TW209245B - - Google Patents

Description

2 Ο Λ 6 η 6 Central Prototype Bureau of the Ministry of Economic Affairs IS; Printed by the Industrial and Consumer Cooperatives 5. Description of the invention () The present invention is in a composition consisting of multiple sequences made by recombinant DNA technology and its contained It is used as a therapeutic agent against chronic viral hepatitis. The present invention also relates to the DNA sequence encoding the multiple sequence and the transfer of infected cells expressing the sequence. There are at least five different viruses, namely A, B, C, D, and E, which cause clinical symptoms of acute hepatitis. Hepatitis A and E transmitted through the intestine are often curable, while Hepatitis B and C (formerly known as non-enteric hepatitis Non-A, and Non-B), and hepatitis D may develop It becomes chronically inflamed and may lead to cirrhosis and primary hepatocellular carcinoma. There are eel hepatitis C and D, and there are relatively few data available on the diagnosis and treatment methods and other viruses. Hepatitis D virus is known as an incomplete RNA virus. Therefore, the virus that needs helpers can develop in patients, and it has only been found in people infected with HBV. It was only recently that the hepatitis C virus was detected and an antibody test (anti-HCV) was developed that can diagnose chronic hepatitis C infection. However, there is still an increasing need for urgent treatment of various diseases. Regarding chronic hepatitis B, the relevant immunological methods have been confirmed, and the onset virus and the virus life cycle and DNA sequence have been better investigated. This disease also has the same situation. If the patient has viral DNA present for more than ten weeks, HBe-antigen (HBeAg) exists for more than 12 times, or if hepatitis B surface antigen (HBsAg) exists for more than six months, etc., the patient is hepatitis B virus Those who have been in the original for a long time. It is roughly estimated that 300 million people are considered to have chronic hepatitis B, and most of them are people living in the Far East. For these people, the main infection crisis occurs during or immediately after the birth, because the mother with the chronic infection transmitted the virus to her (please read the back and the precautions before writing this page) This paper 5IL Standards are used in the China National Standards (CNS) Τ4 specifications (210X297 public *) 3 81. 4. 10, 〇〇〇 Zhang (Η) ς〇9: ^ 〇Λ 6 Π 6 Ministry of Economic Affairs Central Bureau of Standardization Beta Engineering Du Zhong's cooperation with Du Du. V. Description of invention () Newborn. Ninety percent of children who are infected by way of infection will become chronically infected after their lives. In the Western world, infections most often occur late in life, and may be in childhood or even adulthood, mainly through the gut or sex. Of these cases of hepatitis B infection after birth, only 5 to 10 percent of infected people will become long-term carriers. Unexpectedly, the transferred virus does not cause any obvious reaction whether the infected person eliminates the virus or remains in the body for life. For this reason, those who decide the future physical condition seem to be the immune system. The HB virus particles (Dane particles) are composed of different structural proteins, including core proteins and surface proteins. The latter is a translation product of an open cipher composed of a coding sequence with three stuffed S-type domains. The three-ridge S-type domains are all ATG triplet (ATG triplet) whose translation can be initiated within the first time. As the beginning. These three domains are called pre-S1, pre-S2 and S, which are named in order according to the end-to-end order of the molecules. Six proteins can be derived from the ORF: the main protein in glycosylated and non-glycosylated forms (gp27 and p24> (226 p-amino acids); with one or two polysaccharide side chains only translated from the S domain (Respectively gp33 and gp36) intermediate proteins (281 amino acids), which are encoded by the preS2-domain and S-domain; Μ and, glycosylated form (gp42) and non-glycosylated form (Ρ39>) The large protein (389-400 ketoamino acid, which depends on the blood type of the virus>, is formed by the translation of preSl, preS2, and S domains. Its core proteins are HBcAg and HBeAg, which is believed to be the treatment product of HBcAg. The Dane particles, which are the primary particles of the infectious virus, contain the core protein and the surface protein, while the silk body is composed of a mixture of six surface antigens. (Please read the back and the precautions # fill out this page) Using the Chinese Marshmallow (CNS) «P4 specification (210X297 ;;)) 4 81. 4. 10,000 sheets (||) Λ 6 Β6 20924ο V. Description of the invention () S combined itself to form the so-called 20η particles , Article completely non-infectious (please read back and pay attention to item I Item write this page) Ο Patients infected with HB virus will experience several stages of hepatitis before being considered as a long-term HBV-infected person. Immediately after infection, there will be an infectious stage, which is specifically bloody Contains HBeAg. It is difficult to suppress the replication of HBV, and the continuous HBs antigenemia still occurs, showing that the viral DNA sequence has been integrated into the patient's cell genome (genome). The integrated viral sequence does not promote the host cell Synthesize a complete virus. However, hepatocytes with integrated HB V-sequence can only produce HBsAg, which can be detected in the patient's serum and used as an indicator of chronic hepatitis B. The most likely case is that The shaped hepatocytes will not be lysed by cytotoxic T-cells, but will grow and induce long-term hepatitis (CPH) or long-term active hepatitis (CAH), and then may progress to cirrhosis or early hepatocellular carcinoma, Promotes premature death of patients. Printed by the Consumer Labor Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. It has recently been determined that patients with chronic HBV infection will display symptoms of endogenous interferon production defects (Abb et al., 1985: J. Med. Virol 17 Bu 176). This is the theoretical basis for the treatment of chronic hepatitis B patients with HBeAg and HBV-DNA in blood plasma using interferon alpha (IFNa). The results of control trials with a large number of patients showed that those taking dry somatosin a Compared with the control group, the degree of elimination of hepatitis B virus has increased significantly. However, the infected people did not show seroconversion effects on this treatment at birth or during the period after birth. This phenomenon unfortunately makes about 70 % Of the original carriers cannot benefit from IFNa treatment. So far, the correct effect of interferon a on chronic hepatitis B prescription paper "• The standard is used in the home beta (CNS) T4 specification (210x297 mm) -5-81. 4. 10, _ 张 ⑻ C09 : 4〇A 6 Π6 Printed by the A-Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. The description of the invention () is still unclear. Its antiviral activity may be to protect infected cells from infection or reduce the transcription, translation, and replication of viruses in HBV-infected cells. Dry somatostatin can have another immunomodulatory effect by activating T-cells, macrophages and NK-cells, and inducing the expression of MHC class I proteins. Another method for treating chronic hepatitis B is based on the concept of inhibiting the replication of the virus, thus reducing its defense to a sufficient level so that the host immune system can eliminate the virus. This approach has led to attempts to treat patients with chronic HBV infection with antiviral drugs, such as adenosine glucosinolate and adenosine glucosinolate-phosphate. However, no more than half of the patients will respond to the treatment, whether it is continuous or transient seroconversion (HBeAg + to anti-HBeM. Another negative aspect of these antiviral drugs is their immunosuppression Properties. Other drugs that have been tested to treat chronic carriers with M include interferon / 8 | acyclic guano nucleoside, interleukin 2, steroids such as dehydrocortisol, and combinations of the above. But none of them can provide better results than interferon alpha treatment. Only a combination of treatments, including initial use of steroids followed by IFNa, can increase the response rate of all patients. From the existing The technology shows that the orangutans encountered with chronic HBV infection can neither be cured with HBsAg (bound to tetanus toxoid) nor with anti-HBs antibodies. In addition, there are also attempts to immunize patients with chronic HBV infection by taking S peptide M. This treatment does not even encourage these patients to form anti-HBs antibodies. In addition, according to the definition, the specialty of the hepatitis B virus long-term carrier (please read the precautions before you This page) Installation _ Line · This paper standard adopts China's β Family Standard (CNS) T4 specifications (210X297 g *) 6 81. 4. 10,0U0 sheets (Η) 209.4ο Λ 6 ΙΪ6 Central Standards Bureau of the Ministry of Economic Affairs Printed by the Industry and Consumer Cooperatives 5. Description of the invention () HBsAg can be detected in its serum. Therefore, it is absolutely unexpected that the composition of the present invention containing the activated epitope for the activation of the virus S can be induced by the T-cell Hepatitis B virus has a long-term carrier immune function and eventually recovers. Considering the technical status discussed above, the purpose of the present invention is to propose an effective therapeutic agent that can lead to a complete response and use M to treat the disease People with chronic hepatitis disease (ie, can lead to continuous inhibition of HBV replication, reduction of HBV DNA and DWA polymerase, and reduction and final disappearance of HBeAg and HBsAg in patient serum, etc.) According to the present invention, this objective is achieved The method is to combine U) at least one antigenic polypeptide sequence that can contribute to one or more epitopes, and &lt; b &gt; can combine the epitope sequence (a &gt; The multiple sequence (a) By means of adsorption, any chemical bond or secondary valence bond is bound to the carrier (b &gt;. The present invention is also related to the use of this combination in the production of a panacea for the treatment of chronic disease Hepatitis. It is a method for treating chronic viral hepatitis, which is made by taking a patient with the above composition containing the following ingredients: U> At least one tilt can contribute to the antigenicity of one or more epitopes, and &lt; b ) The epitope sequence (a &gt; presented carrier, wherein the multiple sequence (a &gt; can be bound to the carrier (b) via adsorption, any chemical bond or secondary valence bond, etc. Importantly, the multi-sequence, which may be one or more different, is a direct or indirect method that contributes to the antigen resistance of T cell activation epitopes. According to the present invention, the polypeptide sequence (a &gt; can be any type B liver (please read the precautions first and then write this page) Binding and ordering-this paper uses the National Beta Standard (CNS) A 4 specifications (210x297 g *) 81. 4. Ιο, υοο Zhang (Η) 20934ο Λ 6 Π 6 V. Description of invention () Inflammation virus, wait for it to be adw, ayw, adr and ady etc. A combination of two or more of them. Some derived from hepatitis B virus can be HBV, preS, preS2 or S, or the core of HBV. Can be used as multiple sequences (a) except any of the foregoing In addition to peptides> There are also combinations of two or more polyammoniums modified by amino acid deletion, but at least one of them must contain at least six amino acid residues_antigenic determinants, or via N-terminal, or C-terminal plus the remaining amine groups or a combination of two or more chitosans modified in such a way as to insert other base acids in the chimeric sequence U). All these quarrels are necessary to maintain biological activity. The more appropriate, the more sequence u) is the one brewed by Peas. In order to show proper pharmacological activity, the multiple sequence U) in the composition of the present invention must be presented on the carrier (b>. This carrier is composed of a special substance, for example, a hydrophobic polymer Particles, inorganic particles, or polysaccharide particles, etc. Where appropriate, the second type of multi-sequences that form particles during secretion is contained. (B &gt; The particles which are more suitable have a diameter of at least 10 nB (please first Read the back-to-back notes and write this page) Printed more appropriately by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs to form a multi-sequence sequence of particles (t〇 can be taken from: HBV # ·-. 3 peptide, suppress ¥ Core antigen, "^ 抜> Heart antigen, | ^^ surface antigen, 111% surface antigen and HIV core antigen M and poliomyelitis gray surface virus surface antigen, etc. The substantial part of the complete amino acid sequence The more suitable particle-forming carrier (b &gt; is HBV S and / or core. When used as the carrier sequence (b), the above-mentioned multi-cracker can be freely removed by removing amino acids, and one or more Amino acids are substituted or added at the N-terminal or C-terminal Standard (CNS) ^ Specifications (210X297 Minister) 8 81. 4. 10,000 copies (Ji) ς〇9, 4ό Cooperation with the Ministry of Economic Affairs, the Central Bureau of the Ministry of Economic Affairs, Beigongxiao, Du Yinmu V. Invention description () One or more Pour the amino acid, or put a healthy or polyquaternary amino acid in the polypeptide sequence (b) to change the M, but it must maintain its particle forming ability. The more appropriate, the multi-sequence (b) system It is brewed by nutmeg. If the loaded thin &lt; b &gt; is a polypeptide sequence, the sequence (a) and (bh) can be linked together through the following interactions of γ species or multicast, and the hydrophobic fixation (Contributed by the thin bean flesh), the formation of two broken bridges, etc .; or the two sequences can be connected via a bond to form a fusion peptide. In the manner described below, the polypeptide sequence (b) ) Intervening a spacer sequence, which is linked to the two polytopes via a bond. The invention also proposes a recombinant DNA molecule encoding a composition, which can be used to produce a treatment for chronic viral liver disease The drug. The recombinant DNA molecule contains at least one first DNA sequence, suitably contains the second, third and / Or a fourth DNA sequence, wherein &lt; 1 &gt; the at least one first DNA sequence »encodes at least one of the above-defined sequences (a), (u &gt; the second DNA sequence encodes The particle formation is defined by the defined polypeptide sequence (b>, (m) the third DNA sequence encodes the spacer sequence, and (iv &gt; the fourth DNA sequence assembles the selection sequence, and some of them The DNA sequence is controlled by the DNA elements that must be present when expressing, and it has a common code locally. Based on many kinds of amino acids can be compiled by the ginseng on a ΜΜ, therefore, the present invention includes several DNA sequence, »Keoma's above defined sequences (a) and (b &gt;. In addition, the present invention also contains recombinant DNA molecules (please read the precautions before filling in Sun's page). Binding · Order · This paper uses the National Standard (CNS) T4 specification (210x297 male dragon) ) 9 81. 4. 10,000¾ (II) Λ 6 Η 6 Printed by the Employee Consumer Cooperative of the Ministry of Economic Affairs, Pyongyang quasi-bureau V. Invention description (), which differs from the recombinant DNA molecule defined above in how many 30% of the nucleotides can be passed through. Another item of this patent application is to propose host cells that are transfected by recombinant DNA molecules encoding the above-mentioned composition; wherein the composition can be used to treat chronic HBV-infected patients with M. The host cell may be a mammal, a cell other than a mother or a larvae. For the purpose of the present invention, these cells are more suitable as they do not produce any human hemoglobin protein or any other elder hemoglobin protein other than the polyamine contained in the above composition. The term "HBV S 呔" used in the present invention refers to the peptide encoded by the stern region of the HBV gene of the whole crane. The term "HBV 1 ^ -52 呔" used in the present invention refers to the entanglement coded by the pre-S2 and S domains of the HBV genome. The term "HBV pre-Sl 呔" used in the present invention It refers to the polypeptide encoded by the pre-S1, pre-S2, and S domains of the HBV gene. The term "antigenic determinant" used in the present invention refers to the encoded domain of the designated gene. , A sequence containing at least six peddopamine amino acids (for example, "HBV pre-S2 epitope" refers to the sequence encoded by the Pre-S2 domain of the HBV gene, containing at least six amino acids sequence> The term "T-cell epitope" used in the present invention refers to an epitope that can promote or contribute to an immune response by interaction with M on the surface of T-cells. "Antigenicity" used in the present invention means Ability to suppress immune response (i.e., having the role of an antigen), and the ability to promote anti-revenue production (Yi Di, having the role of an antigen) and / or interact with the cell surface by M Ability, etc. The term "HBV" means any type of the virus described in the literature (please read first And the precautions # fill in this page) Binding-Bookbinding-Line. Winter paper standard use 8 Β 家 «Bi (CNS) A 4 specifications (210X297 public *) 81. 4. 10,000 sheets (Π) V. Description of invention Lie, it another! Iadw, ayw »adrfO ayr et al. (Ρ · Valenzuela, Nature Vol. 280, p.815 (1979), Gerlich EP-A-85 111 361, Neurath, EP-A-85 102 250>. Composition Multiple sequences (a &gt; can cause one or more epitopes to be antigenic. Examples of sequences are listed in the "M Sequence Listing" (SEQ ID No. 17-20, 22 &gt;. Preferred embodiments of the present invention include The following composition: a HB S-antigen particle and pre-Sl, pre-S2, and / or core-specific special epitopes (determining factors> -HB core antigen particle and pre-S1, pre- Special antigenic determinants for S2, S-, and / or core antigens (determinant> Hepatitis A antigen particles and Hepatitis B S-, pre-Sl, pre-S2, and / Or core special determinants (determinants) (please read the precautions before writing this page) printed by the Employee Consumer Cooperative of the Bureau of Standards of the Ministry of Economic Affairs is more suitable for use in this issue The specificity of the recombinant DNA molecule contains a sequence that can encode a multiple sequence (a), which is an antigenic substance that can cause one or more T-cell epitopes, and encodes a multiple sequence <b), It is one that can form particles with a diameter of 10 n · or larger when secreted, and both are controlled by appropriate activation factors. An example of the sequence encoding U) may be any sequence with ID numbers 1 to 24 in the "Sequence List". An example of the DNA sequence encoding the multiple sequence (b) can be any of the sequence lists with ID numbers 25 to 27 in the "Sequence List". Any of the above 24 sequences (ID numbers from 24 to 24) can be combined with any sequence disclosed by ID numbers 25 to 27 in the sequence table, including a-b and b-a. The particularly preferred embodiment of the present invention is based on the epitope sequence ID No. The 5K scale of the paper is used in the S-S sleep standard (CNS) A 4 specification (210X297 public; ¢) 11 81. 4.〗 0,000 »(11 ) A 6 Η 6 5. Description of the invention () 28 (The amino acids 9 to 28 in the HBV-S1 sequence are combined with the sequence ID numbers 26 and / or 27 as the particle generator. The hepatitis virus sequence used in the recombinant DNA construct of the present invention can be used to include: isolation and connection of restriction fragments, chemical synthesis of oligonucleotides with a synthesizer (Cyclon, Bio-Search), and PCR method (TJ White , N. Arnleim, Η.E. Erlich, 1989: The Polymerase Chain Reaction, Technical Focus 5 &lt; 6) &gt; and other means to form or difficult to precipitate out of M. The more suitable recombinant DNA molecule is through the synthesis of oligo The polynucleotide was connected to the 5 'Xbal I-Bgl Κ 3' fragment (ID No. 27 &gt;) taken from the S domain of the HBV gene. The fragment was included from the entire value pre-Sb pre-s2-S- BglF-BglE HBV fragment derived from the domain; or formed by connecting to the whole S-domain. The oligo nucleus of this structure is made with Μ The acid picks are listed in the following Table I. (Please read the precautions before filling in this page) Employee consumption cooperation of the Ministry of Economic Affairs of the Ministry of Economic Affairs of the Ministry of Economic Affairs. The paper is printed using the standard of China ’s poor home «quasi (CNS) Τ4 specifications (210x297 male dragon) 12 8i. 4. 1 (), UU0 Zhang (Η) η 6

