a cysteine residue with the amino acid 61 of the same molecule. Sulfide binding, obtaining e antigenicity (M. Nassal and A. Rieger, Journal of Virology, 67; 4307-4315 ^ 1993). From the application of the first 7th cysteine to other amino acids The treatment, etc., resulted in the inability to form the disulfide bond, that is, the fact that the structure of the HBe antigen could not be obtained, and also showed the importance of the disulfide bond. On the other hand, the HBc resistance was from the amino acid No. 1 The site is initially translated, so there is no cysteine residue located in amino acid No. 7 to obtain the HBc structure; two molecules of HBc protein are formed by the semi-photoreceptor residues of the amino acid No. 61. Disulfide combines to form a homodimer, forming Toxic protein sheath (nuclear particle). HBV nuclear-associated antigen in the serum of patients with HBV is roughly divided into HBc antigen (amino acid sequence number: [~183), which mainly constitutes infectious Dane particles, and, although with HBc antigen Almost identical in the amino acid sequence, 7 1330200, but the secretory HBe antigen (amino acid number: -10 to 149) which is different in antigenicity from the HBc antigen. However, TAKAHASHI et al. (Journal of Immunology, 147, 3156-3160, 1991) It has been indicated that other HBe antigens (amino acid number: one 29 to 149) appear to be secondary molecules in the blood before exhibiting HBe antigenicity. The mixed serum is separated by ultracentrifugation, and the protein having HBe antigenicity is purified from the affinity column of the anti-HBe monoclonal antibody and the sequence is determined. The HBe antigen protein line is considered to be present in the blood without forming viral particles as described above. They also determine the sequence of the original sequence without the peeling (destruction) of the coat protein of HBV, that is, the treatment of HBs antigen, or similar treatment of virus particles and nuclear particles: with the evidence The sequence of the HBe antigen (amino acid number: a 29 to 149) is sufficiency in the presence of the system in the serum. On the other hand, 'although it has been reported that there are empty particles containing no DNA in the Dane particles (SAKAMOTO et al. Laboratory Investigation, 48, 678-682, 1983) and HBV containing the shorter ones that are generally spliced. Defective particles of DNA that cannot be replicated (TERRE et al Journal of Virology, 65, 5539_5543, 1991), but no detailed analysis of the HBV protein forming the particles was performed. Thus, the biochemical analysis of HBV nuclear-associated antigens in the blood analyzed in these documents cannot be said to be sufficient, and is considered to be a viral marker of the HBV nuclear-associated antigen reflecting the proliferating/replicating state of HBv in the blood. The existence pattern has been maintained in an unsolved state. [Bright content] 8 1330200 Summary of invention

