说 明 书 Explanation book
一种新的多肽一一人细胞核调控蛋白 56和编码这种多肽的多核苷酸 技 术 领 域 A New Polypeptide-Human Nuclear Regulatory Protein 56 and Polynucleotide Encoding 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一一人细胞 核调控蛋白 56, 以及编码此多肽的多核苷酸序列。 本发明还涉及此多核苷酸和多 肽的制备方法和应用。 背 景 技 术 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a human cell nuclear regulatory protein 56, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
在鼠中克隆到的 jerky基因编码一个 41.7KD的蛋白, 此蛋白称为鼠 jerky蛋 白。 jerky蛋白的部分功能已被发现, 比如, 在鼠的 jerky基因中引入插入突变, 则会造成 jerky基因的失活, 从而导致癫痫综合症; 它还与很多鼠的细胞核调控蛋 白有同源性, 因此推测, jerky蛋白还能够结合于 DNA起到调控作用。 (Nat Genet 1995 Sep ; 11 ( 1 ): 71-5 ) The jerky gene cloned in the mouse encodes a 41.7KD protein, which is called the mouse jerky protein. Some functions of the jerky protein have been discovered. For example, the insertion of a mutation in the jerky gene of a mouse will cause the inactivation of the jerky gene, which will cause epilepsy syndrome. It also has homology with many mouse nuclear regulatory proteins. Therefore, it is speculated that jerky protein can also bind to DNA to play a regulatory role. (Nat Genet 1995 Sep; 11 (1): 71-5)
根据已有的研究, 在人体内已经克隆到几种不同的与鼠的 jerky 基因同源的 基因。 包括从人胎脑 cDNA 中发现的 jerky基因, 荧光原位杂交 (FISH) 的结果表 明, 它定位于人的染色体 8q24.3 位置。 (Biochem Biophys Res Commun 1998 Jul 20; 248 ( 2 ): 307-14 ); 从人的扁桃体 cDNA中克隆到的 jerky基因, 定位于 人染色体 llq21位置, 它在人的绝大多数组织中表达, 特别是脑和骨骼肌中, 它编 码一个 51KD的蛋白, 此外, 它还与着丝粒结合蛋白 -B同源, 因此被推测可以与 DNA 结合起调控作用。 (Biochem Biophys Res 1997 Jul 18; 236 ( 2 ): 389-95 ) 经研究发现, 不同的 jerky 基因均在人的绝大多数组织中表达, 包括脑, 肝, 肺, 脾, 睾丸和卵巢等等。 此外, 在人体内, jerky 基因是剂量敏感性的, jerky 基因表达量的微小改变都会导致严重的结果。 (J Neurosci 1997 Jun 15; 17 ( 12 ): 4562-9 ) According to existing studies, several different genes homologous to the mouse jerky gene have been cloned in humans. Including the jerky gene found in the human fetal brain cDNA, the results of fluorescence in situ hybridization (FISH) indicate that it is located on the human chromosome 8q24.3. (Biochem Biophys Res Commun 1998 Jul 20; 248 (2): 307-14); The jerky gene cloned from human tonsil cDNA is located on the human chromosome llq21, and it is expressed in most human tissues, especially It is in the brain and skeletal muscle. It encodes a 51KD protein. In addition, it is homologous to centromere-binding protein-B, so it is speculated that it can bind to DNA to play a regulatory role. (Biochem Biophys Res 1997 Jul 18; 236 (2): 389-95) It has been found that different jerky genes are expressed in most human tissues, including brain, liver, lung, spleen, testis and ovary, etc. . In addition, in humans, the jerky gene is dose-sensitive, and even small changes in the expression of the jerky gene can lead to serious results. (J Neurosci 1997 Jun 15; 17 (12): 4562-9)
缺失 jerky 等位基因的的动物与野生型相比, 会变得对化学诱导的抽搐易感。 实验显示, 如果在 jerky等位基因中插入 DNA片段来阻活, 会导致鼠的癫痫性抽搐。 Animals lacking the jerky allele are more susceptible to chemically induced convulsions than wild-type animals. Experiments have shown that inserting a DNA fragment into the jerky allele to inactivate it can cause epileptic seizures in mice.
( J Neurosci 1997 Jun 15; 17 ( 12 ): 4562-9 ) (J Neurosci 1997 Jun 15; 17 (12): 4562-9)
根据遗传学的研究, 人类的癫痫性疾病是通过遗传获得的。 通过人与鼠类的 基因组比较, 可以推测 jerky基因在人体内的基本作用, 但是, 要获得对人 jerky 基因的全面了解, 还需要进一步的研究。 According to genetic studies, epilepsy in humans is obtained genetically. By comparing human and mouse genomes, the basic role of the jerky gene in the human body can be speculated, but further research is needed to gain a comprehensive understanding of the human jerky gene.
现将人的 jerky基因的功能总结为: ( 1 ) 调控神经系统的信号传递, jerky蛋
白会导致神经系统的超兴奋性和抽搐, 主要是调控人的原发性癫痫、 青少年肌阵 挛等癫痫性疾病的发生和发作。 (2 ) jerky 基因的表达对内分泌系统也有着重要的 调控功能, 在垂体、 甲状腺、 甲状旁腺、 肾上腺、 胰岛、 性腺、 松果体和胸腺等 内分泌腺中, 由于 jerky基因的表达异常, 可能会导致以上内分泌腺的分泌失常, 从而导致内分泌系统疾病。 ( 3 ) jerky 基因的表达如被完全抑制, 则会造成人个体 和性的发育失常。 (4 )jerky蛋白与着丝粒结合蛋白- B的同源性使我们推测其有 DNA 结合功能。 ( J Neurosc i 1997 Jun 15; 17 ( 12 ): 4562-9 ); ( Biochem Bi ophys Res 1997 Jul 18 ; 236 ( 2 ): 38 -95 ); ( B iochem Biophys Res Commun 1998 Jul 20; 248 ( 2 ): 307-14 ) The functions of the human jerky gene are summarized as follows: (1) Signals that regulate the nervous system, jerky eggs White can cause hyperexcitability and convulsions in the nervous system, mainly regulating the occurrence and onset of epilepsy diseases such as human primary epilepsy and adolescent myoclonus. (2) The expression of the jerky gene also has important regulatory functions on the endocrine system. In endocrine glands such as the pituitary, thyroid, parathyroid, adrenal, islet, gonad, pineal gland, and thymus, due to abnormal expression of the jerky gene, it is possible that Will cause the secretion of the above endocrine glands, resulting in endocrine system disease. (3) If the expression of the jerky gene is completely suppressed, it will cause human and sexual developmental abnormalities. (4) The homology between jerky protein and centromere-binding protein-B makes us speculate that it has a DNA-binding function. (J Neurosc i 1997 Jun 15; 17 (12): 4562-9); (Biochem Bi ophys Res 1997 Jul 18; 236 (2): 38 -95); (B iochem Biophys Res Commun 1998 Jul 20; 248 (2 ): 307-14)
根据氨基酸同源比较的结果, 本发明的多肽被推断鉴定为一种新的人细胞核 调控蛋白 56 ( HJP56 )。 发 明 的 公 开 Based on the results of amino acid homology comparison, the polypeptide of the present invention was inferred and identified as a new human nuclear regulatory protein 56 (HJP56). Public open
本发明的一个目的是提供分离的新的多肽一一人细胞核调控蛋白 56 以及其片 段、 类似物和衍生物。 It is an object of the present invention to provide isolated novel polypeptides-human nuclear regulatory protein 56 and fragments, analogs and derivatives thereof.
