一种新的多肽——有丝分裂激酶 ASK1 - 26和编码这种多肽的多核苷酸 技术领域 A new polypeptide-mitogen kinase ASK1-26 and a polynucleotide encoding the same 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——有 丝分裂激酶 ASK1-26, 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。 技术背景 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, mitogen kinase ASK1-26, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. technical background
有丝分裂激活蛋白 (MAP) 串联激酶能感应于细胞外各种刺激而被激活, 这些细胞外刺激包括生长因子和环境压力。 一个 MAP三激酶 (MAPKKK) , 命名 为 ASK1, 能够激活两个不同的 MAP二激酶 (MAPKK) 亚群, SEK1 (或 MO ) 和 MKK3/MAPKK6 (或 MKK6 ) , 这些 MAP 二激酶依次分别激活压力激活蛋白激酶 ( SAPK ) 和 MAP激酶的 P38亚群。 ASK1 的过度表达能诱导细胞的程序性死亡, 并且 ASK1在细胞内被激活后用来对付胂瘤坏死因子 -α (TNF-α )。 另外, ASK1 的催化非活性形式能抑制 TNF- a诱导的细胞编程性死亡。 AS 可能是环境压 力和细胞因子诱导的细胞编程性死亡机制的关键因素。 Mitogen-activated protein (MAP) tandem kinases are activated by sensing extracellular stimuli, including growth factors and environmental stress. A MAP trikinase (MAPKKK), named ASK1, can activate two different MAP dikinase (MAPKK) subgroups, SEK1 (or MO) and MKK3 / MAPKK6 (or MKK6). These MAP two kinases in turn activate stress activation respectively Protein kinase (SAPK) and P38 subgroups of MAP kinases. ASK1 overexpression can induce programmed cell death, and ASK1 is activated in cells to deal with tumor necrosis factor-α (TNF-α). In addition, the catalytically inactive form of ASK1 can inhibit TNF-a-induced programmed cell death. AS may be a key factor in environmental stress and cytokine-induced programmed cell death mechanisms.
MAP 激酶信号级联作为一个信号转导途径在从酵母细胞到脊推动物细胞都 是高度保守的, 组成该信号途径的几个 MAP激酶包括 MAP激酶 (MAPK) 、 MAPK 激酶(MAPKK )、 MAPKK激酶(MAPKKK) [T. W. Sturgill and J. Wu, Biochim. B iochi m. Biophy. Actal092, 350 (1991); E, Nishida and Y. Gotoh, Trends Biochera. Sci. 18, 128 (1993) ;B.Errede and D. E. Levin, Curr. Opin. Cell Biol.5, 254 (1993); The MAP kinase signal cascade is highly conserved as a signal transduction pathway from yeast cells to spinal impulse cells. Several MAP kinases that make up this signal pathway include MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase. (MAPKKK) [TW Sturgill and J. Wu, Biochim. B iochi m. Biophy. Actal092, 350 (1991); E, Nishida and Y. Gotoh, Trends Biochera. Sci. 18, 128 (1993); B. Errede and DE Levin, Curr. Opin. Cell Biol. 5, 254 (1993);
C. J.Marshall, Curr. Opin. Genet. Dev.4, 82 (1994)]。 MAPKKK磷酸化 MAPKK 从而激活 MA K, 激活后的 MAPKK又依次磷酸化激活 MAPK。 激活的 MAPK可以 转运细胞核并且调节转录因子的激活从而控制基因的表达。 C. J. Marshall, Curr. Opin. Genet. Dev. 4, 82 (1994)]. MAPKKK phosphorylates MAPKK to activate MA K, and activated MAPKK in turn phosphorylates MAPK. Activated MAPK can transport the nucleus and regulate the activation of transcription factors to control gene expression.
命名为 ASK1的三激酶 (MAPKKK) 蛋白由 1375个氨基酸组成, 相对分子量 为 154, 715。 AS 1 的丝氨酸-苏氨酸激酶结构域位于分子的中部, 并且有较长 的 N末端和 C末端侧翼序列, 在 N末端的一个短的氨基酸序列包含一个 FK506 集合蛋白 (FKBP) 型顺反子异构酶结构域。 The three-kinase (MAPKKK) protein named ASK1 is composed of 1375 amino acids and has a relative molecular weight of 154,715. The serine-threonine kinase domain of AS 1 is located in the middle of the molecule and has a long N-terminal and C-terminal flanking sequence. A short amino acid sequence at the N-terminus contains a FK506 collectin (FKBP) cistron Isomerase domain.
通过基因芯片的分析发现, 在胸腺、 睾丸、 肌肉、 脾脏、 肺、 皮肤、 甲状 腺、 肝、 PMA+的 Ecv304 细胞株、 PMA -的 Ecv304细胞株、 未饥饿的 L02 细胞 株、 砷刺激 1小时的 L02细胞株、 砷刺激 6小时的 L02细胞株前列腺、 心、 肺 癌、 胎膀胱、 胎小肠、 胎大肠、 胎胸腺、 胎肌、 胎肝、 胎肾、 胎脾、 胎脑、 胎
肺以及胎心中, 本发明的多肽的表达谱与有丝分裂激酶 ASK1 的表达谱非常近 似, 因此二者功能也可能类似。 本发明被命名为有丝分裂激酶 ASKl-26。 Gene chip analysis revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, arsenic-stimulated L02 L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, fetus In the lung and fetal heart, the expression profile of the polypeptide of the present invention is very similar to the expression profile of the mitogen kinase ASK1, so the functions of the two may also be similar. The present invention is named mitotic kinase ASKl-26.
由于如上所述有丝分裂激酶 ASK1 - 26蛋白在调节细胞分裂和胚胎发育等机 体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有丝分裂激酶 ASK1-26蛋白, 特别是 鉴定这种蛋白的氨基酸序列。 新有丝分裂激酶 ASK1-26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 DNA是非常重要的。 发明目的 As mentioned above, the mitotic kinase ASK1-26 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes. Therefore, there has been a need in the art to identify more involved in these processes The mitogen-kinase ASK1-26 protein, in particular, identifies the amino acid sequence of this protein. The isolation of the new mitotic kinase ASK1-26 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important. Object of the invention
本发明的一个目的是提供分离的新的多肽一 丝分裂激酶 ASK1-26 以及 其片段、 类似物和衍生物。 It is an object of the present invention to provide an isolated novel polypeptide, mitotic kinase ASK1-26, and fragments, analogs and derivatives 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 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有丝分裂激酶 ASK1- 26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 mitotic kinase ASK1-26.
本发明的另一个目的是提供含有编码有丝分裂激酶 ASK1-26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 mitotic kinase ASK1-26.
本发明的另一个目的是提供生产有丝分裂激酶 AS - 26的方法。 Another object of the present invention is to provide a method for producing mitotic kinase AS-26.
本发明的另一个目的是提供针对本发明的多肽——有丝分裂激酶 ASK1 - 26 的抗体。 Another object of the present invention is to provide an antibody against the polypeptide of the present invention, mitotic kinase ASK1-26.
本发明的另一个目的是提供了针对本发明多肽——有丝分裂激酶 ASK1-26 的模拟化合物、 拮抗剂、 激动剂、 抑制剂。 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, mitotic kinase ASK1-26.
