一种新的多肽——脂访氧化酶 10和编码这种多肽的多核苷酸 拮术领域 A New Polypeptide——Lipoxidase 10 and Polynucleotide Encoding This Polypeptide
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——脂肪氧 化酶 10, 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。 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, lipoxygenase 10, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
技术背景 technical background
脂类包含有很多不同种类的化合物, 它们具有以下几种生物学功能: U ) 在 所有细胞中, 膜的主要结构成分为脂类; (2 ) 有些脂类能有效的储存能量; (3 ) 动物中发现的很多维生素和激素是脂类或脂类的衍生物; (4 ) 在消化时, 胆汁酸 有助于溶解其他脂类。 Lipids contain many different types of compounds, which have the following biological functions: U) In all cells, the main structural component of the membrane is lipids; (2) some lipids can effectively store energy; (3) Many vitamins and hormones found in animals are lipids or lipid derivatives; (4) Bile acids help dissolve other lipids during digestion.
花生四烯酸是大多数哺乳动物主要的 C2Q多聚不饱和脂肪酸, 它可以通过酶的 催化变为很多种化合物, 例如, 前列腺素、 凝血恶烷和白三烯等。 在上述的催化 过程中, 脂肪氧化酶起到了重要的作用。 Arachidonic acid is the main C 2Q polyunsaturated fatty acid in most mammals. It can be catalyzed by enzymes into many compounds, such as prostaglandins, thromboxane, and leukotrienes. In the above-mentioned catalytic process, lipoxygenase plays an important role.
脂肪氧化酶是一类铁原子结合的双加氧酶, 它可以催化脂类的氢过氧化作用, 它的催化底物除了花生四烯酸外, 还包括顺, 顺 -1 , 4-戊二烯等等。 脂肪氧化酶 对于脂类的氧化作用有着重要的催化功能, 因此对于脂类的代谢、 脂类及其衍生 物的生理学功能的发挥等都有极其重要的意义。 Lipoxygenase is a type of iron-bound dioxygenase, which can catalyze the hydroperoxidation of lipids. Its catalytic substrate includes cis, cis-1, 4-pentane in addition to arachidonic acid. Ene and so on. Lipoxygenase has an important catalytic function for the oxidation of lipids, so it is of great significance for the metabolism of lipids and the physiological functions of lipids and their derivatives.
编码大豆脂肪氧化酶的基因的核苷酸序列已被得到, 大豆的脂肪氧化酶已经 发现的有三种, L0X1, L0X2 , L0X3. 虽然它们在核苷酸序列上有差异, 但是它们 都具有一个大约为 40个氨基酸残基的保守序列, 此保守序列含有 6个组氨酸和 2 个酪氨酸, 这个保守的序列在大豆的三种脂肪氧化酶中是高度保守的, 并被推测 与可能的铁原子结合有关 ( J Bio l Chem 1988 May 15; 263 (14): 6816-21 )。 The nucleotide sequence of the gene encoding soybean lipoxygenase has been obtained. Soybean lipoxygenase has been found in three types, L0X1, L0X2, L0X3. Although they differ in nucleotide sequence, they all have an approximate It is a conserved sequence of 40 amino acid residues. This conserved sequence contains 6 histidines and 2 tyrosines. This conserved sequence is highly conserved among the three lipoxygenases in soybean, and is speculated to be possible Iron atom binding is involved (J Biol Chem 1988 May 15; 263 (14): 6816-21).
对脂肪氧化酶的结构研究发现, 脂肪氧化酶结合的铁原子由四个配基结合, 其中三个是组氨酸残基。 所有的脂肪氧化酶都具有 6个组氨酸残基, 其中 5个集中 在大约为 40个氨基酸的区域内 (这个区域包括了两个锌指结合配基), 另一个组氨 酸被证明对于脂肪氧化酶的活性有重要作用 ( Boy ington J. C., Gaffney B. J. , Amze l L. M. Sc i ence 260: 1482-1486 (1993) ); ( S teczko J. , Donoho G. P. , C l emens J. C., Di xon J. E. , Axe l rod B. Biochemi s t ry 31 : 4053-4057 (1992) ) e Studies on the structure of lipoxygenase revealed that the iron atom bound by lipoxygenase is bound by four ligands, three of which are histidine residues. All lipoxygenases have 6 histidine residues, 5 of which are concentrated in a region of approximately 40 amino acids (this region includes two zinc finger binding ligands), and another histidine has been shown to Lipoxidase activity plays an important role (Boyington JC, Gaffney BJ, Amzel LM Science 260: 1482-1486 (1993)); (Steczko J., Donoho GP, Clements JC, Dixon JE, Axe l rod B. Biochemi st ry 31: 4053-4057 (1992)) e
综上所述, 在所有生物的脂肪氧化酶中都有一个保守的区域, 大约为 40个氨 基酸残基, 下面所列的是这个保守区域中的两段序列, 第一段包含有 3个组氨酸残 基, 第二段包含有第 4和第 5个组氨酸残基: ( 1 ) H- [EQ] -X (3) -H-χ- [LM] - [NQRC] -
[GST]- H- [LIVMSTAC] (3)- E (其中, 第二个 H和第三个 H结合铁原子); ( 2 ) [LIVMA] -H-P- [LIVM] -x- [KRQ! - [LIVMF] (2) - x- [AP] - H。 这个保守区域的序列是高 度特征的。 因此, 此区域对干脂肪氧化酶发挥其正常的生理学功能有着重要的意 义。 In summary, there is a conserved region in the lipoxygenase of all organisms, which is about 40 amino acid residues. Listed below are two sequences in this conserved region. The first segment contains three groups Amino acid residues, the second paragraph contains the 4th and 5th histidine residues: (1) H- [EQ] -X (3) -H-χ- [LM]-[NQRC]- [GST]-H- [LIVMSTAC] (3)-E (where the second H and the third H are bound to an iron atom); (2) [LIVMA] -HP- [LIVM] -x- [KRQ! -[LIVMF] (2)-x- [AP]-H. The sequence of this conserved region is highly characteristic. Therefore, this region is of great significance for dry lipoxygenase to perform its normal physiological functions.
在植物中, 脂肪氧化酶的作用是与植物的很多生理学过程, 例如生长、 发育、 抗病虫害和损伤修复有关 (Vick B.A., Zimmerman D. C. (In) Biochemistry of plants: A comprehensive treatise, Stumpf P. K. , Ed. , Vol. 9, pp.53-90, Academic Press, New-York, (1987) )9 In plants, the role of lipoxygenase is related to many physiological processes in plants, such as growth, development, resistance to diseases and insect pests, and damage repair (Vick BA, Zimmerman DC (In) Biochemistry of plants: A comprehensive treatise, Stumpf PK, Ed. , Vol. 9, pp.53-90, Academic Press, New-York, (1987)) 9
白三烯是一种极强的肌肉收缩剂, 它能使肺气管有所缩小, 从而对于哮喘的 治疗有重要意义。 此外, 白三烯在白细胞和肥大细胞肿瘤细胞中均有发现。 前列 腺素和凝血恶烷的作用机理还不是很清楚, 但它们对于血液凝固、 腺苷酸环化酶 的活化等过程有着重要的作用。 根据对很多哺乳动物的研究, 脂肪氧化酶与前列 腺素、 凝血恶烷和白三烯等物质的新陈代谢有关。 因此, 脂肪氧化酶可以调控上 述的一些重要的生理过程 ( Needleman P. , Turk J. , Jakschik Β. A. , Morrison A. R., Lefkowith LB. Annu. Rev. Biochem. 55: 69-102 (1986) )0 Leukotriene is a very strong muscle contractor, which can shrink the lung trachea, which is of great significance for the treatment of asthma. In addition, leukotrienes are found in leukocytes and mast cell tumor cells. The mechanism of action of prostaglandins and thromboxane is not clear, but they have important effects on blood coagulation and activation of adenylate cyclase. According to research in many mammals, lipoxygenase is involved in the metabolism of substances such as prostaglandins, thromboxane and leukotriene. Therefore, lipoxygenase can regulate some of the important physiological processes mentioned above (Needleman P., Turk J., Jakschik B. A., Morrison AR, Lefkowith LB. Annu. Rev. Biochem. 55: 69-102 (1986)) 0
进一步研究还发现, 人脂肪氧化酶催化从花生四烯酸到白三烯生成途径的前 两步, 从而对于生物花生四烯酸的代谢有着重要影响, 由此可以间接的调控人的 炎症和过敏性疾病 (Proc Natl Acad Sci U S A 1988 Jan; 85 (2) : 416-20 )0 Further research has also found that human lipoxygenase catalyzes the first two steps of the pathway from arachidonic acid to leukotriene, which has an important effect on the metabolism of biological arachidonic acid, which can indirectly regulate human inflammation and allergies Sexually Transmitted Diseases (Proc Natl Acad Sci USA 1988 Jan; 85 (2): 416-20) 0
由于如上所述脂肪氧化酶 10蛋白在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 脂肪氧化酶 10 蛋白, 特别是鉴定这种蛋白的氨基酸序列。 新脂肪氧化酶 10 蛋白 编码基因的分离也为研究确定该蛋白在健康和疾病状态下的作用提供了基础。 这 种蛋白可能构成开发疾病诊断和 /或治疗药的基础, 因此分离其编码 DM是非常重 要的。 As mentioned above, lipoxygenase 10 protein plays an important role in important functions of the body, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art to identify more lipoxygenase 10 proteins involved in these processes, especially The amino acid sequence of this protein was identified. The isolation of the new lipoxygenase 10 protein encoding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM.