本发明的另一个目的是提供编码该多肽的多核苷酸。 Another object of the invention is to provide a polynucleotide encoding the polypeptide.
本发明的另一个目的是提供含有编码人细胞核调控蛋白 56 的多核苷酸的重组 载体。 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human nuclear regulatory protein 56.
本发明的另一个目的是提供含有编码人细胞核调控蛋白 56 的多核苷酸的基因 工程化宿主细胞。 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human nuclear regulatory protein 56.
本发明的另一个目的是提供生产人细胞核调控蛋白 56的方法。 Another object of the present invention is to provide a method for producing human nuclear regulatory protein 56.
本发明的另一个目的是提供针对本发明的多肽一一人细胞核调控蛋白 56 的抗 体。 Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human nuclear regulatory protein 56.
本发明的另一个目的是提供了针对本发明多肽一一人细胞核调控蛋白 56 的模 拟化合物、 拮抗剂、 激动剂、 抑制剂。 Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention-human nuclear regulatory protein 56.
本发明的另一个目的是提供诊断治疗与人细胞核调控蛋白 56 异常相关的疾病 的方法。 在本发明的第一方面, 提供新颖的分离出的人细胞核调控蛋白 56, 该多肽 是人源的, 它包含: 具有 SEQ ID NO: 2 氨基酸序列的多肽、 或其保守性变异多 肽、或其活性片段、或其活性衍生物、类似物。较佳地,该多肽是具有 SEQ ID NO: 2 氨基酸序列的多肽。 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human nuclear regulatory protein 56. In a first aspect of the present invention, a novel isolated human cell nuclear regulatory protein 56 is provided. The polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or Active fragments, or active derivatives or analogs thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
在本发明的第二方面, 提供编码分离的这些多肽的多核苷酸, 该多核苷酸
包含一核苷酸序列, 该核苷酸序列与选自下组的一种核苷酸序列有至少 70%相 同性: (a)编码上述人细胞核调控蛋白 56 的多核苷酸; ( b)与多核苷酸(a)互补 的多核苷酸。 较佳地, 该多核苷酸编码具有 SEQ ID NO: 2 所示氨基酸序列的多 肽。 更佳地, 该多核苷酸的序列是选自下组的一种: (a)具有 SEQ ID NO: 1 中 787-2322位的序列; 和(b)具有 SEQ ID NO: 1中 1-2350位的序列。 In a second aspect of the invention, a polynucleotide encoding the isolated polypeptides is provided, the polynucleotide Comprising a nucleotide sequence that is at least 70% identical to a nucleotide sequence selected from the group consisting of: (a) a polynucleotide encoding the aforementioned human nuclear regulatory protein 56; (b) a The polynucleotide (a) is a complementary polynucleotide. Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2. More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 787-2322 in SEQ ID NO: 1; and (b) a sequence having 1-2350 in SEQ ID NO: 1 Sequence of bits.
在本发明的第三方面, 提供了含有上述多核苷酸的载体, 以及被该载体转 化或转导的宿主细胞或者被上述多核苷酸直接转化或转导的宿主细胞。 In a third aspect of the present invention, there are provided a vector containing the above polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide.
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。 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。 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. 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, "分离的人细胞核调控蛋白 56" 是指人细胞核调控蛋白 56 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人细胞核调控蛋白 56。 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。 人细胞核调控蛋白 56多肽的纯度能 用氨基酸序列分析。 As used herein, "isolated human nuclear regulatory protein 56" means that human nuclear regulatory protein 56 is essentially free of other proteins, lipids, sugars, or other substances that are naturally associated with it. Those skilled in the art can purify human nuclear regulatory proteins 56 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human nuclear regulatory protein 56 peptide can be analyzed by amino acid sequence analysis.
本发明提供了一种新的多肽一一人细胞核调控蛋白 56, 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。 本发明还包括人细胞核调控蛋白 56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人细胞核调控蛋白 56 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: The present invention provides a new polypeptide, human nuclear regulatory protein 56, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian 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. The invention also includes fragments, derivatives and analogs of human nuclear regulatory protein 56. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the human nuclear regulatory protein 56 of the present invention. A fragment, derivative or analog of the polypeptide of the invention may be:
( I ) 这样一种, 其中一个或多个氨基酸残基被保守或非保守氨基酸残基 (优选的 是保守氨基酸残基) 取代, 并且取代的氨基酸可以是也可以不是由遗传密码子编 码的; 或者 (Π ) 这样一种, 其中一个或多个氨基酸残基上的某个基团被其它基团 取代包含取代基; 或者 ( I I I ) 这样一种, 其中成熟多肽与另一种化合物 (比如延 长多肽半衰期的化合物, 例如聚乙二醇) 融合; 或者 (IV ) 这样一种, 其中附加的
氨基酸序列融合进成熟多肽而形成的多肽序列 (如前导序列或分泌序列或用来纯 化此多肽的序列或蛋白原序列) 通过本文的阐述, 这样的片段、 衍生物和类似物 被认为在本领域技术人员的知识范围之内。 (I) a type in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted amino acid may or may not be encoded by a genetic codon; Or (Π) such a type in which a group on one or more amino acid residues is substituted with another group to include a substituent; or (III) such a type in which the mature polypeptide is mixed with another compound (such as elongation Polypeptide half-life compounds, such as polyethylene glycols) fusions; or (IV) one in which additional A polypeptide sequence formed by fusing an amino acid sequence into a mature polypeptide (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protein sequence). As described herein, such fragments, derivatives, and analogs are considered to be in the art. Within the knowledge of technical staff.