本发明的另一个目的是提供诊断治疗与有丝分裂激酶 ASK1-26异常相关的 疾病的方法。 发明概要 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of mitogen kinase ASK1-26. Summary of invention
本发明涉及一种分离的多肽, 该多肤是人源的, 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. The polypeptide 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;
(b)与多核苷酸(a)互补的多核苷酸; (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2; (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 104-820位的序列; 和(b)具有 SEQ ID NO: 1中 1-1531位的序列。 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 104-820 in SEQ ID NO: 1; and (b) a sequence having 1-1531 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 present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; 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、 拮抗或抑制有丝分裂激酶 ASK1 - 26 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the mitogen kinase ASK1-26 protein, which comprises utilizing the polypeptide of the present invention. The invention also relates to compounds obtained by this method.
本发明还涉及一种体外检测与有丝分裂激酶 ASK1-26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 invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of the mitogen kinase ASK1-26 protein, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof 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恶性胂瘤, 血 液病, 发育紊乱症, HIV 感染, 免疫性疾病和各类炎症或其它由于有丝分裂激 酶 ASK1-26表达异常所引起疾病的药物的用途。 The present invention also relates to the preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of malignant tumors, hematological diseases, developmental disorders, HIV infections, immune diseases and various inflammations or other abnormal expressions of mitotic kinase ASK1-26 Use of medicines that cause disease.
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。 ' 附图说明 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. '' BRIEF DESCRIPTION
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是本发明有丝分裂激酶 ASH- 26和有丝分裂激酶 AS 的基因芯片表达谱 比较图。 上图是有丝分裂激酶 ASH- 26的表达谱折方图, 下图是有1丝分裂激酶 ASK1的表达谱折方图。 其中, 1表示胎肾, 2表示胎大肠, 3表示胎小肠, 4表示 胎肌, 5表示胎脑, 6表示胎膀胱, 7表示未饥饿 L02, 8表示 L02+, lhr, As3+, 9表 示 ECV304 PMA- , 10表示 ECV304 PMA+, 11表示胎肝, 12表示正常肝, 13表示甲 状腺, 14表示皮肤, 15表示胎肺, 16表示肺, 17表示肺癌, 18表示胎脾, 19表 示脾脏, 20表示前列腺, 21表示胎心, 22表示心脏, 23表示肌肉, 24表示睾丸,
25表示胎胸腺, 26表示胸腺。 FIG. 1 is a comparison diagram of gene chip expression profiles of mitogen kinase ASH-26 and mitogen kinase AS of the present invention. The figure is a mitotic kinase expression profile ASH- off side of FIG. 26, the graph is a mitotic kinases ASK1 expression profile of FIG off side. Among them, 1 indicates fetal kidney, 2 indicates fetal large intestine, 3 indicates fetal small intestine, 4 indicates fetal muscle, 5 indicates fetal brain, 6 indicates fetal bladder, 7 indicates non-starved L02, 8 indicates L02 +, lhr, As 3+ , and 9 indicates ECV304 PMA-, 10 means ECV304 PMA +, 11 means fetal liver, 12 means normal liver, 13 means thyroid, 14 means skin, 15 means fetal lung, 16 means lung, 17 means lung cancer, 18 means fetal spleen, 19 means spleen, 20 Means prostate, 21 means fetal heart, 22 means heart, 23 means muscle, 24 means testis, 25 indicates the fetal thymus and 26 indicates the thymus.
图 2 为分离的有丝分裂激酶 AS -26 的聚丙烯酰胺凝胶电泳图 (SDS- PAGE )。 26KDa为蛋白质的分子量。 箭头所指为分离出的蛋白条带。 发明内容 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated mitotic kinase AS-26. 26KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the Invention
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 DNA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。 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 DNA 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、 插入或替换。 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 refers to an amino acid sequence having one or more amino acids or nucleotide changes or a 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 can have "conservative" changes, in which the amino acid substituted 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.
"插入" 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 and to bind specific antibodies in a suitable animal or cell.
"激动剂" 是指当与有丝分裂激酶 ASK1-26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有丝分裂激酶 ASK1- 26的分子。 An "agonist" refers to a molecule that, when combined with the mitogen kinase ASK1-26, causes the protein to change, thereby regulating the activity of the protein. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to the mitotic kinase ASK1-26.
"拮抗剂" 或 "抑制物" 是指当与有丝分裂激酶 ASK1-26结合时, 一种可 封闭或调节有丝分裂激酶 AS -26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有丝分裂激酶 ASK1- 26的分子。
"调节" 是指有丝分裂激酶 ASK1- 26的功能发生改变, 包括蛋白质活性的 升高或降低、 结合特性的改变及有丝分裂激酶 ASK1- 26的任何其它生物学性质、 功能或免疫性质的改变。 An "antagonist" or "inhibitor" refers to a molecule that can block or regulate the biological or immunological activity of mitogen kinase AS-26 when combined with mitogen kinase ASK1-26. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to mitogen kinase ASK1-26. "Regulation" refers to a change in the function of the mitotic kinase ASK1-26, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of the mitogen kinase ASK1-26.
"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有丝分裂激酶 ASKl-26。 基本上纯的有丝分裂激酶' ASK1-26 在非还原性聚丙烯酰胺凝胶上能产生单一的主 带。 有丝分裂激酶 ASK1- 26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 mitotic kinase ASKl-26 using standard protein purification techniques. The essentially pure mitotic kinase 'ASK1-26 produces a single main band on a non-reducing polyacrylamide gel. The purity of mitotic kinase ASK1- 26 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 polynucleotides 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竞争和抑制完 全同源的序列与靶序列在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 the complete mutual complementarity of a sequence with a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to target sequences 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(Hi gg ins, D. G. 和 P. M. Sharp (1988) Gene 73: 237-244) 0 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 identical 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 (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method will check the distance between all pairs by Groups of sequences are arranged in clusters. 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 the following formula:
― 序列 ^与序列 S之间匹配的残基个数 X 11 Λ ()Λ() ― The number of matching residues between the sequence ^ and the sequence S X 1 1 Λ () Λ ()
序列 的残基数 -序列 ^中间隔残基数―序列 ^中间隔残基数 Number of residues in sequence-number of interval residues in sequence ^-number of interval residues in sequence ^
也可以通过 Cl us 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(He in J. , (1990) Methods in enzymology 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。 The percent identity between nucleic acid sequences can also be determined by Cluster method or by methods known in the art, such as Jotun Hein (He in J., (1990) Methods in enzymology 183: 625-645). "" Similarity "is 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 can include aspartic acid and glutamic acid; positively charged Amino acids can include lysine and arginine; have uncharged Amino acids with similar hydrophilicity in the head group may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylpropyl 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. "Antisense strand" refers to a nucleic acid strand that is complementary to a "sense strand."
"衍生物" 是指 HFP或编码其核酸的化学修饰物。 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 HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties 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 性结合有丝分裂激酶 ASK1- 26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 antigenic determinant of mitogen kinase ASK1- 26.
"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系统中 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 is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. 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 part 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 in the natural state .