In the NAT test of HBV with high clinical usefulness, there are problems such as the complicated method of PCR and TMA. In addition, due to the use of the gene amplification method, if the primer for the increase is inconsistent with the target Dna, it will cause a false negative. On the other hand, the immunoassay is simple and inexpensive to measure, but in the current HBe antigen assay as a proliferation marker, it is not possible to form the HBe antigen in the form of an immune complex in the presence of the HBe antibody. The measurement "In the case of a pronuclear mutant or a nuclear promoter variant in which the HBe antigen cannot be produced/secreted, the HBe antigen also becomes negative. Furthermore, the HBe antigen assay system has a correlation with the amount of HBV-DNA, but it cannot be applied clinically because of the complicated pretreatment and insufficient sensitivity. Therefore, the object of the present invention is to perform biochemical analysis of HBV nuclear related antigens which are considered to be clinically applicable in the screening of hepatitis B and the treatment of patients with chronic hepatitis B, and will reflect the blood of the proliferating/replicating state of HBV. The molecular species of the HBV nuclear related antigen were identified. According to the analysis thus made, it is found that a molecular species useful for diagnosis of a patient infected with HBV, and a novel diagnostic drug can be developed. Furthermore, by analyzing the existence form of HBV nuclear-associated antigen and its assignment task, it is possible to link the development of novel HBV vaccines and therapeutic drugs. The invention relates to HBV particles and HBV-associated proteins in blood, which are separated and purified by sucrose density gradient centrifugation, ELISA, electrophoresis, etc., and the molecules forming novel nucleus particles are formed by mass spectrometry. To be identified. One aspect of the present invention is to provide a HBV pronuclear protein comprising all or a portion of a message sequence having the toxic particle forming ability of a disease-like 9 1330200. The term "partially referred to herein" refers to a sequence of one or more residues from the N-terminal side among the message sequences of the 19th residue. On the other hand, the end of the HBV pronuclear protein was cut at any part of the RRRRR after the amino acid No. 150 or later. Further, the message sequence or the HBe sequence portion (amino acid number: -10 to 149) may also contain a variation' as long as the allowable variation is in the range in which the nucleation-like particles can be formed. Preferably, the HBV pronuclei protein is a polypeptide formed from the amino acid sequence of SEQ ID NO: 1. Further, the present invention provides HBV-like core particles or hbv-like virus particles formed by the above HBV pronuclear protein. Further, an HBV diagnostic drug or kit comprising the HBV pronuclear protein, or HBV-like nuclear particles composed of the protein, and HBV-like virus particles is provided. Further, a method for measuring the HBV pronuclear protein, or a diagnostic drug or a diagnostic kit is provided. The HBV pronuclear protein assay may also include the step of exposing the HBv pronuclear protein and the step of binding the HBV pronuclear protein to the recognition probe. However, the step of exposing the HBV pronuclear protein may also be the treatment or addition of the surfactant. The activity of the interface is not limited, and the nonionic surfactant is also effective. The ionic surfactant is particularly suitable. However, when a diionic surfactant is added to an anionic surfactant, further addition of a nonionic surfactant is effective. Further, the probe which binds to the aforementioned HBV pronuclear protein may be combined with the sequence probe of the amino acid number - 28 to 11. The bribe can also be 10 1330200. The anti-HBs antibody is used for immunoprecipitation. By measuring the HBV pronucleus/nuclear protein in the sediment, it is also possible to specifically measure only the particulate HBV pronucleus from which the free HBe antigen has been excluded. / Nuclear protein. This immunosuppression can also be replaced by separation using a probe that binds to the Η B s antigen. According to the above measurement method, when the measured value of the HBV pronuclear protein is included in the measured value of the HBe antigen, the value of the particulate HBV pronuclear protein in the sample can be calculated by subtracting the measured value of the HBe antigen. Determination. Further, the correct value of the particulate HBV pronuclear protein in the sample can also be measured by subtracting the measured value of the HBc antigen from the value. The quantitative value of the determined particulate HBV pronuclei protein can be used as a pathological marker for HBV infection. Further, the present invention provides an assay kit comprising an anionic surfactant and an antibody recognizing the amino acid of the _28 to 15 〇 amino acid of the HBV pronuclear protein, and a diagnostic drug comprising a kit for measuring the HBe antigen. Other aspects of the invention provide HBV vaccines and therapeutic agents comprising HBV pronuclei. In addition to the above, the present invention provides methods for assaying antibodies against particulate HBV pronuclei, or diagnostic agents, diagnostic kits. In the above, the above-mentioned HBV pronuclear protein particles can be used as an antigen. The particles can be purified from density gradient centrifugation, immunoprecipitation, etc. by using a serum such as a density gradient centrifugation or an immunoprecipitation, and the HBV pronucleus can be obtained by removing the coat with a surfactant or the like. The protein antigen is exposed. Alternatively, a recombinant protein can be used to produce a particulate hbv pronuclear protein. By using this antigen, it is possible to measure an antibody against the particulate HBV pronuclear protein which is different from the HBc antibody and the HBe antibody. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram showing the range of HBV pronuclear protein, HBe antigen, HBe antigen having a message sequence, and amino acid range of HBc antigen, and the morphology of each antigen in blood. Fig. 2 is a schematic diagram showing the HBV antigen and HBV-DNA behavior of ΗB e antigen-positive serum separated by sucrose density gradient centrifugation. The HBV nuclear-associated antigen (black square), HBc antigen (#), HBs antigen (◊), HBe antigen (□), and HBV-DNA (〇) are shown in each of the isolated fractions; Do not. Fig. 3 is a schematic diagram showing the behavior of HBV antigen and HBV-DNA when the separated fraction of the isolated Hbv nuclear-associated antigen was separated by sucrose density gradient centrifugation by sucrose density gradient centrifugation. HBV nuclear-associated antigen (black square), HBc antigen (Lu), HBs antigen (◊)', and HBV-DNA (〇) are shown in each fraction; the density of the separated fraction is indicated by a dotted line. Figure 4 is the HBV antigen. A schematic diagram of the act of filtering with a gel. The above figure is the case where the largest separated fraction of the HBV nuclear-associated antigen separated by sucrose density gradient centrifugation is directly applied to the gel filtration without treatment. The figure below shows the same sample treated with 3% NP-40 and then subjected to gel over-reduction. The amount of HBV nuclear-associated antigen (black square) and HBc antigen (♦) in each isolated fraction is shown. The dissolution position of the molecular weight marker is shown on the upper portion. Fig. 5 is an electrophoresis diagram showing the results of Western blot analysis of ΗB e antigen-positive serum and its density gradient centrifugation section, in place of the photo of 12 1330200. The density gradient centrifugation sections 1 to 20 correspond to a sucrose concentration of 10 to 60%. HB50 is a monoclonal antibody that reacts only to HBc and HB91 reacts to both HBc antigen and HBe antigen. Figure 6 is a graphical representation of the ratio of total pronuclear/nuclear protein to the particulate pronuclear protein in each case. The (H) antigen positive example is represented by (_), and the HBe antibody positive example is represented by (〇). [Embodiment 3] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail. A newly identified HBV pronuclear protein in the present invention refers to a protein comprising a message sequence and which can form a viral particle such as HBV. The protein of the present invention in blood, whose main molecule is a p22 antigen formed by auricular abundance of 22,000 amps. A representative amino acid sequence of the p22 antigen is similar to the antigen of the HBV pro-nuclear antigen formed by the 178 amino acid described in the Sequence Number. By obtaining an antibody that specifically recognizes the antigen and combining the pre-treatment methods to construct a measurement system, the HBV nuclear-associated antigen can be measured even in the presence of an antibody. Often referred to as HBV pronuclei/nuclear proteins are HBe antigens (HBV e proteins), HBc antigens (HBV nucleoproteins), and other HBV pronuclear/nuclear gene products. In the case of "HBV pronuclear protein" as used in the present invention, unless otherwise specified, it means a Hbv pronuclear/nuclear gene product, i.e., a protein, which contains a gfl sequence and has particle formation energy. The protein shown in the sequence listing is a representative example thereof. Further, "HBV nuclear-associated antigen" means a HBV pronuclear/nuclear gene product containing HBc antigen (HBV nucleoprotein) and HBe antigen (HBV e protein). 13 1330200 Firstly, patients with hepatitis B were also separated by sucrose density gradient centrifugation. When measuring HBV nuclear-associated antigens in each fraction, a peak was detected near the same density of 1.05 as the HBe antigen, at a density of 117. Another peak can be detected in the vicinity of the separation portion which is substantially identical to the HBc antigen and HBV-DNA. If the peak near the density 117 is further separated by a low gradient sucrose density gradient centrifugation, the HBc antigen and the HBV-DNA show a peak in the same fraction, but the HBV nuclear-associated antigen will be slightly lower. A peak is shown in the separated portion of the density, and a virus foreign protein, that is, an HBs antigen, can be detected in the separated portion. That is, in the serum of patients with hepatitis B, it is considered that in addition to the Dane particles showing the susceptibility, there are viroid-like particles formed by a slightly lighter HBV nuclear-associated antigen. Further, if the fraction containing the particles formed by the HBV nuclear-associated antigen and the Dane particles is subjected to gel filtration, the HBc antigen and the particles formed by the conjugated antigen are separately eluted into the empty body fraction. @, If the same fraction is treated with Nonidet P-40 (NP~4〇: Naealai Tesque), the HBs antigen of the protein is stripped and then condensed and filtered, and the HBc antigen and the antigen associated with HBV are formed. The particles are likewise dissolved separately into the hollow body separation portion. This case shows that the HBc antigen and each particle formed by the HBV-associated antigen are formed, and the strong protein sheath structure is not destroyed even if it is treated with a surfactant which can strip the HBs antigen of the protein. Secondly, from the analysis of the Western blot method, although the HBV nuclear 14 1330200 antigen exhibits a molecular weight of 22,000 amps that is approximately the same as the original HBc, in the C-terminal nucleic acid binding region with the HBc antigen, Specific monoclonal antibodies do not react. That is, the HBV nuclear-associated antigen can be regarded as an uncut pronuclear gene product (p22 antigen) of a message sequence depending on its molecular weight and does not contain a C-terminal nucleic acid binding region. The P22 antigen was separated by SDS-PAGE and excised from polyacrylamide gel. 'Material-assisted laser desorption freeing (MALDI) mass spectrometry (Carr et al. Carrent Protocols in Molecular Biology, John