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 NO: 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 2350个碱基, 其开放读框( 787-2322 )编码了 511 个氨基酸。 根据氨基酸 序列同源比较发现, 此多肽与人的 jerky蛋白有 35%的同源性, 可推断出该人细胞 核调控蛋白 56具有人的 jerky蛋白相似的结构和功能。 The present invention provides an isolated nucleic acid (polynucleotide) consisting essentially of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2350 bases in length and its open reading frame (787-2322) encodes 511 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 35% homology with human jerky protein. It can be deduced that the human nuclear regulatory protein 56 has similar structure and function to human jerky 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。 DM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 NO: 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. DM can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
编码 SEQ ID N0: 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. This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
本发明还涉及与以上所描述的序列杂交的多核苷酸 (两个序列之间具有至少 50%, 优选具有 70%的相同性)。 本发明特别涉及在严格条件下与本发明所述多核苷 酸可杂交的多核苷酸。 在本发明中, "严格条件" 是指: (1)在较低离子强度和较高 温度下的杂交和洗脱, 如 0. 2xSSC, 0. 1%SDS, 60 °C ;或(2)杂交时加用变性剂, 如 50% (v/v)甲酰胺, 0. 1%小牛血清 / 0. l°/。Ficol l , 42 °C等; 或(3)仅在两条序列之间的 相同性至少在 95%以上,更好是 97%以上时才发生杂交。 并且, 可杂交的多核苷酸编 码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功能和活性。 The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) A denaturant was added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° /. Ficol l, 42 ° C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 97% or more. In addition, the hybridizable polynucleotide-encoded polypeptide 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 /或分离编码人细胞核调控蛋白 56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, "nucleic acid tablets A "segment" 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 nucleotides. The nucleic acid fragment is also Nucleic acid amplification techniques (such as PCR) can be used to identify and / or isolate polynucleotides encoding human nuclear regulatory protein 56.
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。 本发明的编码人细胞核调控蛋白 56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 human nuclear regulatory protein 56 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 DNA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 DNA 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(Q i agene)。 而构建 cDM 文库也是通常的方法(Sambrook, et a l. , Mo l ecul ar C loning, A Labora tory Manua l , Co l d Spr ing Harbor Labora tory. New- York , 1989)。 还可得到商业供应的 cDNA 文库, 如 C l ontech 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. Various methods have been developed for mRNA extraction, and kits are also commercially available (Q i agene). And the construction of cDM libraries is also a common method (Sambrook, et al., Molecula Cloning, A Labora tory Manua, Coll d Harbor Labora tory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
可用常规方法从这些 cDNA 文库中筛选本发明的基因。 这些方法包括(但不 限于): (l) DNA- DNA或 DNA- RM杂交; (2)标志基因功能的出现或丧失; (3)测定 人细胞核调控蛋白 56 的转录本的水平; (4)通过免疫学技术或测定生物学活性, 来检测基因表达的蛋白产物。 上述方法可单用, 也可多种方法联合应用。 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RM hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of transcripts of human nuclear regulatory protein 56; (4) Detection of gene-expressed protein products by immunological techniques or determination of 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 generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
在第(4)种方法中, 检测人细胞核调控蛋白 56 基因表达的蛋白产物可用免 疫学技术如 Wes tern印迹法, 放射免疫沉淀法, 酶联免疫吸附法(ELI SA)等。 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA) can be used to detect the protein products of human nuclear regulatory protein 56 gene expression.
应 用 PCR 技 术 扩 增 DM/RNA 的 方 法 (Sa i k i , et a l . Sc i ence 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 DM/RNA片段。 A method for amplifying DM / RNA by using PCR technology (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 amplification method) can be preferably used for PCR. The primers can be appropriately selected based on the polynucleotide sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
如上所述得到的本发明的基因, 或者各种 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 determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
本发明也涉及包含本发明的多核苷酸的载体, 以及用本发明的载体或直接 用人细胞核调控蛋白 56编码序列经基因工程产生的宿主细胞, 以及经重组技术 产生本发明所述多肽的方法。 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human nuclear regulatory protein 56 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
本发明中, 编码人细胞核调控蛋白 56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。 In the present invention, the polynucleotide sequence encoding the human nuclear regulatory protein 56 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 expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
本领域的技术人员熟知的方法能用于构建含编码人细胞核调控蛋白 56 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、 DNA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 DM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。
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。 Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human nuclear regulatory protein 56 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DM 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, 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 adenovirus enhancers. 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, 编码人细胞核调控蛋白 56的多核苷酸或含有该多核苷酸的重组 载体可转化或转导入宿主细胞, 以构成含有该多核苷酸或重组载体的基因工程 化宿主细胞。 术语 "宿主细胞" 指原核细胞, 如细菌细胞; 或是低等真核细胞, 如酵母细胞; 或是高等真核细胞, 如哺乳动物细胞。 代表性例子有: 大肠杆菌, 链霉菌属; 细菌细胞如鼠伤寒沙门氏菌; 真菌细胞如酵母; 植物细胞; 昆虫细 胞如果蝇 S2或 Sf 9 ; 动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。 In the present invention, a polynucleotide encoding human nuclear regulatory protein 56 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.
用本发明所述的 DNA序列或含有所述 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 CaC l 处理, 所用的歩骤 在本领域众所周知。 可供选择的是用 MgC l2。 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 CaCl. The steps used are well known in the art. The alternative is to use MgC l 2 . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
通过常规的重组 DM 技术, 利用本发明的多核苷酸序列可用来表达或生产 重组的人细胞核调控蛋白 56 (Sc ience , 1984 ; 224: 1431)。 一般来说有以下步 骤: Using conventional recombinant DM technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human nuclear regulatory protein 56 (Science, 1984; 224: 1431). Generally, the following steps are taken:
(1) .用本发明的编码人 人细胞核调控蛋白 56 的多核苷酸(或变异体), 或 用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞; (1) using the polynucleotide (or variant) encoding human human nuclear regulatory protein 56 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。 上的疾病之间的关系。 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high Performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Relationship between diseases.