如本文所用, "分离的有丝分裂激酶 ASK1- 26" 是指有丝分裂激酶 ASK1 - 26 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有丝分裂激酶 ASKl-26。 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。 有丝分裂激酶 ASK1-26 多肽的 纯度能用氨基酸序列分析。 As used herein, "isolated mitokinase ASK1- 26" means that the mitogen kinase ASK1-26 is substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify the mitogen kinase ASKl-26 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the mitotic kinase ASK1-26 peptide can be analyzed by amino acid sequence.
本发明提供了一种新的多肽——有丝分裂激酶 ASK1-26 , 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。
本发明还包括有丝分裂激酶 ASK1- 26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有丝 分裂激酶 ASK1- 26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 ( Π Ι ) 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 present invention provides a new polypeptide, mitogen kinase ASK1-26, which basically consists 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. The present invention also includes fragments, derivatives, and analogs of the mitogen kinase ASK1-26. 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 mitotic kinase ASK1-26 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) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (Π Ι) Such a polypeptide sequence in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as the leader or secretory sequence or the sequence used to purify the polypeptide or protease sequence). As set forth 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 1531 个碱基, 其开放读框 104-820编码了 238个氨 基酸。 根据基因芯片表达谱比较发现, 此多肽与有丝分裂激酶 ASK1 有相似的 表达谱, 可推断出该有丝分裂激酶 ASK1- 26 具有有丝分裂激酶 ASK1 相似的功 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. It contains a full-length polynucleotide sequence of 1,531 bases, and its open reading frames 104-820 encode 238 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile as mitogen kinase ASK1, and it can be inferred that the mitogen kinase ASK1- 26 has similar functions as mitogen kinase ASK1.
本发明的多核苷酸可以是 DNA形式或是 RM形式。 DNA形式包括 cDM、 基 因组 DNA 或人工合成的 DNA。 DNA 可以是单链的或是双链的。 DM 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 RM. DNA forms include cDM, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DM can be coded or non-coded. 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 comprising the polypeptide and a polynucleotide comprising 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、 缺失变异体和插入变异体。 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、 缺失或插入, 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. Variants of this polynucleotide can be days Naturally occurring allelic or non-naturally occurring variants. 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 70%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 0)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%, preferably 70% identity, between the two sequences). 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 l, 42 ° C, etc .; or 0) only the same between the two sequences Crosses occur only when the sex 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 /或分离编码有丝分裂激酶 ASK1- 26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 the mitogenic kinase ASK1- 26.
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。 本发明的编码有丝分裂激酶 ASK1-26 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 mitotic kinase ASK1-26 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 DNA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 DM序列以获得所述多肽的双链 DM。 The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DM 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 fflRM 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 Manua l, Cold Spr ing 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 fflRM extraction, and kits are also commercially available (Qiagene). The construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spruing 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): (1) DM- DM 或 DM-RNA 杂交; (2)标志基因功能的出现或丧失; (3)测 定有丝分裂激酶 ASK1-26 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。 These genes can be screened from these cDNA libraries by conventional methods. These methods include (but not (Limited to): (1) DM-DM or DM-RNA hybridization; (2) the appearance or loss of marker gene function; (3) determination of the transcript level of mitogen kinase ASK1-26; (4) through immunological techniques or determination 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 ΜΑ序列。 本发明的基因本身或者片段当然可以 用作探针。 DM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 usually a MA 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. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
在第(4)种方法中, 检测有丝分裂激酶 ASK1-26 基因表达的蛋白产物可用 免疫学技术如 Wes tern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 protein products expressed by the mitotic kinase ASK1-26 gene.
应 用 PCR 技术 扩增 DNA/RNA 的 方 法 (Sa iki , et a l. Sc ience 1985; 230: 1350-1 354)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 DM/RM片段。 A method (Sa iki, et al. Science 1985; 230: 1350-1 354) using PCR technology to amplify DNA / RNA 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, and the primers 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 DM / RM fragment 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 DM 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 e t a l . 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 cDM序列。 The polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be measured 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 needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDM 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 用有丝分裂激酶 ASK1- 26 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 mitotic kinase ASK1- 26 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .
本发明中, 编码有丝分裂激酶 ASK1-26 的多核苷酸序列可插入到载体中, 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 the mitogen kinase ASK1-26 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 a host, any plasmid and vector can be used to construct a recombinant expression vector. Express An important feature of 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有丝分裂激酶 ASK1-26 的 DM 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 DNA 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 DM序列可有效连接到表达载体中的适当启动子上, 以指导 mRM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。 在载体中插入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 an expression vector containing a DM sequence encoding the mitotic kinase ASK1-26 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, Coll Spring Harbor Laboratory. New York, 1989). The DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM 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. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication. ,
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。 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, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
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。 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, 编码有丝分裂激酶 ASK1- 26 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 Sf 9; 动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。 In the present invention, a polynucleotide encoding a mitogen kinase ASK1-26 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 a 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 Sf 9; 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 DM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 0&( 12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 according to the present invention or a recombinant vector containing the DM 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 DNA uptake can be in the exponential growth phase were harvested, treated with 0 & (Method 12, using the procedure well known in the art. Alternatively, it is a MgCl 2. 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, liposome packaging, etc.
通过常规的重组 DM技术, 利用本发明的多核苷酸序列可用来表达或生产 重组的有丝分裂激酶 ASK1-26 (Sc ience , 1984; 224: 1431)。 一般来说有以下 步骤: By the conventional recombinant DM technology, the polynucleotide sequence of the present invention can be used to express or produce the recombinant mitotic kinase ASK1-26 (Science, 1984; 224: 1431). Generally there are the following steps:
(1) 用本发明的编码人 有丝分裂激酶 ASK1-26 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 the human mitogen kinase ASK1-26 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。 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its 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.
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。 The polypeptides of the present invention, as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
有丝分裂激活蛋白 (MAP ) 串联激酶能感应于细胞外各种刺激而被激活, 这些细胞外刺激包括生长因子和环境压力。 一个 MAP三激酶 (MAPKKK ) , 命名 为 ASK1 , 能够激活两个不同的 MAP二激酶 (MAPKK ) 亚群, SEK1 (或 MKK4 ) 和 MKK3/MAPKK6 (或 MKK6 ) , 这些 MAP 二激酶依次分别激活压力激活蛋白激酶 ( SAPK ) 和 MAP激酶的 P38亚群。 ASK1 的过度表达能诱导细胞的程序性死亡, 并且 ASK1在细胞内被激活后用来对付肿瘤坏死因子 - a ( TNF- α )。 另外, ASK1 的催化非活性形式能抑制 TNF- cc诱导的细胞编程性死亡。 ASK1 可能是环境压 力和细胞因子诱导的细胞编程性死亡机制的关键因素。 Mitogen-activated protein (MAP) tandem kinase can be activated by sensing various extracellular stimuli, including extracellular growth factors and environmental stress. One MAP trikinase (MAPKKK), named ASK1, can activate two different MAP dikinase (MAPKK) subgroups, SEK1 (or MKK4) and MKK3 / MAPKK6 (or MKK6). These MAP two kinases activate pressure activation in turn, respectively Protein kinase (SAPK) and P38 subgroups of MAP kinases. ASK1 overexpression can induce programmed cell death, and ASK1 is activated against TNF-a (TNF-α) after being activated in cells. In addition, the catalytically inactive form of ASK1 can inhibit TNF-cc-induced programmed cell death. ASK1 may be a key factor in environmental stress and cytokine-induced programmed cell death mechanisms.