Wiley & Sons, Inc" New York Units, 1997) Determining the amino acid sequence 'is clear that the p 2 2 antigen system is formed by the HBV protein described in the sequence number 丨7-8 amino acid. The amino acid sequence is determined to be a HBV pronuclear protein, but the HBV pronuclear protein of the present invention is the same as the hbv pronucleus previously reported by TAKAHASHI et al. (Journal of Immunology, 147, 3156-3160, 1991). Compared with the protein (pre-HBe antigen), it lacks the N-terminal (No. 29) methionine, and the first 28 glutamine amine is acetylated. In addition, the C terminal is at the end of the CBe antigen C. The 149th amino acid is bound to arginine, at least to No. 150. The HBV pronuclear protein is recognized by the monoclonal antibody HB50 which cannot be identified in the site containing the SPRRR in the HBV nucleic acid binding site. There is a possibility of containing an amino acid up to No. 154. From the HBV pronuclear protein of the present invention, incomplete virions (viral-like particles) are formed, and it is easy to infer that the protein forms a protein sheath, and HBc The situation in which antigens compete to prevent viral replication. The present invention teaches the possibility of using HBV prodrugs of HBV prodrugs. 15 1330200 The HBV pronuclear protein of the present invention forms HBV-like virions in the body of HBV-infected patients. [Additionally, the present invention also teaches the hbV pronuclei. The fact that the protein is present in the complete HBV virion. That is, the HBV pronuclear protein is thought to form the HBV nucleo-capsid together with the HBc antigen. Therefore, the HBV pronuclei protein is determined as hepatitis and cirrhosis. The diagnosis and labeling of pathological changes of lesions or liver cancer are considered to be useful. The HBV pronuclei protein of the present invention is different from the known HBe antigen and HBc antigen in terms of function and amino acid sequence. However, because of its function The approximate nature and the repeat of the amino acid sequence are necessary for the measurement of the HBV pronuclear protein, which is distinguished from the HBe antigen and the HBc antigen. However, as shown in Example 7 to be described later, the HBV is used. The ratio of the amount of pronuclear/nuclear protein to HBc antigen is 12: 1 to 158: 1, and the difference is also 12 times or more. The HBV pronuclei protein of the present invention and the HBe antigen, or the signal peptide reported by Takahashi et al. The HBe antigen is different, and the former forms a nucleus-like particle (viral-like particle). In contrast, the latter exists in a free state in combination with serum proteins in the blood. The function of forming such a nuclear particle (viral-like particle) is The HBV pronuclear protein has approximately the same function as the HBc antigen. The HBV pronuclear protein has the property of forming particles, and it can be speculated that it has a structure closer to the HBc antigen than the HBe antigen, and the amino acid No. 7 The disulfide bond between cysteine and cysteine 61 was not formed. As shown in Example 9 to be described later, according to the HBV nuclear-associated antigen assay system, when the detected HBV nuclear-associated antigen (HBV pronucleus/nuclear protein) is detected by the conventional HBc antigen assay and the HBe antigen assay, The original and HBe 16 1330200 antigens were not detected. This can be considered as the case shown below. The HBV nuclear associated antigen contains a number of HBV pronuclei proteins of the invention. The antibody which binds to the conventionally obtained HBc antigen and the HBe antigen does not bind to the HBV pronuclear protein, and the antigenicity of the HBV pronuclear antigen is different from that of the HBc antigen and the HBe antigen. This case shows that the HBV pronuclei of the present invention can hardly be detected in the conventional HBc antigen assay and HBe antigen assay. That is, it can be considered that the measurement can be carried out by using the HBV nuclear-associated antigen assay of Kimura et al. HBV pronuclei can be detected by HBV nuclear associated antigen. The assay system is characterized by treating a sample containing HBV pronuclear protein with a surfactant and reacting it with an antibody in the presence of a surfactant. Therefore, the epitope on the HBV pronuclear protein must be stable in the presence of the surfactant. In addition, antibodies must also be functionally stable in the presence of a surfactant. Identification of the epitope of such an antibody is based on the linear antigenic epitope of the first 19, 21-40, 31-49, 131-140 amino acid of HBV nucleoprotein, and the constructive antigenic determinant of the first 81 . HBV pronuclei can be assayed by combining antibodies that recognize these epitopes. By combining more than two of these antibodies that recognize the epitope, HBV pronuclei can be detected. A particularly suitable combination is the identification of 21st and 4th, 31st, 19th, 19th, 21st, and 21st. Combination of 40 and 31-49 amino acid epitopes; by adding antibodies recognizing other epitopes to these combinations, the sensitivity is increased. The antibody used at the time of detection must be an antibody which binds to the above-mentioned epitope in the presence of a surfactant. The surfactant used in the HBV pronuclear protein assay 17 1330200 is suitable as an anionic surfactant, but the combination of an anionic surfactant and a diionic surfactant, or an anionic surfactant, When combined with an ionic surfactant and a nonionic surfactant, the sensitivity is improved. Among the anionic surfactants, SDS is most suitable, but other anionic surfactants are also effective. By transposing SDS into other structurally similar anionic surfactants, the above antigenicity of HBV pronuclei can be determined. The site is exposed to form a state in which it can bind to the antibody. Further, the difference between the HBV pronuclei protein of the present invention and the conventional HBe antigen, the HBe antigen having the message peptide reported by Takahashi et al., and the amino acid sequence of the HBc antigen is shown in Fig. 1. HBV pronucleus/nuclear protein As is apparent from the present invention, a representative example is a polypeptide formed from the amino acid sequence of the first 28 to 150. On the other hand, the HBe antigen is a polypeptide composed of the amino acid sequence of the first to 183, and the amino acid sequence of the first 10 to 149 amino acid sequence. According to the above, it is not easy to isolate the HBV pronuclei protein and the HBe antigen of the present invention, the HBe antigen having the message peptide, and the HBc antigen, but it is possible to use whether or not the virion can be formed. The difference between the function and the amino acid sequence is separated. First, in order to isolate the HBV pronuclear protein of the present invention and the HBe antigen or the antigen having the message peptide, the HBV pronuclear protein system is used to form HBV-like HBV particles, and the HBe antigen or the HBe antigen having the message peptide is free. The state (in the present invention, the HBe antigen bound to the antibody is also free) is characteristic. Actually: 18 Measured ~ (1) There is a branch which is measured by the value of 2 measured before the treatment for destroying the virus particles and the measured value measured after the treatment for destroying the virus particles. That is to say, it is possible to measure by subtracting the measured value before the destruction of the virus (4) from the value of the scale after the scale is destroyed. Containing (2) material, there is also a method for separating the (viral) virus particles and then determining the pre-caffeine nucleoprotein. In the method of isolating the (type) virus particles, for example, a method of separating the separated portion of the free antigen and the separated portion of the virus into a difficult portion by density gradient centrifugation is suitable for determining that it is difficult to determine the virus. Part of the HBV pronucleus/nuclear protein was isolated. In addition to the method of centrifuging