发明目的 Object of the invention
本发明的一个目的是提供分离的新的多肽——脂肪氧化酶 10 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, lipoxygenase 10, as well as 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脂肪氧化酶 10的多核苷酸的重组载体。 本发明的另一个目的是提供含有编码脂肪氧化酶 10的多核苷酸的基因工程化 宿主细胞。 It is another object of the present invention to provide a recombinant vector containing a polynucleotide encoding a lipoxygenase 10. Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding lipoxygenase 10.
本发明的另一个目的是提供生产脂肪氧化酶 10的方法。
本发明的另一个目的是提供针对本发明的多肽——脂肪氧化酶 10的抗体。 本发明的另一个目的是提供了针对本发明多肽——脂肪氧化酶 10 的模拟化合 物、 拮抗剂、 激动剂、 抑制剂。 Another object of the present invention is to provide a method for producing lipoxygenase 10. Another object of the present invention is to provide an antibody against lipoxygenase 10, which is a polypeptide of the present invention. Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the lipoxygenase 10 polypeptide of the present invention.
本发明的另一个目的是提供诊断治疗与脂肪氧化酶 10 异常相关的疾病的方 法。 Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of lipoxygenase 10.
发明概要 Summary of invention
本发明涉及一种分离的多肽, 该多肽是人源的, 它包含: 具有 SEQ ID No. 2 氨基酸序列的多肽、 或其保守性变体、 生物活性片段或衍生物。 较佳地, 该多肽 是具有 SEQ ID N0: 2氨基酸序列的多肽。 The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
本发明还涉及一种分离的多核苷酸, 它包含选自下组的一种核苷酸序列或其 变体: The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
(a)编码具有 SEQ ID No. 2氨基酸序列的多肽的多核苷酸; (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;
(b)与多核苷酸(a)互补的多核苷酸; (b) a polynucleotide complementary to polynucleotide (a);
(c)与(a)或(b)的多核苷酸序列具有至少 70%相同性的多核苷酸。 (c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b).
更佳地, 该多核苷酸的序列是选自下组的一种: (a)具有 SEQ I D NO: 1 中 163- 432位的序列; 和(b)具有 SEQ I D NO: 1 中 1-1640位的序列。 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 163-432 in SEQ ID NO: 1; and (b) a sequence having 1-1640 in SEQ ID NO: 1 Sequence of bits.
本发明另外涉及一种含有本发明多核苷酸的载体, 特别是表达载体; 一种用 该载体遗传工程化的宿主细胞, 包括转化、 转导或转染的宿主细胞; 一种包括培 养所述宿主细胞和回收表达产物的制备本发明多肽的方法。 The invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
本发明还涉及一种能与本发明多肽特异性结合的抗体。 The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
本发明还涉及一种筛选的模拟、 激活、 拮抗或抑制脂肪氧化酶 1 0蛋白活性的 化合物的方法, 其包括利用本发明的多肽。 本发明还涉及用该方法获得的化合物。 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of lipoxygenase 10 protein, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.
本发明还涉及一种体外检测与脂肪氧化酶 1 0 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 detecting a disease or disease susceptibility related to abnormal expression of lipoxygenase 10 protein in vitro, which comprises 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癌症、 发育性疾病 或免疫性疾病或其它由于脂肪氧化酶 1 0表达异常所引起疾病的药物的用途。 The present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of lipoxygenase 10.
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 OF THE DRAWINGS
下列附图用于说明本发明的具体实施方案, 而不用于限定由权利要求书所 界定的本发明范围。 The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.
图 1是本发明脂肪氧化酶 1 0在 12-66共 55个氨基酸和结构域脂肪氧化酶特征 蛋白的氨基酸序列同源性比较图。 上方序列是脂肪氧化酶 1 0, 下方序列是脂肪 氧化酶特征蛋白结构域。 Ί " 和 " : " 及 ". " 表示在两个序列间同一氨基酸 出现的概率依次减小。 Fig. 1 is a comparison diagram of amino acid sequence homology of a total of 55 amino acids and domain lipoxygenase characteristic proteins of lipoxygenase 10 at 12-66 of the present invention. The upper sequence is lipoxygenase 10, and the lower sequence is the characteristic protein domain of lipoxygenase. Ί "and": "" and "." Indicate that the probability of the same amino acid appearing between two sequences decreases in sequence.
图 2 为分离的脂肪氧化酶 1 0 的聚丙烯酰胺凝胶电泳图 (SDS- PAGE )。 l OkDa 为蛋白质的分子量。 箭头所指为分离出的蛋白条带。 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated lipoxygenase 10. OkDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
发明内容 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 protein or polynucleotide "variant" 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 substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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" 是指当与脂肪氧化酶 10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
可结合脂肪氧化酶 10的分子。 An "agonist" refers to a molecule that, when combined with lipoxygenase 10, causes a change in the protein to regulate the activity of the protein. Agonists can include proteins, nucleic acids, carbohydrates or any other A molecule that can bind lipoxygenase 10.
"拮抗剂" 或 "抑制物" 是指当与脂肪氧化酶 10结合时, 一种可封闭或调节 脂肪氧化酶 10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脂肪氧化酶 10的分子。 An "antagonist" or "inhibitor" refers to a molecule that, when combined with lipoxygenase 10, can block or regulate the biological or immunological activity of lipoxygenase 10. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind lipoxygenase 10.
"调节" 是指脂肪氧化酶 10的功能发生改变, 包括蛋白质活性的升高或降低、 结合特性的改变及脂肪氧化酶 10的任何其它生物学性质、 功能或免疫性质的改变。 "Regulation" refers to a change in the function of lipoxygenase 10, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of lipoxygenase 10.
"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脂肪氧化酶 10。 基本上纯 的脂肪氧化酶 10在非还原性聚丙烯酰胺凝胶上能产生单一的主带。 脂肪氧化酶 1 0 多肽的纯度可用氨基酸序列分析。 "Substantially pure" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify lipoxygenase 10 using standard protein purification techniques. Substantially pure lipoxygenase 10 produces a single main band on a non-reducing polyacrylamide gel. The purity of the lipoxygenase 10 peptide 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 can 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 Nor thern 印迹等) 来检测。 基本上同源的序列或杂交探针可竟争和抑制完全同源的序列与 靶序列在严格性程度降低的条件下的结合。 这并不意味严格性程度降低的条件允 许非特异性结合, 因为严格性程度降低的条件要求两条序列相互的结合为特异性 或选择性相互作用。 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
"相同性百分率" 是指在两种或多种氨基酸或核酸序列比较中序列相同或相 似的百分率。 可用电子方法测定相同性百分率, 如通过 MEGALIGN程序 (Lasergene sof tware package, DNASTAR, Inc., Mad i son Wi s. )。 MEGALIGN程序可根据不同的 方法如 C lus ter法比较两种或多种序列(H i gg ins , D. G. 和 P. M. Sharp (1988) "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Mad Son Wis.). The MEGALIGN program can compare two or more sequences (Higgins, D. G., and P. M. Sharp (1988) according to different methods, such as the Cluster method.