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 5 0至 5 00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 2 0kb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 Figure resolution 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。 人细胞核调控蛋白 5 6 以有效地治疗和 /或预防 具体的适应症的量来给药。 施用于患者的人细胞核调控蛋白 56 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。 附 图 的 简 要 说 明 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. Human nuclear regulatory protein 5 6 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dose range of human nuclear regulatory protein 56 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. Brief explanation of attached picture
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是本发明人细胞核调控蛋白 5 6和人的 j erky蛋白的氨基酸序列同源性比 较图。 上方序列是人细胞核调控蛋白 5 6, 下方序列是人的 j erky蛋白。 相同氨基 酸在两个序列间用单字符氨基酸表示, 相似氨基酸用 "+" 表示。 Fig. 1 is a comparison diagram of the amino acid sequence homology of the human nuclear regulatory protein 56 and the human jerky protein of the present invention. The upper sequence is human nuclear regulatory protein 5 6 and the lower sequence is human j erky protein. Identical amino acids are represented by single character amino acids between the two sequences, and similar amino acids are represented by "+".
图 2 为分离的人细胞核调控蛋白 56 的聚丙烯酰胺凝胶电泳图 (SDS-PAGE )。 56kDa为蛋白质的分子量。 箭头所指为分离出的蛋白条带。
实 现 本 发 明 的 最 佳 方 式 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human nuclear regulatory protein 56 isolated. 56kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
下面结合具体实施例, 进一步阐述本发明。 应理解, 这些实施例仅用于说明本发 明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法, 通常 按照常规条件如 Sambrook等人, 分子克隆: 实验室手册(New York: Cold Spr ing Harbor Laboratory Pres s, 1989)中所述的条件, 或按照制造厂商所建议的条件。 实施例 1 : 人细胞核调控蛋白 56的克隆 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally performed according to the general conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Harbor Laboratory Pres s, 1989), or according to the conditions Conditions recommended by the manufacturer. Example 1: Cloning of human nuclear regulatory protein 56
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA Isolat ion Ki t Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Quik mRNA Isolat ion Ki t
( Qiegene 公司产品) 从总 RNA中分离 poly (A) mRM。 2ug poly (A) mRNA经逆转录 形成 cDNA。用 Smart cDNA克隆试剂盒(购自 Clontech )将 cDNA片段定向插入到 pBSK (+) 载体(Clontech公司产品)的多克隆位点上, 转化 DH5 oc, 细菌形成 cDNA文库。 用 Dye terminate cycle react ion sequencing ki t (Perkin- Elmer公司产品) 和 ABI 377自 动测序仪(Perkin- Elmer公司)测定所有克隆的 5'和 3'末端的序列。 将测定的 cDNA序 列与已有的公共 DNA序列数据库 (Genebank ) 进行比较, 结果发现其中一个克隆 0675A03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, 0675A03克隆所含的全长 cDNA为 2350bp (如 Seq ID NO: 1 所示), 从第 787bp至 2322bP有一个开放阅读框架(0RF ), 编码一个新的蛋白质(如 Seq ID N0: 2所示) 。 我们将此克隆命名为 pBS- 0675A03 , 编码的蛋白质命名为人细 胞核调控蛋白 56。 实施例 2: cDNA 克隆的同源检索 (Qiegene product) Isolate poly (A) mRM from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 oc. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0675A03 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that the 0675A03 clone contains a full-length cDNA of 2350bp (as shown in Seq ID NO: 1), and has an open reading frame (0RF) from 787bp to 2322b P , which encodes a new protein (such as Seq ID NO: 2). We named this clone pBS-0675A03, and encoded the protein as human nuclear regulatory protein 56. Example 2: Homologous search of cDNA clones
将本发明的人细胞核调控蛋白 56的序列及其编码的蛋白序列, 用 Blas t程序 (Bas iclocal Al ignment search tool) [Al tschul, SF et a l. J. Mol. Biol. 1990; 215: 403-10] , 在 Genbank、 Swi ssport等数据库进行同源检索。 与本发明的人细胞核调控蛋白 56同源性最高的基因是一种已知的人的 jerky蛋白, 其编码的蛋白在 Genbank的准入号为 AF004715。 蛋白质同源结果示于图 1, 两者高度 同源, 其相同性为 35%; 相似性为 51%。 实施例 3: 用 RT-PCR方法克隆编码人细胞核调控蛋白 56的基因 The sequence of the human nuclear regulatory protein 56 and the encoded protein sequence of the present invention were analyzed using the Blas t program (BasiclocalAlignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403 -10], perform homology search in databases such as Genbank, Swissport, etc. The gene with the highest homology to the human nuclear regulatory protein 56 of the present invention is a known human jerky protein, and the accession number encoded by the protein in Genbank is AF004715. The protein homology results are shown in Figure 1. The two are highly homologous, with an identity of 35% and a similarity of 51%. Example 3: Cloning of a gene encoding human nuclear regulatory protein 56 by RT-PCR
用胎脑细胞总 RNA为模板, 以 ol igo-dT为引物进行逆转录反应合成 cDNA,用 CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction.
Qiagene的试剂盒纯化后,用下列引物进行 PCR扩增: After purification of Qiagene's kit, PCR amplification was performed with the following primers:
Pr imerl: 5 '-ACGGTGCTTG ACAATACTATAT-3 ' (SEQ ID NO: 3) Pr imerl: 5 '-ACGGTGCTTG ACAATACTATAT-3' (SEQ ID NO: 3)
Pr imer2: 5,- GGAGAAAGCTTCTCTCGGAGTT- 3, (SEQ ID NO: 4) Pr imer2: 5,-GGAGAAAGCTTCTCTCGGAGTT- 3, (SEQ ID NO: 4)
Pr imerl为位于 SEQ ID NO: 1的 5,端的第 lbp开始的正向序列;
Pr imer2为 SEQ ID NO: 1的中的 3'端反向序列。 Pr imerl is a forward sequence located at the 5th end of bp in SEQ ID NO: 1; Pr imer2 is the 3′-end reverse sequence in SEQ ID NO: 1.