free antigen and (viral) virus particles by two-density, if & method for specifically concentrating (virtual) virus particles, and specific method for removing free antigen, it is suitable for separation The amount of HBV pronucleus/nuclear protein was then determined. Among these methods, for example, an immunosuppression method using an anti-HBS antibody can be mentioned. When immunoprecipitation is carried out using an anti-HBs antibody, it is also possible to specifically measure only the particulate HBV pronucleus/nuclear protein from which the plate antigen is removed by measuring the pre-transcriptional nuclear/nuclear protein in the sediment. However, since the HBV pronuclear protein and the HBc antigen are similar in the function of forming nuclear particles ((viral particles)), it is conceivable to specifically bind to a portion different from the constituent amino acid sequence for the separation measurement. (identifying) the probe to divide it into squares, that is, to determine the HBV pronuclear protein for selection, so that the probe to the ～29~~ acid group is preferred, in order to selectively When the HBc antigen is measured, it is preferred to use a probe which binds to the 151 to 183 amino acid. 1330200 In addition, the amount of antigen of HBV pronuclear protein + HBc antigen is measured using a probe that binds to both, and then the antigen (protein) of any one is specifically measured, and then subtracted, and each antigen (protein) can also be determined. The amount. In fact, in the measurement of HBV pronuclear protein in the sample, the step of destroying the viroid-like particle to expose the HBV pronuclear protein must be performed. In this step, the viral outer cover protein 'i.e., the peeling of the HBs antigen' is used to form a free treating agent for the HBV pronuclear protein. The treatment method is alkaline treatment, and the treatment of NaOH or the like, the HBs antigen is stripped, and the antigen inside HBV is free. Other treatments can use surfactants, such as nonionic surfactants such as Triton X-100 and Nonidet P-40, and anionic surfactants such as SDS or sodium sarcosinate. suitable. Further, a combination of an anionic surfactant as claimed in Japanese Patent No. 3,171, 827 and other surfactants is also effective. If the probe of the No. 29-149 amino acid is not detected by performing the destruction of the virus particle and the exposure of the HBV pronuclear protein, it will become a free HBe antigen or a HBe antigen having a message peptide. It is also subject to measurement. Therefore, when the antigenic protein in the sample is measured without separating the free antigen and the viroid-like particle, the surfactant is not subtracted from the measured value after the treatment with the surfactant. The value obtained by measuring the value of the HBe antigen immediately after the treatment is taken as the measured value of the particulate HBV nucleus/nuclear protein. ^ The destruction of viral particles caused by surfactants and the detection of HBV pronuclear proteins by HBV pre-nuclear eggs. A > u step, using the 20 probe H mosquito HBV pronucleus to identify the first 28~15 amino acid sequence. The probe may be any one of which: I: binds to the pronuclear protein, and antibodies, receptors, primers, ligands and the like can be used. As long as it is a combination of a HBV pronuclear protein and a probe, the test method can be represented by an enzyme antibody immunoassay. In order to isolate the HBV pronuclear protein from the HBc antigen, the HBV pronuclear protein is probed using the identification -28--11, and the HBc antigen is preferably the probe using the identification 151 to 138. However, the HBV pronuclear protein can also be quantified by measuring the total amount of HBV pronuclear protein and HBc antigen by using a probe that recognizes the common region of 1 to 150, and subtracting the total amount from the total amount. EXAMPLES The following examples are illustrative of the invention, but the scope of the invention is not limited thereby. Example 1. The isolated fraction obtained by sucrose density gradient centrifugation of HBV nuclear associated antigen (A) was separated by 10% to 60% sucrose density gradient centrifugation to make sucrose 10%, 20%, 30%, respectively. 40%, 50%, 60% were dissolved in TNE buffer [l〇mM Tris-HCl (pH 7.5), 150 mM NaCl, ImM EDTA], and 1.7 ml were taken from the high density 60% sucrose solution. Carefully overlap them into layers in a 12 ml centrifuge tube and leave at room temperature for 6 hours. A laminate was formed on the sucrose density gradient solution of 1 ml of HBe antigen-positive serum, and centrifuged at 33.4 K rpm and 4 ° C for 15 hours using a Sw40Ti rotor (Beckman), and 300 μl was taken from the top to obtain 40 separations. section. Calculate the density of each fraction, determine HBV nuclear-associated antigen (PCT application: PCT/JP01/06947), HBc antigen (PCT application: PCT/21 1330200 JP01/06947), HBs antigen (Dainabot), HBe antigen (Dainabot In HBV_DNA PCR (Roche Diagnostics), a part of the HBV nuclear-associated antigen shows a peak at the same density as the HBe antigen, but most of the HB V nuclear-associated antigen is detected near the peak of HB V - DNA PCR. Out (Figure 2). (B) The re-separated fraction obtained by low-gradient sucrose density gradient centrifugation, 30%, 40%, 50% sucrose solution, 3.4 ml each from the high density 50% sucrose solution, carefully overlapping The layers were layered in a 12 ml centrifuge tube and allowed to stand at room temperature for 6 hours. The 24th and 25th peak separation portions of (A) were diluted twice with TE buffer [10 mM Tris-HCl (pH 8.0), ImM EDTA], and then laminated on the prepared sucrose density gradient solution. The Sw40Ti rotor (Beckman) was centrifuged at 33.4 Ki: pm for 15 hours at 4 ° C, and 300 separate portions were taken from the top to obtain 40 separated portions. Calculate the density of each fraction, and determine the HBV nuclear-associated antigen (PCT application: PCT/JP01/06947, Example 5), HBe antigen (PCT application: PCT/JP01/06947, Example 6), HBs antigen, HBV - In DNA PCR, the peak of the HBV nuclear-associated antigen appears at a lower density fraction than the peak of the HBV-DNA PCR, which contains the HBs antigen (Fig. 3). After peaking, each peak was stored at 4 °C. Example 2. Gel 遽 treated with NP-40 The peak fraction separated according to (A) of the above Example 1 (24-25) was buffered with 10 mM linalic acid containing 〇_l5M NaCl. When the liquid (pH 7.3) (PBS) was equilibrated with Superose 6 HR (Amersham) for gel filtration, the particles of HBV nuclear-associated antigen and HBc antigen were eluted 22 1330200 into the empty body fraction (Fig. 4) ). On the other hand, NP-40 was added to the peak separation portion (24-25) obtained by the separation of (A) of the above Example 1, so that the final concentration was 3%, and 37 ° C was used for stripping the HBs antigen. Handle for 15 minutes. Uncoated with HBV nuclear-associated antigen and HBc antigen when gel-filtered with Superose 6 HR (Amersham) equilibrated with 10 mM phosphate buffer (pH 7.3) (PBS) containing 0.15 M NaCl The particles are separately eluted to the empty body separation section (Fig. 4, lower panel). The molecular weight separation portion of Superose 6 HR has a molecular weight of 40,000,000 or more, and these molecules are considered to have a viroid-like protein sheath particle structure. Example 3. The analysis obtained by the Western blot method and the method described in (A) of Example 1 were similarly treated with HBe antigen-positive serum to carry out sugarcane density gradient centrifugation, and 600 μl each was taken from the supernatant to obtain 20 Separate parts. SDS-PAGE was performed using this fraction and HBe antigen-positive serum, and after transfer to PVDF membrane (Immobilon, Milli-pore), the C-terminal nucleic acid binding of the monoclonal antibodies HB91 and HBc antigens of both HBc antigen and HBe antigen was identified. In the region, a Western blotting method using a specific monoclonal antibody HB50 was used to detect an HBV nuclear-associated antigen having a molecular weight of about 22,000 amps (Fig. 5). However, the fact that