Gene 73: 237-244)。 C lus ter法通过检査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: Gene 73: 237-244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:
序列 ^与序列 之间匹配的残基个数 The number of residues between the sequence ^ and the sequence
序列 的残基数 -序列 中间隔残基数 -序列 ^中间隔残基数 X Residues sequences - sequences spacer residues - the sequence of residues X ^ interval
也可以通过 C lus ter法或用本领域周知的方法如 Jotun He i n 测定核酸序列之 间的相同性百分率(He in J" (1990) Methods in enzymo logy 183: 625-645) 0
"相似性" 是指氨基醺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';生取代的氨基酸, 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。 May be measured as Jotun He in the percentage of identity between nucleic acid sequences C lus ter method or by methods known in the art (He in J "(1990) Methods in enzymo logy 183: 625-645) 0 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions when the alignment between amino hydrazone sequences is aligned. Used for conservative; amino acid substitutions, such as negatively charged amino acids can include aspartic acid and glutamic acid; positively charged amino acids can include lysine and arginine; have uncharged head groups Amino acids with similar hydrophilicity may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine acid.
"反义"是指与特定的 DNA或 RNA序列互补的核苷酸序列。 "反义链"是指与 "有 义链" 互补的核酸链。 "Antisense" refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence. The "antisense strand" refers to a nucleic acid strand that is complementary to the "sense strand".
"衍生物" 是指 HFP或编码其核酸的化学修饰物。 这种化学修饰物可以是用烷 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 the replacement of a hydrogen atom with an alkyl, 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 结合脂肪氧化酶 10的抗原决定簇。 "Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') 2 and? It can specifically bind to the epitope of lipoxygenase 10.
"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 matter 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 animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system. Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a 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 existing in the natural state. .
如本文所用, "分离的脂肪氧化酶 1 0" 是指脂肪氧化酶 1 0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脂肪氧化酶 1 0。 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。 脂肪氧化酶 1 0多肽的纯度能用氨基酸序列分析。 As used herein, "isolated lipoxygenase 10" means that lipoxygenase 10 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify lipoxygenase 10 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the lipoxygenase 10 polypeptide can be analyzed by amino acid sequence.
本发明提供了一种新的多肽——脂肪氧化酶 10 , 其基本上是由 SEQ ID N0: 2所 示的氨基酸序列组成的。 本发明的多肽可以是重组多肽、 天然多肽、 合成多肽, 优选重组多肽。 本发明的多肽可以是天然纯化的产物, 或是化学合成的产物, 或 使用重组技术从原核或真核宿主(例如, 细菌、 酵母、 高等植物、 昆虫和哺乳动物
细胞)中产生。 根据重组生产方案所用的宿主, 本发明的多肽可以是糖基化的, 或 可以是非糖基化的。 本发明的多肽还可包括或不包括起始的甲硫氨酸残基。 The present invention provides a new polypeptide, lipoxygenase 10, 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 obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammals) using recombinant technology. Cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
本发明还包括脂肪氧化酶 10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脂肪氧化酶 1 0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: U ) 这 样一种, 其中一个或多个氨基酸残基被保守或非保守氨基酸残基 (优选的是保守 氨基酸残基) 取代, 并且取代的氨基酸可以是也可以不是由遗传密码子编码的; 或者 ( Π ) 这样一种, 其中一个或多个氨基酸残基上的某个基团被其它基团取代 包含取代基; 或者 (I I I ) 这样一种, 其中成熟多肽与另一种化合物 (比如延长多 肽半衰期的化合物, 例如聚乙二醇) 融合; 或者 ( IV ) 这样一种, 其中附加的氨 基酸序列融合进成熟多肽而形成的多肽序列 (如前导序列或分泌序列或用来纯化 此多肽的序列或蛋白原序列)。 通过本文的阐述, 这样的片段、 衍生物和类似物被 认为在本领域技术人员的知识范围之内。 The invention also includes fragments, derivatives and analogs of lipoxygenase 10. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the lipoxygenase 10 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: U) a type 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 substituted An amino acid may or may not be encoded by a genetic code; or (Π) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such a Species, wherein the mature polypeptide is fused with another compound (such as a compound that extends 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 a mature polypeptide (such as Leader or secreted 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 1640个碱基, 其开放读框 163-432编码了 89个氨基酸。 此多肽 具有脂肪氧化酶特征蛋白的特征序列, 可推断出该脂肪氧化酶 1 0具有脂肪氧化酶 特征蛋白所代表的结构和功能。 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes a 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 1640 base polynucleotide sequence, and its open reading frame 163-432 encodes 89 amino acids. This polypeptide has a characteristic sequence of a lipoxygenase characteristic protein, and it can be deduced that the lipoxygenase 10 has the structure and function represented by the lipoxygenase characteristic protein.
本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 cDNA、 基因 组 DNA或人工合成的 DM。 DNA可以是单链的或是双链的。 DNA可以是编码链或非 编码链。 编码成熟多肽的编码区序列可以与 SEQ ID N0: 1 所示的编码区序列相同 或者是简并的变异体。 如本发明所用, "简并的变异体" 在本发明中是指编码具有 SEQ ID NO: 2的蛋白质或多肽, 但与 SEQ ID NO: 1所示的编码区序列有差别的核酸 序列。 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DM. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
编码 SEQ I D NO: 2 的成熟多肽的多核苷酸包括: 只有成熟多肽的编码序列; 成熟多肽的编码序列和各种附加编码序列; 成熟多肽的编码序列 (和任选的附加 编码序列) 以及非编码序列。 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加编 码和 /或非编码序列的多核苷酸。 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
本发明还涉及上述描述多核苷酸的变异体, 其编码与本发明有相同的氨基酸 序列的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天然发
生的等位变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺 失变异体和插入变异体。 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 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 The invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between
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, 6(TC;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%Ficoll, 42°C等; 或(3)仅在两条序列之间 的相同性至少在 95%以上,更好是 97%以上时才发生杂交。 并且, 可杂交的多核苷 酸编码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功能和活性。 50%, preferably 70% identity). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) added during hybridization) Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% Above, it is more preferable that the hybridization occurs at 97% or more. Moreover, 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 /或分离编码脂肪氧化酶 10的多核苷酸。 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, 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 nuclei. 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 lipoxygenase 10.
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。 本发明的编码脂肪氧化酶 10 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 lipoxygenase 10 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 DNA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 DNA。 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 genomic DNA; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
上述提到的方法中, 分离基因组 DNA 最不常用。 DNA序列的直接化学合成 是经常选用的方法。 更经常选用的方法是 cDNA序列的分离。 分离感兴趣的 cDNA 的标准方法是从高表达该基因的供体细胞分离 mRNA 并进行逆转录, 形成质粒 或噬菌体 cDNA文库。 提取 mRNA 的方法已有多种成熟的技术, 试剂盒也可从商 业途径获得(Qiagene)。 而构建 cDNA 文库也是通常的方法(Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。还可得到商业供应的 cDM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 mRNA 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 Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDM libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
可用常规方法从这些 cDNA 文库中筛选本发明的基因。 这些方法包括(但不 限于): (l)DNA-DM 或 DNA-RNA 杂交; (2)标志基因功能的出现或丧失; (3)测
定脂肪氧化酶 10 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 are not limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the appearance or loss of marker gene function; (3) measurement Determine the level of lipoxygenase 10 transcripts; (4) Detect gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
在第(1)种方法中, 杂交所用的探针是与本发明的多核苷酸的任何一部分 同源, 其长度至少 10 个核苷酸, 较好是至少 30个核苷酸, 更好是至少 50 个 核苷酸, 最好是至少 100 个核苷酸。 此外, 探针的长度通常在 2000 个核苷酸 之内, 较佳的为 1000 个核苷酸之内。 此处所用的探针通常是在本发明的基因 序列信息的基础上化学合成的 DNA 序列。 本发明的基因本身或者片段当然可以 用作探针。 DNA探针的标记可用放射性同位素, 荧光素或酶(如碱性磷酸酶)等。 In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
在第(4)种方法中, 检测脂肪氧化酶 10 基因表达的蛋白产物可用免疫学技 术如 Western印迹法、 放射免疫沉淀法、 酶联免疫吸附法(ELISA)等。 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the lipoxygenase 10 gene.
应 用 PCR 技术 扩增 DNA/RNA 的 方 法 (Saiki, et al. Science 1985; 230: 1350- 1354)被优选用于获得本发明的基因。 特别是很难从文库中得 到全长的 cDNA时, 可优选使用 RACE法(RACE- cDNA末端快速扩增法),用于 PCR 的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择, 并可用常 规方法合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。 A method using PCR technology to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-rapid cDNA end rapid amplification method) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
如上所述得到的本发明的基因, 或者各种 DNA 片段等的多核苷酸序列可用 常规方法如双脱氧链终止法(Sanger et al. PNAS, 1977, 74: 5463- 5467)测 定。 这类多核苷酸序列测定也可用商业测序试剂盒等。 为了获得全长的 cDNA 序列, 测序需反复进行。 有时需要测定多个克隆的 cDNA 序列, 才能拼接成全 长的 cDNA序列。 The polynucleotide sequence of the gene of the present invention or various DNA 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 cDNA sequence.