V. Description of invention () Table I. Function definition serial number printed by the employee consumption cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. Core ID (core), aa * 59-87 6 core (adw) aa 2-28 7 core (adw) aa- 10-28 8 core (adw) aa 29-58 9 core (adw) aa 1-87 10 core (adw) aa -10-87 11 core (adw) aa 70-110 12 core (adw) aa 80-125 13 core (adw) aa 88-120 15 SI (ayw) aa 9-28 17 SI (ayw) aa 83-103 18 SI (ayw) aa 20-40 19 S1 (ayw) aa 59-94 20 S1 (ayw) aa 94-114 21 SI (ayw) aa 70-105 22 S2 (ayw) aa 2-21 23 S2 (ayw) aa 14-33 24 Amino acid Other suitable DNA molecules are prepared by PCR method The codebook paper standard uses the Chinese National Standard (CNS) Τ4 specification (210x297 mm) 13 81. 4. 10,000 sheets (Η) (please read the precautions before filling in this page) Λ 6 η 6_ V. Invention Description () The core sequence of the τ-cell epitope is connected to the core sequence of HBV (SEQ ID NO 25, as multiple sequence B). The oligonucleotides used to prepare these constructs are listed in Table 1-1. Table H -1 'Function definition SEQ ID number core complete, bp 190 bu 2500 1 core C-terminal_exclude, bp 190 bu 2405 2 core C-terminal deleted and embedded stop code 3 (stop codon) &gt; bp 1901- 2450 core / pre-core 10 aa precore (precore), C-end 4 delete bp 1871-2405 core / pre-core 10 aa precore, C-end delete and embed 5 stop password, bp 1871-2405 core aa (- 10-120) 16 core / pre-core 10 aa pre-core, complete core, 35 bp 1871-2500 The Ministry of Economic Affairs Central Bureau of Employee Employee Consumer Cooperatives printed table Π -2 lists a few examples, which encode T-cell antigens The DNA sequence of the determinant is isolated by restriction fragmentation of the HBV gene body and connected to the DNA sequence encoding the multiple sequence (b) defined above. 81. 4_ 10,000 sheets (11) (please read the precautions and fill in this page first) The paper size is based on the Chinese Standards (CNS) 4 specifications (210X297; ») ς〇9 V. Description of the invention () Boom. 1: 2 Function definition SEQ ID number Core / Front core complete, bp 1403-31 «Μ S2 ay / ad Μ Ν S2 (K) ay / ad S2-S, 7 instrument password is deleted, from 14 The initial code ATG was changed to ΑΤΑ &quot; &quot; the sequence was Galibert, F. et al (1979; Nature 281, 646-650 &gt; and Ono, Υ · et al. (1983; NuCl. Acid Res. 11 (6> , 1747-1757), etc. Table I-3 lists special recombinant DNA molecules. Its composition procedures will be described in more detail in the examples. (Please read the back to the precautions # fill in this page) The Ministry of Economic Affairs, Central Bureau of Pricing and Employee's Cooperative Society printed this paper at 51 scales, and used the Standards (CNS) f 4 specifications (210X297 mm) 15 81. 4. 丨 0,000 sheets (11) V. Description of invention () Table 1-3 Λ 6 It 6 final construct T-cell epitope particle former selection gene-core (-10-120) + SAg + neo core (aa-10-120) S adw / ayw or S / Xbal / B gl I neo MT-Sl (aa 9-28) -S + egpt Sl (aa a-28) ay S adw / ^ yw S / Xl »l / BglE V., k egpt # MT ~ core-neo core / front Core bp 1403-31 core adw neo MT-core (1-87) + HBsAg-neo core (aa / -87) S adw / ayw or S / Xba I / Bgl E neo (please read the notes beforehand (Fill in the wooden page) #egpt = Escherichia coli yellow flower colored guano guanosine nucleophosphoryl nuclear transferase printed by the Employee Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. In addition to coding for more than one in the more suitable recombinant DNA molecule of the present invention ( Outside the domains of a) and (b), it contains additional DNA sequences that encode selection records. In addition, it also contains all the generally necessary elements for performance, such as the starter gene sequence, the start code, and a polygland. Glycosylation signals, etc. Examples of suitable promoters are: methallothionein (MT> when using mammalian larvae as host cells, U2 and H2K promoters, etc. If yeast or bacteria cells are used, appropriate yeast can be used respectively Activate the B-size standard (CNS) 4 specifications (210X297 g; it) 81. 4. Η), 000 sheets (Η) ^ 09 ^ 40 Λ 6 Π 6 Specify () genes, such as the GCN4-promoter and GAL 1/10 promoter, and appropriate bacterial promoters, such as prokaryotic trp- and tac promoters. In order to produce the polyoxa (a &gt; and polyoxo (b) combination of the present invention, recombinant DNA molecules are to be transferred for infection (when using mammalian cells>, transformed (when using cells such as drunk mother and fine sugar), Or other methods to embed in the host cell, as long as the cells of any organism can transcribe and translate the recombinant DNA molecule, for example, cells of mammals, shrubs and yeast can be used as host cells. Appropriate mammalian cells that can be used in the present invention Examples are VER0 cells (a monkey kidney cell strain), 3T3-, C127, and L cells (murine fibroblast cell strain), and CHD (Zhongluo vole oocytes> cells, which are dehydrofolate enzymatic enzymes) Sexual or negative. According to a special embodiment of the present invention, it encodes multiple sequences (a) and multiple sequences (b), the first DNA sequence defined above and the second DNA sequence defined above , It may exist in different recombinant DNA molecules, in this case, it is necessary to transfer the infected DNA with two recombinant DNA molecules at the same time. (Please read the precautions to write this page first)- Binding · Order _ Ministry of Economic Affairs The Central Bureau of Standards and Technology's Beigong Consumer Cooperation Co., Ltd. printed the paper in reverse. + Β National Standard (CNS) T4 specification (210X297) * 81. 4. 10,000 sheets (II) Π Γ) V. Description of invention () (1-8?) «H8sAg-neo MT-cote-neo A 3 games ΜΤΛΙ2 / ϋ2 ΜΤΛΪ2Λί2 ΜΤΛΙ2Λ12 MMI2 / U2 mnm2 MT / II2AJ2 MT / H2 / U2 MT / 1I2AJ2 MT / M2AJ2 MT / lt2AJ2 MT / 1I2AJ2 'Kingko… • ί ___ core i {&gt; (M29-5n) core (Μ-10 · βη) core (ΛΛ 2-2ΒΪ core (M 59-B7) ! 1 i Μ 2 bp 1871-2405 IS? * + Ws + child bp 1B7I -2405 core + front core Ι0ΛΛ; C except core and front core ΙΟΛΛ bp 1901-2405 (= f bp 1901-2405-erbium a dead 〇ir hard 1 'erbium bp 1901 -2500 core, no wattle core-fine touch original vertical base rcn I tip ί-103 * 31 core and front core \ mm entire S aciw / nyw 5 / Xba (^ 〇II | entire S adft / Ttfw 5 / Xb3l / B〇lll entlie S adwA-iyvr S / Xb3l / B〇lll enliie S adw / nyw 5 / Xbat / Bglll eniiie S adw / nyw S / Xbal / Ogl ll core {ndw} core… ㈣ core㈣ core (arfw) core (ndw) core㈣w) particle formation neo / egpt neo / e〇pt neo / eypt neo / egpt neo / egpl neo / egpt 1 neo / egpl neo / egpl neo / e〇p | 丨… ――_ neo / egpt neo / egpl Fine genetic materials and methods!: .........................!! # 2! · ^ * (Please read the back and the precautions # fill in this page) the 5lc standard of this paper is used in "« Home Sample Standard (CNS) 〒 4 specifications (2〗 0 father 297 public goods) 81. 4. 10,000 sheets ( H) 209 4〇Λ 6 Π 6 V. Description of the invention () ϋ: a scorpion zaobao 323 Xue S. 3 Bi Lei book ma lens »a 3 hide secret w» salary 漥. Ministry of Economic Affairs MT-S1 (AA9-28) -S * egpt MT &ore; (-10- + 120) ♦ SAq ♦ neo mmm MT / H2AJ2 MTyH2AJ2 -ΜΤΛΙ2Λί2 MT / H2AJ2 MT / H2AJ2 MT / H2AJ2 MTM2AJ2 MTAI2 / U2 ΜΤΛΪ2ΛΙ2 ΜΤΛΙ2ΛΙ2 MTM2AJ2 MT / H2 / U2 MTVH2 / U2 j ΜΤΛ42ΛΙ2 ΜΤΛΪ2Λί2 ΜΤΛΙ2Λβ MTA12 / U2 promoter i-responder S2- (M14-33) (ay14) -(M9-28) (ay) SI- (ΑΛ 70-105) (ad) SI- (ΛΛ 94-114) (ad) S1- (AA59-9-1) (3y) SI- (ΛΛ 20- 40 ) (ay) S1 *-(ΛΛ S3-103) (ay) S1- (M9-2B) (ay) j ^ 4> (M-10 * SArG4 ΑΛ2-87) core (ΛΑ 88- 120). core ( ΛΛ 80-125) core (M 70-110) core (ΛΛ-10- · 87) T SYN | core (M-10- + 120)-fine ISftM determinant rCR | i S2 ayw mh S ayw / adw S ayw / adw entire S adw / ayw S / Xbal / Bglll entire S adw / ayw S / Xbal / B〇ili core adw entire S adw / ayw 5 / Xbal / B〇m · entire S adw / ayw S / Xbal / BQlll entire S adw / ayw S / Xbal / Bglll · entire S adw / a ^ rw S / Xbal / Bglti entire S adw / ayw SAbaUBglll. entire S adw / ayw S / Xbal / OgHI entire S adw / ayw S ^ baUBgtil · entire S adw / ayw S ^ bai ^ gtl! Entire S adw / ayw S / Xbal / BgHI entire S adw / ayw S / Xbai / QQlll entire S adw / ^ rw. S / XbaUB〇iti. Entire S adw / ayw S / Xbal / B〇HI Particle formation neo / egp! Neo / egpt neo / egpt neo / egpt neo / egpt neo / egpt neo / egpt neo / eopt ...... neo /? Gpt neo / egpt neo / egpt neo / e〇pt neo / egpt neo / egpt neo / egpt neo / egpt neo / egpt materials and methods cr Xie B i) ······························ · · · · · · · · · · · · · · · · · · · · · · · · (Please read the precautions first and then write this page) This paper standard is not used in a B standard (CNS) T4 specifications ( (210X297 public release) 81. 4. 10,000 sheets (II) Printed by Beigong Consumer Cooperative of Central Bureau of Standards, Ministry of Economic Affairs Λ 6 _Π_6__ V. Description of invention () Table HI The appropriate DNA sequence listed combined to give each problem, the DNA construct of the present invention may be comprised. In particular, any of the components shown in this table can be combined into a DNA sequence, which can be used to produce a composition (containing Duo &lt; a) and used for @treatment of chronic viral hepatitis when transferred into a host cell. Medicine. The DNA sequence of the T-cell epitope sequence can be prepared by synthesizing (SYN) with Biosearch Cyclon synthesizer (SYN), or generated by PCR program (PCR), or the viral genome can be cut into fragments using restriction enzymes, etc. It is prepared in a way. For the treatment of patients with chronic viral hepatitis, the combination of the multi-sequence U &gt; and the carrier (b>) can be formulated into any medicinal composition, which can additionally contain a suitable diluent or pharmaceutical carrier material, such as buffer solution The drug administration can be accomplished by any method, for example, parenteral (eg, intravenous or intramuscular), or given orally (eg, encapsulating the active substance with typhoid fever mackerel cells), etc. , The medicinal preparation contains a sufficient concentration of the above-mentioned composition Μ After giving it, it induces f: Λ

