WO2001055165A1

WO2001055165A1 - A novel peptide---dna polymerase iii 12, polynucleotide coding this peptide and the method producing this peptide by dna recombination techniques

Info

Publication number: WO2001055165A1
Application number: PCT/CN2001/000083
Authority: WO
Inventors: Yumin Mao; Yi Xie
Original assignee: Biodoor Gene Technology Ltd. Shanghai
Priority date: 2000-01-28
Filing date: 2001-01-21
Publication date: 2001-08-02
Also published as: CN1319655A; AU3149601A

Abstract

The invention discloses a novel peptide---DNA polymerase III 12, polynucleotide coding this peptide and the method producing this peptide by DNA recombination techniques. This invention also discloses the therapeutical methods for several diseases, such as malignant tumor, haemal illness, HIV infection, immunological illness and all kinds of inflammations. This invention also discloses an antagonist against this peptide and its therapeutical effect. The invention also discloses the use of this polynucleotide coding the novel DNA polymerase III 12.

Description

一种新的多肽一一 DNA聚合酶 ΙΠ12和编码这种多肽的多核苷酸技术领域 A new polypeptide-a DNA polymerase IIII12 and a polynucleotide encoding the polypeptide TECHNICAL FIELD

本发明属于生物技术领域，具体地说，本发明描述了一种新的多肽一一 DNA 聚合酶 ΠΙ12, 以及编码此多肽的多核苷酸序列。本发明还涉及此多核苷酸和多肽的制备方法和应用。背景技术 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a DNA polymerase II12, and a polynucleotide sequence encoding the polypeptide. The invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique

大肠杆菌 DM聚合酶 III (PolIII)全酶（HE)负责细菌染色体的高效而准确的复制 ( Kelman, Z. , and M. O'Donnel.1995. DNA polymeraselll holoenzyme: structure and function of a chromosomal replicating machine. Annu. Rev. Biochem. 64: 171-200) ₀ 该酶是一个多亚基酶，其核心酶同时具有聚合与校对功能。与其它校对聚合酶不同的是，该酶的两个功能是分别由不同的两个亚基完成的。其中 α 亚基具有聚合功能（dnaE 基因产物）， 3'→5，核酸外切酶活性即复制校对功能则由 ε亚基完成（dnaQ基因产物），全酶的三个亚基按照线形的方式排列， α- ε_θ , ε 同时结合 α 和 Θ (Studwel 1-Vaughan, P. S. , and M. O'Donnel 1.1993, DNA polymerase III accessory prote ins. V. Θ encoded by holE. J. Biol. Chem.268: 11785-11791) ₀ 全酶核心亚基之间以及核心亚基与 HE 附属因子之间在结构和功能上的精确反应一直是近期活跃的研究课题 (Bertram, J. G. , L. Β. Β loom, J. Turner, M. O'Donnel 1, J. M. Beechem, and M. F. Goodman.1998. Pre— steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia col i DNA pol ymerase III onto DNA. J. Biol. Chera.273: 24564-24574)。 E. coli DM polymerase III (PolIII) holoenzyme (HE) is responsible for efficient and accurate replication of bacterial chromosomes (Kelman, Z., and M. O'Donnel. 1995. DNA polymeraselll holoenzyme: structure and function of a chromosomal replicating machine Annu. Rev. Biochem. 64: 171-200) ₀ This enzyme is a multi-subunit enzyme whose core enzyme has both polymerization and proofreading functions. Unlike other proofreading polymerases, the two functions of the enzyme are performed by two different subunits. The α subunit has the aggregation function (dnaE gene product), 3 '→ 5. The exonuclease activity, ie, the proofreading function, is performed by the ε subunit (dnaQ gene product). The three subunits of the whole enzyme follow a linear pattern. Permutations, α- ε_θ, ε combine both α and Θ (Studwel 1-Vaughan, PS, and M. O'Donnel 1.1993, DNA polymerase III accessory prote ins. V. Θ encoded by holE. J. Biol. Chem.268: 11785-11791) ₀ Accurate structural and functional responses between core subunits of whole enzymes and between core subunits and HE accessory factors have been recently active research topics (Bertram, JG, L. Β. Β loom, J Turner, M. O'Donnel 1, JM Beechem, and MF Goodman. 1998. Pre—steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia col i DNA pol ymerase III onto DNA. J. Biol. Chera .273: 24564-24574).

通过对大量对 ε亚基基因 dnaQ 突变体的基因研究发现， DnaQ酶的催化活性离不开三个重要的保守核酸外切酶基元序列，同时 ε亚基的 C 末端可能是它与 α亚基反应所必须的。 ε亚基由两个不同的结构域组成，一个是 Ν 末端包含核酸外切酶的酶切位点（也包括 Θ-结合位点），另一个是 C末端这个必须的结构域。 C末端结构域包括 A， B， C三个基元序列。大约有 280个氨基酸左右。在 T7 DM聚合酶和人类 DNA 聚合酶 γ基元序列 B 中，一个酪氨酸具有双脱氧核甘酸敏感性 (Long ley, M. J.， Ropp, P. A. , Lim, S. E. , and Cope land, W. C.1998. Characterization of the native and recomb inant catalytic subunit of human DNA polymerase gamma: I dentif ication of residues critical for exonuc lease activity and. dideoxnucleot ide sens i t ivi ty. Biochemi s try 37: 10529-10539)。 Through a large number of genetic studies of dnaQ mutants of the ε subunit gene, it was found that the catalytic activity of DnaQ enzyme is inseparable from three important conserved exonuclease motifs, and the C-terminus of the ε subunit may be its Required for radical reactions. The ε subunit is composed of two different domains. One is the N-terminus containing an exonuclease cleavage site (also includes the Θ-binding site), and the other is the C-terminus required domain. The C-terminal domain includes three motif sequences of A, B, and C. There are about 280 amino acids. In T7 DM polymerase and human DNA polymerase γ motif sequence B, a tyrosine has dideoxynucleotide sensitivity (Long ley, MJ, Ropp, PA, Lim, SE, and Cope land, WC1998. Characterization of the native and recomb inant catalytic subunit of human DNA polymerase gamma: I dentif ication of residues critical for exonuc lease activity and. dideoxnucleot ide sens it ivi ty. Biochemi s try 37: 10529-10539).

尽管现在人们对 DM 聚合酶的 C 末端结构和功能还不是非常清楚，但是可以肯定的是 DNA聚合酶的 C末端存在着与其它亚基的结合位点，是 DNA聚合酶保持全酶结构所必不可少的多肽片段。如果缺失该片段的话，必然导致 DNA 聚合酶功能部分或完全的丧失。 Although the structure and function of the C-terminus of DM polymerase are still not very clear, it is certain that the C-terminus of DNA polymerase has binding sites with other subunits, which is necessary for DNA polymerase to maintain the entire enzyme structure. Essential peptide fragments. If this fragment is deleted, it will inevitably lead to partial or complete loss of DNA polymerase function.

DNA聚合酶 C末端的表达异常将会导致但不限于以下疾病症状：血细胞的逐步丧失，骨骼***紊乱，生长发育障碍，皮肤色素沉着过度以及素因性癌症。 Abnormal expression of the DNA polymerase C-terminus will cause but is not limited to the following disease symptoms: gradual loss of blood cells, skeletal system disorders, growth and development disorders, hyperpigmentation of the skin, and primed cancer.

本发明人的多肽具有 DNA 聚合酶 C 末端的结构特征，属于该家族，同时推测其具有相似的生物学功能。 The polypeptides of the present inventors have the structural characteristics of the C-terminus of the DNA polymerase and belong to this family, and it is speculated that they have similar biological functions.

由于如上所述 DNA 聚合酶 ΙΠ12 蛋白在机体内重要功能中起重要作用，而且相信这些调节过程中涉及大量的蛋白，因而本领域中一直需要鉴定更多参与这些过程的 DNA聚合酶 III 12蛋白，特别是鉴定这种蛋白的氨基酸序列。新 DNA聚合酶 III 12 蛋白编码基因的分离也为研究确定该蛋白在健康和疾病状态下的作用提供了基础。这种蛋白可能构成开发疾病诊断和 /或治疗药的基础，因此分离其编码 DM是非常重要的。发明的公开 Because the DNA polymerase IIIII protein plays an important role in important functions in the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more DNA polymerase III 12 proteins involved in these processes. In particular, the amino acid sequence of this protein is identified. The isolation of the new DNA polymerase III 12 protein-coding gene also provides the basis for research to determine the role of the protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM. Disclosure of invention

本发明的一个目的是提供分离的新的多肽一一 DNA 聚合酶 ΠΙ 12 以及其片段、 An object of the present invention is to provide an isolated novel polypeptide-DNA polymerase II 12 and fragments thereof,

' 、本发明的另一个目的是提供编码该多肽的多核苷酸。 'Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

本发明的另一个目的是提供含有编码 DNA 聚合酶 ΙΠ 12 的多核苷酸的重组载体。 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a DNA polymerase IIIII12.

本发明的另一个目的是提供含有编码 DNA聚合酶 ΙΠ12 的多核苷酸的基因工程化宿主细胞。 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a DNA polymerase IIIII12.

本发明的另一个目的是提供生产 DNA聚合酶 ΙΠ12的方法。 Another object of the present invention is to provide a method for producing a DNA polymerase IIII12.

本发明的另一个目的是提供针对本发明的多肽一一 DM聚合酶 ΠΙ12的抗体。本发明的另一个目的是提供了针对本发明多肽 __ DNA 聚合酶 ΠΙ 12 的模拟化合物、拮抗剂、激动剂、抑制剂。 Another object of the present invention is to provide an antibody against the polypeptide of the present invention-DM polymerase II12. Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide __ DNA polymerase II 12 of the present invention.

本发明的另一个目的是提供诊断治疗与 DNA 聚合酶 ΙΠ12 异常相关的疾病的方法。 Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of DNA polymerase IIIII12.

本发明涉及一种分离的多肽，该多肽是人源的，它包含：具有 SEQ ID No. 2 氨基酸序列的多肽、或其保守性变体、生物活性片段或衍生物。较佳地，该多肽是具有 SEQ ID NO: 2氨基酸序列的多肽。 The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide Is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

本发明还涉及一种分离的多核苷酸，它包含选自下组的一种核苷酸序列或其变体： The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

(a)编码具有 SEQ ID No. 2氨基酸序列的多肽的多核苷酸； (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

(b)与多核苷酸（a)互补的多核苷酸； (b) a polynucleotide complementary to polynucleotide (a);

(c)与（a)或（b)的多核苷酸序列具有至少 70%相同性的多核苷酸。 (c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b).

更佳地，该多核苷酸的序列是选自下组的一种：（a)具有 SEQ ID NO: 1 中 324- 656位的序列；和（b)具有 SEQ ID NO: 1中 1-1092位的序列。 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 324-656 in SEQ ID NO: 1; and (b) a sequence having 1-1092 in SEQ ID NO: 1 Sequence of bits.

本发明另外涉及一种含有本发明多核苷酸的载体，特别是表达载体；一种用该载体遗传工程化的宿主细胞，包括转化、转导或转染的宿主细胞；一种包括培养所述宿主细胞和回收表达产物的制备本发明多肽的方法。 The invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

本发明还涉及一种能与本发明多肽特异性结合的抗体。 The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.