扩增反应的条件: 在 50 μ 1的反应体积中含有 50讓 oi /L KC1, 10mmol/L Tr i s- CI, (pH8. 5), 1. 5誦 ol/L MgCl2, 200 μ mol/L dNTP, l Opmol引物, 1U的 Taq DNA聚合酶 (C 1 on t ech公司产品)。 在 PE 9600型 DNA热循环仪(Per k i n-E 1 me r公司)上按下列条件 反应 25个周期: 94。C 30sec; 55°C 30sec; 72。C 2min。 在 RT- PCR时同时设 β - act in 为阳性对照和模板空白为阴性对照。 扩增产物用 QIAGEN公司的试剂盒纯化, 用 TA克 隆试剂盒连接到 PCR载体上 (Inv i trogen公司产品) 。 DNA序列分析结果表明 PCR产 物的 DNA序列与 SEQ ID NO: 1所示的 1- 2350bp完全相同。 实施例 4: Northern 印迹法分析人细胞核调控蛋白 56基因的表达: Amplification reaction conditions: 50 μi of reaction volume containing 50 oi / L KC1, 10 mmol / L Tris-CI, (pH 8. 5), 1.5 μl / L MgCl 2 , 200 μ mol in a reaction volume of 50 μ 1 / L dNTP, l Opmol primer, 1U of Taq DNA polymerase (C 1 on tech). The reaction was performed on a PE 9600 DNA thermal cycler (PerkinElmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72. C 2min. During RT-PCR, β-act in 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) using a TA cloning kit. The DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-2350bp shown in SEQ ID NO: 1. Example 4: Northern blot analysis of human nuclear regulatory protein 56 gene expression:
用一步法提取总 RNA [Ana l. Biochem 1987, 162, 156-159]„ 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 ( pt 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 MA沉淀。 将得到的 A沉淀用 70%乙醇洗涤, 干燥并溶于水中。 用 20 g RNA, 在含 20mM 3- ( N-吗啉代) 丙磺酸 (pH7. 0 ) - 5ηιΜ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 c - 32P dATP通过随机引物法制备 32P-标记的 DNA探 针。 所用的 DM探针为图 1所示的 PCR扩增的人细胞核调控蛋白 56编码区序列(787bp 至 2322bp)。 将 32P-标记的探针 (约 2 χ 106cpm/ml ) 与转移了 RNA的硝酸纤维素膜在 一溶液中于 42°C杂交过夜, 该溶液包含 50%甲酰胺 -25mM KH2P04 ( pH7. 4 ) -5 χ SSC-5 x Denhardt's溶液和 200 g/ml鲑精 DNA。 杂交之后, 将滤膜在 1 x SSC-0. 1%SDS中于 55。C洗 30min。 然后, 用 Phosphor Imager进行分析和定量。 实施例 5: 重组人细胞核调控蛋白 56的体外表达、 分离和纯化 Total RNA extraction in one step [Ana l. Biochem 1987, 162, 156-159] This method involves acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidinium isothiocyanate-25 mM sodium citrate, 0. 2M sodium acetate (pt 0) homogenize the tissue, add 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), mix and centrifuge. Aspirate the aqueous layer and add isopropyl alcohol ( 0.8 volume) and the mixture was centrifuged to obtain a MA precipitate. The obtained A precipitate was washed with 70% ethanol, dried and dissolved in water. 20 g of RNA was used in a solution containing 20 mM 3- (N-morpholino) propanesulfonate. Acid (pH7.0)-5ηιΜ sodium acetate-1mM EDTA-2. 2M formaldehyde on a 1.2% agarose gel for electrophoresis. Then transfer to a nitrocellulose membrane. Random primer method using c- 32 P dATP A 32 P-labeled DNA probe was prepared. The DM probe used was the PCR-encoded human nuclear regulatory protein 56 coding region sequence (787bp to 2322bp) shown in Figure 1. The 32P-labeled probe (about 2 x 10 6 cpm / ml) hybridized with RNA-transferred nitrocellulose membrane at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH 7. 4) -5 x SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0. 1% SDS at 55 ° C for 30 min. Then, it was performed with Phosphor Imager Analysis and quantification Example 5: In vitro expression, isolation and purification of recombinant human nuclear regulatory protein 56
根据 SEQ ID NO: 1和图 1所示的编码区序列, 设计出一对特异性扩增引物, 序 列如下: Based on SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:
Pr imer3: 5'-CCCCCATGGATGAATAAGAGAGGGAAATATACA-3' ( Seq ID No: 5 ) Pr imer3: 5'-CCCCCATGGATGAATAAGAGAGGGAAATATACA-3 '(Seq ID No: 5)
Pr imer4: 5,- CCCGGATCCAACGTTCTCTCGGAGTTCCCGTCT- 3, ( Seq ID No: 6 ) 此两段引物的 5'端分别含有 Ncol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, Ncol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- 0675A03质粒为模板, 进行 PCR反应。 PCR反应条件为: 总体积 50 μ 1中含 pBS-0675A03 质粒 10pg、 引物 Pr imer-3和 Pr imer-4分另1 J为 lOpmo l 、 Advantage polymerase Mix
(Clontech公司产品) 1 μ 1。 循环参数: 94°C 20s, 60°C 30s, 68。C 2 min,共 25个 循环。 用 Ncol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 o,在含卡那霉素Pr imer4: 5,-CCCGGATCCAACGTTCTCTCGGAGTTCCCGTCT- 3, (Seq ID No: 6) The 5 'ends of these two primers contain Ncol and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively. The Ncol and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3). The pBS-0675A03 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-0675A03 plasmid, primers Primer-3 and Primer-4, and another 1 J is lOpmo l, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ncol and BamHI were used to double digest the amplified product and plasmid P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed with colibacillus DH5o by the calcium chloride method, containing kanamycin
(终浓度 30 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-0675A03) 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 30 g/mi ) 的 LB 液体培养基中, 宿主菌 BL21 ( pET-0675A03 ) 在 37°C培养至对数生长期, 加入 IPTG 至终浓度 lmmol/L, 继续培养 5小时。 离心收集菌体, 经超声波破菌,离心收集上清, 用能与 6个组氨酸( 6 3 &§)结合的亲和层析柱1^8.8 01 Quick Cartridge( Novagen 公司产品) 进行层析, 得到了纯化的目的蛋白人细胞核调控蛋白 56。 经 SDS-PAGE电 泳, 在 56kDa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 抗人细胞核调控蛋白 56抗体的产生 After the LB plates (final concentration 30 g / ml) were cultured overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (PET-0675A03) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 g / mi), the host bacteria BL21 (pET-0675A03) was cultured at 37 ° C to the logarithmic growth phase, IPTG was added to a final concentration of 1 mmol / L, and continued Incubate for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. Chromatography was performed using an affinity column 1 ^ 8.8 01 Quick Cartridge (product of Novagen) capable of binding to 6 histidines (63 & §). The purified human nuclear regulatory protein 56 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 56 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Production of anti-human nuclear regulatory protein 56 antibody
用多肽合成仪(PE公司产品) 合成下述人细胞核调控蛋白 56特异性的多肽: NH2-Met-Asn-Lys-Arg-Gly-Lys-Tyr-Thr-Thr-Leu-Asn-Leu-Glu-Glu-Lys-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, 方法参 见: Avrameas,et al. Immunochemistry, 1969; 6: 43。 用 4mg上述血蓝蛋白多肽复合 物加上完全弗氏佐剂免疫家兔, 15天后再用血蓝蛋白多肽复合物加不完全弗氏佐剂 加强免疫一次。 采用经 15yg/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 SePhar0Se4B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人细胞核调控蛋白 56结合。 工 业 实 用 性 The following peptides specific for human nuclear regulatory protein 56 were synthesized using a peptide synthesizer (product of PE): NH 2 -Met-Asn-Lys-Arg-Gly-Lys-Tyr-Thr-Thr-Leu-Asn-Leu-Glu -Glu-Lys-COOH (SEQ ID NO: 7). The polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively. For the method, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with 15yg / ml bovine serum albumin polypeptide complex was used as an ELISA to determine the antibody titer in rabbit serum. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Se P ha r0Se 4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human nuclear regulatory protein 56. Industrial applicability
本发明的多肽在人的绝大多数组织中表达, 包括脑, 肝, 肺, 脾, 骨骼肌, 睾丸和卵巢等等。 本发明的多肽由于其剂量敏感性, 因此, 人体内的 jerky蛋白表 达水平的微小变化都可引起神经信号传导的异常、 内分泌腺的激素分泌失调, 从 而造成神经系统和内分泌系统的疾病。 The polypeptide of the present invention is expressed in most human tissues, including brain, liver, lung, spleen, skeletal muscle, testis, ovary and the like. Due to the dose sensitivity of the polypeptide of the present invention, even small changes in the expression level of jerky protein in the human body can cause abnormal neural signal transmission and hormonal imbalance of endocrine glands, thereby causing diseases of the nervous system and endocrine system.