Description of the deception case Figure I shows a DNA construct encoding a value-initiating gene, a stoichiometric sequence and a slap selection gene (explained in Example 3/4). Figure Π shows a DNA-gene construct, containing a promoter gene, an epitope with an integer HB-S-Ag, and an exhaustive selection gene (explained in Example 3/18). Figure K shows a DNA construct, presenting a prokaryotic gene, a copy of the paper standard for easy use in a family standard (CNS) A 4 specifications (210X297 public; tt) 81. 4. 1〇, 〇〇〇 Zhang (π) (Please read the notes before filling in and fill out this page) Outfit-Line-Printed by the Employee Consumption Cooperation of the Central Bureau of Economic Affairs of the Ministry of Economic Affairs Λ 6 __ _ Π 6_ V. Description of the invention () τ with particle formation -Cell epitope, and a selective gene (explained in Example 3/21). Bin IV lists the AST value of Black Budgie 1 during Hepa-Care treatment (explained in Example 10/1 Of>. 圏 V showed the antigen value of chimpanzee 1 during Hepa-Care treatment (explained in Example 10/1). 圏 VI showed that chimpanzee 1 had liver after three intensive treatments with Hepa-Care The values of both enzymes ALT and GGT (explained in Example 10/2). Figure V »shows chimpanzee 2 during Hepa-Care treatment The values of liver enzymes ALT, AST, and GGT, and their antigens. (Explained in Example 10/3). Mission VI lists the non-treated control chimpanzee's hepatic liver "vegetarian value (¥ (Explained in Example 10/3). Figure IX & X shows the antigenic valence and anti-rollet valence of Patient 1 during Hepa-Care treatment (explained in Example 11). Yuan XI &amp; XI shows the patient 2 Antigen potency and anti-odor valence during Hepa-Care treatment (explained in Example 11). Figure XI & XIV shows patient 2's antigen potency and anti-manual force during Hepa-Care treatment Price (described in Example 11). The following examples of the present invention will be described more specifically. Example 1 1. Staged precipitation with polyethylene glycol (PEG) from HBV protein-producing cultures After collecting the clear liquid, separate it into pieces (please read the back-end precautions # fill out this page) and install it. This paper is used in the national standard (CNS) Ή specifications (210X297; ¢) 81. 4. 10,000 sheets (H) 209> 4〇Λ 6 116 Printed by the Beigong Consumer Cooperative of the Central Bureau of Economic Affairs of the Ministry of Economic Affairs It is part of 2400 ml. In each part, add 144 g of PEG 6000 (Serua> and stir at room temperature for 20 minutes to dissolve, then stir in 4C bighead for 6 hours. Then, put it in a 500 ml bottle , Centrifuged at 9,000rpn (15,000xg) for 30 minutes at 10¾ in a GS 3 turntable. Collect the resulting clear solution, add 144 grams of PEG 6000 and dissolve as described above. The resulting solution was placed at 41C and stirred for 3 hours. The precipitate of the solution was collected in the above manner, except that the centrifugation operation continued for 60 minutes. 2. Gel chromatography separation method The material obtained by PEG precipitation is redissolved in 20 ml of PBS, and then subjected to gel chromatography separation on A-5m (Biora &lt; 〇. The column size is 25X 1000 mm, and the bed volume is 480 ml. In a typical liquid separation operation, 10 to 15 ml of a solution containing 1,000 mg of PEG in HBV protein was poured, and eluted with PBS at a rate of M6 drops / min (<18 ml / h). The mass system was eluted at the first chromatographic peak. The collected liquid fraction was separated into tj »CsCl gradient sedimentation. 3. Separation of CsCl gradient sedimentation Separate about 30 mL fractions containing purified HBV protein covered by the first chromatographic peak of A-5 · Column Chromatography Separation into about 100 mL. The solution was adjusted with CsCl to a density of 1.30 g / cm3, and then transferred to a PolyalloBer tube fitted into a SW 27/28 turntable. The gradient is formed by placing 4 ml of CsCl solution with a density of 1.35 g / cm3 at the bottom of the tube, then covering with 4 ml of CsCl solution with a density of 1.25 g / cm3, and finally coating 4 ml of CsCl solution with a density of 1.20 g / cm3 . This gradient M 28,000rp · was operated at 101C for 50 hours. Afterwards, divide the gradient into sections (please read the precautions before filling in this page) to install-line. This paper * standard is used in the B country «quasi (CNS) T4 specification (2) 0χ297 public *) 81. 4 . 10,000 sheets (Η) 209H〇Λ 6 Π 6 Printed in collaboration with Zhengong Consumers, Bureau of Standards, Ministry of Economic Affairs. V. Invention description () Separate, collect purified HBV floating in 1.20 g / cm3 density layer protein. This solution was placed in a dialysis bag and dialyzed against water three times to remove salt. Example 2: Quantitative determination of HBV protein 1. Using radioimmunoassay in AUSRIA 11-125 "sandwich" radioimmunoassay (the equipment can be purchased from Abbot), the guinea pig will be coated against type B Beads of anti-hepatitis (anti-HBs) of hepatitis antigens are brought into contact with blood plasma, plasma, or purified proteins and appropriate controls, etc. Any HBsAg contained will bind to the solid-phase antibody. Extract unbound substances After aspirating and washing the beads, human iaeT-anti-HBs are used to react with the anti-hepatite-antigen complex on the beads. Then, the beads are washed to remove unbound 18βΤ-anti-HBs.)-Anti -HBs HBsAg) -anti-HBs · HBsAg 1ββΤ-anti-HBs) -anti-HBs · HBsAg ♦… T-anti-HBs The radioactivity remaining on the beads is counted by M 2. Use ELISA in Enzygnost HBsAg withdrawal "sandwich" analysis (the equipment can be purchased from Behring), each hole is coated with anti-HBs. Then serum, plasma or purified protein and appropriate control samples, etc. Add to the hole separately and give K to receive the injection. After washing, M peroxidase-sassaying pair HBsAg's anti-glucose is used to react with the remaining antigenic determinants. Then, the unbound enzyme (please read the WJ back first and note #fill ¾ this page) is installed &lt; Order · Line &lt; This paper size Use the Chinese National Standard (CNS) T4 specifications (210x297 g *) 81. 4. 10,000 sheets (II) 1 ° ** 9 ο Printed by the employee consumption cooperation of the Central Bureau of Standards of the Ministry of Economic Affairs