MAP歡酶信号级联简略过程是 MAPKKK磷酸化 MAPKK从而激活 MAPKK, 激活 后的 MAPKK又依次磷酸化激活 MAPK。 激活的 MAPK可以转运细胞核并且调节转 录因子的激活从而控制基因的表达。
本发明的多肽的表达谱与人有丝分裂激活蛋白的三激酶 ASK1 的表达谱相 一致, 两者具有相似的生物学功能。 本发明的多肽在体内有着各种重要的作用, 主要感应于生长因子和环境压力, 从而调节机体的多种生理功能, 本发明的多 肽的过度表达能诱导细胞的程序性死亡, 并且本发明的多肽在细胞内被激活后 用来对付肿瘤坏死因子 - α。另外,本发明的多肽的催化非活性形式能抑制 TNF- α诱导的细胞编程性死亡。 它可能是环境压力和细胞因子诱导的细胞编程性死 亡机制的关键因素。 因此, 其表达异常通常与肿瘤发生、 胚胎发育紊乱、 生长 发育障碍、 炎症、 免疫调节紊乱的发生密切相关, 并产生相关的疾病。 The simple process of the MAP enzyme signal cascade is that MAPKKK phosphorylates MAPKK and activates MAPKK. After activation, MAPKK activates MAPK in turn. Activated MAPK can transport the nucleus and regulate the activation of transcription factors to control gene expression. The expression profile of the polypeptide of the present invention is consistent with the expression profile of the trikinase ASK1 of human mitotic activation protein, and both have similar biological functions. The polypeptide of the present invention has various important functions in the body, mainly sensing growth factors and environmental pressure, thereby regulating various physiological functions of the body. The overexpression of the polypeptide of the present invention can induce programmed cell death, and the present invention Peptides are activated in cells to fight tumor necrosis factor-α. In addition, the catalytically inactive form of the polypeptide of the present invention can inhibit TNF-α-induced programmed cell death. It may be a key factor in environmental stress and cytokine-induced programmed cell death mechanisms. Therefore, its abnormal expression is usually closely related to the occurrence of tumors, embryonic developmental disorders, growth and development disorders, inflammation, and immune regulation disorders, and produce related diseases.
由此可见, 本发明的有丝分裂激酶 ASK1-26 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、 炎症、 免疫性疾病, 这些疾病包括但不限于: It can be seen that the abnormal expression of the mitogen kinase ASK1-26 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, growth and development disorders, inflammation, and immune diseases. These diseases include, but are not limited to:
各种组织的胂瘤: 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, 膀胱癌, 子官癌, 子宫内膜癌, 胸腺肿瘤, 鼻咽癌, 喉癌, 气管肿瘤, 纤维瘤, 纤维肉瘤, 脂肪瘤, 脂肪肉瘤 Tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Neurofibromas, Colon Cancer, Melanoma, Bladder Cancer, Child Cancer, Endometrial Cancer, Thymic Tumor, Nasopharyngeal Cancer, Laryngeal Cancer, Tracheal Tumor, Fibroma, Fibrosarcoma, Lipoma, Liposarcoma
胚胎发育紊乱症: 先天性流产, 腭裂, 肢体缺如, 肢体分化障碍, 房间隔 缺损, 神经管缺陷, 先天性脑积水, 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, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
生长发育障碍性疾病: 精神发育迟缓, 脑发育障碍, 皮肤、 脂肪和肌肉发 育不良性疾病, 骨与关节发育不良性疾病, 各种代谢缺陷病, 呆小症, 侏儒症, 库兴综合症, 性发育迟缓症 Growth and development disorders: mental retardation, brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing syndrome, Sexual retardation
炎症: 慢性活动性肝炎, 结节病, 多肌炎, 慢性鼻炎, 慢性胃炎, 脑脊髓 多发性硬化, 肾小球性肾炎, 心肌炎, 心肌病, 动脉粥样硬化, 胃溃疡, 子宫 颈炎, 各种感染性炎症 Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
免疫性疾病: 系统性红斑狼疮, 类风湿性关节炎, 支气管哮喘, 荨麻疹, 特异性皮炎, 感染后心肌炎, 硬皮病, 重症肌无力, 格林-巴利综合症, 普通 易变免疫缺陷病, 原发性 Β淋巴细胞免疫缺陷病, 获得性免疫缺陷综合症 Immune diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
本发明的有丝分裂激酶 ASK1- 26 的表达异常还将产生某些遗传性, 血液性 疾病等。 Abnormal expression of the mitotic kinase ASK1-26 of the present invention will also produce certain hereditary, hematological diseases and the like.
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、 炎症、 免疫性疾病, 某些遗传性, 血液性疾病等。
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)有丝分裂 激酶 ASK1-26 的药剂的方法。 激动剂提高有丝分裂激酶 ASK1-26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有丝分裂激酶 ASK1-26 的膜制 剂与标记的有丝分裂激酶 ASK1-26 —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, inflammation, and immunity. Sexual diseases, certain hereditary, blood diseases, etc. The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) mitotic kinase ASK1-26. Agonists enhance biological functions such as mitotic kinase ASK1-26 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, a mammalian cell or a membrane preparation expressing the mitotic kinase ASK1-26 can be cultured with the labeled mitogen kinase ASK1-26 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined. '
有丝分裂激酶 ASK1- 26 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。 有丝分裂激酶 ASK1-26 的拮抗剂可以与有丝分裂激酶 ASK1-26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 mitogen kinase ASK1-26 include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of the mitogen kinase ASK1-26 can be combined with the mitogen kinase ASK1-26 to eliminate its function, or to inhibit the production of the polypeptide, or to bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
在筛选作为拮抗剂的化合物时, 可以将有丝分裂激酶 ASK1-26 加入生物分 析测定中, 通过测定化合物对有丝分裂激酶 ASK1- 26 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。 能与有丝分裂激酶 ASK1-26 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, 一般应对有丝分裂激酶 ASK1- 26分子进行标记。 In screening compounds that act as antagonists, mitotic kinase ASK1-26 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between mitogen kinase ASK1- 26 and its receptor. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to mitotic kinase ASK1-26 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the mitogen-kinase ASK1- 26 molecule 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 了针对有丝分裂激酶 ASK1- 26 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 invention also provides antibodies against the mitotic kinase ASK1- 26 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有丝分裂激酶 ASK1- 26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。 制备有丝分裂激酶 ASK1- 26 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 tein. 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)。 而已有 的生产单链抗体的技术(U. S. Pa t No. 4946778)也可用于生产抗有丝分裂激酶 ASK1-26的单链抗体。 Polyclonal antibodies can be produced by injecting mitotic kinase ASK1- 26 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 mitotic kinase ASK1- 26 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV -Hybridoma technology, etc. Chimeric antibodies that bind human constant regions to non-human-derived variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851). The existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against mitogen-kinase ASK1-26.
抗有丝分裂激酶 ASK1- 26 的抗体可用于免疫组织化学技术中, 检测活检标 本中的有丝分裂激酶 ASK1- 26。 Antibodies against mitotic kinase ASK1- 26 can be used in immunohistochemical techniques to detect mitotic kinase ASK1- 26 in biopsy specimens.