本发明也涉及包含本发明的多核苷酸的载体, 以及用本发明的载体或直接 用脂肪氧化酶 10 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 a polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a lipoxygenase 10 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
本发明中, 编码脂肪氧化酶 10 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 al. 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。
本领域的技术人员熟知的方法能用于构建含编码脂肪氧化酶 10 的 DNA 序 列和合适的转录 /翻译调控元件的表达载体。 这些方法包括体外重组 DNA 技术、 腿 合成技术、 体内重组技术等(Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。 所述的 DNA序列可有效连接到表达载体中的适当启动子上, 以指导 mRNA合成。 这些启动子的代表性例子有: 大肠杆菌的 lac 或 trp 启动子; λ噬菌体的 PL 启动子; 真核启动子包括 CMV 立即早期启动子、 HSV 胸苷激酶启动子、 早期和 晚期 SV40启动子、 反转录病毒的 LTRs 和其它一些已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。 In the present invention, the polynucleotide sequence encoding the lipoxygenase 10 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. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements. Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding lipoxygenase 10 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, leg synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, and 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 and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
此外, 表达载体优选地包含一个或多个选择性标记基因, 以提供用于选择 转化的宿主细胞的表型性状, 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗 性以及绿色荧光蛋白(GFP), 或用于大肠杆菌的四环素或氨苄青霉素抗性等。 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, 编码脂肪氧化酶 10 的多核苷酸或含有该多核苷酸的重组载体 可转化或转导入宿主细胞, 以构成含有该多核苷酸或重组载体的基因工程化宿 主细胞。 术语 "宿主细胞" 指原核细胞, 如细菌细胞; 或是低等真核细胞, 如 酵母细胞; 或是高等真核细胞, 如哺乳动物细胞。 代表性例子有: 大肠杆菌, 链霉菌属; 细菌细胞如鼠伤寒沙门氏菌; 真菌细胞如酵母; 植物细胞; 昆虫细 胞如果蝇 S2或 Sf9; 动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。 In the present invention, a polynucleotide encoding a lipoxygenase 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Fly S2 or Sf9; animal cells such as CH0, COS or Bowes melanoma cells.
用本发明所述的 DNA序列或含有所述 DNA序列的重组载体转化宿主细胞可 用本领域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时, 能 吸收 DNA 的感受态细胞可在指数生长期后收获, 用 CaCl2法处理, 所用的步骤 在本领域众所周知。 可供选择的是用 MgCl2。 如果需要, 转化也可用电穿孔的 方法进行。 当宿主是真核生物, 可选用如下的 DNA转染方法: 磷酸钙共沉淀法, 或者常规机械方法如显微注射、 电穿孔、 脂质体包装等。 Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
通过常规的重组 DNA技术, 利用本发明的多核苷酸序列可用来表达或生产
重组的脂肪氧化酶 1 0 (Sc i ence , 1984 ; 224: 1431)。 一般来说有以下步骤:By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used for expression or production Recombinant lipoxygenase 10 (Science, 1984; 224: 1431). Generally there are the following steps:
(1)用本发明的编码人 脂肪氧化酶 1 0 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 a polynucleotide (or variant) encoding human lipoxygenase 10 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 the antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.
脂肪氧化酶是一类铁原子结合的双加氧酶, 它可以催化脂类的氢过氧化作用, 它的催化底物除了花生四烯酸外, 还包括顺, 顺 -1, 4-戊二烯等等。 它对于脂类 的代谢、 脂类及其衍生物的生理学功能的发挥等都有极其重要的意义。 花生四烯 酸可通过脂肪氧化酶的催化转变为很多种化合物, 例如, 前列腺素、 凝血恶垸和 白三烯等。 对于调控人的炎症和过敏性疾病有重要作用 (Proc Na t l Acad Sc i USA 1988 Jan; 85 (2) : 416- 20 )。 Lipoxygenase is a type of iron-bonded dioxygenase, which can catalyze the hydroperoxidation of lipids. In addition to arachidonic acid, its catalytic substrate includes cis, cis-1, 4-pentane Ene and so on. It is of great significance to the metabolism of lipids and the physiological functions of lipids and their derivatives. Arachidonic acid can be converted into many compounds by the catalysis of lipoxygenase, such as prostaglandins, thromboxane, and leukotrienes. It plays an important role in regulating human inflammation and allergic diseases (Proc Na t l Acad Sci USA 1988 Jan; 85 (2): 416-20).
前列腺素是人体内分布最广、 效应最大的生物活性物质, 对生殖、 心血管、 呼吸、 消化及神经等系统均有作用, 它在机体内不作为激素起作用, 而是通过对 某些激素的调节起作用。 在人体中前列腺素的代谢异常, 可引起子宫收缩不良性 难产, 持久性黄体, 血管收缩舒张障碍相关性心血管疾病, 支气管痉挛, 胃酸过 多引起的胃溃疡, 脏器血供障碍等。 白三烯涉及各类炎症反应、 凝血、 哮喘等生 理病理过程。 凝血恶烷也涉及凝血过程。 Prostaglandin is the most widely distributed and most effective biologically active substance in the human body. It has effects on the reproductive, cardiovascular, respiratory, digestive, and nervous systems. It does not act as a hormone in the body, but through certain hormones. The adjustment works. Abnormal metabolism of prostaglandins in the human body can cause uterine dysfunction, dystocia, persistent corpus luteum, vasodilation-related cardiovascular disease, bronchospasm, gastric ulcer caused by excessive gastric acid, and impaired blood supply to the organ. Leukotriene is involved in various physiological and pathological processes such as inflammatory response, coagulation, and asthma. Thromboxane is also involved in the coagulation process.
由此可见, 特异的脂肪氧化酶 mot i f 的表达异常, 将致使含此 mot i f 的多肽 的功能异常, 从而导致脂类及其衍生物的代谢障碍, 影响花生四烯酸的代谢, 并
产生前列腺素、 凝血恶烷和白三烯的代谢异常, 并产生相关的疾病如脂类代谢障 碍性疾病、 炎症、 凝血障碍呼吸系统疾病、 前列腺素代谢异常相关疾病等。 It can be seen that abnormal expression of the specific lipoxygenase mot if will cause abnormal function of the polypeptide containing the mot if, resulting in metabolic disorders of lipids and their derivatives, affecting the metabolism of arachidonic acid, and It produces abnormal metabolism of prostaglandins, thromboxane and leukotriene, and produces related diseases such as lipid metabolism disorders, inflammation, coagulopathy, respiratory diseases, and diseases related to abnormal prostaglandin metabolism.