V. Description of the invention () The anti-galvanized washing is removed, and the enzyme activity remaining on the solid phase is measured. The enzyme-catalyzed reaction between hydrogen oxide and Fasikang is terminated by adding dilute sulfuric acid to M. The color intensity is proportional to the HBsAg concentration of the sample, and it is the one who compares the color intensity of the unknown sample with the negative intensity of the hidden sample and the color intensity M photometer of the hidden control blood volume. Window M3 The present invention contains a promoter, a desired antigen sequence, and the preparation of a gene construct such as a selective gene 1. Isolation of the MT-promoter gene The quality pBPV-342-12 (ATCC 37224) is endonucleated with nucleic acid llBgll And Ba «H g number to decompose. Generate three DNA molecules. The desired fragment contains the νl 'Θ methalloiliionein promoter gene and the PBR322 sequence with 4.5 kb, which can be easily detected from other fragments (2.0 kb and 7.6 kb). The reaction was carried out in a solution containing 200 U1 anti-bin-grade flushing liquid with a final concentration of 0.5 / i g // i 1 DNA and 100 units of restriction enzymes. After being left at 37 ° C for 3 hours, the completion of the decomposition reaction was checked by agarose gel swimming method M0.8% agarose gel. The reaction was stopped by adding 4 / il 0.5M EDTA to M. Next, the 4.5 kb fragment was separated from other fragments using a preparative 1.2% agarose gel «swimming method. Put this piece of jellyfish gel on DE-81 Whatman filter paper, and elute the DNA with high-salt grade liquid. The DNA was then purified by K-phenol extraction and two ethanol precipitations. 2. Connect the 1.8kb fragment encoding HBV-core antigen from the DNA containing HBV to the 1.8kb BamH I containing the HBV-core code domain (please read the back-end notes #fill this page) to install- Thread · Original paper »Standard Xiaozhong Standard (CNS) Grade 4 (210X297);": "81. 4. 10,000¾ (II) -09: 4〇Λ 6 116 Employee Consumption of the Central Standards Bureau of the Ministry of Economic Affairs Printed by the cooperative. V. Description of invention () Bam-HI fragments are isolated. This fragment was then ligated with a 4.5 kbH segment (explained in 1) containing the MT-promoter and the rest of the PBR. Mix 2w 1 of the 1.8kb fragment with 3 «1 of the 4.5kb fragment, and react in 14¾ reaction overnight in 1〇 &lt; / 1 ligation solution containing 2 units of T4-DNA ligation and 2.M ATP Build together. Next, the ligation mixture was added to 150 / ul of the appropriate bacterial cell suspension to ingest DNA. After DNA uptake, the ratio of cell volume of fine cell M to 50 to 300 per cell plate was spread on LB agar plates containing 50 / i 1 / »1 ampicillin. The agar plate was then placed at 37t: overnight. Finally, the colonies of solitary mackerel were screened to obtain the pluton with the desired fragment. 3. Screen the fine _ colony containing the desired texture. Isolate the colony to pick up the stomach with a toothpick and place it in a test tube (5 ml) containing LB-Ampicillin-based food base. If; test f is placed at 37C , Incubate overnight in a rapid moldy environment. Afterwards, a small plastid preparation was performed for each partridge suspension. A variety of different DNAs were decomposed using the restriction endonuclease Bgll. The expected target was 400bP fragments And 5.9kbii | H-segment molecules. The decomposition reaction is carried out by agarose gel «swimming method to give W minutes ft. In this way, the quality eel DNA is isolated from the fine cells. The mass fraction obtained in (3) above was decomposed with the restriction enzyme EcoRI to linearize it. This reaction was performed by the total product of M50 // 1 and the final concentration of 1 mass fraction DNA of 1 / ig / w. Add 50 After the unit of EcoRr, let it stand at 37t for 3 hours, and then verify the decomposition reaction by the method of gel skimming. Then, add 1 / u 1 0.5M EDTA to stop the reaction, and the final splash of 0.3M brewing acid Accept (please read the back and attention matters # fill in this page) to install ·-line · The paper size is not in the national B standard (CNS) A 4 specifications (210X297 public ¢) 81, 4. 10,000 sheets (II) 2〇9 Λ 6 Π 6 Printed by the Ministry of Economics + Central Central Bureau of Employee Consumer Cooperative Fifth, the description of invention () and the fermentation of 3-4% of the yeast added, Put it at -80t, 30 minutes of bighead carp to precipitate DNA. Then Δdissolve the precipitated DNA in 50W 1 distilled water. Dissolve 1 linearized cockroach with 3 // 1 containing nethallothionein promoter βΗΓ 新 徽 素 逸 择DNA fragments (such as PMT-ne〇-E (available from ATCC / Exogene), which is decomposed with endonuclease EcoRI, and the isolated one is a 3.9 kb fragment) are mixed and ligated together. Fine sugar colonies were screened by SM to obtain those containing the desired minerals. 5. Isolate the H segment containing the U2 promoter sequence and use pUC-8-42 (available from Exogene) with restriction endonucleases EcoR I and Apal Decompose. Two types of DNA molecules are produced. The desired fragment containing the U2-promoter has 340 bp and is easy to probe from another fragment (<3160 bp). The decomposition reaction was carried out in 200 «1 total staghornis, at a final concentration of 0.5Wg // il DNA, and containing 100 units of Hebei restriction enzyme, etc. Three hours after leaving at 371D S, the completeness of the decomposition reaction was checked by agarose gel electrophoresis using 0.7% agar gel. The reaction was stopped by adding 4W 1 0.5M EDTA to M. Then, a 340 bp fragment was isolated from the genomic DNA by preparative 1.2% agarose gel electrophoresis. Next, the oligosaccharide gel containing the fragment was placed on DE-81 hlhaUan filter paper, and the DNA was extracted with a high-salt-level flush solution. Finally, the DNA was inactivated by phenol / chloroform extraction and two ethyl acetate precipitations. 6. Mosaic the fragment containing the promoter sequence into the multi-linker fermented brew will contain the polylinker sequence (polylinker sequence) ^ contains the following restriction sites: EcoRI, Sac I, S ^ a I &gt; Aoa I &gt; BaiH I , Bgl I, Sal I, Pst I, Hindi &gt; the quality of pSP165 (available from (please read the first WI back and the precautions # fill out this w))-Threading · This paper size is not used in the ea family 榣Standard (CNS) T 4 specifications (210X297 g *) 81. 4. 10,000 sheets (H) Printed by the Employee Consumer Cooperative of the Ministry of Economic Affairs, Gwangyang, Ministry of Economic Affairs V. Description of invention () Pro ^ ega Biotec &gt; Use restricted enzymes for M Linearization. The anti-condensation system is carried out in a solution of 50 «1 total product and the final concentration of lwg /« 1 qualitative DNA. Add 50 units of EcoRI and place at 371C for three hours. The decomposition reaction was verified by the gel-swimming method. The reaction was stopped by adding 1 to 1 0.5 M EDTA to M, and then M 0.3 M sodium hydroxide at a final concentration and 3-4 volumes of ethanol at -801C for 30 minutes. Precipitate the DNA. Then, dissolve the precipitated DNA in 50 // 丨 distilled «water. 2w 1 plastid DNA and 10« 1 fragment DN containing U2 promoter gene Mix A, and then ligate them together in a ligation buffer containing 2 units of T4-DNA ligase and 2 · M ATP, 25% of total tares, at 14Ϊ :, and connect them together overnight. After that, extract with phenol / chloroform Then, after the DNA was purified by two ethyl acetate precipitations, dissolve 10 μM in distilled water. The sticky ends of EcoRI and MI must be converted into blunt ends \ r: 'and then connected. The sticky ends are carefully prepared! ^ 豆Those reacted by acid-decomposing enzymes and converted into blunt ends are as follows: add 25 units of enzyme to 25 m 1 of the reaction entanglement solution containing DNA (1 «g / w 1 irrigation) to make it contain 1% The final concentration of glycerol and the final reaction volume of 35W1. After 30 minutes at 30C, the DNA was purified by extraction with phenol-nitroform followed by two ethyl acetate precipitations. The DNA was then dissolved in 5 «1 distilled water. The resulting blunt ends were ligated together in a 15wl reaction solution containing 10 times more T4 ligase and 2 mM ATP, etc. than used above, after 14TC. Next, the ligation reaction mixture was added to 150 μ 1 of appropriate fine cells DNA ingestion of M in the suspension. After DNA ingestion, apply a volume of 50 to 300 w 1 of fine sugar cells M per plate on LB agar plate containing 50w 8 / · 1 ampicillin. Then, place the agar plate at 371D overnight. Afterwards, (please read the precautions before filling in this page) Installation-Order ·- Thread-This paper standard is used in beta «Family Standard (CNS) A 4 specifications (210X297 male dragon) * 21 — 81. 4. 10,000 sheets (Η) Printed by the Central Bureau of Industry and Commerce β Industrial Consumption Cooperative Print

V. Description of the invention () Sieve the single-isolated scallion colonies to obtain the plutonium containing the desired H2-promoting gene H segment. Finally, the resulting plastids were precipitated from the fine cells and determined by the restriction enzyme analysis method. 7. Synthesize oligo-DNA-nucleoside ether 89 (SEQ ID No .: 30) and MT-promoter gene fragment (4.5 kb) to link together the 4.5 kb fragment containing the MT-promoter and pBR remainder (described above 1 Middle> Synthetic oligonucleotide 89 (SEQ ID No .: 30> ligated together. The ligation reaction mixture was added to 150 / il appropriate fine cell suspension for DNA uptake. Then * from single chick The bacteria are screened to find out the ones with the desired quality. The new quality mule is proved by the restriction endonuclease EcoRI and Xbal decomposed by M. It is expected that there are two kinds of molecules, one is 2.Okb, the other It is 2.6kb. 8. Link the synthetic oligonucleotide 101 (5 £ 1110 ^ 〇.: 32) with the quality || (the one described in 7 above). Connect the quality Qiang (the one described in 7) with Bgll Decompose with BaiHI to remove the 13-nucleotide-containing fragment (as described in 1>. The first fragment oligonucleotide 89 (SEQ ID No: 30) and the oligonucleotide l〇l ( SEQ ID No .: 32), which is a fragment of Bgl Π-BamH I, ligated together. After the DNA is taken up, the isolated colony cells containing the desired screen are screened out. The new trade is endonucleated Enzymes EcoR I and, or EcoR I and Bg in decomposition proof passer. 9. Connect the synthetic DNA-oligonucleotide 99 (SEQ ID No .: 31> to the 4.5 kb fragment (described in 1). 4. The 4.5 kb fragment (Bgl E -Ba ^ HI ) And DNA oligonucleotide 99 (SEQ (please read the precautions before filling out this page) This paper "standard is used in a β home" quasi (CNS) Τ4 specifications (210x297 public goods) 81. 4. 10,000 Zhang (Π) 5. Description of the invention ()

The MID No.: 31 of the Employee Consumer Cooperative of the Bureau of Standards of the Ministry of Economic Affairs is linked together. After screening isolated bacterial colonies containing different DNA, MEcoRI was decomposed to obtain two fragments of 1.9 kb and 2.7 kb, and positive linearization analysis was performed with Bgl II or BamHI to identify the desired plastid. Then use Pst I and Ba «HI to decompose the new plastid. The anticipator has two molecules, one is 2.6 kb, which contains the pBR residue, the MT-promoter and the oligonucleotide, and the other is 2.0 kb, which contains the pBR residue. The 2.6 kb fragment was isolated. 10. The plastid fragment of 2.6 kb described in 9 and the fragment isolated from plastid (the one described in «I 8) are linked together. DNA oligonucleotides 89 and 10 are contained <SEQ ID No. .: 30 and 32) Decompose with PstI and BglE. It is expected to have two slaps. One is a 2.5 kb fragment containing the PBR residue and MT-promoter, and the other is a 2.2 kb fragment containing the pBR residue and two oligonucleotides. Among them, the 2.2 kb fragment and the 2.6 kb fragment containing the PBR remainder, MT-initiator, and oligonucleotide 99 &lt; SEQ ID No .: 31) are linked together. After the desired quality has been screened out , Bgl Π -Xbal restriction endonuclease digestion for identification. Two fragments are expected, one for the 270bpj = j segment of the oligo-DNA-nucleic acid and the other 4.5kb containing the MT-promoter and the rest of the pBR. 11.2.3kb Ligation of HBV BglE-Bglll Fragments The 2.3kb Bgl E-Bgl E fragments containing HBV pre-Sl, Pre-S2 and S coding domains were isolated from HBV-containing DNA. Connect the 2.3 kb fragment to the 4.5 kbH segment containing the methollathionein promoter (the one obtained from 1) (please read the notes before Ml and write this page on this page) CNS) 4 specifications (210X297 g *) 81. 4. 1 (), () 00 sheets (||) Λ 6η 6 Produced by the Beigong Bureau of Economics, Ministry of Economic Affairs, Beigong Consumer Cooperation Co., Ltd. V. Description of invention () in together. After mixing the 2.3kb fragment of 2 / i 1 with the 4.5kbH segment of 3 «1, in a total of 10 mL / ul) reaction mixture, containing 2 units of T4-DNA ligase and 2. In the case of ATP, they are connected together at 14TC overnight. Then, add the ligation reaction mixture to 150 / ul of appropriate cell suspension for DNA uptake. After DNA uptake, the cells were spread on LB agar plates containing 50 / z g / «l ampicillin at a volume of 50 to 300« 1 per plate. The agar plate was incubated overnight at 37C. After that, the isolated bacterial colonies were screened to find the ear body containing the desired fragment. 12_ Part of the HBV-gene sequence is transformed with HBV-core epitope. The haze obtained in 11 above is decomposed with M by the endonuclease Bgll and Xbal. Two molecules are expected, one is a 550 bp fragment and the other is a 6.25 kb fragment, which is isolated after dipping on an agarose gel. Connect the 6.25 kb fragment to the 270bP plastid H segment of the HBV-core gene epitope partially described in 10 (after Bgll and Xbal decomposition and fragment isolation as described above) ;. The ligation mixture is added to the fine sugar cell suspension of 150 suitable for DNA uptake. Then, the single-isolated barley colony is screened to obtain the desired plastid. The new substance is decomposed by Ba «HI Μ proved that. There are three kinds of molecules expected: 950bp, 450bp and 5150bp and other three fragments. 13. Preparation of "medium" quality scorpion will decompose the quality snakehead mentioned in 11 with EcoRI and Xbal. M is expected to have two molecules, one It is a 2450bP fragment and another 4,350bp fragment, which is isolated after condensing and swimming. (Please read the back to the precautions # fill out this page) This paper is used in the β B home «Bi (CNS) T4 specification (210X297 public *) 81. 4. 10J00 Zhang (II) ;: 4〇Λ 6 Η6 V. Invention description Printed by the Employee Consumer Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs. The 4.35bpH section and HBV-S- The oligo-DNA-nucleotide 39 encoded by the DNA sequence from ATG to Xba I in the gene (SEQ ID No .: 29, of which ATG Replaced by ΑΤΑ) connected together. 14. The entire core-epitope upstream segment of the HBV-S gene, and then, the resulting "medium" is brewed with Pst I and Xbal to decompose, it is expected that there are two molecules, one is 600bp quality The remaining segment of 雠 and another 3,850 bp fragment. The latter after isolation is connected with the 2,800 bp (2,700 bp) PstI -Xbal fragment precipitated by the decomposition of the mass muslin described in 10. After sieving out the desired plastid, use Restriction endonucleases such as EcoR I and Xba I, EcoR I and Bgll and BaiHI are decomposed and identified by M. 15. Selecting the embedding of 溷 志 之 to linearize the resulting substance 14 with «EcoR I. The reaction is at 50 wl. # Total 1 / Z g / tf 1 quality DNA was performed at the final temperature. After adding 50 units of EcoR I and set at 37P # for 3 hours, the decomposition reaction was detected by A. lucidum gel electrophoresis. T Afterwards, the reaction was stopped by adding 0.5MEDTA of lwl, and the DNA was precipitated with -300M sodium acetate and 3-4 surface of the yeast under the conditions of -801, 30 minutes, etc. The precipitated DNA was dissolved in 50 / i 1 steamed water. Take 2 / il linearized masses and 3 // 丨 DNA slices containing the gene of the new ethenothionein promoter of Teng ethallothionein (The ones described in 4) Mix and connect them together. Separate the fine plaques from the single-fine sugar sieve, and isolate, purify, and identify them. --- ¾ Each of the genes mentioned above The object can also be constructed with U2-Qipan \ _, by which, after decomposition with EcoRI and Bgin, it will contain MT-start