本发明还涉及一种筛选的模拟、激活、拮抗或抑制 DNA聚合酶 III 12蛋白活性的化合物的方法，其包括利用本发明的多肽。本发明还涉及用该方法获得的化合物。 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the DNA polymerase III 12 protein, which comprises using the polypeptide of the invention. The invention also relates to compounds obtained by this method.

本发明还涉及一种体外检测与 DM 聚合酶 ΠΙ 12 蛋白异常表达相关的疾病或疾病易感性的方法，包括检测生物样品中所述多肽或其编码多核苷酸序列中的突变，或者检测生物样品中本发明多肽的量或生物活性。 The present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of DM polymerase III 12 protein in vitro, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a biological sample. The amount or biological activity of a polypeptide of the invention.

本发明也涉及一种药物组合物，它含有本发明多肽或其模拟物、激活剂、拮抗剂或抑制剂以及药学上可接受的载体。 The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.

本发明还涉及本发明的多肽和 /或多核苷酸在制备用于治疗癌症、发育性疾病或免疫性疾病或其它由于 DNA聚合酶 III 12表达异常所引起疾病的药物的用途。 The present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of DNA polymerase III 12.

本发明的其它方面由于本文的技术的公开，对本领域的技术人员而言是显而易见的。本说明书和权利要求书中使用的下列术语除非特别说明具有如下的含义： "核酸序列" 是指寡核苷酸、核苷酸或多核苷酸及其片段或部分，也可以指基因组或合成的 DM或 RNA, 它们可以是单链或双链的，代表有义链或反义链。类似地, 术语 "氨基酸序列" 是指寡肽、肽、多肽或蛋白质序列及其片段或部分。当本发明中的 "氨基酸序列" 涉及一种天然存在的蛋白质分子的氨基酸序列时，这种 "多肽" 或 "蛋白质" 不意味着将氨基酸序列限制为与所述蛋白质分子相关的完整的天然氨基酸。 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. The following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DM or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

蛋白质或多核苷酸 "变体" 是指一种具有一个或多个氨基酸或核苷酸改变的氨基酸序列或编码它的多核苷酸序列。所述改变可包括氨基酸序列或核苷酸序列中氨基酸或核苷酸的缺失、 ***或替换。变体可具有 "保守性" 改变，其中替换的氨基酸具有与原氨基酸相类似的结构或化学性质，如用亮氨酸替换异亮氨酸。变体也可具有非保守性改变，如用色氨酸替换甘氨酸。 A "variant" of a protein or polynucleotide is one that has one or more amino acid or nucleotide changes Amino acid sequence or polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants may have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

"缺失" 是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的缺失。 "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

"***" 或 "添加" 是指在氨基酸序列或核苷酸序列中的改变导致与天然存在的分子相比，一个或多个氨基酸或核苷酸的增加。 "替换" 是指由不同的氨基酸或核苷酸替换一个或多个氨基酸或核苷酸。 "Insertion" or "addition" means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

"生物活性" 是指具有天然分子的结构、调控或生物化学功能的蛋白质。类似地，术语 "免疫学活性" 是指天然的、重组的或合成蛋白质及其片段在合适的动物或细胞中诱导特定免疫反应以及与特异性抗体结合的能力。 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.

"激动剂" 是指当与 DM 聚合酶 ΙΠ12 结合时，一种可引起该蛋白质改变从而调节该蛋白质活性的分子。激动剂可以包括蛋白质、核酸、碳水化合物或任何其它可结合 DM聚合酶 III 12的分子。 An "agonist" refers to a molecule that, when combined with DM polymerase IIII, can cause the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind DM polymerase III 12.

"拮抗剂"或 "抑制物"是指当与 DNA聚合酶 III12结合时，一种可封闭或调节 DNA 聚合酶 ΙΠ 12的生物学活性或免疫学活性的分子。拮抗剂和抑制物可以包括蛋白质、核酸、碳水化合物或任何其它可结合 DM聚合酶 ΙΠ12的分子。 An "antagonist" or "inhibitor" refers to a molecule that, when combined with DNA polymerase III12, can block or regulate the biological or immunological activity of DNA polymerase IIIII12. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind DM polymerase III.

"调节" 是指 DM聚合酶 ΙΠ12的功能发生改变，包括蛋白质活性的升高或降低、结合特性的改变及 DM聚合酶 III 12的任何其它生物学性质、功能或免疫性质的改变。 "Regulation" refers to a change in the function of DM polymerase IIII12, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of DM polymerase III12.

"基本上纯' '是指基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化 DNA聚合酶 11112。基本上纯的 DNA 聚合酶 ΙΠ 12 在非还原性聚丙烯酰胺凝胶上能产生单一的主带。 DM 聚合酶 ΠΙ 12 多肽的纯度可用氨基酸序列分析。 "Substantially pure '" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify DNA polymerase 11112 using standard protein purification techniques. Essentially pure DNA polymerase II 12 can produce a single main band on a non-reducing polyacrylamide gel. The purity of DM polymerase II 12 polypeptide can be analyzed by amino acid sequence.

"互补的" 或 "互补" 是指在允许的盐浓度和温度条件下通过碱基配对的多核苷酸天然结合。例如，序列 "C- T- G- A" 可与互补的序列 "G- A- C- T" 结合。两个单链分子之间的互补可以是部分的或全部的。核酸链之间的互补程度对于核酸链之间杂交的效率及强度有明显影响。 "Complementary" or "complementary" refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

"同源性" 是指互补的程度，可以是部分同源或完全同源。 "部分同源" 是指一种部分互补的序列，其至少可部分抑制完全互补的序列与靶核酸的杂交。这种杂交的抑制可通过在严格性程度降低的条件下进行杂交（Southern印迹或 Northern 印迹等）来检测。基本上同源的序列或杂交探针可竟争和抑制完全同源的序列与靶序列在的严格性程度降低的条件下的结合。这并不意味严格性程度降低的条件允许非特异性结合，因为严格性程度降低的条件要求两条序列相互的结合为特异性或选择性相互作用。 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be achieved by hybridization under conditions of reduced stringency (Southern blotting or Northern Blot, etc.) to detect. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.

"相同性百分率" 是指在两种或多种氨基酸或核酸序列比较中序列相同或相似的百分率。可用电子方法测定相同性百分率，如通过 MEGALIGN程序（Lasergene sof tware package, DNASTAR, Inc. , Madi son Wi s. )。 MEGALIGN程序可根据不同的方法如 Clus ter法比较两种或多种序列（Higg ins, D. G. 和 P. M. Sharp (1988) Gene 73: 237-244)。 Clus ter法通过检查所有配对之间的距离将各组序列排列成簇。然后将各簇以成对或成组分配。两个氨基酸序列如序列 A和序列 B之间的相同性百分率通过下式计算： "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:

序列 A与序列 B之间匹配的残基个数 X 100 Number of matching residues between sequence A and sequence X 100

(序列 A的残基数一序列 A中间隔残基数一序列 B中间隔残基数）也可以通过 Clus ter法或用本领域周知的方法如 Jotun Hein 测定核酸序列之间的相同性百分率（Hein J. , (1990) Methods in emzumology 183: 625-645)。 (The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B) The percent identity between nucleic acid sequences can also be determined by the Clus ter method or by a method known in the art such as Jotun Hein ( Hein J., (1990) Methods in emzumology 183: 625-645).

"相似性 " 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或保守性取代的程度。用于保守性取代的氨基酸例如，带负电荷的氨基酸可包括天冬氨酸和谷氨酸；带正电荷的氨基酸可包括赖氨酸和精氨酸；具有不带电荷的头部基团有相似亲水性的氨基酸可包括亮氨酸、异亮氨酸和缬氨酸；甘氨酸和丙氨酸；天冬酰胺和谷氨酰胺；丝氨酸和苏氨酸；苯丙氨酸和酪氨酸。 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

"反义" 是指与特定的 DNA或 RNA序列互补的核苷酸序列。 "反义链"是指与 "有义链" 互补的核酸链。 "Antisense" refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence. The "antisense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

"衍生物" 是指 HFP或编码其的核酸的化学修饰物。这种化学修饰物可以是用垸基、酰基或氨基替换氢原子。核酸衍生物可编码保留天然分子的主要生物学特性的多肽。 "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.

"抗体" 是指完整的抗体分子及其片段，如 Fa、？(^') ₂及？^ 其能特异性结合 DNA聚合酶 ΙΠ 12的抗原决定簇。 "Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and? ^ It can specifically bind to the epitope of DNA polymerase IIII 12.

"人源化抗体" 是指非抗原结合区域的氨基酸序列被替换变得与人抗体更为相似，但仍保留原始结合活性的抗体。 A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

"分离的" 一词指将物质从它原来的环境（例如，若是自然产生的就指其天然环境）之中移出。比如说，一个自然产生的多核苷酸或多肽存在于活动物中就是没有被分离出来，但同样的多核苷酸或多肽同一些或全部在自然***中与之共存的物质分开就是分离的。这样的多核苷酸可能是某一载体的一部分，也可能这样的多核苷酸或多肽是某一组合物的一部分。既然载体或组合物不是它天然环境的成分，它们仍然是分离的。 The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally). For example, the presence of a naturally occurring polynucleotide or polypeptide in a living animal is It is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances with which it coexists in natural systems. Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

如本发明所用， "分离的" 是指物质从其原始环境中分离出来（如果是天然的物质，原始环境即是天然环境）。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。 As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .

如本文所用， "分离的 DNA 聚合酶 ΠΙ 12" 是指 DNA聚合酶 ΠΙ12 基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化 DNA 聚合酶 11112。基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。 DNA聚合酶 ΙΠ12多肽的纯度能用氨基酸序列分析。 As used herein, "isolated DNA polymerase II 12" means that DNA polymerase II 12 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify DNA polymerase 11112 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the DNA polymerase IIIII polypeptide can be analyzed by amino acid sequence.

本发明提供了一种新的多肽一一 DNA聚合酶 III 12 , 其基本上是由 SEQ ID NO: 2 所示的氨基酸序列组成的。本发明的多肽可以是重组多肽、天然多肽、合成多肽，优选重组多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主（例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞）中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。 The present invention provides a new polypeptide DNA polymerase III 12 which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

本发明还包括 DNA 聚合酶 ΙΠ12 的片段、衍生物和类似物。如本发明所用，术语 "片段"、 "衍生物" 和 "类似物" 是指基本上保持本发明的 DM 聚合酶 III 12 相同的生物学功能或活性的多肽。本发明多肽的片段、衍生物或类似物可以是：（I ) 这样一种，其中一个或多个氨基酸残基被保守或非保守氨基酸残基（优选的是保守氨基酸残基）取代，并且取代的氨基酸可以是也可以不是由遗传密码子编码的；或者（Π )这样一种，其中一个或多个氨基酸残基上的某个基团被其它基团取代包含取代基；或者（I I I )这样一种，其中成熟多肽与另一种化合物（比如延长多肽半衰期的化合物，例如聚乙二醇）融合；或者（IV )这样一种，其中附加的氨基酸序列融合进成熟多肽而形成的多肽序列（如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列）通过本文的阐述，这样的片段、衍生物和类似物被认为在本领域技术人员的知识范围之内。 The invention also includes fragments, derivatives and analogs of the DNA polymerase IIIII12. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the DM polymerase III 12 of the present invention. A fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (Π) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.