与有丝分裂激酶 ASK1-26 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 mitotic kinase ASK1-26 can also be labeled with radioisotopes. Its location and distribution can be tracked into the body. 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。 如有丝分裂激酶 Antibodies can also be used to design immunotoxins that target a particular part of the body. Mitogen kinase
ASK1-26 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有丝 分裂激酶 ASK1- 26阳性的细胞。 ASK1-26 high affinity monoclonal antibodies 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 the antibody with a thiol cross-linking agent such as SPDP. Through the exchange of disulfide bonds, 'bind the toxin to the antibody. This hybrid antibody can be used to kill mitotic kinase ASK1- 26 positive cell.
本发明中的抗体可用于治疗或预防与有丝分裂激酶 ASK1- 26相关的疾病。 给予适当剂量的抗体可以刺激或阻断有丝分裂激酶 ASK1- 26的产生或活性。 The antibodies of the present invention can be used to treat or prevent diseases related to the mitogen kinase ASK1-26. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of the mitogen kinase ASK1-26.
本发明还涉及定量和定位检测有丝分裂激酶 ASK1- 26 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 所检测的有丝分裂激酶 ASK1- 26水平, 可以用作解释有丝分裂激酶 ASK1- 26在 各种疾病中的重要性和用于诊断有丝分裂激酶 ASK1-26起作用的疾病。 The invention also relates to a diagnostic test method for quantitative and localized detection of the levels of mitotic kinase ASK1- 26. These tests are well known in the art and include FISH assays and radioimmunoassays. The levels of mitotic kinase ASK1- 26 detected in the test can be used to explain the importance of mitogen kinase ASK1- 26 in various diseases and to diagnose diseases in which mitogen kinase ASK1-26 plays a role.
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.
编码有丝分裂激酶 ASK1- 26 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有丝分裂激酶 ASK1- 26 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 表达变异的有丝分裂激酶 ASK1-26 , 以抑制内源性的有丝分裂激酶 ASK1-26 活 性。 例如, 一种变异的有丝分裂激酶 ASK1-26 可以是缩短的、 缺失了信号传导 功能域的有丝分裂激酶 ASK1- 26 , 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有丝分裂激酶 AS - 26 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有丝分裂激酶 ASK1-26 的多核 苷酸转移至细胞内。 构建携带编码有丝分裂激酶 ASK1- 26 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, 重组编码有丝分裂 激酶 ASK1-26的多核苷酸可包装到脂质体中转移至细胞内。 Polynucleotides encoding the mitotic kinase ASK1- 26 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of mitotic kinase ASK1- 26. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated mitogen kinase ASK1-26 to inhibit endogenous mitogen kinase ASK1-26 activity. For example, a mutated mitogen kinase ASK1-26 may be a shortened mitogen kinase ASK1--26 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of mitogen kinase AS-26. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the polynucleotide encoding the mitogen kinase ASK1-26 into the cell. Methods for constructing a recombinant viral vector carrying a polynucleotide encoding a mitotic kinase ASK1-26 can be found in the existing literature (Sambrook, et al.). In addition, the polynucleotide encoding the recombinant mitotic kinase ASK1-26 can be packaged into liposomes and transferred into cells.
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。 Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide 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.
抑制有丝分裂激酶 ASK1- 26 mRNA的寡核苷酸(包括反义 MA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 RM 特异性杂交后进行核酸内切作用。 反义 的 RNA和 DNA及核酶可用已有的任何 RM或 DM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已广泛应用。反义 RNA分子可通过编码该 RM 的 DM序列在体外或体内转录获得。 这种 DM序列已整合到载体的 RM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 MA and DNA) and nucleus that inhibit mitotic kinase ASK1- 26 mRNA Enzymes are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any existing RM or DM synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DM sequence encoding the RM. This DM sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
编码有丝分裂激酶 ASK1-26 的多核苷酸可用于与有丝分裂激酶 ASH- 26的 相关疾病的诊断。 编码有丝分裂激酶 ASK1-26 的多核苷酸可用于检测有丝分裂 激酶 ASK1-26 的表达与否或在疾病状态下有丝分裂激酶 ASK1-26 的异常表达。 如编码有丝分裂激酶 ASK1-26 的 DNA序列可用于对活检标本进行杂交以判断有 丝分裂激酶 ASK1-26 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。 用有丝分裂激酶 ASK1-26特异的引物进行 RM -聚合酶 链反应(RT-PCR)体外扩增也可检测有丝分裂激酶 ASK1-26的转录产物。 The polynucleotide encoding the mitogen kinase ASK1-26 can be used for the diagnosis of diseases related to the mitogen kinase ASH-26. The polynucleotide encoding the mitotic kinase ASK1-26 can be used to detect the expression of the mitogen kinase ASK1-26 or the abnormal expression of the mitogen kinase ASK1-26 in a disease state. For example, the DNA sequence encoding mitotic kinase ASK1-26 can be used to hybridize biopsy specimens to determine the expression of mitogen kinase ASK1-26. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for differential expression analysis and gene diagnosis of genes in tissues. RM-polymerase chain reaction (RT-PCR) in vitro amplification using mitogen-kinase ASK1-26-specific primers can also detect the transcript of mitogen-kinase ASK1-26.
检测有丝分裂激酶 ASK1- 26 基因的突变也可用于诊断有丝分裂激酶 ASK1- 26相关的疾病。 有丝分裂激酶 ASK1- 26突变的形式包括与正常野生型有丝分裂 激酶 ASH- 26 DNA 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。 Detection of mutations in the mitotic kinase ASK1- 26 gene can also be used to diagnose mitotic kinase ASK1- 26-related diseases. Mitokinase ASK1-26 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type mitotic kinase ASH-26 DNA 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, so 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 染色体具体位置并且可以与其杂交。 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 于标记染色体位置。 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. This sequence will specifically target 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。
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。 In short, PCR primers (preferably 15-35 bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. 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. PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, by a similar method, 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 hybrid pre-selection 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体位置, 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 V. Mckus ick, Mendel ian Inher i tance in Man (可通过与 Johns Hopkins Univers i ty Welch Medica l Library联机获 得)。 然后可通过连锁分析, 确定基因与业已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 V. Mckusick, Mendel ian Inherance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been 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。 有丝分裂激酶 ASK1-26 以有效地治疗和 /或预 防具体的适应症的量来给药。 施用于患者的有丝分裂激酶 ASK1- 26 的量和剂量
7 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。 实施例 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. The mitogen kinase ASK1-26 is administered in an amount effective to treat and / or prevent a specific indication. Amount and dose of mitotic kinase ASK1- 26 administered to patients 7 The scope 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. Examples
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 Spr ing Harbor Laboratory Pres s, 1989)中所述的条件, 或按照制造厂 商所建议的条件。 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. The experimental methods in the following examples are not marked with specific conditions, usually according to conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Harbor Laboratory Pres s, 1989), or according to the conditions described in Conditions recommended by the manufacturer.