The size of this paper is used in the BS home standard (CNS) T4 specification (210X297 g; tt) (please read the precautions before filling the nest page) Packing-line · Ψ 81. 4. 10,000 sheets (11) 09 . Λ 6 η 6 Printed by the Employee Consumer Cooperative of the Bureau of Standards, Ministry of Economic Affairs V. Invention description () The gene @ ΝΑ segment can be replaced with the U2-kai DNA fragment obtained by the decomposition and isolation of EcoRI and Bgll. 16. Escherichia coli yellow flower essence black dung dung phosphatidyl ribosyl transferase (egpt &gt; segregation of selection genes, the PMSG PM quality is decomposed and isolated by Ba * HI and BglH and the fragment containing the egpt selection gene (1.8kb) and containing The 4.5 kb fragment of the MT-promoter (Bgl E-BanHI, described in 1) is connected together. After the sieve is irrigated with the desired quality, it is isolated, purified and finally converted from its BaaiHI site to an EcoRI site 17. Isolate the desired DNA sequence by PCR method and isolate a DNA fragment (400bp) after gel electrophoresis treatment. The specific oligonucleotides 131 and 132 (SEQ ID No .: 33 and 34> are used as guides (? | ^ «61 *) produced by the? 0! ^ Method (the one described in Example 5). The DNA fragment was decomposed with the endonucleases Ba« HI and Xba I to M, and then condensed waist « It was purified by electrophoresis. The isolated PCR-fragment was then combined with the 6.25kb fragment obtained by the decomposition and isolation of the masses described in 13 with Bgll and Xbal. Quality. 18. Introduce the shovel of the selection of Zhizhi Zhi into 17 and add the quality of the genus to the final selection of the gene (according to 15). 19. H2K start The H2K promoter is isolated from pSP65H2 (available from Exogene &gt; M EcoRI and Bgl I fragments (2kb). In all the constructs mentioned above, all promoter genes can be pre-treated with EcoRI / Bgll fragments K 置 捵. (Please read the precautions and fill in this page first) Binding-Binding-Line · This paper size is applicable to a 漸 楳 楳 准 (CNS) T4 specification (2 丨 0X297 public address) 81. 4. 10,000 sheets (||) Λ 6 H6 Central Ministry of Economic Affairs, Central Bureau of Industry and Commerce, KK industrial and consumer cooperation du printing 5. Description of the invention () 20. Part of the transformation of HBV-gene sequence The endonuclease BglH obtained from the above 11 pawns And Xbal are decomposed. Two molecules are expected, one of which is 6.250kb fragment, isolated after swimming in agarose gel. The 6.250kb fragment is oligo-DNA-nucleotide 23 (SEQ ID No. : 28 &gt; Connect together. Then, add the ligation mixture to 150 μl of fresh larval cell suspension for DNA uptake. Then, sieve the single isolated larval colony to extract the desired substance. The new substance It was proved by the endonucleases EcoRI and Bgll [decomposed. Two molecules are expected, one is 1.9kb Another 4.450kb. 21. Egpt infiltrate the mines of 抷 志 臨. The 20-mass mass was linearized with EcoRI. The reaction was based on 100 // 1 total mole and 0.6Wg / al plastid DNA. Under other conditions. 60 units of EcoRI were added, and after three hours at 3710¾, K agarose gel electrophoresis was used to verify the decomposition reaction. After that, 2 wl of 0.5 MEDTA was added to stop the reaction, and DNA was precipitated out at -80¾ with an final concentration of 0.3 M sodium sugar acid and 4 mg of ethanol at -80¾ for one hour. The precipitated DNA was redissolved in 50 / i 1 of distilled water. The decomposition of 2 «1 linearized chrysanthemum and 3 in 1 by £ 〇: 〇111 contains this obituary &amp; 11〇-1 ^ 1〇116 111 promoter and 68? 1 ^ gene (the one described in 16> ; 0 "-fragment (3.7kb> mixed and ligated together. After that, sieve S isolated colonies to find the desired quality. Each of the gene constructs listed in Table I can be used in the same way as described above Prepared by Μ. 傭 玉 锎 Λ (please read the precautions before filling in this page) The paper size is free to use the Chinese National Standard (CNS) 4 specifications (210X297 public *) 81. 4.] 0,000 sheets ( li), 09 ^ 40 A 6 η 6 V. Description of the invention () Printed by the Ministry of Economic Affairs, Bureau of Economics and Trade, Beigong Consumer Cooperative to transfer HBV infected with mammalian cells using the structure of the invention in order to make the structure of the invention HBV If it can be secreted in a substantial amount, the DNA construct of the present invention must be used to transfer and infect mammalian cells. The co-transfer infection is a two-step method (different transfer infection for each constituent) or a single step * method &lt; i.e., one-time metastasis infection using the preparation of the two-pile composition &gt; Different selections are used to determine the epidemiology, or by immunoassay to detect the secretion of the expression product of the two constituents of the two bridges. It can also be determined by M. In addition, the sequence of the present invention that encodes the HBV sequence can be determined with the code. The Tsurube sequence of whole-bed S, or core, or white matter, etc. is combined in a single structure. Example 5 Polymerase bond reaction (PCR) _ Polymerase bond type inversion can be used to combine known gene sequence The specific DNA nucleotide sequence of a certain localized area is in uitro (in uitro> expansion of the first 100 times) (Thoiaas J. White, Nornan Arnlei ·, Henry A · Erlich 1989: The Polynerase Chain reaction (polymerase Key type reaction) ° Technical Focus, Vol. 5. No. 6; S. Kwok and R. Higuchi 1989: Avoiding false positive with PCR. Nature, Vol. 339, pp 237-238>. Separated from cells DNA or DNA treatment of DNA M separates its complementary strands. Then some of the strands are used in excess, chemically synthesized to match the sequence separated by X nucleotides (here X is usually between 50 and 2,000_ pairs) Between two DNA oligonucleotides (each 20-25 pairs in length) Anneal (please read the back-end precautions #fill in this page) Binding-Order _ This paper is used in the standard "« 極 楳 准 (CNS) T4 specifications (210X297 public; it) 81. 4. 10,000 Zhang (Η) Λ 6 Η 6 The Ministry of Economic Affairs, Central Bureau of Industry and Commerce #Cooperate with Du Yin, V. Description of the invention () Two types of oligonucleotides were created as specific guides for DNA polymerization DNA synthesis to replicate the DNA between the sequences aligned by the two oligonucleotides. If the two guide oligonucleotides contain the correct sequence, the new decomposition site may be created at and. After multiple cycles of inversion, a large number of DNA fragments of desired length are obtained, purified by gel-swimming method and decomposed by restriction enzyme decomposition and agar gel electrophoresis. This expanded and purified DNA fragment and other fragments are then used. For example, the qualitative table can be connected together. The PCR-DNA fragment is made of blunt ore. To get sticky (for ligation procedures), the fragment must be digested with the necessary endonuclease_ and purified. The PCR reaction can be operated for 20 to 30 cycles. One partial cycle can be divided into three tilting steps, each with different reaction time and different reaction degrees, all controlled by PCR-therao-cycler. The first step is the "denaturation" of the base-DNA (1 minute shovel-95¾), the second step is the "mixing" of the matrix DNA and the leader (1 minute pupil / 55¾), followed by the "polymerization" (2 minutes / 72Ό &gt;. The final volume of an analysis is, for example, 30 μl, which contains the following final concentration: PCR-grade balance solution (Κ>, a nucleotide mixture with four nucleotides each of 200 «Μ , Each of the two guides of 200ng in 30u 1 contains 0.5 units of Tag-polymerase per 30W of 1 aqueous solution. Fen case R Transfer of infected cells to the cultured meal M secreted protein will be received cells (C127- or CH0- cells , Obtained from ATCC) inoculated in the positive (please read the back and the precautions # fill out this page) This paper standard is used in the BS home «quasi (CNS) Τ4 specifications (210x297 male dragon) 81. 4. 10,000 sheets (Η ) Γ Λ 6 Η 6 5. Description of the invention (DMEM + 10% fetal bovine blood, glucose and glutamine) printed on the constant growth medium (DMEM + 10% fetal bovine blood) by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. Shallow culture dishes, <1-2Χ10β cells / dish, Φ 10 cm). Remove the culture dishes on the next day (in the DNA sink) 4 hours before adding the material to the cells), and wash the cells twice with 1 X PBS. Then, add 8 ml of DMEM without FCS, and after 4 hours, add the DNA pellet (prepared as described below) to On top of the cells, similarly, after 4 hours, the medium was removed, and 3 ml of glycerol-mixture &lt; 50 ml of 2X TBS buffer solution, 30 ml of glycerol, 1.0 ml of distilled water was added. After 3 minutes, the glycerol-mixture was removed and the cells were washed with IX PBS. The cells were then cultured overnight with 8 ml of DMEM containing 10% FCS. After 48 hours, trypsin-EDTA-solution (0.025% trypsin + 1 · Μ EDTA) treatment to recover the cells from the dish. Afterwards, wash trypsin-EDTA with lx PBS to remove the trypsin-EDTA from the cells, and then suspend the cells in DMEM containing 10% FCS and distribute to 24Costar- In the hole-plate (the cells contained in one dish are divided into four-value 24-hole-plates). When the cells grow well, add the selected food base (concentration of 0.5-1 mg / ml of neo-embroidin, or: Yellow flower essence (250 wg / Bl>, sub-yellow flower essence (15 «g / nl> or glandular glomerulus (25« g / 臞 1), thymidine (10w 8 / dirty 1) Jidie (2 // g / stuffed, emblemphenolic acid &lt; 25ju g / el &gt;, for, for example). The meal base should be changed every week. After 2 weeks You can see the colonies of the first healthy growing cells. In 10 «g-quality DNA and 20 // g carrier-DNA (salmon sperm DNA, calf thymus DNA>), add TE-buffer &lt; 10ibM Tris-HCl, leM MDTA, 戸 117.05) to the final volume of 440 ^ £ 1, and then mixed with 60 «12 ^! [&amp; (: 18 mixed in (please first« please note #fill ¾ this page) installed. Line. This paper * The standard is used in the "Mingjia Sample Standard (CNS) T4 specification (210x297mm: ¢) 81. 4. 10,00051c (II) 209.4b Λ 6 II 6 V. Ministry of Economic Affairs + Central Standards Bureau Beigong Consumer Cooperation Print the invention description () together. Next, add the same amount of 2XTBS (Hepes 50aM, NaCl 280 »&gt; 1," 11Porpo4 1.5 «^ ,?» 17.05) and mix thoroughly. After putting the precipitation solution at 37¾ for 30 minutes, add it directly to the desired Transferred to infected cells. Example 7: Preparation of purified particle adjuvant In an antigen solution of the desired concentration, suspended in saline-free saline, adding 1: 10,000 thiaerosol, 1/10 AK Zhen has been filtered Reduced 0.2M KA1 (S0 «> 12Hβ0. After adjusting its pH value to 5.0 with sterile IN NaOH, the suspension was stirred in the chamber» for three hours. After that, it was centrifuged at 2, OOOrpn for 10 minutes to recover the cast Precipitated antigen, and resuspended in normal water containing 1:10, Thi Beroso, and distributed under sterile conditions. Example 8 Purification of Hepatitis B Core Antigen HB-core antigen secreting cells The cells were collected and concentrated by M ultrafiltration method. Then, the splashing condensate was centrifuged at 20,000 γρ for 15 minutes in a Becknan SW28 centrifuge at 4¾. The particles were formed via Becknan SW28. In a centrifuge, centrifuge at 28,000 rp · 41 at 41C with an M sugar density (0-45 % Cheap sugar) 24 hours to be tested. After that, the gradient is divided into several parts, and each single part is subjected to Elisa analysis. Fenyu Example 9 The following table lists the immunogenic particles of the present invention obtained by E analysis Some results are as follows: I Table IV lists any HBV-sequence constructs of the present invention, including pre-Sl (please read back and forth before filling in this page) Binding-Binding · Thread-This paper content The ββ family quasi (CNS) T4 specification (2 丨 0X297); ":) 81. 4 · 10,000 sheets (ΙΪ) 209 Η. Λ 6 Η 6. V. Description () Produced by epitopes and S domain etc. The purified HBs-transgenic particles were subjected to Eli sa analysis using anti-Pre-Si «single-anti-anti-MA 18/7 and anti-HBs single anti-G 2 2. v Table IV lists CsCl The result of the part (21) collected after the density gradient settled. Table IV -1