本发明提供了分离的核酸（多核苷酸），基本由编码具有 SEQ ID NO: 2 氨基酸序列的多肽的多核苷酸组成。本发明的多核苷酸序列包括 SEQ ID N0: 1 的核苷酸序列。本发明的多核苷酸是从人胎脑组织的 cDNA 文库中发现的。它包含的多核苷酸序列全长为 1092个碱基，其开放读框 324- 656编码了 110个氨基酸。此多肽具有 DNA聚合酶 C末端家族的特征序列，可推断出该 DNA聚合酶 ΙΠ 12具有 DNA聚合酶 C末端家族所代表的结构和功能。 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. Polynucleoside The full length of the acid sequence is 1092 bases, and its open reading frames 324-656 encode 110 amino acids. This polypeptide has the characteristic sequence of the C-terminal family of the DNA polymerase, and it can be deduced that the DNA polymerase III has the structure and function represented by the C-terminal family of the DNA polymerase.

本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 cDNA、基因组 DM或人工合成的 DM。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与 SEQ ID N0: 1 所示的编码区序列相同或者是简并的变异体。如本发明所用， "简并的变异体" 在本发明中是指编码具有 SEQ ID NO: 2的蛋白质或多肽，但与 SEQ ID NO: 1所示的编码区序列有差别的核酸序列。 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DM, or synthetic DM. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

编码 SEQ ID NO: 2 的成熟多肽的多核苷酸包括：只有成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列（和任选的附加编码序列）以及非编码序列。 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加编码和 /或非编码序列的多核苷酸。 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.

本发明还涉及上述描述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片断、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和***变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或***，但不会从实质上改变其编码的多肽的功能。 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

本发明还涉及与以上所描述的序列杂交的多核苷酸（两个序列之间具有至少 50%, 优选具有 7 W的相同性）。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中， "严格条件" 是指：（1)在较低离子强度和较高温度下的杂交和洗脱，如 0. 2xSSC, 0. 1%SDS, 60°C;或（2)杂交时加用变性剂，如 50% (v/v)甲酰胺， 0. 1%小牛血清 /0. l%Ficol l , 42 °C等；或（3)仅在两条序列之间的相同性至少在 95%以上，更好是 97%以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功能和活性。 The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50% identity between the two sequences, preferably 7 W identity). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

本发明还涉及与以上所描述的序列杂交的核酸片段。如本发明所用， "核酸片段"的长度至少含 10个核苷酸，较好是至少 20- 30个核苷酸，更好是至少 50- 60个核苷酸，最好是至少 100个核苷酸以上。核酸片段也可用于核酸的扩增技术（如 PCR) 以确定和 /或分离编码 DNA聚合酶口12的多核苷酸。 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding DNA polymerase port 12.

本发明中的多肽和多核苷酸优选以分离的形式提供，更佳地被纯化至均质。本发明的编码 DNA聚合酶 ΠΙ12 的特异的多核苷酸序列能用多种方法获得。例如，用本领域熟知的杂交技术分离多核苷酸。这些技术包括但不局限于： 1) 用探针与基因组或 cDNA 文库杂交以检出同源的多核苷酸序列，和 2)表达文库的抗体筛选以检出具有共同结构特征的克隆的多核苷酸片段。 The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the DNA polymerase III12 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

本发明的 DM片段序列也能用下列方法获得： 1)从基因组 DM分离双链 DNA 序列； 2)化学合成 DNA序列以获得所述多肽的双链 DM。 The DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from genomic DM; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DM of the polypeptide.

上述提到的方法中，分离基因组 DNA 最不常用。 DNA 序列的直接化学合成是经常选用的方法。更经常选用的方法是 cDNA序列的分离。分离感兴趣的 cDNA 的标准方法是从高表达该基因的供体细胞分离 mRNA并进行逆转录，形成质粒或噬菌体 cDNA 文库。提取 mRNA 的方法已有多种成熟的技术，试剂盒也可从商业途径获得（Qiagene)。而构建 cDNA 文库也是通常的方法（Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。还可得到商业供应的 cDNA文库，如 Clontech公司的不同 cDNA 文库。当结合使用聚合酶反应技术时，即使极少的表达产物也能克隆。 Of the methods mentioned above, genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene). The construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

可用常规方法从这些 cDNA 文库中筛选本发明的基因。这些方法包括（但不限于）：（l)DNA- DNA 或 DNA- RNA 杂交；（2)标志基因功能的出现或丧失；（3)测定 DNA 聚合酶 ΙΠ12 的转录本的水平；（4)通过免疫学技术或测定生物学活性，来检测基因表达的蛋白产物。上述方法可单用，也可多种方法联合应用。 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or loss of marker gene function; (3) determination of the level of the transcript of DNA polymerase III12; (4) by Immunological techniques or assays for biological activity to detect gene-expressed protein products. The above methods can be used singly or in combination.

在第（1)种方法中，杂交所用的探针是与本发明的多核苷酸的任何一部分同源，其长度至少 10个核苷酸，较好是至少 30个核苷酸，更好是至少 50个核苷酸，最好是至少 100个核苷酸。此外，探针的长度通常在 2000个核苷酸之内，较佳的为 1000个核苷酸之内。此处所用的探针通常是在本发明的基因序列信息的基础上化学合成的 DNA序列。本发明的基因本身或者片段当然可以用作探针。 DNA探针的标记可用放射性同位素，荧光素或酶（如碱性磷酸酶）等。 In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

在第（4)种方法中，检测 MA 聚合酶 ΠΙ12 基因表达的蛋白产物可用免疫学技术如 Western印迹法，放射免疫沉淀法，酶联免疫吸附法（ELISA)等。 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the MA polymerase II12 gene.

应用 PCR 技术扩增 DNA/RNA 的方法（Saiki, et al. Science 1985; 230: 1350 - 1354)被优选用于获得本发明的基因。特别是很难从文库中得到全长的 cDNA 时，可优选使用 RACE法（RACE - cDNA末端快速扩增法），用于 PCR 的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。 A method using PCR technology to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-Rapid Amplification of cDNA Ends) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

如上所述得到的本发明的基因，或者各种 DNA 片段等的多核苷酸序列可用常规方法如双脱氧链终止法（Sanger et al. PNAS , 1977 , 74： 5463- 5467)测定。这类多核苷酸序列测定也可用商业测序试剂盒等。为了获得全长的 cDNA序列，测序需反复进行。有时需要测定多个克隆的 cDNA 序列，才能拼接成全长的 cDNA 序列。 Polynucleotide sequences of the gene of the present invention obtained as described above, or various DNA fragments can be used It is determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或直接用 DNA聚合酶 III 12 编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a DNA polymerase III 12 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .

本发明中，编码 DM聚合酶 III 12 的多核苷酸序列可***到载体中，以构成含有本发明所述多核苷酸的重组载体。术语 "载体" 指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其它载体。在本发明中适用的载体包括但不限于：在细菌中表达的基于 T7启动子的表达载体（Rosenberg, et a l. Gene, 1987, 56: 125)；在哺乳动物细胞中表达的 pMSXND表达载体（Lee and Nathans, J Bio Chem. 263: 3521, 1988)和在昆虫细胞中表达的来源于杆状病毒的载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用于构建重组表达载体。表达载体的一个重要特征是通常含有复制起始点、启动子、标记基因和翻译调控元件。 In the present invention, a polynucleotide sequence encoding DM polymerase III 12 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

本领域的技术人员熟知的方法能用于构建含编码 D1 聚合酶 ΠΙ12的 DNA序列和合适的转录 /翻译调控元件的表达载体。这些方法包括体外重组 MA技术、 DNA 合成技术、体内重组技术等（Sambroook, et a l. Molecular Cloning, a Laboratory Manua l , co ld Spr ing Harbor Laboratory. New York, 1989)。所述的 DNA序列可有效连接到表达载体中的适当启动子上，以指导 mRNA合成。这些启动子的代表性例子有：大肠杆菌的 lac或 trp启动子； λ噬菌体的 PL启动子；真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启动子、早期和晚期 SV40 启动子、反转录病毒的 LTRs和其它一些巳知的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。在载体中***增强子序列将会使其在高等真核细胞中的转录得到增强。增强子是 DNA表达的顺式作用因子，通常大约有 10到 300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的 100 到 270个碱基对的 SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。 Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding D1 polymerase II12 and appropriate transcription / translation regulatory elements. These methods include in vitro recombination MA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, cold Harbor Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polytumor enhancers on the late side of the origin of replication, and adenoviral enhancers.

此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白（GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性等。本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件（如启动子、增强子等）和选择性标记基因。 In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture. And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli. Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

本发明中，编码 DNA聚合酶 ΙΠ 12 的多核苷酸或含有该多核苷酸的重组载体可转化或转导入宿主细胞，以构成含有该多核苷酸或重组载体的基因工程化宿主细胞。术语 "宿主细胞" 指原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；细菌细胞如鼠伤寒沙门氏菌；真菌细胞如酵母；植物细胞；昆虫细胞如果蝇 S2或 Sf9 ; 动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。 In the present invention, a polynucleotide encoding a DNA polymerase IIIII or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9; animal cells such as CH0, COS or Bowes melanoma cells.

用本发明所述的 DNA序列或含有所述 DNA序列的重组载体转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收 DNA 的感受态细胞可在指数生长期后收获，用 CaCl₂法处理，所用的步骤在本领域众所周知。可供选择的是用 MgCl₂。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的 DNA 转染方法：磷酸钙共沉淀法，或者常规机械方法如显微注射、电穿孔、脂质体包装等。 Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Alternatively, MgCl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

通过常规的重组 DNA 技术，利用本发明的多核苷酸序列可用来表达或生产重组的 DNA聚合酶 ΠΙ 12 (Science, 1984; 224： 1431)。一般来说有以下步骤： Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant DNA polymerase II 12 (Science, 1984; 224: 1431). Generally there are the following steps:

(1) .用本发明的编码人 DNA聚合酶 III 12的多核苷酸（或变异体），或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞； (1) using the polynucleotide (or variant) encoding human DNA polymerase III 12 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2) .在合适的培养基中培养宿主细胞； (2) culturing host cells in a suitable medium;

(3) .从培养基或细胞中分离、纯化蛋白质。 (3) Isolate and purify protein from culture medium or cells.

在步骤（ 2 ) 中，根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法（如温度转换或化学诱导）诱导选择的启动子，将细胞再培养一段时间。 In step (2), depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

在步骤（ 3 ) 中，重组多肽可包被于细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法包括但并不限于：常规的复性处理、蛋白沉淀剂处理（盐析方法）、离心、渗透破菌、超声波处理、超离心、分子筛层析（凝胶过滤) '、吸附层析、离子交换层析、高效液相层析（HPLC)和其它各种液相层析技术及这些方法的结合。附图的简要说明 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration) ', adsorption chromatography, Ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods. Brief description of the drawings

下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。 The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.