由此可见, 本发明的脂肪氧化酶 1 0 的表达异常将产生各种疾病尤其是脂类 代谢障碍性疾病、 炎症、 凝血障碍、 呼吸系统疾病、 前列腺素代谢异常相关疾病, 这些疾病包括但不限于: It can be seen that abnormal expression of lipoxygenase 10 of the present invention will produce various diseases, especially lipid metabolic disorders, inflammation, coagulopathy, respiratory diseases, and diseases related to abnormal prostaglandin metabolism. These diseases include but are not Limited to:
脂类代谢障碍性疾病。 这些疾病包括但不限于: Disorders of lipid metabolism. These diseases include, but are not limited to:
1. 脂肪沉积性疾病: 脂肪肝, 脂肪变性性心肌病, 脂肪变性性肾病 1. Fatty deposition disease: fatty liver, steatosis cardiomyopathy, steatosis nephropathy
2. 心血管疾病: 冠状动脉粥样硬化性心脏病如隐匿性心脏病、 心绞痛、 心肌 梗塞、 瘁死性冠心病, 高血压 2. Cardiovascular diseases: coronary atherosclerotic heart disease such as occult heart disease, angina pectoris, myocardial infarction, dying coronary heart disease, hypertension
3. 固醇衍生物 〔如胆汁酸、 性激素 (睾酮、 雌二醇、 雌三醇、 孕酮) ) 代谢 障碍性疾病: ( 1 )胆汁酸代谢障碍性疾病如胆汁性肝硬化, 胆石症 (2 ) 生长发 育阶段的性发育障碍: 性早熟, 性发育延迟, 性分化障碍, 其它外生殖器发育缺 陷 (3 ) 内分泌及代谢综合征: 肾上腺皮质功能亢进病如 Cush ing综合征、 醛固酮 增多症, 肾上腺皮质功能低下病如急性肾上腺皮质功能低下症、 慢性肾上腺皮质 功能低下症 3. Sterol derivatives (such as bile acids, sex hormones (testosterone, estradiol, estriol, progesterone)) Metabolic disorders: (1) Bile acid metabolic disorders such as biliary cirrhosis, cholelithiasis ( 2) Sexual developmental disorders in the growth and development stages: precocious puberty, delayed sexual development, sexual differentiation disorders, other defects in external genital development (3) endocrine and metabolic syndromes: hyperadrenocortical diseases such as Cushing syndrome, hyperaldosteronism, Adrenal insufficiency diseases such as acute adrenal insufficiency and chronic adrenal insufficiency
4. 相关肿瘤: 脂肪瘤、 脂肪母细胞瘤、 脂肪肉瘤, 乳腺癌 4. Related tumors: lipoma, lipoblastoma, liposarcoma, breast cancer
各种炎症: 变应性反应、 风湿样关节炎、 类风湿样关节炎、 骨关节炎、 胆囊 炎、 肾小球性肾炎、 免疫复合物型肾小球肾炎、 急性前葡萄膜炎、 骨质疏松症、 皮肤肌炎、 荨麻疹、 特异性皮炎、 血色素沉着症、 多肌炎、 阿狄森氏病、 格雷夫 斯氏病、 慢性活动性肝炎、 肠应急性综合症、 萎缩性胃炎、 系统性红斑狼疮、 重 症肌无力、 脑脊髓多发性硬化、 格林-巴利综合症、 颅内肉芽肿、 Wegener 肉芽肿 病、 自体免疫甲状腺炎、 自身免疫性间质性肾炎、 溃疡性结肠炎、 贫血、 胰腺炎、 节段性回肠炎、 心肌炎、 动脉粥样硬化、 多发性硬皮病、 以及感染和外伤引起的 炎症 Various inflammations: allergic reactions, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex glomerulonephritis, acute anterior uveitis, bone Porphyria, dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, polymyositis, Addison's disease, Graves' disease, chronic active hepatitis, bowel emergency syndrome, atrophic gastritis, system Lupus erythematosus, myasthenia gravis, cerebrospinal multiple sclerosis, Guillain-Barre syndrome, intracranial granulomatosis, Wegener granulomatosis, autoimmune thyroiditis, autoimmune interstitial nephritis, ulcerative colitis, anemia , Pancreatitis, segmental ileitis, myocarditis, atherosclerosis, multiple scleroderma, and inflammation caused by infections and trauma
呼吸系统疾病: 支气管哮喘、 过敏性肺炎、 成人呼吸窘迫综合症、 肺嗜酸粒 细胞增多症、 结节病 Respiratory diseases: bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, pulmonary eosinophilia, sarcoidosis
前列腺素代谢异常相关疾病: 子宫收缩不良性难产, 持久性黄体, 血管收缩 舒张障碍相关性心血管疾病, 支气管痉挛, 胃酸过多引起的胃溃疡, 脏器血供障 碍 Diseases related to abnormal prostaglandin metabolism: dystocia, dystocia, persistent corpus luteum, vasoconstriction, vasodilation-related cardiovascular disease, bronchospasm, gastric ulcer caused by hyperacidity, impaired blood supply
本发明的脂肪氧化酶 1 0的表达异常还将产生某些遗传性、 免疫性疾病等。 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脂类代谢障碍性疾病、 炎症、 凝血障碍、 呼吸
系统疾病、 前列腺素代谢异常相关疾病, 某些遗传性、 免疫性疾病等 Abnormal expression of the lipoxygenase 10 of the present invention will also cause certain hereditary and immune diseases. 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 lipid metabolic disorders, inflammation, coagulopathy, respiratory Systemic diseases, diseases related to abnormal prostaglandin metabolism, certain hereditary and immune diseases, etc.
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)脂肪氧化 酶 10 的药剂的方法。 激动剂提高脂肪氧化酶 10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脂肪氧化酶 10的膜制剂与标记的脂肪氧化酶 10 一起培养。 然后测定药物提高或阻遏此相互作用的能力。 The invention also provides methods for screening compounds to identify agents that increase (agonist) or inhibit (antagonist) lipoxygenase 10. Agonists increase biological functions such as lipoxygenase 10, which stimulates cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing lipoxygenase 10 can be cultured with labeled lipoxygenase 10 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
脂肪氧化酶 10 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。 脂肪氧化酶 10 的拮抗剂可以与脂肪氧化酶 10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 lipoxygenase 10 include antibodies, compounds, receptor deletions, and the like that have been screened. An antagonist of lipoxygenase 10 can bind to lipoxygenase 10 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
在筛选作为拮抗剂的化合物时, 可以将脂肪氧化酶 10 加入生物分析测定 中, 通过测定化合物对脂肪氧化酶 10 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。 能与脂肪氧化酶 10 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 对脂肪氧化酶 10分子进行标记。 When screening compounds as antagonists, lipoxygenase 10 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between lipoxygenase 10 and its receptor. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to lipoxygenase 10 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 10 molecules of lipoxygenase should 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 了针对脂肪氧化酶 10 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 directed against a lipoxygenase 10 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脂肪氧化酶 10 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。 制备脂肪氧化酶 10 的单克隆抗体的技术包括但不限于杂交瘤技术(Kohler and Milstein. 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(Morrison et al , PNAS, 1985, 81: 6851) 0 而已有的生产单链抗 体的技术(U.S. Pat No.4946778)也可用于生产抗脂肪氧化酶 10的单链抗体。 Polyclonal antibodies can be produced by injecting lipoxygenase 10 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 lipoxygenase 10 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridoma Technology, etc. Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pat No. .4946778) can also be used to produce single chain antibodies against lipoxygenase 10.
抗脂肪氧化酶 10 的抗体可用于免疫组织化学技术中, 检测活检标本中的 脂肪氧化酶 10。 Anti-lipoxygenase 10 antibodies can be used in immunohistochemistry to detect lipoxygenase 10 in biopsy specimens.
与脂肪氧化酶 10 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 有转移。 Monoclonal antibodies that bind to lipoxygenase 10 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method There is metastasis due to the localization and judgment of tumor cells.
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如脂肪氧化酶 10 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脂肪氧化酶 10阳性的细胞。 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, lipoxygenase 10 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 an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill lipoxygenase 10-positive cells.
本发明中的抗体可用于治疗或预防与脂肪氧化酶 10 相关的疾病。 给予适 当剂量的抗体可以刺激或阻断脂肪氧化酶 10的产生或活性。 The antibodies in the present invention can be used to treat or prevent diseases related to lipoxygenase 10. Administration of an appropriate amount of antibody can stimulate or block the production or activity of lipoxygenase 10.
本发明还涉及定量和定位检测脂肪氧化酶 10 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 脂肪氧化酶 10水平, 可以用作解释脂肪氧化酶 10在各种疾病中的重要性和用 于诊断脂肪氧化酶 10起作用的疾病。 The invention also relates to a diagnostic test method for quantitative and localized detection of lipoxygenase 10 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of lipoxygenase 10 detected in the test can be used to explain the importance of lipoxygenase 10 in various diseases and to diagnose diseases in which lipoxygenase 10 functions.
本发明的多肽还可用作肽谱分析, 例如, 多肽可用物理的、 化学或酶进行 特异性切割, 并进行一维或二维或三维的凝胶电泳分析,更好的是进行质谱分 析。 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
编码脂肪氧化酶 10 的多核苷酸也可用于多种治疗目的。 基因治疗技术可 用于治疗由于脂肪氧化酶 10 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脂肪 氧化酶 10, 以抑制内源性的脂肪氧化酶 10 活性。 例如, 一种变异的脂肪氧化 酶 10 可以是缩短的、 缺失了信号传导功能域的脂肪氧化酶 10, 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脂肪氧 化酶 10 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脂肪氧化 酶 10 的多核苷酸转移至细胞内。 构建携带编码脂肪氧化酶 10 的多核苷酸的重 组病毒载体的方法可见于已有文献(Sambrook, et al.)。 另外重组编码脂肪氧 化酶 10的多核苷酸可包装到脂质体中转移至细胞内。 Polynucleotides encoding lipoxygenase 10 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 lipoxygenase 10. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant lipoxygenase 10 to inhibit endogenous lipoxygenase 10 activity. For example, a mutated lipoxygenase 10 may be a shortened lipoxygenase 10 lacking a signal transduction domain. Although it can bind to downstream substrates, it lacks signal transduction activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of lipoxygenase 10. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding lipoxygenase 10 into a cell. Methods for constructing recombinant viral vectors carrying a polynucleotide encoding lipoxygenase 10 can be found in the literature (Sambrook, et al.). Alternatively, a recombinant polynucleotide encoding lipoxygenase 10 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.