the HBV nuclear-associated antigen reacts to HB91 but does not react with HB50 becomes clear (Fig. 5, lower panel). The HB50 antibody is analyzed by the epitope, although it can be understood that it recognizes the 168-176 amino acid (5 (^?1^1^), in addition to its 141-159 amino acid (STLPETTVVRRRGRSPRRR) And the 150-167 amino acid (RRRGRSPRRRTPSPRRRR) is also reactive. The common sequence of these amino acids is SPRRR, and at least the sequence 23 1330200 is necessary in combination with the HB50 antibody. That is, since the 155th The SPRRR sequence is present, so the HBV pronucleus/nuclear protein of the present invention is considered to be a sequence lacking after 155. This fact can also be derived from the peak in the HBc antigen assay system using the HB50 antibody as a marker antibody alone. It does not overlap with the peak of the HBV pronucleus/nuclear protein (Fig. 2, Fig. 3). Example 4. Analysis of the amino acid sequence obtained by MALDI mass analysis will be based on the examples. The HBV nuclear-associated antigen isolation fraction obtained by the method of (A) was gelled using Superose 6 HR (Amersham) equilibrated with 10 Mm phosphate buffer (pH 7.3) (PBS) containing 0.15 M NaCl. When filtering, HBV nuclear-associated antigen and HBc antigen The formed particles were eluted into the hollow body separation portion. The hollow body fraction was laminated to TNE buffer containing 1% NP-40, 20% sucrose [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM On the EDTA], the particles containing the HBV nuclear-associated antigen particles were recovered by centrifugation for 15 hours at 33.4 Krpm and 4 ° C using a Sw40Ti rotor (Beckman). The particles were separated by SDS-PAGE and the HBV of 22 kDa was cut out. The region of the nuclear-associated antigen was subjected to MALDI-TOF mass analysis by Voyager-DE STR (Applied Biosystems) after trypsin digestion. As a result, sequences corresponding to sequence numbers 2, 3, 4, 5 and 6 were detected. 5 amino acids, wherein the terminal of the imidate of SEQ ID NO: 2 is acetylated. Example 5. Detection and assay of HBV nuclear associated antigen (pronuclear/nuclear protein) Anti-HBV nuclear antigen monoclonal antibody ΗΒ44 (identification site 31-49 amino acid), ΗΒ61 (identification site 131-140 amino acid) and 1^114 (identification site 1-81 amino acid), 0.5 Μ NaCl, 0.1 Μ carbonate buffer (ΡΗ9 .6) Dilute to 24 1330200 Final concentration is 2, 1,1 pg/m b. Each black 96-well microplate (Nunc) Inject 1 〇〇哗/well and leave at 4 〇c for one night. Washed twice with 0.4 ml of ΙΟπιΜ sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl, and added a blocking solution (0.5% casein sodium, 3% sucrose, i5 mM mM NaCl, 10 mM phosphate buffer (pH 7.4). )) 〇.4ml ' Further allowed to stand at room temperature for 2 hours. After removing the blocking solution, vacuum drying was performed. The serum 1〇〇μ1_addition treatment solution (15% SDS, 2% Tween 60) 50μb was treated at 70 ° C for 30 minutes, and 5 μμ1 was taken as the measurement sample. The reaction buffer was 1 μμ1. 5 μμ of the test sample was added to the above well, which was reacted at room temperature for 2 hours. After washing with a washing solution (0.05% Tween 20 '0.15Μ NaC Bu 10 mM acid natrate buffer (pH 7.4)) 0.4 ml, 'Alkaline Phosphotase (AP) labeled monoclonal antibody HB91 (identification site 1-19 amino acid) and HB110 (identification site 21-40 amino acid) were diluted to 〇.1μ§/ m b 0.5pg/m b and added to ΙΟΟμΙ/well to make it react at room temperature. Lj, hour. After washing 6 times with 0.4 ml of the washing solution, a CDP-Star with Emerald II (TROPIX) solution was added as a light-emitting substrate, and the mixture was allowed to react at room temperature for 20 minutes, and then the luminescence was measured. Example 6. Determination of HBc antigen (A) Preparation of antibody solid phase culture plate Three kinds of monoclonal antibodies HB44 (identification site 31-49 amino acid) and HB61 which are both reactive to both HBc antigen and HBe antigen (identification) The sites 131-140 amino acid and HB114 (identification sites 1-81) were sensed into the 25 1330200 wells of the microtiter plate and allowed to solidify. After washing with PBS, it was blocked with a solution containing casein, and the solution was removed and dried. (B) Pre-treatment of mixed 50μ1 of pretreatment solution (15% sodium dodecyl sulfate [SDS], 3% CHAPS 1% / Hexadodecyltri-methyl ammonium bromide) The sample of 1〇〇μΐ (serum, plasma, etc.) was treated in sputum for 30 minutes. (C) One-time reaction A reaction buffer of 1 〇 0 μl (ΡΗ 8.0) and 50 μl of the pretreated sample were stably stirred at room temperature for 2 hours. (D) Two-time reaction After the well was washed, a solution containing a single antibody of an alkaline phosphatase marker (50 (identification site 168-176 amino acid) was added and squirmed, and allowed to react at room temperature for 1 hour. (Ε) Matrix reaction After the wells were washed, 'CDP Star with Emerald II (Applied Biosystems) 100 μl ' was added and reacted at room temperature for 2 minutes, and the amount of luminescence of each well was measured with a Micro-plate Luminometer. Example 7. Measurement of HBV pronuclear protein, HBe antigen, HBc antigen in the sample and calculation of the ratio The serum 7 sample was separated by sucrose density gradient centrifugation according to the method of the above Example 1 (A). In each sample, a separate fraction containing a low-density free HBe antigen (for example, in Figure 1, in the 1-19 separation portion 26, 1313030 minutes or so) and a separation portion containing a high-density virus particle (for example, The measurement of each antigen was carried out in the same manner as in Example 5 and Example 6 except that 'the 20th separated portion or more' was separated in Fig. 1 . When the low-density separated fraction was measured by the method of Example 5, the separated fraction was determined to be a free HBe antigen free of viral particles. When the high-density separated fraction was measured by the method of Example 5, since the measurement method was carried out by combining the HBc antigen and the particulate pronuclear protein, the value of Example 6 was subtracted from the value measured in Example 5. The value of the particulate HBV pronuclear protein can be calculated from the value. The ratio of HBe antigen, HBc antigen, and HBV pronuclear protein measured in this manner in each sample was calculated (Table 1). Table 1. Sample HBV pronuclear protein/HBe antigen HBV pronuclear protein/HBc antigen BBI PHM935A-14 13.437 27.236 BBI PHM935A-16 6.321 158.352 BBI PHJ201-04 0.589 22.070 BBI PHJ201-07 1.367 44.853 BBI PHJ201-13 0.296 33.743 ProMedDex #101499 0.025 12.031 ProMedDex#999077 1.365 12.779 Average 3.384 44.438 HBV pronuclear protein: HBe antigen is 13 : 1~1 : 40 'Depending on any of the samples containing a large amount of antigen, protein will be different. On the other hand, HBV pronuclear protein: HBc antigen in the range of 12: 1 to 158: 1 'all samples contain a large amount of HBV pronuclear protein. 27 1330200 Sample name supernatant A pgtal HBc antigen woven B Pg^nl Fiber ratio B/(A+B) % HBV pronuclear/nuclear protein supernatant woven ratio A Β Β/(Α·+Β) pg/ml Pgtol % BBIPHM901-06 5 69 93% 2,151 948 31% BBIPHM907-10 5 207 98% 29,728 2^19 7% BBIPHM921-06 3 76 96% 2,723 1,307 32% BBIPHM922-12 46 749 94% 20,025 10,364 34% BCPHBV6278- 11 5,238 118,840 96% 4,958,186 941,077 16% Nabi SBO408-J 4,296 116,834 96% 1,382,054 1,829,461 57% BBIPHM935A-14 8,671 188,535 96% 1,636,481 3,370,973 67% BBIPHM935A-16 959 7,800 89% 482,713 591,319 55% ProMedDx #9990776 6,264 52,270 89% 1,646,640 1,194,289 42% ProMedDx #10149975 477 4,522 90% 2,094,278 146,459 7% 011670 2,877 17,930 98% 540,397 177,755 25% 01 1341 535 32,615 98% 2,168,349 414,203 16% 01 3567 1,409 40,800 97% 5,325,365 325,486 6% 01 2296 198 5,319 96% 1,036,210 59,531 5% 01 1188 23 692 97% 5,171 2,151 29% 01 2877 555 11,692 95% 161,907 126,510 44% particulate pronuclear protein ratio E/(C+D) % ^28% 6% 31% 32% 14% 53 % 64% 54% 40% 6% 6% 15% 5% 5% 20% 40% Table 2. Obtained by density gradient centrifugation of HBe antigen, the HBc antigen, as well as the nucleoprotein were measured before the particulate HBV