图 1是本发明 DNA聚合酶 ΙΠ 12和 DNA聚合酶 C末端家族功能结构域的氨基酸序列比较图。 Fig. 1 is a comparison diagram of the amino acid sequences of the functional domains of the DNA polymerase III and the C-terminal family of the DNA polymerase of the present invention.

图 2为分离的 DNA聚合酶 ΙΠ 12的聚丙烯酰胺凝胶电泳图（SDS- PAGE )。 12KDa 为蛋白质的分子量。箭头所指为分离出的蛋白条带。实现本发明的最佳方式 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated DNA polymerase IIII. 12KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如 Sambrook 等人，分子克隆：实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件，或按照制造厂商所建议的条件。实施例 1 : DNA聚合酶口12的克隆 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually performed according to the conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory conditions (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions. Example 1: Cloning of DNA polymerase port 12

用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。用 Quik mRNA I solat ion Ki t ( Qiegene 公司产品）从总 RNA中分离 poly (A) mRNA。 2ug poly (A) mRNA经逆转录形成 cDNA。用 Smart cDNA克隆试剂盒（购自 Clontech )将 cDNA片段定向***到 pBSK (+) 载体 (Clontech公司产品）的多克隆位点上，转化 DH5 a , 细菌形成 cDNA文库。用 Dye terminate cycle react ion sequencing ki t (Perkin - Elmer公司产品) 和 ABI 377 自动测序仪 (Perkin- Elmer公司）测定所有克隆的 5'和 3'末端的序列。将测定的 cDNA 序列与已有的公共 DNA序列数据库（Genebank ) 进行比较，结果发现其中一个克隆 0914g02的 cDNA序列为新的 DNA。通过合成一系列引物对该克隆所含的*** cDNA片段进行双向测定。结果表明， G914_g02克隆所含的全长 cDNA为 1092bp (如 Seq ID N0: l 所示），从第 324bp至 656bp有一个 333bp的开放阅读框架（ 0RF ) , 编码一个新的蛋白质 (如 Seq ID NO: 2所示）。我们将此克隆命名为 pBS-0914g02 , 编码的蛋白质命名为 DNA聚合酶 11112。实施例 2: cDNA 克隆的结构域分析 Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with an existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0914g02 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the full-length cDNA contained in the G914 _g 02 clone was 1092bp (as shown in Seq ID N0: l), and there was a 333bp open reading frame (0RF) from 324bp to 656bp, encoding a new protein (such as Seq ID NO: 2). We named this clone pBS-0914g02, and the encoded protein was named DNA polymerase 11112. Example 2: Domain analysis of cDNA clones

将本发明的 DNA聚合酶 ΠΙ 12的序列及其编码的蛋白序列，用 GCG中的 prof i le scan程序 (Bas icloca l Al ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] , 在 pros i te等数据库进行结构域分析。本发明的 DNA聚合酶 III 12与结构域 DNA聚合酶 C末端家族有同源，同源结果示于图 1。实施例 3: 用 RT- PCR方法克隆编码 DM聚合酶 ΙΠ 12的基因 The sequence of the DNA polymerase III 12 of the present invention and the protein sequence encoded by the same are used in a profiling scan program (Basicloca l Al ignment search tool) in GCG [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], performing domain analysis in databases such as Prote. The DNA polymerase III 12 of the present invention is homologous to the C-terminal family of the domain DNA polymerase, and the homology results are shown in FIG. 1. Example 3: Cloning of a gene encoding DM polymerase II 12 by RT-PCR

用胎脑细胞总 MA为模板，以 ol igo- dT为引物进行逆转录反应合成 cDNA,用 CDNA was synthesized using fetal brain cell total MA as a template and ol igo-dT as a primer for reverse transcription reaction.

Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增： After purification of Qiagene's kit, PCR amplification was performed with the following primers:

Pr imerl: 5'- GGGATGGATTCCACCCGCCCGCCC—3' (SEQ ID NO: 3) Pr imerl: 5'- GGGATGGATTCCACCCGCCCGCCC-3 '(SEQ ID NO: 3)

Pr imer2: 5 - ATGAAGTCTCCATTTATTCTGGGA -3' (SEQ ID NO: 4) Pr imer2: 5-ATGAAGTCTCCATTTATTCTGGGA -3 '(SEQ ID NO: 4)

Pr imerl为位于 SEQ ID NO: 1的 5，端的第 lbp开始的正向序列； Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;

Primer2为 SEQ ID NO: 1的中的 3'端反向序列。 Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.

扩增反应的条件：在 50 μ 1的反应体积中含有 50mmol/L KCl, 10mmol/L Tr i s- Cl, (pH8. 5) , 1. 5mmol/L MgCl₂, 200 μ mol/L dNTP, l Opmol引物， 1U的 Taq DNA聚合酶（Clontech公司产品）。在 PE9600型 DNA热循环仪（Perkin- Elmer公司）上按下列条件反应 25个周期： 94°C 30sec; 55°C 30sec; 72。C 2min。在 RT- PCR时同时设 β -act in 为阳性对照和模板空白为阴性对照。扩增产物用 QIAGEN公司的试剂盒纯化，用 TA 克隆试剂盒连接到 pCR载体上（Invi trogen公司产品）。 DNA序列分析结果表明 PCR 产物的 DNA序列与 SEQ ID NO: 1所示的 1- 1092bp完全相同。实施例 4: Northern 印迹法分析 DNA聚合酶 ΙΠ12基因的表达： Conditions for the amplification reaction: 50 mmol / L KCl, 10 mmol / L Tris-Cl, (pH 8.5.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, l Opmol primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min. During RT-PCR, set β-act in as a positive control and template blank as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit. The DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-1092bp shown in SEQ ID NO: 1. Example 4: Northern blot analysis of the expression of DNA polymerase ΙΠ12 gene:

用一步法提取总 RNA [Anal. Biochera 1987, 162, 156-159] _a 该法包括酸性硫氰酸胍苯酚 -氯仿抽提。即用 4M异硫氰酸胍- 25m 柠檬酸钠， 0. 2M乙酸钠（pH4. 0 ) 对组织进行匀浆，加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇（49 : 1 ) , 混合后离心。吸出水相层，加入异丙醇（0. 8体积）并将混合物离心得到 RNA沉淀。将得到的 RNA沉淀用 70%乙醇洗涤，干燥并溶于水中。用 20 μ _§ RNA, 在含 20mM 3- ( N- 吗啉代）丙磺酸（ PH7. 0 ) - 5mM乙酸钠 - ImM EDTA-2. 2M甲醛的 1. 2%琼脂糖凝胶上进行电泳。然后转移至硝酸纤维素膜上。用 a - ³²P dATP通过随机引物法制备 ³²P-标记的 DNA探针。所用的0 探针为图1所示的？01扩增的0 聚合酶111 12编码区序列 (324bp至 656bp)。将 32P-标记的探针（约 2 χ 10⁶cpm/ml ) 与转移了 RNA的硝酸纤维素膜在一溶液中于 42°C杂交过夜，该溶液包含 50%甲酰胺 - 25mM KH₂P0₄ ( pH7. 4 ) -5 x SSC- 5 x Denhardt's溶液和 2Q0 g/ml鲑精 DNA。杂交之后，将滤膜在 1 x SSC- 0. 1%SDS中于 55°C洗 30min。然后，用 Phosphor Imager进行分析和定量。实施例 5: 重组 DM聚合酶 ΙΠ12的体外表达、分离和纯化 Total RNA extraction in one step [Anal. Biochera 1987, 162, 156-159] _a This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25m sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 μ _§ RNA, perform electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (PH7. 0)-5 mM sodium acetate-1 mM EDTA-2. 2M formaldehyde . It was then transferred to a nitrocellulose membrane. A- ³² P dATP was used to prepare ³² P-labeled DNA probes by random primers. The 0 probe used is shown in Figure 1? 01 amplified 0 polymerase 111 12 coding region sequence (324bp to 656bp). A 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH ₂ P0 ₄ (pH7. 4)-5 x SSC- 5 x Denhardt's solution and 2Q0 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 5: In vitro expression, isolation and purification of recombinant DM polymerase IIII12

根据 SEQ ID NO: 1和图 1所示的编码区序列，设计出一对特异性扩增引物，序列如下： Based on SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:

Primer3: 5'- CCCCATATGATGAGACACCTTGACCAATATGCT -3' ( Seq ID No: 5 ) Primer 4: 5'- CATGGATCCCTAGTTCATGAAGCCCCAACACTT -3' (Seq ID No: 6 ) 此两段引物的 5'端分别含有 Mel和 BamHI酶切位点，其后分别为目的基因 5'端和 3'端的编码序列， Ndel和 BamHI酶切位点相应于表达载体质粒 pET28b(+) (Novagen 公司产品， Cat. No.69865.3)上的选择性内切酶位点。以含有全长目的基因的 pBS - 0914g02质粒为模板，进行 PCR反应。 PCR反应条件为：总体积 50 μ 1中含 pBS- 0914g02 质粒 10pg、引物 1：1[1½3：-3和？；1：^6]：-4分另!]为10 11101、 Advantage polymerase Mix (Clontech公司产品） 1 μ1。循环参数： 94。C 20s, 60°C 30s, 68。C 2 min,共 25个循环。用 Mel和 BamHI分别对扩增产物和质粒 pET- 28 (+)进行双酶切，分别回收大片段，并用 T4连接酶连接。连接产物转化用氯化钙法大肠杆细菌 DH5a,在含卡那霉素 (终浓度 30μ_§/ηι1 ) 的 LB平板培养过夜后，用菌落 PCR方法筛选阳性克隆，并进行测序。挑选序列正确的阳性克隆（pET- 0914g02)用氯化钙法将重组质粒转化大肠杆菌 BL21(DE3)plySs (Novagen公司产品）。在含卡那霉素（终浓度 30μ_§/πι1 ) 的 LB 液体培养基中，宿主菌 BL21 (pET- 0914g02) 在 37°C培养至对数生长期，加入 IPTG 至终浓度 1瞧 ol/L, 继续培养 5小时。离心收集菌体，经超声波破菌，离心收集上清，用能与 6个组氨酸（ 6His- Tag ) 结合的亲和层析柱 His. Bind Quick Cartridge (Novagen公司产品）进行层析，得到了纯化的目的蛋白 DNA聚合酶 11112。经 SDS- PAGE 电泳，在 12KDa处得到一单一的条带（图 2) 。将该条带转移至 PVDF膜上用 Edams水解法进行 N-端氨基酸序列分析，结果 N-端 15个氨基酸与 SEQ ID NO: 2所示的 N-端 15 个氨基酸残基完全相同。实施例 6 抗 DNA聚合酶 ΠΙ12抗体的产生 Primer3: 5'- CCCCATATGATGAGACACCTTGACCAATATGCT -3 '(Seq ID No: 5) Primer 4: 5'- CATGGATCCCTAGTTCATGAAGCCCCAACACTT -3' (Seq ID No: 6) The 5 'ends of these two primers contain Mel and BamHI restriction sites, respectively , Followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI restriction sites correspond to the selective endonucleases on the expression vector plasmid pET28b (+) (Novagen, Cat. No. 69865.3). Enzyme site. The PCR reaction was performed using the pBS-0914g02 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-0914g02 plasmid, primers 1: 1 [1½3: -3 and? 1: ^ 6]: -4 points and another!] Is 10 11101, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. The amplified product and plasmid pET-28 (+) were double-digested with Mel and BamHI, respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into the colibacillus DH5a by the calcium chloride method. After being cultured on an LB plate containing kanamycin (final concentration 30 μ _§ / ηι1) overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0914g02) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30μ _§ / πι1), the host bacteria BL21 (pET-0914g02) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 ol / L , Continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (Novagen) was used to obtain 6 histidines (6His-Tag). Purified the target protein DNA polymerase 11112. After SDS-PAGE electrophoresis, a single band was obtained at 12 KDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Production of anti-DNA polymerase III12 antibody