CsU-gradient part of the number of Elisa Lan (E = 492) single plant anti-Mian 18/7 13 0.092 14 0.210 15 0.388 16 1.662 17 2.604 18 0.648 19 0.031 (please read the backing notes first ) Binding-Ordering-Printed Paper of Beigong Consumer Cooperatives, Central Bureau of Standards, Ministry of Economics, Standard Side Use «Home» Standard (CNS> A4 Specification (210x297mm) 81. 4, 10,000 sheets (II)

V. Description of Invention () Table W-2

CsH-gradient part number Elisa measurement (E = 492) individual strains of anti-Mule G022, 13 0. 136 14 0.426 15 0.822 16 1.970 17 2.954 18 0.967 19 0.076 (please read back and attention beforehand # fill in this page ) Table V shows purified HB-core-antigen particles produced by any HB-core sequence constructs of the present invention. Elisa was performed with multiple anti-HB-core antibodies and single anti-pus G022 against HB-S-Ag Analyze the data. The paper printed by the Employee Consumer Cooperative of the Central Bureau of Economic Affairs of the Ministry of Economic Affairs is printed on the Λ scale, using the Chinese B-standard (CNS) T4 specifications (210x297 g *) 81. 4, 10,000 sheets (H) r V. Invention description () Λ (i Β 6 Table V -1 Μ 耱 Gradient part number El isa measurement (Ε = 492) Monoclonal antibody 6 0.25 7 0.922 8 1.423 9 1.5 10 1.5 11 1.28 12 0.466 Table V -2 (Please read the precautions first #Pot write this page) Packing-Order _ Gradient part number of the sugarcane gradient printed by the staff cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Elisa measurement (Ε = 492) monoclonal antibody G022 6 0.020 7 0.024 8 0.018 9 0.011 10 0.015 11 0.020 12 0.022 Thread-This Paper "scale is used in China National Standards (CNS) A 4 specifications (210X297 male dragon) 81. 4. 10,000¾ (II) 09.4 ο Printed by the employees of the Central Standards Bureau of the Ministry of Economy 3. Description of the invention () Fen M 10 The study of chimpanzees taking Hepa-Care: Hepa-Care is a particle made by mixing Hepatitis B surface antigen (S1 and S) into a special formula (ratio 50:50), It is a long-term carrier of hepatitis virus treated with M. . Experiment 1 Hepa-Care was administered with a dose of 18 «g per injection at 0, 4, and 8 weeks when the chimpanzee carrier of the hepatitis B carrier was buried 1 (intramuscular injection). The liver was monitored during the treatment Enzymes (circle IV) and their hepatitis B antigen levels (circle V &gt;. Experiment 2 Chimpanzees 1 treated as described above were given intensive treatment at 30, 34, and 38. The results are shown in the graph. In VI. Experiment 3 Chimpanzee 2 was treated with MHepa-Care, but it is different from Chimpanzee 1 in that it is administered intravenously. Its amount is 2 mg. The results are shown in Figure VI. The chimpanzee 3 in the control group was monitored for liver enzymes, and the results are listed in the circle. Jian Yuli 11 treated with Hepa-Care: (The definition is described in Example 10) Patient 1 (Male, age = 65 years old, already 2 years of illness): Hepatitis B parameters: HBsAg Μ-anti-HBs negative (please read the back of the notes and write this page)

The size of this paper is easy to use China Sleepless Standard (CNS) 4 specifications (21〇297297 *) 81. 4. 10,000 sheets (II) V. Description of invention () HBeAg negative Anti-HBe positive Anti-HBc negative

Hepa-Care was used for M treatment (intramuscular injection) at 0, 1, 6, and 7 months. The antigen and anti-crocodile antibody results are listed in 圏 Κ and X ° Patient 2 (Female, age = 48 years, has been sick for 12 years>: Hepatitis B parameters: HBsAg positive HBeAg negative anti-HBs negative anti- HBe anti-HBc anti-HBc is treated with Hepa-Care (intramuscular injection) at Ο, 1, and 6 months. Its antigen and anti-medium results are listed in gardens XI and XI. Patient 3 (female , Age = 41 years, has been ill for 5 years &gt;: Hepatitis B parameters: HBsAg positive HBeAg negative anti-HBs negative anti-HBe exposure (please read back to the precautions # fill out this page) installed. -SJ . Line. Printed by Hepa-Care (intramuscular injection) for Hepa-Care printed in the Pei Gong Consumer Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs. Listed in ^ XI and XIV, the paper is Λ-scale, easy-to-use, and homey, "Bi (CNS) specifications (210X297 g *) 81. 4. 10,000 sheets (II) dagger 〇9, V. Description of invention () A β It 6 SEQ ID N0: 1 SEQ category: nucleotide sequence length: 558bp chain strand: single strand form: linear divide Category: DNA gene feud original to the sea: HBV core direct experimental Source: PCR- big throw into confusion

ATG GAC ATT GAC CCT TAT AAA GAA GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACTCTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA. TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA TCT CAA TGT TAG (please read the notes on the back # fill in this page ) Binding-Order _-Line. This paper is used in the standard BB family "Standard (CNS) A 4 specifications (210x297 mm) 81. 4. 10,000 sheets (II) 9 ο 40 66 ΛΒ V. Description of invention () SEQ ID N0: 2 SEQ category: nucleoside Sequence length: 504bp Stranded strands: Single strand form: Linear molecular category: Gene striated DNA Original source: HBV core direct experimental source: PCR-amplification (please read back to the precautions #fill this page) Ministry of Economic Affairs Standards Bureau ts: Printed by industrial and consumer cooperatives

ATG GAC ATT GAC CCT TAT! AAA GAA ^ »r * m. GGA GCT ACT C-TG GAG TTA CTC TC3 mm TTC- CCT TC? GAC \ T: C TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC .. Bu TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CC7 CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC GGG GAA mmQ ATG AC? CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTC AAT TAT GTT. \ AT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT ccc TCG CCT CGC AGA CGT Specification (210x297; ¢) 81. 4. 10,000 sheets ⑻ .0¾, 4b V. Description of the invention () SEQ ID NO: 3 SEQ category: nucleotide sequence length: 504bP bond strand: single strand form: linear molecular category : Gene Huan DNA Original source: HBV core direct experimental source : PCR-Expansion (please read WI's notes beforehand, Sun fills this page) Printed by Beigong Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs

ATG GAC ATT GAC CCT TAT AAA GAA ΤΤΊ * GGA GCi1 ACT gig GAG TTA CTC TCG -nmm TTG CCT TCT GAC TTC fTi m rrt CCT TCC GTA CGA GAT CTC CTA GAC ACC Guangr · TCA Guang &lt; »m Λ. CTG TA ? CGA GAA GCC TTA GAG CCT GAG CAT TGC TCA CCT CAC CAT AC? GCA CTC •-• Lrvj CAA C-CC ATT CTC TGC TGG GGG GAA TTG ATG AC? CTA C-CT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TC-G TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATTGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC- * AGA CGT r- Original paper »Standard selection Zhongyinjia Standard (CNS) Grade A 4 specifications (210x297 g; Jt) 81. 4. 10,000 »(Η) binding. Binding-line. 5. Description of invention () Λ 6 Π 6 SEQ ID NO: 4 SEQ category: nuclear Glycine sequence length: 534bp bond strand: single strand morphology: linear molecular category: gene clam DNA Original source: HBV core Direct experiment source: PCR-Amplification (Please read the back-to-back precautions #fill this page) Printed by the Central Standards Bureau of the Ministry of Economic Affairs Μ 工 消 # Cooperative

TCC AAC CTG TGC CX *! * GGG TGG CTT TGG GGC ATG GAC ATT GAC Γ r TAT AAA GAA TTT GGA GCT ACT GTG GAG ^ TA CTC TCG mm 上 丄 TTG CCT rp wide ”GAC TTC r *» r · rr « CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG CGA GAA GCC TTA GAG TCT Guang Guang-1 V · »V · #-GAG CAT TGC TCA CCT CAC CAT AC7 GCA Guang mr * · L Thai AGG CAA GCC ATT CTC TGC GGG GAA TTG ATG AC? CTA GCT ACC TGG GTG &lt; t \ 's? V3 丄 AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG ZXk CTA TTG TGG TTT CAT ATA TCT TGC CTT rvC * TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCt CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT This paper standard is used in the "B Family Standard (CNS) T4 specification (210x297 male dragon) 46 81. 4. 10,000 sheets ⑻

SEQ ID NO: 5 SEQ category: nucleotide sequence length: 534bp strand strand: single strand morphology: linear molecule category: genomic DNA original source: HBV core direct experimental source: PCR-amplification (please read back to the first note Please fill in this page again) TCC AAC CTG TGC CTT GGG TGG CTT. TGG GGC ATG GAC ATT GAC CCT ΊΑ? AAA GAA GGA GCT ACT C-TG GAG TTA CTC TCG IT? TTG /-r · m TCT GAC TTC CCT TCC QTA CGA GA7 CTC CTA GAC ACC GCC TCA GCT CTG ΊΑ? CGA gaa GCC TTA GAG TCT Guangr · Vw Jr GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC GGG GAA ATG ACT CTA GCT ACC TGG GTG * r ^ * r * \ S \ SX AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA QTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CCG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT TCG CCT CGC AGA CGT: installed &lt; In this paper, the standard size of the paper is used in the Ming Dynasty (CNS) T4 specification (210x297 g *) 81. 4. 10,000 sheets (II) V. Description of the invention (SEQ ID NO: 6 SEQ category: nucleotide sequence length ： 87bp 鐽 Share: single strand form: linear molecular category: genomic DNA Original source: HBV core direct experimental source: chemical synthesis

ATC CTC TGC TGG GGG GAA TGG ACC TGG GTG GGC AAT AAT TTG TCT AGG GAC CTT GTA GTA AAT Λ 6 Η 6 (please first «please note #fill in this page) Employee Consumer Cooperative of the Ministry of Economic Affairs Printed SEQ ID NO: SEQ category: Sequence length: Strand strand: Morphology: Molecule category: Original source: Direct experiment source: 7 nucleotides 81bp Single strand linear genomic DNA HBV core chemical synthesis GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG .---, this paper "standard" uses the Chinese National Standard `` quasi (CNS) A 4 specifications (2 丨 0x297 public *)

81. 4. 10,000 »W ........ ^ ..... 玎…-. 线 · ^ 〇9-4b V. Description of the invention () Λ 6 Η6 SEQ ID NO: 8 SEQ category: core Glycoside Sequence Length: 114bp Strand Strand: Single Strand Form: Linear Molecule Type: Gene Chong DNA Original Source: HBV Core Direct Experimental Source: Chemical Synthesis (please read the precautions before filling in this page)

TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT ΆΑΑ GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG 81. 4. 10, 〇〇〇 Zhang (H ) This paper is used in the standard β Beta Family Standard (CNS) T4 specification (210x297 g *). Printed by the Ministry of Economic Affairs, Central Bureau of Precision Industry, Beta-Consumer Cooperation SEQ ID NO: 9 SEQ Category: Nucleotide Sequence Length: 90bp Spreadability: Single strand morphology: Linear molecular type: Gene mule DNA Original source: HBV core direct experimental source: Chemical synthesis

GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GGT V. Description of invention () Λ 6 η 6 Employee rape co-operatives of the Central Standards Bureau of the Ministry of Economic Affairs Printed SEQ ID NO: 10 SEQ category: nucleotide sequence length: 261 bp strands: single-strand morphology: linear molecule category: gene ray DNA original source: HBV core direct experimental source = chemical synthesis

ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG. TCT CCT GAG CTA TGC . CCT CAC CAT ACT GCA CTC AGG CAA GGT ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC CTT (Gm GTA AAT (please read the precautions first -Fill in this I) -Installation- Order_ -Line · The standard of the paper used in the Chinese standard (CNS) &gt; P4 specification (210X297; it) 81. 4. ΙΟ, ΟΟΟϑ (II) 209 V. Description of invention () SEQ ID NO: 11 SEU category: Nucleotide sequence length: 291bp 铧 strand property: single strand morphology: linear molecule category: genomic DNA Original source: HBV core direct experimental source: chemical synthesis

TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT GCC TTA GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GGT ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC · 6TT GTA GTA AAT (Please read the precautions before filling in this page)

T printed by the Central Ministry of Education and Consumers Cooperatives of the Central Ministry of Labor and Welfare Cooperative Standards "National Standard (CNS) Τ4 Specification (210x297) *) 81. 4. 10,000 sheets (II) 209 ^^^ V. Description of invention () SEQ ID NO: 12 SEQ category: nucleotide sequence length: 123bp strand strand: single strand morphology: linear molecule category: genomic DNA original source: HBV core direct experimental source: chemical synthesis Λ 6 Π 6 (please first Read the notes to the contrary #fill this page)

ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG- GGT TTA AAG ATC AGG CAA CTA • TTG TGG TTT CAT ΑΤΑ TCT TGC CTT ACT 'TTT Binding-Order Economy Department DuPont printed by the Central Bureau of Consumer Affairs SEQ ID NO: 13 SEQ category: nucleotide sequence length: 138bP bond strand: single strand form: linear molecule category: gene cDNA DNA original source: HBV core direct experimental source: chemistry synthesis

GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA 'CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG To use the "Standard Standards (CNS) T4 Specification (2 丨 0X297) *) 4. 10,000 sheets (II)-line-Λ 6 li 6 5. Description of invention ()

SEQ ID N0: 14 SEQ category: nucleotide sequence length: 822bp strand strand: single strand morphology: linear molecule category: gene DNA original source: HBV S2 ayw / adw direct experiment source: HBV DNA (please read the back first Note (fill in this page) Central Ministry of Economic Affairs Bei Gong Xiao # Cooperative Du Printed ATG CAG TGG AAT TCC AGA ACC TTC CAC CAA Acr CTG CAA GAT CCC AGA GTG AGA GGC CTG TAT TTC nr'-y 'W — C-CT C-GT GGC TCC AGT TCA GGA ACA GTA AAC CCT GTT CTG ACT ACT GCC TCT CCC TTA TCG TCA ATC TTC TCC- AGG ΑΤΑ GAG AAC ATC ACA TCA GGA TTC CTA GGA CCC CTT CTC G? G CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC Λ0Λ n 丄 A CCC- CAG AGT CTA GAC TCG II-GG AC? TCT C? C .-Lrv x TTT CTA GGnJ ACT ACC GT3? GT CTT GGC CAA AAT TC3 OG TCC TCA ACC 7CC ϋ l, CAC TCA CCA ACC rn wide pn ^ d. TGT CCA ACT TC-? CCT GGT TA? CGC TC-G ATG TC-T CTG CGG CGT? T? ATC ATC TTC CTC TTC ATC CTG CTG CTA TC-C CTC ATC ΤΓΓ: ^ TG GTT cxr CTG GAC ΤΛΤ CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA TCC TCA ACA ACC AGC ACG GGA CCA TGC CGG ACC TGC ATG ACT ACT GCT CAA GGA ACC TC? ATG TAT CCC TCC TGT TC-C TGT ACC ΛΑΑ CCT TCG GAC C-GA AAT TGC ACC TGT ATT CCC ATC CCA TCA TCC TGG GCT TTC GGA ΛΑ TTC CTA TGG GAG TGG GCC TCA GCC CGT TTC TCC TGG CTC AGT TTA CTA GTG CCA TT GTT CAG TGG TTC GTA GGG CTT TCC CCC ACT GTT TGG CTT TCA GTT ATA TGG ATG ATG TGG TAT TGG GGG CCA AGT CTG TAC AGC ATC TTG CCC TTT TTA CCG CTG TTA CCA ATT TTC TTT TGT CTT TGG GTA TAC ATT r This paper scale is used in the national «quasi (CNS) T4 specification (210X297 male dragon) 81. 4. 10, 〇〇〇 sheets (|| )% Λ 6 It 6 V. Description of the invention () SEQ ID N0: 15 SEQ category: nucleotide sequence length: 99bp strand strands: single strand form: linear molecule category: gene DNA original source: HBV core direct experimental source : Chemical Synthesis

TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT • ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT &quot; \ ttg GTC (please read the notes on the back # fill in this page) Installation · Thread · Printed on the paper standard for easy use by the Consumer Cooperatives of the Central Bureau of Economy, Ministry of Economic Affairs a Η Family Standard (CNS) Ή Specification (210x297 g; ¢) 81. 4.] 0,000 sheets (Η) V. Description of invention () SEQ ID NO: 16 SEQ category: Nucleotide sequence length: 390bp 鐽 strand property: single strand morphology: linear molecular category: genomic DNA Original source: HBV core direct experimental source: PCR-amplification (please read the back side first Please pay attention to this matter and fill out this page) Printed by Beigongxiaot Cooperative Society, Bureau of Standards, Ministry of Economic Affairs

TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT GCC TTA GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA &quot; 'TAT TTG GTC Pack-This paper scale is used in the B country «quasi (CNS) T4 specifications ( 210x297 male *) 81. 4. 10,000 sheets (11) V. Description of the invention () SEQ ID NO: 17 SEQ category: nucleotide and corresponding protein sequence length: 60 bp stranding: single strand form: linear molecule category: Gene DNA Original source: HBV SI ay Direct source: Chemical synthesis AAT CC? CTG GGA TTC TTT ccc GAT CAC CAG TTG GAT CCA GCC TTC AGA C-CA AAC ACC GCA Asn Pro Leu Gly? He Phe Pro Asp His GinLeu Asp Pro Ala? He Arg Ala Asn Thr Ala V, ..: 〆 (please read the precautions and fill in this page first) Binding-Order-Line-KX industrial and consumer cooperation du printed version of the Central Standards Bureau of the Ministry of Economic Affairs Paper-scale Chinese National Standard (CNS) T4 specifications (210X297 mm) for free use 81. 4. 10,000 ^ (1!) V. Description of invention Λ (i Β 6 SEQ ID Ν0: 18 SEQ category: nucleotide and Corresponding protein sequence length: 63bp 鐽 strand property: single strand form: linear molecule category: genomic DNA original source: HBV SI ay direct experiment source: chemical synthesis (please read the precautions before filling this page) CC7 TCC ACC AAT CAG TCA GGA Λ Guang — .-LVjvy CAG-CCZ ACC CCG dagger · θ CCA »· ρ Ff« AOS. AAC Pro Ala Ser Thr Asn Arg Gin Ser C-ly Arg Gin Pro Thr Pro He Ser Pro Pro Leu Asn * 81. 4. The 10,000 sheets (Η) of this paper are printed in Chinese National Standard (CNS) Τ4 specifications (210 &gt; &lt; 297 public address). Printed by the Ministry of Economic Affairs, quasi-bureau of quasi-bureau ΚΧ 工 Consumer Cooperatives. Description of the invention () SEQ ID NO: 19 SEQ category: nucleotide and corresponding Protein sequence length: 63bp Bond strand: Single strand form: Linear molecule category: Genotype DNA Original source: HBV SI ay Direct source of verification: Chemical synthesis (please read back to the precautions # fill in this page) GAT CCA GCC TTC AGA GCA? ΛΟ ACC C-CA ΛΑΤ CCA GAT TGG GAC TTC CCC AAC AAG GAC Λ / &quot; r * AS? Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Tiir 81. 4. 10,000 sheets (Η) of this paper are used in China National Standards (CNS) T4 specification (2 丨 0X297); ») Printed by the employees of the Central Bureau of Standards of the Ministry of Economic Affairs & Cooperatives 5. Description of invention () Λ 6 It 6 SEQ ID N0: 20 SEQ category: Nucleotide and corresponding protein sequence length: 108bp Leng strand: single strand morphology: linear molecule category: Gene Huan DNA Original source: HBV SI ay Direct experiment source: Chemical synthesis CCG CAC Guangsan 'CTT TTG r · m «AC-C CCT CAG Guangyi CAG ΑΤΑ CTA ΟΛΑ ACT TTG CCA GCA AAT rr · ^ v〇v *» CCT C-CC TCC ACC ^ &gt; rn CC-C CAG TCA AGG * CAG CCT Pro His Gly Gly Leu Leu Gly. Trp Ser Pro Gin Ala Gin Gly lie Leu Glu Thr Leu Pro Ala Asn Pro Pro Pro Ala Ser Thr Asn Arg Gin Ser Gly Arg Gin Pro Λ &quot; (please read the notes on the back # fill in this page) Employees of the Central Bureau of Standards of the Ministry of Economic Affairs have cooperated with Du to print this paper. "8 Standards (〇 阳) 肀 4 Specifications (210 father 297 public;« :) 81. 夂 10,000 sheets (H) V. Description of invention () SEQ ID NO: 21 SEQ category: nucleotide sequence length: 63bP strand strand: single strand morphology: linear molecule category: genomic DNA source: HBV SI ay direct experiment source: chemical synthesis Λ 6η 6 (please (First read the back-end notes # 塡 write this page)

CCT GCC ACC AA? T &quot; * ^ CAG TCA GGA AC-C- CAG CCT ACT CCC ATC TCT1 CCA CCT CTA C-AC-X Pack-Printed by the Ministry of Economic Affairs, Bureau of Standards, Beigong Consumer Cooperative Cooperative, paper size China S Home Standards (CNS) T4 specification (210X297 g *) 81. 4.! 0,000 sheets (H) 209 ^ 43 Printed by the Ministry of Economic Affairs Central Bureau of Precision Industry Beigong Consumer Cooperative V. Description of invention () SEQ ID NO: SEQ Category: Sequence Length: Molecular Weight: Morphology: Molecular Category: Original Source: Direct Experimental Source: 22 nucleotides and corresponding protein 108 bp single strand linear genomic DNA HBV SI V ^ · Chemical Synthesis (please read WI first Matters needing attention # fill in this page)

cc. \ CAC C * GT CAG Guang ATA TTG CCT TCC ACC

ATT C-GG TGG ACC ACA GTC- TCA AAT r * Lurvj CAG TCA

Pro His Gly Gly Ala Gin Gly lie Pro Pro Pro Ala Arg Gin Pro

He Leu Gly Trp Leu Thr Thr Val Ser Thr Asn Arg AGC CAG GCT ACA ATT production V »· CCT GGA • \ r-r * Λ \ Σ \ 3 CAG r Ser Pro Gin Ser Thr lie Gin Ser Gly

Installation · Line · This paper standard is used in "« Home Standard ^ "&lt;? 4 specifications (2 丨 0 father 29 '/ male;« :) 81. 4. 10,000 »(II) Λ fi η 6 V. Description of the invention () SEQ ID NO: SEQ category: Sequence length: Chain strand: Morphology: Molecular category: Original source: Direct experimental source

CAG TGG AAT

23 nucleotides 60bp single strand, linear genomic DNA HBV SI ay chemical synthesis TCC AGA ACC TT.C CAC CAA ACT CTG (please read back and note beforehand #pot write this page) Installation · Central Ministry of Economic Affairs Cooperate with Duzheng for printing

CAA GAT CCC AGA GTG AGA GGC C ^ G. *** \ TAT-X SEQ ID NO: 24 SEQ category: nucleotide sequence length: 60bp bond strand: single strand morphology: linear molecule category: genomic DNA original source : HBV SI ay Direct experiment source: Chemical synthesis GAT CCC AGA GTG GGT GGC TCC AGT AGA GGC CTG TAT TTC CCT TCA GGA ACA GTA AAC-X-ray. This paper is used in the "H home standard (CNS) T4 specification (210X297 Public; «:) 81. 4. 10,000 (11) 2〇924〇Λ 6 Η 6 V. Description of the invention () SEQ ID NO: 25 SEQ category: nucleotide sequence length: 558bp strand strand: single strand form ： Linear molecular category: Gene DNA Original source: HBV core adw Direct source of verification: PCR-Amplification (please read the back and the precautions # fill out this page) Printed by Beigongxiaot Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs

ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT: GAG CAT TGC CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATCGG CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG. GAA TCT CAA TGT TAG installation and line. This paper is used in the "national" «Standard (CNS) T4 specifications (210X297; Jt) 81. 4. 10,000 sheets (Η) 〇〇924〇Λ 6 __Π6 V. Description of the invention () SEQ ID NO: 26 SEQ category: nucleotide sequence length: 678bp Key strands: single strand : Linear

Molecule category: Gene Molecule DNA Original source: HBV S adw / ayw

Direct experiment source: HBV DNA (please read the back-to-back notes # fill in this page) Employee Consumers 1V-Co-op Print