E=DB pg/ml 879~ 2,012 U31 9,615 822,237 1,712,627 3,182,438 583,519 1,132,020 141,397 39,825 381,588 284,686 54,212 1,460 114,818 The HBc antigen, HBV nuclear associated antigen assay value of each fraction and the particulate pronucleus calculated from these values The protein concentration is shown in Table 2. In all of the quantifiable examples, more than 90% of the HBc antigen is present in the high density separation fraction. The HBV pronucleus/nuclear protein present in the low-density fraction is HBe antigen; the HBV pronucleus/nuclear protein in the high-density fraction is the hbc antigen and the particulate HBV pronuclear protein. Therefore, the concentration of particulate pronuclear protein can be calculated by subtracting the HBc antigen concentration from the high-density HBV pronucleus/nucleus protein concentration. Example 8. Separate measurement of the free and particulate HBV pronucleus/nuclear protein and HBc antigen in the sample and the ratio of the HBV Direct-Mag Kit (JSR Corporation) "reaction buffer" 50μ1, "HBV supplement" The particles (anti-HBs antibody-phased magnetic gas particles) 5 〇 μ1, and the sample 200 μb were shaken at room temperature for 3 , minutes to bind the 28 1330200 HBV-related particles containing the HBs antigen. The magnetic particles are separated by a strong magnet, and the supernatant is removed. The supernatant contained free HBe antigen. 60% of a 15% SDS solution was added to the residual magnetic particles, and heated at 70 ° C for 10 minutes to dissolve the bound particulate HBV pronucleus/nuclear protein. The magnetic particles are separated by a strong magnet, and the extract is taken out. The HBV pronucleus/nuclear protein and HbcAg in the supernatant and the precipitated extract were measured by the methods of Example 5 and Example 6, respectively, and the HBe antigen, HBc antigen, and particulate HBV pronuclear protein in the sample were calculated. The respective concentrations and ratios. The results are shown in Table 3. In all of the quantifiable examples, more than 90% of the HBc antigen was present in the precipitate. That is, the particles coated with the HBs antigen are substantially all immunosuppressed. The HBV pronucleus/nuclear protein in the supernatant is HBe antigen, and the HBV pronucleus/nuclear protein in the sink is HBc antigen and particulate HBV pronuclei. Therefore, the concentration of particulate pronuclear protein can be calculated by subtracting the HBc antigen concentration from the pre-nuclear/nuclear protein concentration in the precipitate. The concentrations and ratios were very similar to those obtained by density gradient centrifugation of Example 7, and it was shown that both of these assays can be used for the determination of particulate pronuclei. Example 9: According to the difference in the morbid state of the particulate HBV pronuclear protein, in the same manner as in the case of the eighth embodiment, a total of 81 samples of the blood samples of the four tissues of the HBV-bearing hepatitis were measured. The free and particulate HBV pronucleus/nuclear protein, as well as the HBc antigen, were compared to the ratio of intact pronuclear/nuclear protein (antigen + HBc antigen + particulate HBV pronuclear protein) relative to the particulate HBV pronuclear protein. The results are shown in Figure 6. 29 1330200 In the case of HBe antigen-positive cases, 'the tendency to appear high in the case of acute hepatitis' tends to be low in cases of no symptomatic carriers and cirrhosis. In the case of the HBe antibody-positive case, the case of high incidence in chronic hepatitis and liver cancer tends to be low after acute hepatitis healing and in cirrhosis. Based on these results, a useful method for the diagnosis and treatment of hepatitis B can be obtained by measuring the particulate pronuclear protein, which can further be used in the pathological explanation of ΗB V and hepatitis B. Based on the knowledge thus obtained, it is also possible to obtain useful knowledge in the development of therapeutic drugs for hepatitis. Example 10 Comparison of Sample Processing Method In each sample pretreatment liquid, in the same manner as in Example 6, only the pretreatment liquid was changed, and the pretreatment of HBV positive serum 5 and the measurement of HBc antigen were performed. Compare the effects of each treatment method. Among them, SDS, N-Lauroyl sarcosin-Na, Deoxycholic Acid-Na are anionic surfactants, and n-Decyl trimethyl ammonium-Br is a cationic surfactant. The final concentration of the active agent in the sample treatment becomes one third of the pretreatment liquid, and the final concentration in the first reaction in the HBc antigen measurement system becomes 1 / 9. Table 4. Reaction of HBc antigen in ELISA (luminescence amount) in each treatment method No 15% of pretreatment liquid SDS 15% N-Lauroyl sarcosin-Na 7.5% Deoxycholic Acid-Na 15% n-Decyl trimethyl ammonium-Br Negative 180 154 158 156 503 Sample 1 364 46656 4208 1375 1381 Sample 2 212 4249 510 459 357 Sample 3 151 2157 1323 141 311 Sample 4 167 2487 430 143 292 Sample 5 153 1609 300 180 378 Based on this result, SDS , N-Lauroyl sarcosin-Na, 30 1330200