用多肽合成仪（PE公司产品）合成下述 DM聚合酶 ΠΙ12特异性的多肽： A peptide synthesizer (product of PE company) was used to synthesize the following DM polymerase-specific peptides:

NH2-Met-Arg-His-Leu-Asp-Gln-Tyr-Ala-Leu-Gly-Asp-Gly-Leu-Leu-His- C00H (SEQ ID NO: 7)。将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合，方法参见： Avrameas, et al. Immunochemistry, 1969; 6: 43。用 4mg上述血蓝蛋白多肽复合物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完全弗氏佐剂加强免疫一次。采用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定板做 ELISA测定兔血清中抗体的滴度。用蛋白 A-Sepharose从抗体阳性的家兔血清中分离总 IgG。将多肽结合于溴化氰活化的 Sepharose4B柱上，用亲和层析法从总 IgG 中分离抗多肽抗体。免疫沉淀法证明纯化的抗体可特异性地与 DNA聚合酶 III 12结合。实施例 7: 本发明的多核苷酸片段用作杂交探针的应用 NH2-Met-Arg-His-Leu-Asp-Gln-Tyr-Ala-Leu-Gly-Asp-Gly-Leu-Leu-His-C00H (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Antibody A positive rabbit serum with protein A-Sepharose Total IgG was isolated in. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method demonstrated that the purified antibody could specifically bind to DNA polymerase III 12. Example 7: Use of a polynucleotide fragment of the present invention as a hybridization probe

从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的用途，如用该探针可与不同来源的正常组织或病理组织的基因组或 cDNA文库杂交以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一步还可用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理组织细胞中的表达是否异常。 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.

本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1 中挑选出合适的寡核苷酸片段用作杂交探针，并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核苷酸序列或其同源的多核苷酸序列。滤膜杂交方法包括斑点印迹法、 Southern 印迹法、 Northern 印迹法和复印方法等，它们都是将待测的多核苷酸样品固定在滤膜上后使用基本相同的步骤杂交。这些相同的步骤是：固定了样品的滤膜首先用不含探针的杂交缓冲液进行预杂交，以使滤膜上样品的非特异性的结合部位被载体和合成的多聚物所饱和。然后预杂交液被含有标记探针的杂交缓冲液替换，并保温使探针与靶核酸杂交。杂交步骤之后，未杂交上的探针被一系列洗膜步骤除掉。本实施例利用较高强度的洗膜条件（如较低盐浓度和较高的温度），以使杂交背景降低且只保留特异性强的信号。本实施例选用的探针包括两类：第一类探针是完全与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段；第二类探针是部分与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。本实施例选用斑点印迹法将样品固定在滤膜上，在较高强度的的洗膜条件下，第一类探针与样品的杂交特异性最强而得以保留。 The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. In this example, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

一、探针的选用 First, the selection of the probe

从本发明的多核苷酸 SEQ ID NO: 1中选择寡核苷酸片段用作杂交探针，应遵循以下原则和需要考虑的几个方面： The selection of oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:

1 , 探针大小优选范围为 18- 50个核苷酸； 1. The preferred range of probe size is 18-50 nucleotides;

2 , GC含量为 30%- 70%, 超过则非特异性杂交增加; 2.GC content is 30% -70%, non-specific hybridization increases when it exceeds;

3 , 探针内部应无互补区域； 3, there should be no complementary regions inside the probe;

4 , 符合以上条件的可作为初选探针，然后进一步作计算机序列分析，包括将该初选探针分别与其来源序列区域（即 SEQ ID NO: 1 ) 和其它已知的基因组序列及其互补区进行同源性比较，若与非靶分子区域的同源性大于 85%或者有超过 15个连续碱基完全相同，则该初选探针一般就不应该使用； 4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences The column and its complementary region are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used generally

5, 初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确定。 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

完成以上各方面的分析后挑选并合成以下二个探针： After completing the above analysis, select and synthesize the following two probes:

探针 1 (probel)，属于第一类探针，与 SEQ ID NO: 1 的基因片段完全同源或互补 (41Nt)： Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

5'- TGAGACACCTTGACCAATATGCTTTGGGTGACGGTCTCCTA -3' ( SEQ ID NO: 8 ) 探针 2 (probe2)，属于第二类探针，相当于 SEQ ID NO: 1 的基因片段或其互补片段的替换突变序列（41Nt): 5'- TGAGACACCTTGACCAATATGCTTTGGGTGACGGTCTCCTA -3 '(SEQ ID NO: 8) Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:

5- TGAGACACCTTGACCAATATCCTTTGGGTGACGGTCTCCTA -3' ( SEQ ID NO: 9 ) 与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文献： DNA PROBES G. H. Kel ler; Μ· Μ· Manak; Stockton Press, 1989 (USA)以及更常用的分子克隆实验手册书籍如《分子克隆实验指南》（ 1998 年第二版） [美]萨姆布鲁克等著，科学出版社。 5- TGAGACACCTTGACCAATATCCTTTGGGTGACGGTCTCCTA -3 '(SEQ ID NO: 9) For other commonly used reagents and their preparation methods not related to the following specific experimental steps, please refer to the literature: DNA PROBES GH Kel ler; Μ · Μ · Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning laboratory manuals, such as the Guide to Molecular Cloning Experiments (Second Edition 1998) [US] Sambrook et al., Science Press.

样品制备： Sample Preparation:

1, 从新鲜或冰冻组织中提取 DNA 1.Extract DNA from fresh or frozen tissue

步骤： 1) 将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS) 的平皿中。用剪刀或手术刀将组织切成小块。操作中应保持组织湿润。 2) 以 lOOOg离心切碎组织 10分钟。 3 )用冷匀浆缓冲液 ( 0.25mol/L蔗糖； 25mmol/L Tris-HCl,pH7.5; 25醒 ol/LnaCl; 25mmol/L MgCl₂ ) 悬浮沉淀（大约 10ml/g )。 4) 在 4°C用电动匀浆器以全速匀浆组织悬液，直至组织被完全破碎。 5) lOOOg 离心Procedure: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the precipitate (about 10 ml / g) with a cold homogenization buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 ol / LnaCl; 25 mmol / L MgCl ₂ ). 4) Homogenize the tissue suspension at 4 ° C at full speed with an electric homogenizer until the tissue is completely broken. 5) 1000g centrifuge

10分钟。 6)用重悬细胞沉淀（每 0. lg最初组织样品加 l-5ml), 再以 lOOOg离心10 minutes. 6) Resuspend the cell pellet (l-5ml per 0.1 g of the initial tissue sample), and centrifuge at 1,000 g

10分钟。 7)用裂解缓冲液重悬沉淀（每 O. lg最初组织样品加 1ml), 然后接以下的苯酚抽提法。 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

2, DNA的苯酚抽提法 2, DNA phenol extraction method

步骤： 1)用 1- 10ml 冷 PBS洗细胞， lOOOg离心 10分钟。 2)用冷细胞裂解液重悬浮沉淀的细胞（lxlO⁸细胞 /ml) 最少应用 lOOul 裂解缓冲液。 3)加 SDS 至终浓度为 1%，如果在重悬细胞之前将 SDS直接加入到细胞沉淀中，细胞可能会形成大的团块而难以破碎，并降低的总产率。这一点在抽提〉10⁷细胞时特别严重。 4 )加蛋白酶 K至终浓度 200ug/ml。 5) 50°C保温反应 1小时或在 37°C轻轻振摇过夜。 6)用等体积苯酚：氯仿：异戊醇（ 25: 24： 1)抽提，在小离心机管中离心 10分钟。两相应清楚分离，否则重新进行离心。 7)将水相转移至新管。 8)用等体积氯仿：异戊醇（24: 1)抽提，离心 10分钟。 9)将含 DNA的水相转移至新管。然后进行 DNA的纯化和乙醇沉淀。 Steps: 1) Wash the cells with 1-10 ml of cold PBS and centrifuge at 1,000 g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (1x10 ⁸ cells / ml). Use a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) use Extract an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the DNA-containing aqueous phase to a new tube. The DNA was then purified and ethanol precipitated.

3, DNA的纯化和乙醇沉淀 3, DNA purification and ethanol precipitation

步骤： 1 ) 将 1 0体积 2mol/L醋酸钠和 2倍体积冷 100%乙醇加到 DNA溶液中，混匀。在- 20°C放置 1小时或至过夜。 2) 离心 10分钟。 3)小心吸出或倒出乙醇。 4)用 70%冷乙醇 500ul洗涤沉淀，离心 5分钟。 5)小心吸出或倒出乙醇。用 500ul冷乙醇洗涤沉淀，离心 5分钟。 6)小心吸出或倒出乙醇，然后在吸水纸上倒置使残余乙醇流尽。空气干燥 10-15 分钟，以使表面乙醇挥发。注意不要使沉淀完全干燥，否则较难重新溶解。 7) 以小体积 TE或水重悬 DNA沉淀。低速涡旋振荡或用滴管吹吸，同时逐渐增加 TE, 混合至 DNA充分溶解，每 1- 5 χ 10^ό细胞所提取的大约加 lul。 Steps: 1) Add 10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, every ^1- 5 χ 10 ό extracted cells plus about lul.

以下第 8-13步骤仅用于必须除去污染时，否则可直接进行第 14步骤。 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8 )将 RNA酶 A加到 DNA溶液中，终浓度为 100ug/ml， 37°C保温 30分钟。 9 )加入 SDS和蛋白酶 K, 终浓度分别为 0.5½和 100ug/mL 37°C保温 30分钟。 10)用等体积的苯酚：氯仿：异戊醇（ 25: 24: 1 )抽提反应液，离心 10 分钟。 11)小心移出水相，用等体积的氯仿：异戊醇（24: 1) 重新抽提，离心 10 分钟。 12) 小心移出水相，加 1/10体积 2mol/L醋酸钠和 2.5 体积冷乙醇，混匀置 - 20°C 1 小时。 13)用 70%乙醇及 100%乙醇洗涤沉淀，空气干燥，重悬核酸，过程同第 3- 6步骤。 14) 测定 A_26fl和 Α_28β以检测 DNA的纯度及产率。 15 )分装后存放于 - 20°C。样膜的制备： 8) Add RNase A to the DNA solution to a final concentration of 100 ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K with final concentrations of 0.5½ and 100ug / mL at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, and mix well-20 ° C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-5. 14) A _26fl and A _{28β were measured} to detect the purity and yield of DNA. 15) Store at -20 ° C after dispensing. Preparation of sample film:

1)取 4 x 2 张适当大小的硝酸纤维素膜（NC膜），用铅笔在其上轻轻标出点样位置及样号，每一探针需两张 NC膜，以便在后面的实验步骤中分别用高强度条件和强度条件洗膜。 1) Take 4 x 2 pieces of nitrocellulose membrane (NC membrane) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are needed for each probe for later experiments In the step, the film is washed with high-strength conditions and strength conditions, respectively.