抑制脂肪氧化酶 10 mRNA 的寡核苷酸(包括反义 RNA和 DNA)以及核酶也在 本发明的范围之内。 核酶是一种能特异性分解特定 RNA 的酶样 RM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 RNA或 DNA合成技术获得, 如固相磷酸酰胺化学 合成法合成寡核苷酸的技术已广泛应用。反义 RM分子可通过编码该 RNA的 DNA 序列在体外或体内转录获得。 这种 DM序列已整合到载体的 RNA聚合酶启动子 的下游。 为了增加核酸分子的稳定性, 可用多种方法对其进行修饰, 如增加两 侧的序列长度, 核糖核苷之间的连接应用磷酸硫酯键或肽键而非磷酸二酯键。 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit lipoxygenase 10 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RM 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 RNA or DNA synthesis technology. For example, the technique of solid phase phosphoramidite chemical synthesis to synthesize oligonucleotides has been widely used. Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the RNA 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.
编码脂肪氧化酶 10 的多核苷酸可用于与脂肪氧化酶 10 的相关疾病的诊 断。 编码脂肪氧化酶 10 的多核苷酸可用于检测脂肪氧化酶 10 的表达与否或在 疾病状态下脂肪氧化酶 10的异常表达。 如编码脂肪氧化酶 10的 DNA序列可用 于对活检标本进行杂交以判断脂肪氧化酶 10 的表达状况。 杂交技术包括 Southern 印迹法、 Northern 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。 用脂肪氧化酶 10 特异 的引物进行 RNA-聚合酶链反应(RT- PCR)体外扩增也可检测脂肪氧化酶 10 的转 录产物。 Polynucleotides encoding lipoxygenase 10 can be used to diagnose diseases related to lipoxygenase 10. Polynucleotides encoding lipoxygenase 10 can be used to detect the expression of lipoxygenase 10 or the abnormal expression of lipoxygenase 10 in a disease state. For example, a DNA sequence encoding lipoxygenase 10 can be used to hybridize biopsy specimens to determine the expression of lipoxygenase 10. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, and related kits are commercially available. A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with lipoxygenase 10 specific primers can also detect lipoxygenase 10 transcript products.
检测脂肪氧化酶 10基因的突变也可用于诊断脂肪氧化酶 10相关的疾病。 脂肪氧化酶 10突变的形式包括与正常野生型脂肪氧化酶 10 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、 Western印迹法可间接判断基因有无突变。 Detection of mutations in the lipoxygenase 10 gene can also be used to diagnose lipoxygenase 10-related diseases. Forms of lipoxygenase 10 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type lipoxygenase 10 DNA sequences. Mutations can be detected using existing techniques such as Southern imprinting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具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。 In short, PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
体细胞杂合细胞的 PCR定位法, 是将 DNA定位到具体染色体的快捷方法。 使 用本发明的寡核苷酸引物, 通过类似方法, 可利用一组来自特定染色体的片段 或大量基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位 杂交、 用标记的流式分选的染色体预筛选和杂交预选, 从而构建染色体特异的
cDNA库。 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, 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 chromosome localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybrids to construct chromosome-specific cDNA library.
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 Manual of Basic Techniques, Pergamon Press, 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 Manual of Basic Techniques, Pergamon Press, 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. Mckusick,Mendelian Inheritance in Man (可通过与 Johns Hopkins University Welch Medical 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, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch 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。 脂肪氧化酶 10 以有效地治疗和 /或预防具体的 适应症的量来给药。 施用于患者的脂肪氧化酶 10 的量和剂量范围将取决于许 多因素, 如给药方式、 待治疗者的健康条件和诊断医生的判断。 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Lipoxidase 10 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of lipoxygenase 10 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.
实施例 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 Sarabrook 等人, 分子克隆: 实验室手册(New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件, 或按照制造厂商所建议 的条件。 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 without specific conditions are not indicated in the following examples. It is usually according to the conditions described in Sarabrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.
实施例 1 脂肪氧化酶 10的克隆 Example 1 Cloning of Lipoxygenase 10
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA Isolation Kit (Qiegene 公司产品) 从总 RNA中分离 poly (A) mRNA. 2ug poly (A) mRNA经逆转录 形成 cDM。用 Smart cDNA克隆试剂盒(购自 Clontech ^cDNA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 α , 细菌形成 cDNA文库。 用 Dye terminate cycle reaction sequencing kit (Perkin- Elmer公司产品) 和 ABI 377 自动测序仪(Perkin-Elmer公司)测定所有克隆的 5'和 3'末端的序列。将测定的 cDNA 序列与已有的公共 DNA序列数据库 (Genebank)进行比较, 结果发现其中一个克隆 1050e06的 cDNA序列为新的 DNA。 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, 1050e06克隆所含的全长 cDNA为 1640bp (如 Seq IDN0: l 所示) , 从第 163bp至 432bp有一个 264bp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-1050e06 , 编码的蛋白 质命名为脂肪氧化酶 10。 实施例 2 cDNA 克隆的结构域分析 Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (product of Qiegene). 2ug poly (A) mRNA was reverse transcribed to form cDM. Smart cDNA cloning kit (purchased from Clontech ^ cDNA fragment was inserted into the multiple cloning site of pBSK (+) vector (Clontech)) to transform DH5 α to form a cDNA library. Dye terminate cycle reaction sequencing kit ( (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) to determine the 5 'and 3' ends of all clones. Compare the determined cDNA sequence with the existing public DNA sequence database (Genebank), It was found that the cDNA sequence of one of the clones 1050e06 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragment of the clone in both directions. The results showed that the full-length cDNA contained in the 1050e06 clone was 1640bp (such as Seq IDN0: l), there is a 264bp open reading frame (0RF) from 163bp to 432bp, encoding a new protein (as shown in Seq ID NO: 2). We named this clone pBS-1050e06, the encoded protein Named lipoxygenase 10. Example 2 Domain analysis of cDNA clones
将本发明的脂肪氧化酶 10的序列及其编码的蛋白序列, 用 GCG中的 profile scan程 序 (Basic local alignment search tool) [Altschul, SF et a 1. J.Mol. Biol.1990; 215: 403-10] , 在 prosite等数据库进行结构域分析。 本发明的 脂肪氧化酶 10在 12-66与结构域脂肪氧化酶特征蛋白有同源, 同源结果示于图 1, 同源率为 0.26, 得分为 13.99; 阈值为 13.92。 实施例 3 用 RT-PCR方法克隆编码脂肪氧化酶 10的基因 The sequence of the lipoxygenase 10 of the present invention and the protein sequence encoded by the lipoxygenase 10 of the present invention were profiled using the GCG profile scan program (Basic local alignment search tool) [Altschul, SF et a 1. J. Mol. Biol. 1990; 215: 403 -10], perform domain analysis in databases such as prosite. The lipoxygenase 10 of the present invention is homologous with the domain lipoxygenase characteristic protein at 12-66, and the homology result is shown in FIG. 1. The homology rate is 0.26, and the score is 13.99; the threshold value is 13.92. Example 3 Cloning of a gene encoding lipoxygenase 10 by RT-PCR
用胎脑细胞总 RNA为模板,以 oligo-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 total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
Primerl: 5'-GATCATAGAACTATTGCATAAAAC-3' (SEQ ID NO: 3) Primerl: 5'-GATCATAGAACTATTGCATAAAAC-3 '(SEQ ID NO: 3)
Primer2: 5-AGTTTTGTTTGTTTAATGAATAAT-3' (SEQ ID NO: 4) Primer2: 5-AGTTTTGTTTGTTTAATGAATAAT-3 '(SEQ ID NO: 4)
Primerl为位于 SEQ ID NO: 1的 5'端的第 lbp开始的正向序列; Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
Primer2为 SEQ ID NO: 1的中的 3'端反向序列。 Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
扩增反应的条件: 在 50μ1的反应体积中含有 50mmol/L C1, 10mmol/L
Tris-HCl, pH8.5, 1.5mmol/L gCl2, 200 raol/L dNTP, lOpmol引物, 1U的 Taq DMA 聚合酶(Clontech公司产品)。 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 β-actin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 ( Invitrogen公司产品) 。 DNA序列分析结 果表明 PCR产物的 DNA序列与 SEQ ID NO: 1所示的 l-1640bp完全相同。 实施例 4 Northern 印迹法分析脂肪氧化酶 10基因的表达 Amplification conditions: 50 mmol / L C1, 10 mmol / L in a 50 μl reaction volume Tris-HCl, pH 8.5, 1.5 mmol / L gCl 2 , 200 raol / L dNTP, lOpmol primer, 1U Taq DMA polymerase (Clontech). PE9600 DM thermal cycler (Perkin-Elmer) reacts for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, β-actin was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1640bp shown in SEQ ID NO: 1. Example 4 Northern blot analysis of lipoxygenase 10 gene expression