An anionic surfactant such as De-oxycholic Acid-Na is suitable. Among them, SDS is especially suitable. On the other hand, when the cationic surfactant such as n-Decyl trimethyl ammonium-Br is used alone, it is not suitable for the measurement system because of its high background value in the negative sample (Table 4). . Reaction of HBc antigen in ELISA (luminescence amount) in each treatment method Pretreatment liquid 15% SDS 15% SDS, 1.5% CHAPS 15% SDS, 1.5% CHAPS 0.3% Triton-Xl〇〇 negative 29 26 42 Sample 1 35009 45368 50818 Sample 2 2323 3739 4316 Sample 3 541 903 1046 Sample 4 1327 2048 2158 Sample 5 532 690 773 Based on this result, CHAPS of an amphoteric surfactant was added to 15% SDS or further added to nonionic When the surfactant, Triton-X 100 or the like is used as the pretreatment liquid, the effect of the pretreatment becomes higher (Table 5). Example 11. Antigenicity of particulate HBV pronuclear protein The anti-HBc monoclonal antibody (Anti-HBc(0)2A22 specific immunization of ΐμβ/ml was diluted with 0.5 M NaCl, 0.1 M carbonate-Na buffer (pH 9, 6). The institute was injected into a 100 μl well to 96-well microplate (NUNC) and allowed to stand overnight at 4 °C. After washing with PBS, the solution containing casein was blocked, and after removing the solution, the sample solution was added to ΙΟΟμΙ/well, and allowed to react at room temperature for 1 hour. After washing with a washing solution, Ipg/ml of biotin-labeled 2A22 monoclonal antibody was added to ιοομΐ/well, and allowed to react at room temperature for 1 hour. After washing with a washing solution, 1 〇, 〇〇〇-diluted peroxidase 31 1330200 labeled avidin (Vector) was added to ΙΟΟμΙ/well, and allowed to react at room temperature for 1 hour. After washing with a washing solution, 0PD (SIGMA) / hydrogen peroxide solution was added to ΙΟΟμΙ/well, and subjected to a color reaction at room temperature for 30 minutes. After the reaction was stopped by adding 1 M sulfuric acid to ΙΟΟμΙ/well, the absorbance at 420 nm was measured. In this assay system, approximately 2 ng/ml of HBcAg can be detected. Example 1 (A) The peak separation portion of the BV nuclear-associated antigen was treated with 1% NP-40 to remove the coat, diluted 10-fold (HBV nuclear-associated antigen content was about 150 ng/ml) and then subjected to the above determination. No HBc antigen was detected. Further, although the same sample was measured by the clinically used HBe antigen measuring system (Dainabot), the HBe antigen was not detected. Therefore, the particulate HBV pronuclear protein of the present invention is considered to have a different antigenicity from the HBc antigen and the HBe antigen. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram showing the HBV pronuclear protein, the HBe antigen, the HBe antigen having a message sequence, and the amino acid range of the HBc antigen, and the morphology of each antigen in the blood. Fig. 2 is a schematic diagram showing the HBV antigen and HBV-DNA behavior of ΗB e antigen-positive serum separated by sucrose density gradient centrifugation. The HBV nuclear-associated antigen (black square), HBc antigen (#), HBs antigen (◊), HBe antigen (□), and HBV-DNA (〇) are shown in the respective fractions; the density of the separated fraction is indicated by a broken line. Fig. 3 is a schematic diagram showing the HBV antigen and HBV-DNA behavior when the separated fraction of the isolated HBV nuclear-associated antigen was separated by sucrose density gradient centrifugation by sucrose density gradient centrifugation at 32 1330200. The HBV nuclear-associated antigen (black square), HBc antigen (Φ), HBs antigen (◊), and HBV-DNA (〇) are shown in the respective fractions; the density of the separated fractions is indicated by a broken line. Figure 4 is a schematic representation of the behavior of gel filtration of HBV antigen. The above figure is the case where the largest separated fraction of the HBV nuclear-associated antigen separated by sucrose density gradient centrifugation is directly applied to the gel filtration without treatment. The figure below shows the same sample treated with 3 % N P - 40 and then filtered by gel. The amount of HBV nuclear-associated antigen (black square) and HBc antigen (φ) in each separated portion is shown. The dissolution position of the molecular weight marker is shown on the upper portion. Fig. 5 is an electrophoresis diagram showing the results of western blot analysis of HBe antigen-positive serum and its density gradient centrifugation section, in place of the photograph of the figure. The density gradient centrifugation sections 1 to 20 correspond to a sucrose concentration of 10 to 6 % 〇 Β Β 50 which is a monoclonal antibody which reacts only to HBc, and HB91 which reacts with both HBc antigen and HBe antigen. Figure 6 is a graphical representation of the ratio of total pronuclear/nuclear protein to the particulate pronuclear protein in each case. A positive example of HBe antigen is indicated by (Lu), and a positive example of HBe antibody is indicated by (〇). [Graphic main element ^ 牛表符表] (none) 33 1330200 Sequence Listing <110> Orthodox Life Science Research Institute Co., Ltd. <120> HBV pronuclear protein with particle formation energy (130) 1024582 &lt;160&gt; <210> 1 <211>178