2) 吸取及对照各 15微升，点于样膜上，在室温中晾干。 2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.

3 )置于浸润有 0. Imol/LNaOH, 1.5mol/LNaCl的滤纸上 5分钟 (两次），晾干置于浸润有 0.5mol/L Tris-HCl ( pH7.0 ), 3mol/LNaCl的滤纸上 5分钟 (两次 ), 晾干。 3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl Allow to dry for 5 minutes (twice).

4)夹于干净滤纸中，以铝箔包好， 60- 80°C真空干燥 2小时。 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.

探针的标记 Labeling of probes

1 ) 3 μ lProbe ( 0.10D/10 μ 1 )，加入 2 μ IKinase缓冲液， 8-10 uCi y-³²P-dATP+2U Kinase, 以补加至终体积 20 μ 1。 1) 3 μl Probe (0.10D / 10 μ 1), add 2 μ IKinase buffer, 8-10 uCi y- ³² P-dATP + 2U Kinase, to make up to a final volume of 20 μ 1.

2) 37 °C 保温 2小时。 3 )加 1/5体积的溴酚蓝指示剂 ( BPB )。 2) Incubate at 37 ° C for 2 hours. 3) Add 1/5 volume of bromophenol blue indicator (BPB).

4 )过 Sephadex G- 50柱。 4) Pass Sephadex G-50 column.

5 ) 至有 ³²P-Probe洗出前开始收集第一峰（可用 Moni tor监测）。 5) Start collecting the first peak before ³² P-Probe washes out (can be monitored by Moni tor).

6 ) 5滴 /管，收集 10-15管。 6) 5 drops / tube, collect 10-15 tubes.

7 )用液体闪烁仪监测同位素量 7) Monitor the amount of isotope with a liquid scintillator

8 ) 合并第一峰的收集液后即为所需制备的 ³²P- Probe (第二峰为游离 γ-³²Ρ- dATP )₀ 8) were collected after the first peak were combined to ³² P- Probe (second peak to prepare the desired free γ- ³² Ρ- dATP) ₀

预杂交 Pre-hybridization

将样膜置于塑料袋中，加入 3-1 Omg预杂交液（ 1 OxDenhardt- s； 6xSSC, 0. lmg/ml CT DNA (小牛胸腺 DM )。），封好袋口后， 68°C水洛摇 2小时。 Put the sample film in a plastic bag, add 3-1 Omg pre-hybridization solution (1 OxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM).), Seal the bag, 68 ° C Shui Luo shake for 2 hours.

杂交 Cross

将塑料袋剪去一角，加入制备好的探针，封好袋口后， 42°C水浴摇过夜。洗膜： Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake it at 42 ° C in a water bath overnight. Wash film:

高强度洗膜： High-intensity washing film:

1 )取出已杂交好的样膜。 1) Take out the hybridized sample membrane.

2 ) 2xSSC, 0. 1%SDS中 , 40°C洗 15分钟（ 2次）。 2) 2xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

3 ) 0. lxSSC, 0. 1°細中， 40。C洗 15分钟（2次）。 3) 0.1xSSC, 0.1 ° fine, 40. C wash for 15 minutes (twice).

4 ) 0. lxSSC, 0. 1°/。SDS中, 55°C洗 30分钟（ 2次），室温晾干。 4) 0.1xSSC, 0.1 ° /. Wash in SDS at 55 ° C for 30 minutes (twice) and dry at room temperature.

低强度洗膜： Low-intensity washing film:

1 )取出已杂交好的样膜。 1) Take out the hybridized sample membrane.

2 ) 2xSSC, 0. 1%SDS中， 37°C洗 15分钟（ 2次）。 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

3 ) 0. lxSSC, 0. l°/oSDS中， 37。C洗 15分钟（ 2次）。 3) 0.1xSSC, 0.1 ° / oSDS, 37. C Wash for 15 minutes (twice).

4 ) 0. lxSSC, 0. 1%SDS中, 40°C洗 15分钟（ 2次）, 室温晾干。 4) Wash in 0.1xSSC, 0.1% SDS at 40 ° C for 15 minutes (twice), and dry at room temperature.

X -光自显影： X-ray autoradiography:

-70°C, X-光自显影（压片时间根据杂交斑放射性强弱而定）。 -70 ° C, X-ray autoradiography (pressing time depends on the radioactivity of the hybrid spot).

实验结果： Experimental results:

釆用低强度洗膜条件所进行的杂交实验，以上两个探针杂交斑放射性强弱没有明显区别；而釆用高强度洗膜条件所进行的杂交实验，探针 1 的杂交斑放射性强度明显强于另一个探针杂交斑的放射性强度。因而可用探针 1 定性和定量地分析本发明的多核苷酸在不同组织中的存在和差异表达。工业实用性本发明的多肽以及该多肽的拮抗剂、激动剂和抑制剂可直接用于疾病治疗，例如，可治疗恶性肿瘤、肾上腺缺乏症、皮肤病、各类炎症、 HIV感染和免疫性疾病等。釆 The hybridization experiments performed under low-intensity membrane washing conditions did not differ significantly in the radioactivity of the above two probe hybrid spots; while the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of hybridization spots of probe 1 was obvious. Stronger in radioactivity than the hybridization spot of another probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues. Industrial applicability The polypeptide of the present invention, as well as its antagonists, agonists and inhibitors, can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.

DNA 聚合酶 III ( Pol III ) 可负责染色体的高效而准确的复制。 DNA 聚合酶 III具有 α、 ε、 Θ亚基。 ε亚基基因 dnaQ的突变可使 DnaQ酶的催化活性丧失或下降，同时 ε亚基的 C末端特征性序列是形成其活性所必需。 DNA聚合酶的 C末端缺失将导致 DNA聚合酶功能部分或完全的丧失。 DNA polymerase III (Pol III) is responsible for efficient and accurate replication of chromosomes. DNA polymerase III has α, ε, and Θ subunits. Mutations of the ε subunit gene dnaQ can cause the loss or decrease of the catalytic activity of the DnaQ enzyme, and the characteristic sequence of the C-terminal end of the ε subunit is necessary for its activity. Deletion of the C-terminus of DNA polymerase will result in partial or complete loss of DNA polymerase function.

本发明的多肽是含 DNA聚合酶 ΠΙ家族的特征性 C末端序列的多肽，其表达异常将导致染色体复制功能的异常，使信息传递发生异常，并产生相关的疾病。 The polypeptide of the present invention is a polypeptide containing a characteristic C-terminal sequence of the DNA polymerase III family. Abnormal expression of the polypeptide will cause abnormal chromosome replication function, cause abnormal information transmission, and cause related diseases.

由此可见，本发明的 DNA聚合酶 ΙΠ 12 的表达异常将产生各种疾病尤其是各种肿瘤、胚胎发育紊乱症、生长发育障碍性疾病、内分泌疾病，这些疾病包括但不限于： It can be seen that the abnormal expression of the DNA polymerase III 12 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, growth and development disorders, and endocrine diseases. These diseases include, but are not limited to:

各种组织的肿瘤：胃癌，肝癌，肺癌，食管癌，乳腺癌，白血病，淋巴瘤，甲状腺肿瘤，子宫肌瘤，神经细胞瘤，星形细胞瘤，室管膜瘤，胶质细胞瘤，神经纤维瘤，结肠癌，黑色素瘤，肾上腺癌，膀胱癌，骨癌，骨肉瘤，骨髓瘤，骨髓癌，子宫癌，子宫内膜癌，胆囊癌，结肠癌，胸腺肿瘤，鼻腔及鼻窦肿瘤，鼻咽癌，喉癌，气管肿瘤，纤维瘤，纤维肉瘤，脂肪瘤，脂肪肉瘤，平滑肌瘤 Tumors of various tissues: stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal and sinus tumors, nose Pharyngeal cancer, Laryngeal cancer, Tracheal tumor, Fibroma, Fibrosarcoma, Lipoma, Liposarcoma, Leiomyoma

胚胎发育紊乱症：先天性流产，腭裂，肢体缺如，肢体分化障碍，透明膜病，肺膨胀不全，多囊肾，隐睾，先天性***，双子宫， ***闭锁，尿道下裂，两性畸形，房间隔缺损，室间隔缺损，肺动脉狭窄，动脉导管未闭，神经管缺陷，先天性脑积水，虹膜缺损，先天性青光眼或白内障，先天性耳聋 Fetal developmental disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, androgynous Malformation, Atrial septal defect, Ventricular septal defect, Pulmonary stenosis, Arterial duct occlusion, Neural tube defect, Congenital hydrocephalus, Iris defect, Congenital glaucoma or cataract, Congenital deafness

生长发育障碍性疾病：精神发育迟缓，脑性瘫痪，脑发育障碍，智力障碍，家族性脑神经核发育不全综合症，斜视，皮肤、脂肪和肌肉发育不良性疾病如先天性皮肤松弛症、白化病、早老症、先天性角化不良，骨与关节发育不良性疾病如软骨发育不全、骨骺发育不良、代谢性骨病，各种代谢缺陷病如各种氨基酸代谢缺陷症，呆小症，侏儒症，库兴综合这征，性发育迟缓症 Growth disorders: mental retardation, cerebral palsy, mental retardation, mental retardation, familial cerebellar dysplasia, strabismus, skin, fat, and muscular dysplasias such as congenital skin relaxation, albinism , Alzheimer's disease, congenital keratosis, bone and joint dysplasia diseases such as cartilage dysplasia, epiphyseal dysplasia, metabolic bone disease, various metabolic defects such as various amino acid metabolic defects, dementia, dwarfism Cushing syndrome, sexual retardation

内分泌疾病：尿崩症，性早熟症，巨人症和肢端肥大症，侏儒症，垂体瘤，单纯性甲状腺肿，甲状腺炎，甲状腺功能亢进症，甲状腺功能减退症，甲状旁腺机能亢进症，甲状旁腺机能减退症，皮质醇增多症、原发性醛固酮增多症，肾上腺皮质功能减退症，肾上腺肿瘤，糖尿病，胰岛素瘤，胃泌素瘤，卵巢病：经前期紧张症，经绝期综合征，卵巢发育不全，闭经，男性性腺功能减退症，男性性早熟本发明的 DNA聚合酶 ΙΠ 12 的表达异常还将产生某些遗传性，血液性疾病及免疫***疾病等。 Endocrine diseases: diabetes insipidus, precocious puberty, giant disease and acromegaly, dwarfism, pituitary tumors, simple goiter, thyroiditis, hyperthyroidism, hypothyroidism, hyperparathyroidism, Hypoparathyroidism, Cortisol, Primary Aldosterone, Adrenal Insufficiency, Adrenal Tumor, Diabetes, Insulinoma, Gastrinoma, Ovarian Disease: Premenstrual Tension, Menopausal Syndrome Symptoms, ovarian hypoplasia, amenorrhea, hypogonadism, precocious puberty The abnormal expression of the DNA polymerase III 12 of the present invention will also produce certain hereditary, hematological and immune system diseases.