本发明的多肽可用于诊断和治疗很多疾病, 包括神经系统、 内分泌系统和一 些免疫系统疾病。 The polypeptides of the invention are useful in the diagnosis and treatment of many diseases, including nervous system, endocrine system, and some immune system diseases.
神经系统疾病包括:
( 1 ) 神经系统退行性疾病: 癫痫症, 老年痴呆症, 帕金森氏病, 舞蹈症, 抑郁症, 健忘症, 亨延顿病, 偏头痛、 痴呆症, 多发性硬化症; Neurological diseases include: (1) Degenerative diseases of the nervous system: epilepsy, dementia, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, migraine, dementia, multiple sclerosis;
( 2 ) 精神性疾病: 精神分裂症, 抑郁症, 偏执狂, 焦虑症, 强迫症, 恐惧症, 神 经衰落; (2) mental illness: schizophrenia, depression, paranoia, anxiety, obsessive-compulsive disorder, phobia, neuronal decline;
( 3 ) 神经皮肤综合症: 神经纤维瘤病, 结节性硬化症、 脑三叉神经血管瘤病、 共济失 调毛细血管扩张症; (3) neurocutaneous syndrome: neurofibromatosis, tuberous sclerosis, trigeminal neurohemangioma, ataxia telangiectasia;
( 4 ) 周围神经疾病: 三叉神经痛、 面神经麻痹、 延髓麻痹、 坐骨神经痛、 格林-巴利 综合症。 (4) Peripheral nerve disease: trigeminal neuralgia, facial paralysis, bulbar palsy, sciatica, Guillain-Barre syndrome.
内分泌系统疾病包括: 促甲状腺激素释放激素、 生长素释放激素、 促甲状腺素、 生长 素、 肾上腺素、 去甲肾上腺素等激素的分泌失调。 Diseases of the endocrine system include: thyroid hormone-releasing hormone, ghrelin, thyrotropin, auxin, epinephrine, norepinephrine and other hormones.
免疫系统疾病包括: 免疫反应的无反应性, 或异常免疫反应, 或宿主防卫无 效。 本发明的多肽和其抗体对免疫组织的损伤、 缺陷或失调类疾病也有作用, 特 别是对于造血系统疾病 (如恶性贫血), 皮肤病 (如牛皮癣), 自身免疫病 (如类 风湿性关节炎), 放射性疾病以及免疫淋巴细胞的生成和调节有极为密切的关系。 Diseases of the immune system include: non-reactivity of the immune response, or abnormal immune response, or ineffective host defense. The polypeptides and antibodies of the present invention also have effects on damage, defects or disorders of immune tissues, especially for diseases of the hematopoietic system (such as malignant anemia), skin diseases (such as psoriasis), and autoimmune diseases (such as rheumatoid arthritis). ), Radiation diseases and the production and regulation of immune lymphocytes are extremely closely related.
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)人细胞核 调控蛋白 56 的药剂的方法。 激动剂提高人细胞核调控蛋白 56 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人细胞核调控蛋白 56 的膜制剂与标 记的人细胞核调控蛋白 56 —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 suppress (antagonist) human nuclear regulatory protein 56. Agonists enhance human nuclear regulatory proteins 56 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing human nuclear regulatory protein 56 can be cultured with labeled human nuclear regulatory protein 56 in the presence of a drug. The ability of the drug to increase or block this interaction is then measured.
人细胞核调控蛋白 56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。 人细胞核调控蛋白 56 的拮抗剂可以与人细胞核调控蛋白 56 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 human nuclear regulatory protein 56 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human nuclear regulatory protein 56 can bind to human nuclear regulatory protein 56 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that the polypeptide cannot perform biological functions.
在筛选作为拮抗剂的化合物时, 可以将人细胞核调控蛋白 56加入生物分析 测定中, 通过测定化合物对人细胞核调控蛋白 56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。 能与人细胞核调控蛋白 56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, 一般应对人细胞核调控蛋白 56分子进行标记。 In screening compounds that act as antagonists, human nuclear regulatory protein 56 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 human nuclear regulatory protein 56 and its receptor. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human nuclear regulatory protein 56 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 56 molecules of human nuclear regulatory protein 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 了针对人细胞核调控蛋白 56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 an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against the human nuclear regulatory protein 56 epitope. These antibodies include (but are not limited to): many Cloned antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
多克隆抗体的生产可用人细胞核调控蛋白 56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。 制备人细胞核调控蛋白 56 的单克隆抗体的技术包括但不限于杂交瘤 技术(Kohl er and Mi l s t e in. Nature, 1975, 256: 495-497) , 三瘤技术, 人 Β-细 胞杂交瘤技术, EBV-杂交瘤技术等。 将人恒定区和非人源的可变区结合的嵌合 抗体可用已有的技术生产(Morr i son et a l , PNAS, 1985, 81: 6851) 0 而已有的生 产单链抗体的技术(U. S. Pa t No. 4946778)也可用于生产抗人细胞核调控蛋白 56 的单链抗体。 Polyclonal antibodies can be produced by injecting human nuclear regulatory protein 56 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 human nuclear regulatory protein 56 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions to 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 Pa t No. 4946778) can also be used to produce single chain antibodies against human nuclear regulatory protein 56.