<212>TRP <400> 1

Gin Leu Phe His Leu Cys h ue ne Knowing that you are gambling 5 10 15

Gin Ala Ser Lys Leu Cys Leu Qy Trp Leu Tip Qy Asp fle Asp 20 25 30

PtO Tyr Lys CHu Phe Qy Ala Sa Val Glu Leu Leu Ser Phe Leu Pto 35 40 45

Ser Asp Phe Fhe Pro Ser He Arg Asp Leu Leu Asp Thr Ala So* Ala 50 55 60

Leu Tyr Aig Glu Ala Leu Glu Ser Pro Glu His Cys Ser Fto His His 65 70 75 80

Thr Ala Lai Aig Qn Ala De Leu Cys Tip Gly Glu Leu Met Asn Leu 85 90 95

Ala Thr Tip Val Gly Ser Aai Lea Glu Asp fto Ala So* Aig Glu Leu 100 105 HO

Val Val Ser Tyr Val Asn Thr Asn Md; Gly Leu Lys He Aig Gin Leu 115 120 125

Lai Tip Fhe Ms De Ser Cys Leu Thr Phe Qy Aig Qu Thr Val Leu 130 135 140 1330200

Glu Tyr Leu Val Ser Phe Qy Val Tip De Aig Thr Pro Ito Ala Tyr 145 150 155 160

Aig Pro Pro Asn Ala Pro De Lai Ser Thr Leu Pro Qu Thr Thr Val 165 170 175

Val Aig <210>2 <211>20 <212>PRT <400> 2

Gin Leu Phe His Leu Cys Leu He He Sea* Cys So: Cys Ro Thr Val 5 10 15

Gin Ala So* Lys 20 <210>3 &lt;211&gt; 11 <212>PRT <400> 3

Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg 5 10 <210>4 <211>17 &lt;212&gt; PRT &lt;400 )4

Glu Ala Lai Qu So· Pro Qu His Cys Sea* Pro His His Thr Ala Lai 5 10 15

Aig <210>5 <211>14 1330200 <212>PRT <400> 5

Glu Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 5 10 <210>6 &lt;211&gt; 23 <212>ΡΚΓ <400> 6

Thr Pro Pro Ala Tyr Aig Pro Pro Asn Ala Pro He Leu Ser Thr Leu 5 10 15

Pro Glu Thr Thr Val Val Aig 20 3

Claims (1)

1330200 u] F; 绦 (more) is replacing page No. 93101988 Patent application for special phoenix replacement main __ &amp; April picking up, patent application scope: 1. An isolated HBV pronuclear protein with HBV The nucleus-like particles form energy and contain the amino acid sequence set forth in SEQ ID NO: 1. 2. An isolated HBV (class) nuclear particle or HBV (type) viral particle comprising the HBV pronuclear protein as in claim 1 of the patent application. 3. An HBV vaccine containing a HBV pronuclear protein as in claim 1 of the scope of the patent application. 4. A therapeutic agent for HBV comprising a HBV pronuclear protein as in claim 1 of the patent application. 5. An HBV diagnostic drug containing HBV pronuclear protein as in claim 1 of the scope of the patent application. 6. A diagnostic kit containing HBV pronuclear protein as in claim 1 of the scope of the patent application. 7. A method for assaying an HBV pronuclear protein according to claim 1 in vitro, comprising the steps of: stripping a viral coat protein from a HBV (viral) virion as in claim 2; That is, the HBs antigen), which binds the HBV pronuclear protein; and binds to a probe containing an antibody, a receptor, an aptamer (Aptamer) or a ligand which specifically recognizes the HBV pronuclear protein. 8. The method of claim 7, wherein the step of freeing the HBV pronucleus from the (type) virion is a treatment or addition of a surfactant. 9. The method of claim 8, wherein the surfactant is 1330200 anionic surfactant, or an anionic surfactant and a diionic interface/toner, or an anionic surfactant, Any of an ionic surfactant and a nonionic surfactant. 10. The method according to any one of claims 7 to 9, which comprises a probe system which binds to the aforementioned HBV pronuclear protein, a receptor 'heterosome or a ligand, and the like One of the HBV pronuclear proteins binds specifically, but not to the HBe antigen or HBc antigen. η· As for the m method in the seventh to the ninth aspect of the patent application, the probe which binds to the nuclear protein of HBV is an antibody. 12. The method of claim 1, wherein the probe that binds to the pronuclear protein is an antibody. 13. The method according to any one of claims 7 to 9, wherein the HBV pronuclei protein in the sediment is determined by (immuno) sedimentation of a probe that binds to the HBs antigen by an anti-HBs antibody. By. 14. The method of claim 1 (), wherein the HBV pronuclear protein in the sediment is determined by (immuno) deposition by a probe that binds to the "HBs antigen-binding antibody". Patent application (4) is a method for determining the HBV pronuclear protein in the sediment by the (immuno) sedimentation of the anti-hBs antibody binding to the HBs antigen. The anti-ms antibody binds to the HBs antigen for (immune) sedimentation to determine the HBV pronuclear protein in the sediment. 17. The method of using HBV pronuclear protein, the patent scope will be The quantification of the HBV pronuclei protein determined by the method of any one of the items 2 to 1330200 7 to 16 is regarded as a pathological marker of HBV infection or hepatitis B. 18. - In vitro detection of particulate HBV pronuclear protein The method of the antibody is to use the HBV pronuclear protein as the antigen user in the first paragraph of the patent application. 19. An assay kit for determining an antibody containing a particulate HBV pronuclear protein of a ΗBV pronuclear protein, and Contains the HBV prenucleus as in item 1 of the scope of the patent application White as an antigen.