本发明的多肽以及该多肽的拮抗剂、激动剂和抑制剂可直接用于疾病治疗，例如，可治疗各种疾病尤其是各种肿瘤、胚胎发育紊乱症、生长发育障碍性疾病、内分泌疾病，某些遗传性，血液性疾病及免疫***疾病等。本发明也提供了筛选化合物以鉴定提高（激动剂）或阻遏（拮抗剂） DNA 聚合酶 ΙΠ 12的药剂的方法。激动剂提高 DM聚合酶 ΠΠ 2刺激细胞增殖等生物功能，而拮抗剂阻止和治疗与细胞过度增殖有关的紊乱如各种癌症。例如，能在药物的存在下，将哺乳动物细胞或表达 DM聚合酶 III 12 的膜制剂与标记的 DM聚合酶 ΠΙ 12—起培养。然后测定药物提高或阻遏此相互作用的能力。 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various tumors, embryonic development disorders, growth and development disorders, endocrine diseases, Certain hereditary, hematological and immune system diseases. The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) DNA polymerase IIIII. Agonists increase the biological functions of DM polymerase II and stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing DM polymerase III 12 can be cultured together with labeled DM polymerase II 12 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.

DNA 聚合酶 ΙΠ 12 的拮抗剂包括筛选出的抗体、化合物、受体缺失物和类似物等。 DNA聚合酶 ΠΙ 12 的拮抗剂可以与 DM聚合酶 III12结合并消除其功能，或是抑制该多肽的产生，或是与该多肽的活性位点结合使该多肽不能发挥生物学功能。 Antagonists of DNA polymerase II 12 include screened antibodies, compounds, receptor deletions, and the like. An antagonist of DNA polymerase III 12 can bind to DM polymerase III12 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform a biological function.

在筛选作为拮抗剂的化合物时，可以将 DNA聚合酶 ΙΠ12加入生物分析测定中，通过测定化合物对 MA聚合酶 ΠΙ 12 和其受体之间相互作用的影响来确定化合物是否是拮抗剂。用上述筛选化合物的同样方法，可以筛选出起拮抗剂作用的受体缺失物和类似物。能与 DNA聚合酶 ffi l2 结合的多肽分子可通过筛选由各种可能组合的氨基酸结合于固相物组成的随机多肽库而获得。筛选时，一般应对 DNA聚合酶 ΙΠ 12分子进行标记。 When screening compounds as antagonists, DNA polymerase IIII 12 can be added to a bioanalytical assay to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between MA polymerase IIII and its receptor. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to DNA polymerase ffi l2 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, DNA polymerase III molecules should generally be labeled.

本发明提供了用多肽，及其片段、衍生物、类似物或它们的细胞作为抗原以生产抗体的方法。这些抗体可以是多克隆抗体或单克隆抗体。本发明还提供了针对 DM聚合酶 ΙΠ 12抗原决定簇的抗体。这些抗体包括 (但不限于）：多克隆抗体、单克隆抗体、嵌合抗体、单链抗体、 Fab 片段和 Fab 表达文库产生的片段。 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The present invention also provides antibodies against the DM polymerase IIIII epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

多克隆抗体的生产可用 DM聚合酶 ΙΠ 12直接注射免疫动物（如家兔，小鼠，大鼠等）的方法得到，多种佐剂可用于增强免疫反应，包括但不限于弗氏佐剂等。制备 DNA聚合酶 ΙΠ 12的单克隆抗体的技术包括但不限于杂交瘤技术（Kohler and Mi l s te in. Nature, 1975, 256: 495-497) , 三瘤技术，人 Β-细胞杂交瘤技术， EBV-杂交瘤技术等。将人恒定区和非人源的可变区结合的嵌合抗体可用已有的技术生产（Morr i son et al , PNAS, 1985, 81: 6851) ₀ 而已有的生产单链抗体的技术（U. S. Pat No. 4946778)也可用于生产抗 DNA聚合酶 III 12的单链抗体。 Polyclonal antibodies can be produced by injecting DM polymerase III 12 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. . Techniques for preparing monoclonal antibodies to DNA polymerase IIII 12 include, but are not limited to, hybridoma technology (Kohler and Miles in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human variable regions are available Some technologies produce (Morr i son et al, PNAS, 1985, 81: 6851) ₀ and the existing technologies for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against DNA polymerase III 12 .

抗 DNA聚合酶 ΙΠ 12 的抗体可用于免疫组织化学技术中，检测活检标本中的 DNA聚合酶 III 12。 Antibodies to DNA polymerase II 12 can be used in immunohistochemistry to detect DNA polymerase III 12 in biopsy specimens.

与 DNA聚合酶 ΠΙ 12结合的单克隆抗体也可用放射性同位素标记，注入体内可跟踪其位置和分布。这种放射性标记的抗体可作为一种非创伤性诊断方法用于肿瘤细胞的定位和判断是否有转移。 Monoclonal antibodies that bind to DNA polymerase II 12 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

抗体还可用于设计针对体内某一特殊部位的免疫毒素。如 DNA 聚合酶 ΙΠ 12 高亲和性的单克隆抗体可与细菌或植物毒素（如白喉毒素，蓖麻蛋白，红豆碱等）共价结合。一种通常的方法是用巯基交联剂如 SPDP , 攻击抗体的氨基，通过二硫键的交换，将毒素结合于抗体上，这种杂交抗体可用于杀灭 DM 聚合酶 ΠΙ 12 阳性的细胞。 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, a DNA polymerase II 12 high affinity monoclonal antibody can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill DM polymerase II 12 positive cells.

本发明中的抗体可用于治疗或预防与 DNA聚合酶 ΙΠ 12相关的疾病。给予适当剂量的抗体可以刺激或阻断 DNA聚合酶 ΙΠ 12的产生或活性。 The antibodies of the present invention can be used to treat or prevent diseases related to DNA polymerase IIIII. Administration of an appropriate dose of antibody can stimulate or block the production or activity of DNA polymerase IIIII.

本发明还涉及定量和定位检测 DNA聚合酶 ΠΙ 12水平的诊断试验方法。这些试验是本领域所熟知的，且包括 FISH测定和放射免疫测定。试验中所检测的 DNA 聚合酶 ΠΙ 12水平，可以用作解释 DNA聚合酶 ΠΙ 12在各种疾病中的重要性和用于诊断 DNA聚合酶 III 12起作用的疾病。 The invention also relates to a diagnostic test method for quantitative and localized detection of the level of DNA polymerase II 12. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of DNA polymerase II 12 detected in the test can be used to explain the importance of DNA polymerase II 12 in various diseases and to diagnose diseases in which DNA polymerase III 12 functions.

本发明的多肽还可用作肽谱分析，例如，多肽可用物理的、化学或酶进行特异性切割，并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分析。 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.

编码 DNA聚合酶 III 12 的多核苷酸也可用于多种治疗目的。基因治疗技术可用于治疗由于 DNA 聚合酶 ΠΙ 12 的无表达或异常 /无活性表达所致的细胞增殖、发育或代谢异常。重组的基因治疗载体 (如病毒载体)可设计用于表达变异的 DNA 聚合酶 ΙΠ12 , 以抑制内源性的 DNA聚合酶 ΠΙ 12活性。例如，一种变异的 DNA聚合酶 ΙΠ 12 可以是缩短的、缺失了信号传导功能域的 MA聚合酶 11112，虽可与下游的底物结合，但缺乏信号传导活性。因此重组的基因治疗载体可用于治疗 DNA 聚合酶 ΙΠ12表达或活性异常所致的疾病。来源于病毒的表达载体如逆转录病毒、腺病毒、腺病毒相关病毒、单纯疱疹病毒、细小病毒等.可用于将编码 DNA 聚合酶 ΙΠ 12 的多核苷酸转移至细胞内。构建携带编码 DNA聚合酶 ΙΠ12的多核苷酸的重组病毒载体的方法可见于已有文献（Sambrook, et a l. )。另外重组编码 DNA聚合酶 ΙΠ 12的多核苷酸可包装到脂质体中转移至细胞内。多核苷酸导入组织或细胞内的方法包括：将多核苷酸直接注入到体内组织中；或在体外通过载体 (如病毒、噬菌体或质粒等）先将多核苷酸导入细胞中，再将细胞移植到体内等。 Polynucleotides encoding DNA polymerase III 12 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormalities in cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of DNA polymerase III 12. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated DNA polymerase II 12 to inhibit endogenous DNA polymerase II 12 activity. For example, a mutated DNA polymerase III 12 may be a shortened MA polymerase 11112 lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of DNA polymerase III. Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, parvoviruses, etc. can be used to transfer polynucleotides encoding DNA polymerase II 12 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a DNA polymerase III12 can be found in the existing literature (Sambrook, et al.). In addition, the polynucleotide encoding the recombinant DNA polymerase IIII 12 can be packaged into liposomes and transferred into cells. Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid) and then transplanting the cell Into the body and so on.

抑制 DNA聚合酶 ΠΙ 12 mRNA的寡核苷酸（包括反义 RNA和 DNA)以及核酶也在本发明的范围之内。核酶是一种能特异性分解特定 RNA 的酶样 RNA分子，其作用机制是核酶分子与互补的靶 RNA特异性杂交后进行核酸内切作用。反义的 RM 和 DNA及核酶可用已有的任何 RNA或 DNA合成技术获得，如固相磷酸酰胺化学合成法合成寡核苷酸的技术巳广泛应用。反义 A分子可通过编码该 RNA的 DNA 序列在体外或体内转录获得。这种 DNA序列已整合到载体的 RNA聚合酶启动子的下游。为了增加核酸分子的稳定性，可用多种方法对其进行修饰，如增加两侧的序列长度，核糖核苷之间的连接应用磷酸硫酯键或肽键而非磷酸二酯键。 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit DNA polymerase II 12 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RM, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the solid-phase phosphate amide chemical synthesis method for oligonucleotide synthesis, which is widely used. Antisense A molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.