实施例 1 有丝分裂激酶 ASK1- 26的克隆 Example 1 Cloning of mitotic kinase ASK1--26
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA Isolat 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 cDM片段定向插入到 pBSK (+) 载体 (Clontech公司产品)的多克隆位点上, 转化 DH5 a , 细菌形成 cDM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 cDM 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 0301gl 0的 cDNA序列为新的 DM。 通过合成一系列引物对该克隆所含的插入 cDNA片 段进行双向测定。结果表明, 0301g10克隆所含的全长 cDNA为 1531bp (如 Seq ID NO: 1 所示) , 从第 l ( bp至 820bp有一个 717bp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-0301g10, 编码的蛋白 质命名为有丝分裂激酶 ASK1- 26。 实施例 2 用 RT- PCR方法克隆编码有丝分裂激酶 ASK1-26的基因 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 Isolat 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 CDM fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a CDM library. Dye terminate cycle reaction 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 cDM sequence was compared with an existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0301gl 0 was a new DM. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the 0301 g 10 clone contained a full-length cDNA of 1531 bp (as shown in Seq ID NO: 1), from the l (bp to 820 bp has a 717 bp open reading frame (0RF), encoding a new protein ( (Shown in Seq ID NO: 2). We named this clone pBS-0301 g 10 and the encoded protein was named mitogen kinase ASK1- 26. Example 2 Cloning of the gene encoding mitogen kinase ASK1-26 by RT-PCR method
用胎脑细胞总 RNA为模板,以 ol igo- dT为引物进行逆转录反应合成 cDNA,用 Qiagene的试剂盒纯化后,用下列引物进行 PCR扩增: CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
Primerl: 5'- GGATTTAATATGAAGCACAGAAGC -3, (SEQ ID NO: 3) Primerl: 5'- GGATTTAATATGAAGCACAGAAGC -3, (SEQ ID NO: 3)
Pr imer 2: 5,- TGGTTGACAGTTTATTAAATACAC -3, (SEQ ID NO: 4) Pr imer 2: 5,-TGGTTGACAGTTTATTAAATACAC -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;
Pr imer2为 SEQ ID NO: 1的中的 3'端反向序列。 Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
扩增反应的条件: 在 50μ1的反应体积中含有 50mmol/L KCl, 10mmol/L Tri s-HCl pH8. 5, 1. 5mmol/L MgCl2, 20(^mol /L dNTP, l Opmol引物, 1U的 Taq DNA聚合酶 (Clontech公司产品)。 在 PE9600型 DM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 (3 -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) 。 DM序列分析结果表明 PCR 产物的 DNA序列与 SEQ ID NO: 1所示的 1- 1531bp完全相同。 实施例 3 Northern 印迹法分析有丝分裂激酶 ASK1-26基因的表达 用一步法提取总 RNA [Anal. Bi ochem 1987, 162, 156-159] 0 该法包括酸性硫 氰酸胍苯酚 -氯仿抽提。 即用 4M异硫氰酸胍- 25 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 RM沉淀。 将 得到的 RM沉淀用 70%乙醇洗涤, 干燥并溶于水中。 用 20μ§ 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 DM探针为图 1所示的 PCR扩增的有丝分裂激酶 ASK1-26编码区序 列(104bp至 820bp)。 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 -25niM KH2P04 ( pH7. 4 ) - 5 x SSC- 5 χ Denhardt's溶液和 20(^g/ml鲑精 DNA。 杂交之后, 将滤膜在 l 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 4 重组有丝分裂激酶 ASK1- 26的体外表达、 分离和纯化 Amplification reaction conditions: containing 5 0mmol / L KCl in a reaction volume of 50μ1, 10mmol / L Tri s-HCl pH8 5, 1. 5mmol / L MgCl 2, 20 (^ mol / L dNTP, l Opmol primer. 1U Taq DNA polymerase (Clontech). PE9600 DM thermal cycler (Perkin-Elmer) according to the following conditions. Response 25 cycles: 94. C 30sec; 55 ° C 30sec; 72. C 2min. During RT-PCR, (3-act in was used as a positive control and template blank was used as a negative control. The amplified product was purified using a QIAGEN kit and the TA cloning kit was connected to a pCR vector (Invitrogen)). The results of DM sequence analysis showed that the DNA sequence of the PCR product was exactly the same as 1- 1531bp shown in SEQ ID NO: 1. Example 3 Northern blot analysis of the expression of mitogen kinase ASK1-26 gene Total RNA was extracted in one step [Anal. Bi ochem 1987, 162, 156-159] 0 This method includes acid guanidine thiocyanate phenol-chloroform extraction. 4M guanidine isothiocyanate-25 sodium citrate, 0.2 M sodium acetate (pH 4.0) Perform homogenization, add 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 volume) and centrifuge the mixture The RM precipitate was obtained. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water. Using 20 μ § RNA, in 20mM 3- (N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate -ImM EDTA- 2. 2M formaldehyde on a 1.2% agarose gel for electrophoresis. Then transfer to nitrate Acid cellulose membrane. A- 32 P dATP was used to prepare 32 P-labeled DNA probes by random primers. The DM probe used was the PCR-amplified mitotic kinase ASK1-26 coding region sequence (104bp) shown in Figure 1. To 820bp). 32P-labeled probe (approximately 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-25niM KH 2 P0 4 (pH7. 4)-5 x SSC- 5 x Denhardt's solution and 20 g / ml salmon sperm DNA. After hybridization, the filter was washed in lx SSC- 0.1% SDS at 55 ° C. 30min. Then, Phosphor Imager was used for analysis and quantification. Example 4 In vitro Expression, Isolation and Purification of Recombinant Mitotic Kinase ASK1- 26
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:
Pr imer3: 5'- CATGCTAGCATGGCAGAAATTAGTCGAATTCAG -3, ( Seq ID No: 5 ) Pr imer4: 5'- CATGGATCCCTAGCGGCGAAACCAGAGCCAGCT -3, ( Seq ID No: 6 ) 此两段引物的 5'端分别含有 Nhel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, Nhel和 BamHI酶切位点相应于表达载体质粒 pET- 28b (+) (Novagen公司产品, Ca t. No. 69865. 3)上的选择性内切酶位点。 以含有全长 目的基因的 pBS- 0301gl 0质粒为模板, 进行 PCR反应。 PCR反应条件为: 总体积 50μ1 中含 pBS-0301gl O质粒 10pg、 引物 Pr imer—3和 Pr imer—4分别为 l Opmol、 Advantage polymerase Mix ( Clontech公司产品) 1μ1。 循环参数: 94°C 20s, 60°C 30s, 68°C 2 min,共 25个循环。 用 Nhel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 DH5 cc,在
含卡那霉素 (终浓度 3(^g/ml ) 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-0301glO )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-0301glO ) 在 37°C培养至对数生 长期, 加入 IPTG至终浓度, lmmol/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( 6Hi s-Tag )结合的亲和层析柱 His. Bind Quick Cartr idge ( Novagen公司产品)进行层析, 得到了纯化的目的蛋白有丝分裂激酶 ASK1- 26。 经 SDS-PAGE电泳, 在 KDa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 5 抗有丝分裂激酶 ASK1-26抗体的产生 Pr imer3: 5'- CATGCTAGCATGGCAGAAATTAGTCGAATTCAG -3, (Seq ID No: 5) Pr imer4: 5'- CATGGATCCCTAGCGGCGAAACCAGAGCCAGCT -3, (Seq ID No: 6) The 5 'ends of these two primers contain Nhel and BamHI digestion sites, respectively Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively. The Nhel and BamHI digestion sites correspond to the expression vector plasmid pET-28b (+) (Novagen Corporation product, Ca t. No. 69865. 3) Selective endonuclease site. The pBS-0301gl 0 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-0301gl O plasmid, primers Primer-3 and Primer-4 were 1 Opmol, Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed using the calcium chloride method for coliform bacteria DH5 cc. After the LB plates containing kanamycin (final concentration 3 (^ g / ml)) were cultured overnight, the positive clones were screened by colony PCR and sequenced. The positive clones with the correct sequence (pET-0301glO) were selected by the calcium chloride method. the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (Novagen Co.). in containing kanamycin (final concentration of 30μ § / πι1) in LB liquid medium, host strain BL21 (P ET-0301glO) at 37 ° C. Cultivate to the logarithmic growth phase, add IPTG to the final concentration, 1 mmol / L, and continue the cultivation for 5 hours. Centrifuge to collect the bacterial cells, sonicate the bacteria, centrifuge to collect the supernatant, and use 6 histidine (6Hi s- Tag) affinity chromatography column His. Bind Quick Cartr idge (Novagen) was used to perform chromatography, and the purified target protein mitotic kinase ASK1- 26 was obtained . After SDS-PAGE electrophoresis, a single strip was obtained at KDa. Band (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, 15 N-terminal amino acids and 15 N-terminal amino acid residues shown in SEQ ID NO: 2 The groups are identical. Example 5 Anti-mitotic kinase ASK1-26 Production of antibodies