用一步法提取总 RNA[Anal. Biochem 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 -25mM柠檬酸钠, 0.2M乙酸钠 ( pH4.0 ) 对组织进行匀浆, 加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇 (49: 1 ) , 混合 后离心。 吸出水相层, 加入异丙醇 (0.8体积) 并将混合物离心得到 RNA沉淀。 将 得到的 RNA沉淀用 70°/。乙醇洗涤, 干燥并溶于水中。 用 20μ8 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 ex- 32P dATP通过随机引物法制备 32P-标记 的 DNA探针。所用的 DNA探针为图 1所示的 PCR扩增的脂肪氧化酶 10编码区序列(163bp 至 432bp)。 将 32P-标记的探针 (约 2 X 106cpm/ml ) 与转移了 RNA的硝酸纤维素膜在 一溶液中于 42。C杂交过夜, 该溶液包含 50%甲酰胺 -25mM KH2P04 ( pH7.4 ) -5 χ SSC- 5 χ Denhardt's溶液和 20(^g/ml鲑精 DNA。 杂交之后, 将滤膜在 1 χ SSC- 0.1°/。SDS中 于 55°C洗 30min。 然后, 用 Phosphor Imager进行分析和定量。 实施例 5 重组脂肪氧化酶 10的体外表达、 分离和纯化 Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0 This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA was precipitated at 70 ° / °. Wash with ethanol, dry and dissolve in water. Using 20 μ8 RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation 32 P- DNA probe labeled with ex- 32 P dATP by random priming method. The DNA probe used was the PCR-amplified lipoxygenase 10 coding region sequence (163bp to 432bp) shown in FIG. 1. A 32P-labeled probe (approximately 2 × 10 6 cpm / ml) and a nitrocellulose membrane to which RNA was transferred were placed in a solution at 42 ° C. C hybridization overnight, the solution contains 50% formamide-25mM KH 2 P0 4 (pH7.4) -5 χ SSC-5 χ Denhardt's solution and 20 (^ g / ml salmon sperm DNA. After hybridization, the filter membrane is at 1 χ SSC-0.1 ° /. Wash in SDS for 30 min at 55 ° C. Then, analyze and quantify with Phosphor Imager. Example 5 In vitro expression, isolation and purification of recombinant lipoxygenase 10
根据 SEQ ID N0: 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: According to SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers was designed, the sequence is as follows:
Primer 3: 5'- CCCCATATGATGATTTTAAACAGCATTTCCATT- 3' ( Seq ID No: 5 ) Primer4: 5-CATGGATCCCTAAATGTATGGCTGACACTGTAC-3' ( Seq ID No: 6 ) 此两段引物的 5'端分别含有 Ndel和 BamHI酶切位点, 其后分别为目的基因 5'端 和 3'端的编码序列 , Ndel和 BamHI酶切位点相应于表达载体质粒 pET - 28b(+) (Novagen公司产品, Cat. No.69865.3)上的选择性内切酶位点。 以含有全长 目的基因的 pBS- 1050e06质粒为模板, 进行 PCR反应。 PCR反应条件为: 总体积 50μ1 中含 pBS- 1050e06质粒 10pg、 引物 Primer- 3和 Pr imer- 4分别为 lOpmol、 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 Ndel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 C ,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- 1050e06 )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§/ηι1 ) 的 LB液体培养基中, 宿主菌 BL21 ( PET-1050e06 ) 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,离心收集上清液, 用能与 6个组氨酸 (6H i s-Tag ) 结合的亲和层析柱 Hi s. Bind Quick Cart r idge ( Novagen公司产品) 进行层析, 得到了纯化的目的蛋白脂肪氧 化酶 10。 经 SDS- PAGE电泳, 在 l OkDa处得到一单一的条带 (图 2 ) 。 将该条带转移 至 PVDF膜上用 Edams水解法进行 N-端氨基酸序列分析,结果 N-端 15个氨基酸与 SEQ ID NO: 2所示的 N-端 15个氨基酸残基完全相同。 实施例 6 抗脂肪氧化酶 10抗体的产生 Primer 3: 5'- CCCCATATGATGATTTTAAACAGCATTTCCATT- 3 '(Seq ID No: 5) Primer4: 5-CATGGATCCCTAAATGTATGGCTGACACTGTAC-3' (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively. The coding sequences for the 5 'and 3' ends of the gene of interest are followed, respectively. The Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site. PCR was performed using the pBS-1050e06 plasmid containing the full-length target gene as a template. PCR reaction conditions were: 1 in a total volume of 50 μ plasmid pBS- 1050e06 containing 10pg, primer Primer- 3 and Pr imer- 4 are lOpmol, Advantage polymerase Mix (Clontech Products) 1 μ 1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel 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 into Escherichia coli DH5 C by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 (g / ml)), positive clones were selected by colony PCR method and sequenced. The correct positive clone (pET-1050e06) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method. Cultured in LB liquid containing kanamycin (final concentration 30 μ§ / ηι1) the group, the host bacteria BL21 (P ET-1050e06) cultured at 37 ° C to logarithmic phase, IPTG was added to a final concentration of lmmol / L, incubation was continued for 5 hours. harvested by centrifugation, broken by ultrasonic bacteria harvested by centrifugation The supernatant was chromatographed using an affinity chromatography column His s. Bind Quick Cart r idge (product of Novagen) capable of binding to 6 histidines (6H s-Tag), and the purified target protein was oxidized. Enzyme 10. After SDS-PAGE electrophoresis, a single band was obtained at 1 OkDa (Figure 2). The band was transferred to a PVDF membrane and analyzed by N-terminal amino acid sequence using Edams hydrolysis method. 15 amino acids at the N-terminus of SEQ ID NO: 2 Group is identical. 10 antibodies produced in Example 6 Anti-lipoxygenase embodiment
用多肽合成仪 (PE公司产品) 合成下述脂肪氧化酶 10特异性的多肽: A peptide synthesizer (product of PE company) was used to synthesize the following lipoxygenase 10 specific peptides:
NH2-Met-I le-Leu-Asn-Ser-I le-Ser-I le-Phe-Leu-Pro-Met-Met-Cys-Leu-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 a l . Immunochemi s try, 1969; 6: 43。 用 4mg上述 jk蓝蛋白多肷 复合物加上完全弗氏佐剂免疫家兔, 15天后再用血蓝蛋白多肽复合物加不完全弗 氏佐剂加强免疫一次。 采用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定板做 ELISA测定兔血清中抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家兔血清中分 离总 IgG。 将多肽结合于溴化氰活化的 Sepharose4B柱上, 用亲和层析法从总 IgG中 分离抗多肽抗体。 免疫沉淀法证明纯化的抗体可特异性地与脂肪氧化酶 10结合。 实施例 7 本发明的多核苷酸片段用作杂交探针的应用 NH 2 -Met-I le-Leu-Asn-Ser-I le-Ser-I le-Phe-Leu-Pro-Met-Met-Cys-Leu-COOH (SEQ ID NO: 7). The polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned jk cyanoprotein complex and complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex and incomplete Freund's adjuvant were used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method demonstrated that the purified antibody could specifically bind to lipoxygenase 10. Example 7 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 cDNA文库杂交 以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列,进一步还可 用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理 组织细胞中的表达是否异常。 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1 中挑选出合适的寡核苷 酸片段用作杂交探针, 并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核
苷酸序列或其同源的多核苷酸序列。 滤膜杂交方法包括斑点印迹法、 Southern 印 迹法、 Northern 印迹法和复印方法等, 它们都是将待测的多核苷酸样品固定在滤 膜上后使用基本相同的步骤杂交。 这些相同的步骤是: 固定了样品的滤膜首先用 不含探针的杂交缓冲液进行预杂交, 以使滤膜上样品的非特异性的结合部位被载 体和合成的多聚物所饱和。 然后预杂交液被含有标记探针的杂交缓冲液替换, 并 保温使探针与靶核酸杂交。 杂交步骤之后, 未杂交上的探针被一系列洗膜步骤除 掉。 本实施例利用较高强度的洗膜条件 (如较低盐浓度和较高的温度), 以使杂交 背景降低且只保留特异性强的信号。 本实施例选用的探针包括两类: 第一类探针 是完全与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段; 第二类探 针是部分与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实施 例选用斑点印迹法将样品固定在滤膜上, 在较高强度的的洗膜条件下, 第一类探 针与样品的杂交特异性最强而得以保留。 The purpose of this embodiment is to select suitable oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes, and to identify whether some tissues contain the multinucleus of the present invention by using a filter hybridization method. Nucleotide sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, 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 SEQ ID NO: 1 中选择寡核苷酸片段用作杂交探针, 应遵 循以下原则和需要考虑的几个方面: The selection of oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
1, 探针大小优选范围为 18-50个核苷酸; 1. The preferred range of probe size is 18-50 nucleotides;
2 , GC含量为 30%-70%, 超过则非特异性杂交增加; 2.GC content is 30% -70%, non-specific hybridization increases when it exceeds;
3 , 探针内部应无互补区域; 3, there should be no complementary regions inside the probe;
4 , 符合以上条件的可作为初选探针, 然后进一步作计算机序列分析, 包 括将该初选探针分别与其来源序列区域 (即 SEQ ID NO: 1 ) 和其它已知的基因组 序列及其互补区进行同源性比较, 若与非靶分子区域的同源性大于 85%或者有超过 15个连续碱基完全相同, 则该初选探针一般就不应该使用; 4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences 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, 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。 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.