编码 DNA聚合酶 ΙΠ12的多核苷酸可用于与 DNA聚合酶 III12的相关疾病的诊断。编码 DNA聚合酶 ΠΙ 12的多核苷酸可用于检测 DNA聚合酶 ΠΙ12的表达与否或在疾病状态下 DNA聚合酶 ΙΠ12的异常表达。如编码 DNA聚合酶 ΙΠ 12的 DNA序列可用于对活检标本进行杂交以判断 DNA聚合酶 ΠΙ12 的表达状况。杂交技术包括 Southern印迹法， Nor thern印迹法、原位杂交等。这些技术方法都是公开的成熟技术，相关的试剂盒都可从商业途径得到。本发明的多核苷酸的一部分或全部可作为探针固定在微阵列（Microarray)或 DNA芯片（又称为 "基因芯片" ）上，用于分析组织中基因的差异表达分析和基因诊断。用 DNA聚合酶 ΠΙ 12 特异的引物进行 RNA-聚合酶链反应（RT-PCR)体外扩增也可检测 DNA聚合酶 ΠΙ 12的转录产检测 DM 聚合酶 ΙΠ 12 基因的突变也可用于诊断 DM 聚合酶 III12 相关的疾病。 DNA聚合酶 ΠΙ12突变的形式包括与正常野生型 DNA聚合酶 ΙΠ12 DM序列相比的点突变、易位、缺失、重组和其它任何异常等。可用已有的技术如 Southern 印迹法、 DNA序列分析、 PCR和原位杂交检测突变。另外，突变有可能影响蛋白的表达，因此用 Northern印迹法、 Wes tern印迹法可间接判断基因有无突变。 The polynucleotide encoding DNA polymerase III12 can be used for the diagnosis of diseases related to DNA polymerase III12. The polynucleotide encoding the DNA polymerase III12 can be used to detect the expression of the DNA polymerase III12 or the abnormal expression of the DNA polymerase III12 in a disease state. For example, the DNA sequence encoding DNA polymerase IIII can be used to hybridize biopsy specimens to determine the expression of DNA polymerase IIII12. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly known and mature, and related kits are commercially available. Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microarray) or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. DNA polymerase II 12 specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect the transcription of DNA polymerase II 12 detection of DM polymerase II II 12 mutations can also be used to diagnose DM polymerization Enzyme III12 related diseases. The form of DNA polymerase III12 mutation includes point mutation, translocation, deletion, recombination, and any other abnormalities compared to the normal wild-type DNA polymerase IIIII12 DM sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

本发明的序列对染色体鉴定也是有价值的。该序列会特异性地针对某条人染色体具***置且并可以与其杂交。目前，需要鉴定染色体上的各基因的具体位点。现在，只有很少的基于'实际序列数据（重复多态性）的染色体标记物可用于标记染色***置。根据本发明，为了将这些序列与疾病相关基因相关联，其重要的第一步就是将这些 DNA序列定位于染色体上。 The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on 'actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

简而言之，根据 cDNA制备 PCR引物（优选 15- 35bp) , 可以将序列定位于染色体上。然后，将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。只有那些含有相应于引物的人基因的杂合细胞会产生扩增的片段。 In short, PCR primers (preferably 15-35bp) are prepared based on cDNA, which can be used to position the sequence for staining. Physically. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.

体细胞杂合细胞的 PCR定位法，是将 DNA定位到具体染色体的快捷方法。使用本发明的寡核苷酸引物，通过类似方法，可利用一组来自特定染色体的片段或大量基因组克隆而实现亚定位。可用于染色体定位的其它类似策略包括原位杂交、用标记的流式分选的染色体预筛选和杂交预选，从而构建染色体特异的 cDNA库。 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.

将 cDNA克隆与中期染色体进行荧光原位杂交（FISH) , 可以在一个步骤中精确地进行染色体定位。此技术的综述，参见 Verma等， Human Chromosomes: a Manua l of Bas ic Techniques, Pergamon Pres s, New York (1988)。 Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manua l of Basic Techniques, Pergamon Pres s, New York (1988).

一旦序列被定位到准确的染色***置，此序列在染色体上的物理位置就可以与基因图数据相关联。这些数据可见于例如， V. Mckus ick, Mendel ian Inher i tance in Man (可通过与 Johns Hopk ins Univers i ty We l ch Medica l Library联机获得）。然后可通过连锁分析，确定基因与业巳定位到染色体区域上的疾病之间的关系。 Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inherance in Man (available online with Johns Hopk ins University Welch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.

接着，需要测定患病和未患病个体间的 cDNA或基因组序列差异。如果在一些或所有的患病个体中观察到某突变，而该突变在任何正常个体中未观察到，则该突变可能是疾病的病因。比较患病和未患病个体，通常涉及首先寻找染色体中结构的变化，如从染色体水平可见的或用基于 cDNA序列的 PCR可检测的缺失或易位。根据目前的物理作图和基因定位技术的分辨能力，被精确定位至与疾病有关的染色体区域的 cDNA, 可以是 50至 500个潜在致病基因间之一种（假定 1兆碱基作图分辨能力和每 20kb对应于一个基因）。 Next, the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

可以将本发明的多肽、多核苷酸及其模拟物、激动剂、拮抗剂和抑制剂与合适的药物载体组合后使用。这些载体可以是水、葡萄糖、乙醇、盐类、缓冲液、甘油以及它们的组合。组合物包含安全有效量的多肽或拮抗剂以及不影响药物效果的载体和赋形剂。这些组合物可以作为药物用于疾病治疗。 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

本发明还提供含有一种或多种容器的药盒或试剂盒，容器中装有一种或多种本发明的药用组合物成分。与这些容器一起，可以有由制造、使用或销售药品或生物制品的政府管理机构所给出的指示性提示，该提示反映出生产、使用或销售的政府管理机构许可其在人体上施用。此外，本发明的多肽可以与其它的治疗化合物结合使用。 The invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

药物组合物可以以方便的方式给药，如通过局部、静脉内、腹膜内、肌内、皮下、鼻内或皮内的给药途径。 DNA聚合酶 ΙΠ12 以有效地治疗和 /或预防具体的适应症的量来给药。施用于患者的 DNA聚合酶 ΙΠ12 的量和剂量范围将取决于许多因素，如给药方式、待治疗者的健康条件和诊断医生的判断。 The pharmaceutical composition can be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, Subcutaneous, intranasal or intradermal route of administration. DNA polymerase III12 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dose range of DNA polymerase III12 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Claims

杈利要求书 Book

I、一种分离的多肽 - DNA 聚合酶 ΠΙ 12 , 其特征在于它包含有： SEQ ID NO: 2 所示的氨基酸序列的多肽、或其多肽的活性片段、类似物或衍生物。 I. An isolated polypeptide-DNA polymerase III, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog, or derivative thereof.

2、如权利要求 1 所述的多肽，其特征在于所述多肽、. 类似物或衍生物的氨基酸序列具有与 SEQ ID NO: 2所示的氨基酸序列至少 95%的相同性。 2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.

3、如权利要求 2所述的多肽，其特征在于它包含具有 SEQ ID NO: 2所示的氨基酸序列的多肽。 3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.

4、一种分离'的多核苷酸，其特征在于所述多核苷酸包含选自下组中的一种： 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

(a) 编码具有 SEQ ID NO: 2 所示氨基酸序列的多肽或其片段、类似物、衍生物的多核苷酸； (a) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;

(b) 与多核苷酸（a ) 互补的多核苷酸；或 (b) a polynucleotide complementary to polynucleotide (a); or

(c) 与（a )或（b ) 有至少 70%相同性的多核苷酸。 (c) A polynucleotide that is at least 70% identical to (a) or (b).

5、如权利要求 4所述的多核苷酸,其特征在于所述多核苷酸包含编码具有 SEQ ID NO: 2所示氨基酸序列的多核苷酸。 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 2.

6、如权利要求 4所述的多核苷酸，其特征在于所述多核苷酸的序列包含有 SEQ ID NO: 1中 324-656位的序列或 SEQ ID NO: 1中 1-1092位的序列。 6. The polynucleotide according to claim 4, characterized in that the sequence of said polynucleotide comprises the sequence of positions 324-656 in SEQ ID NO: 1 or the sequence of positions 1-1092 in SEQ ID NO: 1. .

7、一种含有外源多核苷酸的重组载体，其特征在于它是由权利要求 4-6 中的任一杈利要求所述多核苷酸与质粒、病毒或运载体表达载体构建而成的重组载体。 7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is constructed from the polynucleotide according to any one of claims 4 to 6 and a plasmid, virus or vector expression vector Recombinant vector.

8、一种含有外源多核苷酸的遗传工程化宿主细胞，其特征在于它是选自于下列一种宿主细胞： 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞；或 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

(b) 用杈利要求 4- 6中的任一权利要求所述多核苷酸转化或转导的宿主细胞。 (b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.

9、一种具有 DM聚合酶 ΙΠ12活性的多肽的制备方法，其特征在于所述方法包括： (a) 在表达 DNA聚合酶 ΙΠ12条件下，培养权利要求 8所述的工程化宿主细胞；9. A method for preparing a polypeptide having DM polymerase IIIII12 activity, characterized in that the method comprises: (a) culturing the engineered host cell of claim 8 under the condition of expressing DNA polymerase IIIII12;

(b) 从培养物中分离出具有 DNA聚合酶 ΠΙ12活性的多肽。 (b) Isolating a polypeptide having DNA polymerase II12 activity from the culture.

10、一种能与多肽结合的抗体,其特征在于所述抗体是能与 DNA聚合酶 ΠΙ 12特异性结合的抗体。 10. An antibody capable of binding to a polypeptide, characterized in that the antibody is an antibody capable of specifically binding to a DNA polymerase III12.

II、一类模拟或调节多肽活性或表达的化合物，其特征在于它们是模拟、促进、拮抗或抑制 DNA聚合酶 ΠΙ12的活性的化合物。 II. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of a DNA polymerase II12.

12、如权利要求 11所述的化合物，其特征在于它是 SEQ ID N0: 1所示的多核苷酸序列或其片段的反义序列。 12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.

13、一种权利要求 11所述化合物的应用，其特征在于所述化合物用于调节 DNA聚合酶 ΙΠ12在体内、体外活性的方法。 13. Use of a compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of DNA polymerase IIII12 in vivo and in vitro.

14、一种检测与杈利要求 1-3 中的任一权利要求所述多肽相关的疾病或疾病易感性的方法，其特征在于其包括检测所述多肽的表达量，或者检测所述多肽的活性，或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。 14. A method for detecting a disease or susceptibility to a polypeptide related to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the Activity, or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

15、如权利要求 1-3 中的任一权利要求所述多肽的应用，其特征在于它应用于筛选 DNA 聚合酶 ΠΙ12 的模拟物、激动剂，拮抗剂或抑制剂；或者用于肽指紋图谱鉴定。 15. Use of a polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of DNA polymerase II12; or for peptide fingerprinting Identification.

16、如权利要求 4-6 中的任一权利要求所述的核酸分子的应用，其特征在于它作为引物用于核酸扩增反应，或者作为探针用于杂交反应，或者用于制造基因芯片或微阵列。 16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray.

17、如权利要求 1-6及 11 中的任一杈利要求所述的多肽、多核苷酸或化合物的应用，其特征在于用所述多肽、多核苷酸或其模拟物、激动剂、拮抗剂或抑制剂以安全有效剂量与药学上可接受的载体组成作为诊断或治疗与 DNA 聚合酶 ΙΠ12 异常相关的疾病的药物组合物。 17. Use of a polypeptide, polynucleotide or compound as claimed in any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist The agent or inhibitor is composed of a safe and effective dose and a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with a DNA polymerase IIII12 abnormality.

18、权利要求 1-6及 11中的任一权利要求所述的多肽、多核苷酸或化合物的应用，其特征在于用所述多肽、多核苷酸或化合物制备用于治疗如恶性肿瘤，血液病， HIV 感染和免疫性疾病和各类炎症的药物。 18. The use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Diseases, HIV infection and immune diseases and drugs of various inflammations.