ΑΤΑ GAG AAC ATC ACA TCA GGA TTC CTA GGA CCC CTT CTC GTG TTA CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC ACA ATA CCG CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC ΑΑΤ TTT CTA GGG GGA ACT ACC GT CAA AAT TCG CAG tCc TCA ACC TCC AAT CAC TCA CCA ACC TCT TGT CCT CCA ACT TGT CCT GGT TAT CGC TGG ATG TGT CTG CGG CGT TTT ATC ATC TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TTG TTG GTT CTT CTG CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA TCC TCA ACA ACC AGC ACG GGA CCA TGC CGG ACC TGC ATG ACT ACT GCT CAA GGA ACC TCT ATG TAT CCC TCC TGT TGC TGT ACC AAA CCT TCG GAC • GGA AAT TGC ACC ATT CCC ATC CCA TCA TCC TGG GCT TTC GGA AAA TTC CTA TGG GAG TGG GCC TCA GCC CGT TTC TCC TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GGG CTT TCC CCC ACT GTT TGG CTT TCA GTT TATA TGG ATG ATGTG TAT TGG * GGG CCA AGT CTG TAC AGC ATC TTG AGT CCC TTT TTA CCG CTG TTA CCA ATT TTC TTT TGT CTT TGG GTA TAC ATT Binding · Ordering · j · Line · This paper scale is used in the "B home" standard (CNS ) T4 regulation Grid (210X297; ¢) 81. 4. 10,000 sheets (H)-ο, '^ 920 66 ΛΠ V. Description of invention ()

SEQ ID NO: 27 SEQ category: nucleotide sequence length: 585bp strand strand: single strand morphology: linear molecule category: gene pus DNA original source: HBV S adw / ayw direct experiment source: HBV DNA (please read the back first Zhi Note #fill this page) Central Ministry of Economic Affairs / ¾ Employee Consumption Cooperation Du Print

CTA C-AC TCG TC-G TGG ACT TCT CTC AAT pt &lt; pn m • Up CTA GGG GGA TCT CCC GTG m / * ·· 71 CTT Guang CAA ΑΛΤ TCG CAG TCC CCA ACC TCC ΛΑΤ CAC TCA CCA ACC TCC TGT Guang ww — 〇v—Λ ATT? GT CCT production 1 TAT cc-c m ^ i. \ Z \ 2 ATG m ^ CTG CGG ΤΤΊ * ATC ATA TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TTA TTG GTT CTT CTG GAT TAT CAA GGT ATG TTG CCC .GTT TGT CCT CTA ATT CCA GGA TCA ACA ACA ACC AGT ACG GGA CCA TGC AAA ACC TGC ACG AC? CCT GCT CAA C-GC AAC TCT ATG TTT CCC TCA TGT TGC TGT ACA AAA CCT ACG GAT GGA AAT TGC ACC TGT ATT CCC ATC CCA TCG TCC TGG GCT TTC GCA AAA TAC CTA TGG GAG TGG GCC TCA GTC CGT TTC TCT TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GGG CTT TCC ccc ACT GTT TGG CTT ATA TGG ATG ATG TGG TAT TGG GGG CCA AGT CTG TAC AGC ATC GTG AGT CCC TTT ATA CCG CTG TTA CCA ATT 'TTC TTT TGT CTC TGG GTA TAG ATT, &gt; Pack. This paper scale is used in a B home «Bi ( CHS) Ή specifications (210X297 g *) 81. 4. 10,000 sheets (Η) 〇〇9 ^ b Ministry of Economic Affairs Central Standards Bureau KX Engineering Printed by Fei Cooperative Society 5. Description of the invention () SEQ ID NO: SEQ category: Sequence length: Chain strand: Morphology: Molecular category: Original source: Direct experiment source 28 nucleotides 106bp Single strand linear gene mule DNA HBV S1 Chemical synthesis 3 · -GAT-CTT-TAA-CAT-GGA-GAA-CAA-TCC-TCT-G CG-A77-C7T-TCC-CGA-TCA-CCA-3TT-GGA-TCC-A GC-CTT-CAG-AGC -AAA-CAC-CSC-AAA-TCC-AGA-T TS-GGA-CTT-CAA-TCC-CAG- (7) -3 'SEQ ID NO: SEQ category: Sequence length: Chain strand: Morphology: Molecular category : Original source: Direct experiment source: 29 nucleotide 115bp single stranded linear gene mule DNA HBV S Chemical synthesis 5'-AAT-TCT-A6A-CTC-GA6-TCT-EAA-CA7-AGA-G AA-CAT-CAC —AT C-AGG-ATT —CCT-AGG—ACC—CCT —T CT-CGT—GTT—ACA ~ GGC ~ 5 * 3G-GTT—TTT—CTT-GTTt ^ AC-AAG-AAT-CCT-CAC-AATthCC -GCA-GAG- (OVV · This paper) Standard "National Sample Standard (CNS) T4 Specification (210X297):« :) 81. 4, 10,000 sheets (II) (Please read the precautions before filling in (This page) V. Description of the invention (SEQ ID NO: SEQ category: Sequence length: Chain strands: Form: points Subcategory: Original source: 30 nucleotides 108 bp single-stranded linear gene DNA HBV core Λ 6 B 6 Direct experimental source: chemical synthesis 3, a GAT-CTT-TTA-AAG-SGA ^ T 匚 C-TCT- ( 2CT- .: 5SS-.3 gG-ί 二 .A Ding Yi 5SA_TGA-C: TC_ Ding AG-CTA-CCT-r.SG-TGG-G CA_A.TA_ATT_TGG—AAG-ATC_C: AG_CAT_C: TA_33 {B-AC匚 一 TTG—TAS-TAA—ATC-TAG—AC-: A) -3, 〆〆… (please read the back-up notes first # fill in this page) SEQ ID NO is printed by the employee consumer cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs : SEQ category: Sequence length: Stranding: Morphology: Molecular category: Original source: Direct experimental source

31 nucleotide 106bp single stranded linear genomic DNA HBV core chemical synthesis 5-Ding &quot; &quot; C &quot; TC_C36-Ding TC-GT (3-{3GG-CAT_, Z'GA-two AT-Ding GA- CC.C-TTA—Ding AA—AGA — ATT — TSG—AGC: — Ding AC — T m «· — — · · ·-* ~. Η — ^ I —HU. Ί 一 L ·-; —. · Γ 丨! One ◆ · Two Uu-I I. One I-U /; 2: This paper scale is easy to use and difficult to use 掳 楳 樳 (CNS) A 4 specifications (210X297 public; Ϊ) 81. 4.] 0,000 sheets⑻

It 6 V. Description of the invention () Co-printed by the Ministry of Economic Affairs, Central Bureau of Standards, Migong Consumers SEQ ID NO: 32 SEQ Category: Nucleotide Sequence Length: 89bp Strand Strand: Single Strand Form: Linear Molecule Class: Gene Mole Original DNA source: HBV core direct experimental source: Chemical synthesis 5 '—SAT-C D C-CTA—GAC-ACC-GCC—TCA—SCT-GTS—T AT-C5A-GAA-, 3CC—Ding A_GAG—D ! 1 了 _ * 二 [: 丁 一 3AG-CA 丁 一 T _5C ^ TCA-C 匚 一 CAC- 匚 AT-ACT-0CA- · 二 二 TC-AGS-CAA—3-(G) -3 * SEQ ID N0: 33.) SEQ category: nucleotide sequence length: 25bp strand strand: single strand morphology: linear molecule category: gene mule DNA original source: HBV core direct experimental source: chemical synthesis j'-TT-3—SAT -CCT-CCA-ACC-TST—Ξ · 2Z-Τ ~ ·-This paper size is used in the Bffl family standard (CNS) T4 specification (210X297; ¢) 81. 4. 10,000¾ (II) (Please read first Contrary note #fill in this page) 5. Description of the invention () SEQ ID NO: SE (i category: sequence length: bond strand: shape: molecular category: original source: straight Access to the experiment source: 34 nucleotides 25bp single strand linear genomic DNA HBV core chemical synthesis (please read the precautions before filling this page) Pack · Order _ Line-Central Ministry of Economic Affairs Bureau of Industrial Engineering Cooperative The size of the paper used for printing is easy to use. The Chinese ďzhun (CNS) «P 4 specifications (210X297 ;; :) 81. 4. 10,000 sheets (| .i) η 6 V. Description of invention () SEQ ID NO: 35 SEQ category: Nucleotide sequence length: 588bP Leng strand: single strand form: linear molecular category: gene sickle DNA original source: HBV core direct experiment source: PCR-Mining University (please read the back to the precautions # fill in this Page) Printed by the β-Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG ATA GCC TTA GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATC TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA TCT CAA TGT TAG Used in the national standard (CNS) 〒4 specifications (2 丨 0X297 male:-70-81. 4. 10,000 »(Η)

Claims (1)

A7 B7 C7 D7 : Seven〆Amendment date: April 4, 82, patent application No. 8 1 102989. 1. A pharmaceutical composition for viral hepatitis 4 which includes a combination of the following components: (a) At least one polypeptide sequence that has an amino acid sequence consisting of one or more members selected from the group consisting of: pre SI, pre S2 and hepatitis B (HB) virus S wins It and HB core antigens, the (among) multiple sequences are selectively modified in the following manner: (i) arbitrarily deleted (amino acid residue 1), as long as it can be preserved containing at least 6 Simply construct the epitope of the amino acid residue * (ii) replace one or several amino acids • or (iii) add an additional amino acid sequence to the (among) polypeptide sequence ( a) the N-terminus or C- 绱, or insert it into the (equal) polypeptide sequence (a); the (equal) polypeptide sequence (a) has one or more tilted activated T-cells Antigenic epitope; and (b) —A carrier that can display the epitope sequence U) in the (etc.) * Where the (multiple) sequence U) is shared by Bond or hydrophobic bond is bound to the support (b) on. 2. A pharmaceutical composition as claimed in items 1, 2, 3 or 4 of the patent scope, characterized in that the (among others) sequence (U) is spatified by a mystic crocodile. 3. The pharmaceutical composition as claimed in item 1 of the patent scope, the special feature is that the carrier (b) is all a polysaccharide · a hydrophobic polymer or a granular body with the size of the snail in accordance with the Chinese National Standard (CNS ) A 4 specifications (210 X 297 mm) _ 1 I II ------------- ^ ------ installed ------. 玎 ----- A (please Read the precautions on the back first and then this page) Printed 1Ϊ A7 B7 C7 D7____ in the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. 6. Inorganic molecules applying for patent paradigm. 4. The pharmaceutical composition as claimed in item 3 of the patent application scope is characterized in that the carrier (b) is a second polypeptide sequence. 5. The pharmaceutical composition as claimed in item 4 of the patent application, the particularity is that the polypeptide sequence (b) forms a particle with a diameter of at least 10 nra during secretion. 6. The pharmaceutical composition as claimed in item 4 or 5 of the patent application, whose contribution lies in that the polycyclic sequence (b) is a substantial part or complete amino acid of one of the polycyclic groups selected from the following group Sequence: HBV S-A, HBV core antigen, HAV core antigen, H IV core antigen, and surface antigen of poliovirus, H AV or H IV. 7. If the pharmaceutical composition of the 4th or 5th patent application scope, its special advice is that the multiple sequence (b) can be modified in the following ways: (i) arbitrarily deleted (as long as the amino acid residue can be preserved The ability to form particles is acceptable, (H) one or more amino acids are replaced, or (iii) an additional amino acid sequence is added to the N-pin or C-pin of the polypeptide sequence (b) * Or insert it within the multi-sequence sequence (&gt;). S. As for the metaphysical composition of item 4 or 5 of the patent application, its special emblem is that the multiple sequence (b) is hydroformylated by myristic acid. * ~ &Quot; Printed by the Rongong Consumer Cooperative of the Central Equatorial Bureau of the Ministry of Eel and Economy 9. The pharmaceutical composition as claimed in item 1 or 4 of the patent application scope, whose joy lies in the polypeptide sequence (a) and the polypeptide (B) It is pseudo-connected via a disulfide bridge. 10. Such as the pharmaceutical composition of the first or fourth patent scope of the patent application, the special feature lies in the (etc.) polypeptide sequence U) and the poly (b) customary via `` hydrophobic solid grate 2 (please read the back Please pay attention to this page and then fill in this page.) Species of the Caishui Sample Standard (CNS) A4 (210 X 297 mm) A7 209 ^ 4o b7 C7 ____D7___ 6. Application for a patent for aphooric acid (hydrophobic aichoring). Facilitate) to be connected. 1 1 · The pharmaceutical composition as claimed in item 1 or 4 of the patent scope, the special feature is that the (etc.) polypeptide sequence (a) and the poly (b) are connected via a bond, of which one The spacer sequence is inserted between the (equal) polypeptide sequence (a) and the multiple sequence (b). The spacer sequence is connected to the (equal) multiple sequence (a) and the multiple sequence via a bond Sequence (b). 1 2. The pharmaceutical composition as claimed in item 1 of the patent scope &gt; wherein the composition is intended for administration by intravenous or intramuscular injection. (Please read the precautions on the back before writing this page) Binding and ordering. 丨 ί Printed by the Central Standards Bureau of the Ministry of Economic Affairs of the People ’s Republic of China 8. Printed by the Industrial and Commercial Cooperatives ¾ 3 The paper size is based on China® Home Standards (CNS) A 4 specifications (210 X 297 mm)