用多肽合成仪(PE公司产品)合成下述有丝分裂激酶 ASK1 - 26特异性的多肽: NH2-Met-Ala-Glu-I le-Ser-Arg-I l e-G 1 n-Tyr-G 1 u- Me t -G 1 u-Tyr-Thr-G 1 u-COOH (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物, 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 15μ πι1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 Sepharos B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有丝分裂激酶 ASK1 - 26 结合。 实施例 6 本发明的多核苷酸片段用作杂交探针的应用 Polypeptide synthesizer (product of PE company) was used to synthesize the following mitogen-kinase ASK1- 26-specific peptides: NH2-Met-Ala-Glu-I le-Ser-Arg-I l eG 1 n-Tyr-G 1 u- Me t -G 1 u-Tyr-Thr-G 1 u-COOH (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex. 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. The titer of antibodies in rabbit serum was determined by ELISA using a 15 μm bovine serum albumin peptide complex-coated titer plate. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharos B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to the mitotic kinase ASK1-26. Example 6 Application of the 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 cDM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。 The suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects. For example, the probes can be used to hybridize to the genome or CDM library 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 using a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern blotting Trace method, Northern blotting method and copying method, etc., are all used to fix the polynucleotide sample to be tested on the filter membrane and then hybridize using basically the same steps. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals. 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 embodiment, 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 SBQ 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 SBQ ID NO: 1 of the present invention 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 )和其它巳知的基因组序列 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 unknown genomic sequences and their complements The regions 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, then the primary probe should not be used;
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。 完成以上各方面的分析后挑选并合成以下二个探针: 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments. 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'- TGGCAGAAATTAGTCGAATTCAGTACGAAATGGAATATACT -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 (41M ): 5'- TGGCAGAAATTAGTCGAATTCAGTACGAAATGGAATATACT -3 '(SEQ ID NO: 8) Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41M) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
5'- TGGCAGAAATTAGTCGAATTCAGTACGAAATGGAATATACT -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: DM PROBES G. H. Kel ler; M. M. Manak; Stockton Pres s, 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'- TGGCAGAAATTAGTCGAATTCAGTACGAAATGGAATATACT -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: DM PROBES GH Kel ler; MM Manak; Stockton Pres s, 1989 (USA) and more commonly used molecular cloning experiment manuals such as the "Molecular Cloning Experiment Guide" (1998 Second Edition) [US] Sambu Luke waiting, Science Press.
样品制备: Sample Preparation:
1 , 从新鲜或冰冻组织中提取 DM 1.Extract DM 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; 25mmol/L NaCl; 25mmol/L MgCl2 ) 悬浮沉淀(大约 lOml/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 10分钟。 6)用重悬细胞沉淀(每 0. lg最初组织样品加 1- 5ml), 再以 lOOOg离心 10分钟。 7)用裂解缓冲液重悬沉淀(每 0. lg最初组织样品加 lral), 然后接以下 的苯酚抽提法。 Steps: 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 pellet (approximately 10 ml / g) with cold homogenization buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 mmol / L NaCl; 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) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (1 to 5 ml per 0.1 g of the original tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet in lysis buffer (lral is added every 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, phenol extraction method for DNA
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 (l x lO8细胞 /nil) 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。 Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000 g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (lx 10 8 cells / nil). 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)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。 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) Extract with 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 1/10体积 2mol/L醋酸钠和 2倍体积冷 100%乙醇加到 DM溶液 中, 混匀。 在- 20°C放置 1小时或过夜。 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- 5χ 10ό细胞所 提取的大约加 lul。 Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DM 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- 5χ 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 RM酶 A加到 DM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 0.5%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 A26。和 A28。以检测 DM的纯度及产率。 15)分装后存放于 -20° (:。 8) Add RM enzyme A to the DM solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9 ) Add SDS and proteinase K to the final concentration of 0.5% and 100ug / ml. Incubate 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 pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Determine A 26 . And A 28 . To check the purity and yield of DM. 15) Store at -20 ° (:) after packing.
样膜的制备: Preparation of sample film:
1)取 4x2 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 4x2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are required for each probe, so that they can be used in the following experimental steps. The film was 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. lmol/L NaOH, 1.5raol/L NaCl的滤纸上 5分钟 (两次), 晾干置于浸润有 0.5raol/L Tris-HCl (pH7.0 ), 3mol/L NaCl的滤纸上 5分钟 (两 次), 晾干。 ' 3) Place on filter paper infiltrated with 0.1mol / L NaOH, 1.5raol / L NaCl for 5 minutes (twice), dry and put in 0.5raol / L Tris-HCl (pH7.0), 3mol / L NaCl filter paper for 5 minutes (twice) and allowed to dry. '
4)夹于干净滤纸中, 以铝箔包好, 60-S0°C真空干燥 2小时。 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-S0 ° C for 2 hours.
探针的标记 Labeling of probes
1) 3μ1 Probe ( 0. IOD/Ιθμΐ ), 加入 2μ1 Kinase缓冲液, 8-10 uCi γ- 32P - dATP+2U Kinase, 以补加至终体积 20μ1。 1) 3μ1 Probe (0.1OD / Ιθμΐ), add 2μ1 Kinase buffer, 8-10 uCi γ- 32 P-dATP + 2U Kinase, to make up to a final volume of 20μ1.
2) 37 °C 保温 2小时。 2) Incubate at 37 ° C for 2 hours.
3)加 1/5体积的溴酚蓝指示剂 (BPB)。 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).
4 ) 过 Sephadex G-50柱。 4) Pass through a Sephadex G-50 column.
5 ) 至有 32P- Probe洗出前开始收集第一峰(可用 Monitor监测)。 5) Before the 32 P-Probe is washed out, start collecting the first peak (can be monitored by Monitor).
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 )。 8) combining the first peak was collected after 32 P-Prob e is prepared as required (the second peak to the free γ- 32 Ρ- dATP).