完成以上各方面的分析后挑选并合成以下二个探针: After completing the above analysis, select and synthesize the following two probes:
探针 1 ( probel ), 属于第一类探针, 与 SEQ ID NO: 1 的基因片段完全同源 或互补 ( 41Nt ) Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt)
5 -TGATTTTAAACAGCATTTCCATTTTCCTTCCCATGATGTGT-3' ( SEQ ID NO: 8 ) 探针 1 ( probe2 ), 属于第二类探针, 相当于 SEQ ID NO: 1 的基因片段或其 互补片段的替换突变序列 (41Nt ): 5 -TGATTTTAAACAGCATTTCCATTTTCCTTCCCATGATGTGT-3 '(SEQ ID NO: 8) Probe 1 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
5'-TGATTTTAAACAGCATTTCCATGTTCCTTCCCATGATGTGT-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.Keller;M.M.Manak; Stockton Press, 19890JSA)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'-TGATTTTAAACAGCATTTCCATGTTCCTTCCCATGATGTGT-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 text Contributions: DM PROBES GHKeller; MMManak; Stockton Press, 19890JSA) and more commonly used molecular cloning experiment manuals such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambrook and others, Science Press.
样品制备: Sample Preparation:
1, 从新鲜或冰冻组织中提取 DNA 1. Extract DNA from fresh or frozen tissue
步骤: 1 ) 将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS) 的平皿中。 用剪刀或手术刀将组织切成小块。 操作中应保持组织湿润。 2) 以 lOOOg离心切碎组织 10分钟。 3)用冷匀浆缓冲液 (0.25mol/L蔗糖; 25讓 ol/L Tris-HCl, pH7.5; 25mraol/L NaCl; 25mmol/L MgCl2 ) 悬浮沉淀(大约 1 Oml/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 1000g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 lml ), 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 with cold homogenization buffer (0.25 mol / L sucrose; 25 ol / L Tris-HCl, pH 7.5; 25 mraol / L NaCl; 25 mmol / L MgCl 2 ) (about 1 Oml / g). 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 (add 1-5 ml per 0.1 g of the original tissue sample), and centrifuge at 1000 g for 10 minutes. 7) Resuspend the pellet with lysis buffer (add 1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.
2, DNA的苯酚抽提法 2, DNA phenol extraction method
步骤: 1 ) 用 1- 10ml冷 PBS洗细胞, 1000g离心 10分钟。 2 )用冷细胞裂解 液重悬浮沉淀的细胞 (l x 108细胞 /ml ) 最少应用 lOOul 裂解缓冲液。 3)加 SDS 至终浓度为 1%, 如果在重悬细胞之前将 SDS 直接加入到细胞沉淀中, 细胞可能会 形成大的团块而难以破碎, 并降低总产率。 这一点在抽提 >107细胞时特别严重。 4) 加蛋白酶 K至终浓度 200ug/ml。 5 ) 50°C保温反应 1小时或在 37°C轻轻振摇过夜。 6)用等体积苯酚: 氯仿: 异戊醇 ( 25: 24: 1)抽提, 在小离心机管中离心 10分 钟。 两相应清楚分离, 否则重新进行离心。 7) 将水相转移至新管。 8) 用等体积 氯仿: 异戊醇 (24: 1)抽提, 离心 10分钟。 9)将含 DNA的水相转移至新管。 然 后进行 DM的纯化和乙醇沉淀。 Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (lx 10 8 cells / ml) and apply a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 7 cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) 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. Purification of DM and ethanol precipitation were then performed.
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 DNA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 DM 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。
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。 Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DM 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. The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
8 )将 RNA酶 A加到 DNA溶液中, 终浓度为 100ug/ml, 37°C保温 30分钟。 9) 加入 SDS和蛋白酶 K, 终浓度分别为 0.5%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 0体积 2mol/L醋酸纳和 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 A26fl和 Α28β以检测 DM的纯度及产率。 15)分装后存放于 - 20°C。 8) Add RNase A to the DNA solution to a final concentration of 100 ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K 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, re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase, add 10 vol. 2 mol / L sodium acetate and 2.5 vol. 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) A 26fl and A 28β were measured to check the purity and yield of DM. 15) Store at -20 ° C after dispensing.
样膜的制备: Preparation of sample film:
1 )取 4 x 2 张适当大小的硝酸纤维素膜(NC膜), 用铅笔在其上轻轻标出点 样位置及样号, 每一探针需两张 NC膜, 以便在后面的实验步骤中分别用高强度条 件和强度条件洗膜 。 1) Take 4 x 2 pieces of nitrocellulose membrane (NC membrane) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are needed for each probe for later experiments. In the step, the film is washed with high-strength conditions and strength conditions, respectively.
2) 吸取及对照各 15微升, 点于样膜上, 在室温中晾干。 2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.
3 ) 置于浸润有 0. lmol/L NaOH, 1.5mol/L NaCl的滤纸上 5分钟 (两次), 晾 干置于浸润有 0.5mol/LTris- HC1 ( pH7.0 ), 3raol/LNaCl的滤纸上 5分钟(两次), 晾干。 3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), and dry in 0.5 mol / LTris-HC1 (pH 7.0), 3raol / L NaCl Leave on filter paper for 5 minutes (twice) and dry.
4) 夹于干净滤纸中, 以铝箔包好, 60-80°C真空干燥 2小时。 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.
探针的标记 Labeling of probes
1 ) 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 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) Collect the first peak before 32 P-Probes are washed out (monitorable).
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- 32P - dATP )。 8) The 32 P-Probe (the second peak is free γ- 32 P-dATP) is prepared after the collection solutions of the first peak are combined.
预杂交 Pre-hybridization
将样膜置于塑料袋中,加入 3- 10mg预杂交液( lOxDenhardfs; 6xSSC, 0. lrag/ml CT DNA (小牛胸腺 DNA) )。 封好袋口后, 68。C水浴摇 2小时。 Place the sample membrane in a plastic bag and add 3-10 mg of prehybridization solution (lOxDenhardfs; 6xSSC, 0.1 lrag / ml CT DNA (calf thymus DNA)). After closing the bag, 68. C water bath for 2 hours.
杂交
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: Cross Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake at 42 ° C in water overnight. Wash film:
高强度洗膜: High-intensity washing film:
1 )取出已杂交好的样膜。 1) Take out the hybridized sample membrane.
2 ) 2xSSC, 0. l°/oSDS中, 40°C洗 15分钟 ( 2次)。 2) 2xSSC, 0.1 ° / oSDS, wash at 40 ° C 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% SDS, 55. Wash for 30 minutes (twice) and dry at room temperature. Low-intensity washing film:
1 )取出已杂交好的样膜。 1) Take out the hybridized sample membrane.
2 ) 2xSSC, 0. 1%SDS中, 37°C洗 15分钟 ( 2次)。 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).
3 ) 0. l xSSC , 0. 1%SDS中, 37°C洗 15分钟 ( 2次)。 3) Wash in 0.1 x SSC, 0.1% SDS at 37 ° C for 15 minutes (twice).
4 ) 0. l xSSC , 0. 1 /。SDS中, 40°C洗 15分钟 ( 2次), 室温晾干。 4) 0.1 xSSC, 0.1 / 1 /. In SDS, wash 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 (compression 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。
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.