抗人细胞核调控蛋白 56的抗体可用于免疫组织化学技术中, 检测活检标本 中的人细胞核调控蛋白 56。 Antibodies against human nuclear regulatory protein 56 can be used in immunohistochemical techniques to detect human nuclear regulatory protein 56 in biopsy specimens.
与人细胞核调控蛋白 56结合的单克隆抗体也可用放射性同位素标记, 注入 体内可跟踪其位置和分布。 这种放射性标记的抗体可作为一种非创伤性诊断方 法用于肿瘤细胞的定位和判断是否有转移。 Monoclonal antibodies that bind to human nuclear regulatory protein 56 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如人细胞核调控蛋 白 56 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人细胞核 调控蛋白 56阳性的细胞。 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human nuclear regulatory protein 56 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (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 human nuclear regulatory protein 56 positive cells.
本发明中的抗体可用于治疗或预防与人细胞核调控蛋白 56相关的疾病。 给 予适当剂量的抗体可以刺激或阻断人细胞核调控蛋白 56的产生或活性。 The antibodies of the present invention can be used to treat or prevent diseases associated with human nuclear regulatory protein 56. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human nuclear regulatory protein 56.
本发明还涉及定量和定位检测人细胞核调控蛋白 56水平的诊断试验方法。 这些试验是本领域所熟知的, 且包括 FI SH测定和放射免疫测定。 试验中所检测 的人细胞核调控蛋白 56 水平, 可以用作解释人细胞核调控蛋白 56 在各种疾病 中的重要性和用于诊断人细胞核调控蛋白 56起作用的疾病。 The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human nuclear regulatory protein 56. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of human nuclear regulatory protein 56 detected in the test can be used to explain the importance of human nuclear regulatory protein 56 in various diseases and to diagnose diseases in which human nuclear regulatory protein 56 plays a role.
本发明的多肽还可用作肽谱分析, 例如, 多肽可用物理的、 化学或酶进行 特异性切割, 并进行一维或二维或三维的凝胶电泳分析,更好的是进行质谱分 析。 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
编码人细胞核调控蛋白 56的多核苷酸也可用于多种治疗目的。 基因治疗技 术可用于治疗由于人细胞核调控蛋白 56 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 变异的人细胞核调控蛋白 56 , 以抑制内源性的人细胞核调控蛋白 56 活性。 例
如, 一种变异的人细胞核调控蛋白 56可以是缩短的、 缺失了信号传导功能域的 人细胞核调控蛋白 56 , 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人细胞核调控蛋白 56 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人细胞核调控蛋白 56 的多核苷酸转移至细胞 内。 构建携带编码人细胞核调控蛋白 56 的多核苷酸的重组病毒载体的方法可见 于已有文献(Sambrook,et a l. )。 另外重组编码人细胞核调控蛋白 56 的多核苷 酸可包装到脂质体中转移至细胞内。 The polynucleotide encoding human nuclear regulatory protein 56 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 human nuclear regulatory protein 56. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human nuclear regulatory protein 56 to inhibit endogenous human nuclear regulatory protein 56 activity. example For example, a mutated human nuclear regulatory protein 56 may be a shortened human nuclear regulatory protein 56 that lacks a signal transduction domain. Although it can bind to downstream substrates, it lacks signal transduction activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human nuclear regulatory protein 56. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human nuclear regulatory protein 56 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding human nuclear regulatory protein 56 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human nuclear regulatory protein 56 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.
抑制人细胞核调控蛋白 56 mRNA 的寡核苷酸(包括反义 RNA 和 DM)以及核 酶也在本发明的范围之内。 核酶是一种能特异性分解特定 RNA的酶样 RNA分子, 其作用机制是核酶分子与互补的靶 RNA 特异性杂交后进行核酸内切作用。 反义 的 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 RNA分子可通过编码该 RNA 的 DM序列在体外或体内转录获得。 这种 DM序列已整合到载体的 RM聚合酶 启动子的下游。 为了增加核酸分子的稳定性, 可用多种方法对其进行修饰, 如 增加两侧的序列长度, 核糖核苷之间的连接应用磷酸硫酯键或肽键而非磷酸二 酯键。 Oligonucleotides (including antisense RNA and DM) and ribozymes that inhibit human nuclear regulatory protein 56 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This DM sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
编码人细胞核调控蛋白 56 的多核苷酸可用于与人细胞核调控蛋白 56 的相 关疾病的诊断。 编码人细胞核调控蛋白 56 的多核苷酸可用于检测人细胞核调控 蛋白 56 的表达与否或在疾病状态下人细胞核调控蛋白 56 的异常表达。 如编码 人细胞核调控蛋白 56的 DNA序列可用于对活检标本进行杂交以判断人细胞核调 控蛋白 56 的表达状况。 杂交技术包括 Southern 印迹法, Nor thern 印迹法、 原 位杂交等。 这些技术方法都是公开的成熟技术, 相关的试剂盒都可从商业途径 得到。本发明的多核苷酸的一部分或全部可作为探针固定在微阵列(Mi croarray) 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。 用人细胞核调控蛋白 56特异的引物进行 RNA-聚合酶链反应(RT-PCR) 体外扩增也可检测人细胞核调控蛋白 56的转录产物。 The polynucleotide encoding human nuclear regulatory protein 56 can be used for the diagnosis of diseases related to human nuclear regulatory protein 56. The polynucleotide encoding human nuclear regulatory protein 56 can be used to detect the expression of human nuclear regulatory protein 56 or the abnormal expression of human nuclear regulatory protein 56 in a disease state. For example, the DNA sequence encoding human nuclear regulatory protein 56 can be used to hybridize biopsy specimens to determine the expression of human nuclear regulatory protein 56. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. A part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues. Human nuclear regulatory protein 56 specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect human nuclear regulatory protein 56 transcripts.