预杂交 Pre-hybridization
将样膜置于塑料袋中,加入 3-1 Omg预杂交液( lOxDenhardt-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。 The sample membrane was placed in a plastic bag, and 3-1 Omg pre-hybridization solution (lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM)) was added. After closing the bag, 68. C. Water 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水洛摇过夜。 洗膜:
高强度洗膜: Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake at 42 ° C in water overnight. Wash film: 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, 40. C Wash for 15 minutes (twice).
3 ) 0. lxSSC, 0. 1%SDS中, 40°C洗 15分钟 ( 2次)。 3) Wash in 0.1xSSC, 0.1% SDS at 40 ° C for 15 minutes (twice).
4 ) 0. lxSSC, 0. 1°/。SDS中, 55°C洗 30分钟 ( 2次), 室温晾干。 低强度洗膜: 4) 0.1xSSC, 0.1 ° /. Wash in SDS at 55 ° C for 30 minutes (twice) and dry at room temperature. Low-intensity washing film:
1 )取出巳杂交好的样膜。 1) Take out the sample membrane of hybridization.
2 ) 2xSSC, 0. Γ/oSDS中, 37。C洗 15分钟 ( 2次)。 2) 2xSSC, 0. Γ / oSDS, 37. C wash for 15 minutes (twice).
3 ) 0. lxSSC, 0. 1 SDS中 , 37°C洗 15分钟 ( 2次)。 3) Wash in 0.1xSSC, 0.1 SDS at 37 ° C 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:
采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。 实施例 7 DNA Microarray The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger. To the radioactive intensity of the hybridization spot of another probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1. Example 7 DNA Microarray
基因芯片或基因微矩阵 (DNA Microarray )是目前许多国家实验室和大制药 公司都在着手研制和开发的新技术, 它是指将大量的靶基因片段有序地、 高密度 地排列在玻璃、 硅等载体上, 然后用荧光检测和计算机软件进行数据的比较和分 析, 以达到快速、 高效、 高通量地分析生物信息的目的。 本发明的多核苷酸可作 为靶 DNA 用于基因芯片技术用于高通量研究新基因功能; 寻找和筛选组织特异性 新基因特别是肿瘤等疾病相关新基因; 疾病的诊断, 如遗传性疾病。 其具体方法 步骤在文献中巳有多种报道, 如可参阅文献 DeRi s i, J. L. , Lyer, V. &Brown, P. 0. (1997) Science 278, 680-686.及文献 Hel l e, R. A. , Schema, M., Chai, A., Shalom, D. , (1997) PNAS 94: 2150-2155. Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature. For example, see the literature DeRi si, JL, Lyer, V. & Brown, P. 0. (1997) Science 278, 680-686. And the literature Hel le, RA, Schema , M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
(一) 点样 (A) spotting
各种不同的全长 cDM共计 4000条多核苷酸序列作为靶 DM,其中包括本发明
的多核苷酸。 将它们分别通过 PCR 进行扩增, 纯化所得扩增产物后将其浓度调到 500ng/ul左右, 用 Cartes ian 7500点样仪(购自美国 Cartes ian公司)点于坡璃介 质上, 点与点之间的距离为 280μΐη。 将点样后的玻片进行水合、 干燥, 置于紫外 交联仪中交联, 洗脱后干燥使 DNA 固定在玻璃片上制备成芯片。 其具体方法步骤 在文献中巳有多种报道。 本实施例的点样后处理步骤是: A total of 4,000 polynucleotide sequences of various full-length cDM as target DM, including the present invention Polynucleotide. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a sloped glass medium using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 μΐη. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slide to prepare a chip. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
1. 潮湿环境中水合 4小时; 1. Hydration in a humid environment for 4 hours;
2. 0. 2%SDS洗涤 1分钟; 2. 0.2% SDS was washed for 1 minute;
3. ddH20洗涤两次, 每次 1分钟; 3. Wash twice with ddH 2 0 for 1 minute each time;
4. NaBH4封闭 5分钟; 4. NaBH 4 is blocked for 5 minutes;
5. 95°C水中 2分钟; 5. 95 ° C water for 2 minutes;
6. 0. 2%SDS洗涤 1分钟; 6. Wash with 0.2% SDS for 1 minute;
7. dd 0冲洗两次; 7. dd 0 flush twice;
8. 凉干, 25°C储存于暗处备用。 8. Dry and store at 25 ° C in the dark for future use.
(二)探针标记 (Two) probe marking
用一步法分别从人体混合组织与机体特定组织 (或经过刺激的细胞株) 中抽 提总 m丽, 并用 Ol igotex mRNA Midi Ki t (购自 QiaGen公司)纯化 mRNA,通过反转 录分另!]将焚光试亦 j Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye, 购自 Amersham Phamacia Biotech公司)标记 人体混合组织的 mRNA, 用荧光试剂 Cy5dUTP (5- Amino-propargyl-2'- deoxyuridine 5'-triphate coupled to Cy5 f luorescent dye, 购自 Amersham Phamacia Biotech 公司)标记机体特定组织 (或经过刺激的细胞株) mRNA, 经纯化后制备出探针。 具 体步囅参照及方法见: Total mili was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen). ] Cyclone 3DUTP (5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP was used. (5- Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of specific tissues (or stimulated cell lines) of the body, and probes were prepared after purification. . For detailed steps and methods, see:
Schena, M. , Shalon, D,, Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614-10619. Schena, M. , Shalon, Dari. , Davi s, R. W. (1995) Science. 270 (20): 467-480. Schena, M., Shalon, D ,, Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614-10619. Schena, M., Shalon, Dari., Davi s, RW ( 1995) Science. 270 (20): 467-480.
(三)杂交 (Three) cross
分别将来自 以上两种组织的探针与芯片一起在 UniHyb™ Hybridizat ion Solut ion (购自 TeleChem公司)杂交液中进行杂交 16 小时, 室温用洗涤液 (l x SSC, 0. 2%SDS ) 洗涤后用 ScanArray 3000扫描仪 (购自美国 General Scanning公 司)进行扫描, 扫描的图象用 Imagene软件(美国 Biodi scovery公司)进行数据 分析处理, 算出每个点的 Cy3/Cy5比值。 The probes from the above two tissues and the chip were respectively hybridized in a UniHyb ™ Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
以上机体特定组织 (或经过刺激的细胞株)分别为胸腺、 睾丸、 肌肉、 脾脏、 肺、 皮肤、 甲状腺、 肝、 PMA+的 Ecv304细胞株、 PMA -的 Ecv304细胞株、 未饥饿的
L02细胞株、 砷刺激 1小时的 L02细胞株、 砷刺激 6小时的 L02细胞株前列腺、 心、 肺 癌、 胎膀胱、 胎小肠、 胎大肠、 胎胸腺、 胎肌、 胎肝、 胎肾、 胎脾、 胎脑、 胎肺 以及胎心。 根据这 26个 Cy3/Cy5比值绘出折方图 (图 1 ) 。 由图可见本发明所述的 有丝分裂激酶 ASK1- 26和有丝分裂激酶 ASK1表达谱很相似。
The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen , Fetal brain, fetal lung, and fetal heart. Draw a graph based on these 26 Cy3 / Cy5 ratios (Figure 1). It can be seen from the figure that the expression profiles of mitotic kinase ASK1- 26 and mitogen kinase ASK1 according to the present invention are very similar.