检测人细胞核调控蛋白 56 基因的突变也可用于诊断人细胞核调控蛋白 56 相关的疾病。 人细胞核调控蛋白 56突变的形式包括与正常野生型人细胞核调控 蛋白 56 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。 另夕卜, 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 human nuclear regulatory protein 56 gene can also be used to diagnose human nuclear regulatory protein 56-related diseases. Human nuclear regulatory protein 56 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human nuclear regulatory protein 56 DNA sequence. Available already Some techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization detect mutations. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具体位置且并可以与其杂交。 目前, 需要鉴定染色体上的各基因的具体 位点。 现在, 只有很少的基于实际序列数据(重复多态性)的染色体标记物可用 于标记染色体位置。 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. The sequence specifically targets a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
简而言之, 根据 cDNA制备 PCR引物(优选 15-35bp), 可以将序列定位于染色 体上。 然后, 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。 只 有那些含有相应于引物的人基因的杂合细胞会产生扩增的片段。 In short, PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the 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, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
将 cMA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 cMA 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。 这些数据可见于例如, V.Mckusick, Mendel ian Inheritance in Man (可通过与 Johns Hopkins Univers ity 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
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, for example, in V. Mckusick, Mendelian Inheritance in Man (available online with the Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine if genes and genes have been mapped to chromosomal regions
(1)一般信息: (1) General information:
(ii)发明名称: 人细胞核调控蛋白 56及其编码序列(ii) Title of invention: Human nuclear regulatory protein 56 and its coding sequence
(iii)序列数目: 7 (iii) Number of sequences: 7
(2)SEQ ID NO: 1的信息: (2) Information of SEQ ID NO: 1:
(i)序列特征: (i) Sequence characteristics:
(A)长度: 2350bp (A) Length: 2350bp
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 双链 (C) Chain: double strand
(D)拓朴结构: 线性 (D) Topological structure: linear
( )分子类型: cDNA () Molecular type: cDNA
(xi)序列描述: SEQ ID NO: 1:
(xi) Sequence description: SEQ ID NO: 1:
1081 AGAGGCGTGGAGCTTCAGGCTGCTGCAGAGAGATTTGCACGGTGTTTTGGGCGAACAGAT 1081 AGAGGCGTGGAGCTTCAGGCTGCTGCAGAGAGATTTGCACGGTGTTTTGGGCGAACAGAT
20 1 GATTATAGAGAAATTCTTGAAAAATGTGGGGAGTTGGAAACTAAGTTGGATGATGATAGG 20 1 GATTATAGAGAAATTCTTGAAAAATGTGGGGAGTTGGAAACTAAGTTGGATGATGATAGG
2341 AGCTTTCTCC 2341 AGCTTTCTCC
(3) SEQ ID NO: 2的信息: (3) Information of SEQ ID NO: 2:
(i)序列特征: (i) Sequence characteristics:
(A)长度: 511个氨基酸 (A) Length: 511 amino acids
(B)类型: 氨基酸 (B) Type: Amino acid
(D)拓扑结构: 线性 (D) Topological structure: linear
(Π)分子类型: 多肽 (Π) Molecular type: Polypeptide
(xi)序列描述: SEQ ID NO: 2: (xi) Sequence description: SEQ ID NO: 2:
1 Met Asn Lys Arg Gly Lys Tyr Thr Thr Leu Asn Leu Glu Glu Lys 16 Met Lys Val Leu Ser Arg lie Glu Ala Gly Arg Ser Leu Lys Ser 31 Val Met Asp Glu Phe Gly lie Ser Lys Ser Thr Phe Tyr Asp lie
dsy dsy 3¾ "^丄 9Md "ID 96171 Met Asn Lys Arg Gly Lys Tyr Thr Thr Leu Asn Leu Glu Glu Lys 16 Met Lys Val Leu Ser Arg lie Glu Ala Gly Arg Ser Leu Lys Ser 31 Val Met Asp Glu Phe Gly lie Ser Lys Ser Thr Phe Tyr Asp lie dsy dsy 3¾ "^ 丄 9Md" ID 9617
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311 1。W noq sAq uiO Old "ID ΙΒΛ usy "TO ISI 311 1. W noq sAq uiO Old "ID ΙΒΛ usy" TO ISI
•I3S 1¾Λ ngq Ι^Λ "ID "TO ^19 ^10 §iy usy ΐϋ 9il• I3S 1¾Λ ngq Ι ^ Λ "ID" TO ^ 19 ^ 10 §iy usy ΐϋ 9il
911 siH usy §iy SJV ^丄 ^10 ^丄 BIV III 911 siH usy §iy SJV ^ 丄 ^ 10 ^ 丄 BIV III
dsy 丄 8jy ^IO S D n\ gjy nIO ¾IV 901dsy 丄 8jy ^ IO SD n \ gjy n IO ¾IV 901
¾IV uiO ngq "TO ^ΐθ ¾1V gjy sAq 16¾IV uiO ngq "TO ^ ΐθ ¾1V gjy sAq 16
"10 "10 丄 丄 i dsy dsy dsy 丄 9L"10" 10 10 丄 i dsy dsy dsy 丄 9L
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OStOO/OOMD/I3d 81 08€8£/10 OAV
(4) SEQ ID NO: 3的信息 OStOO / OOMD / I3d 81 08 € 8 £ / 10 OAV (4) Information of SEQ ID NO: 3
(i)序列特征 (i) Sequence characteristics
(A)长度: 22碱基 (A) Length: 22 bases
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(D)拓扑结构: 线性 (D) Topological structure: linear
(Π)分子类型: 寡核苷酸 (Π) Molecular type: Oligonucleotide
(xi)序列描述: SEQ ID NO: 3: (xi) Sequence description: SEQ ID NO: 3:
5,- ACGGTGCTTGACAATACTATAT- 3, 5,-ACGGTGCTTGACAATACTATAT- 3,
(5) SEQ ID NO: 4的信息 (5) Information of SEQ ID NO: 4
(i)序列特征 (i) Sequence characteristics
(A)长度: 22碱基 (A) Length: 22 bases
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(D)拓扑结构: 线性 (D) Topological structure: linear
(i i)分子类型: 寡核苷酸 (i i) Molecular type: Oligonucleotide
(x i)序列描述: SEQ ID NO: 4: (x i) Sequence description: SEQ ID NO: 4:
5'- GGAGAAAGCTTCTCTCGGAGTT- 3, 5'- GGAGAAAGCTTCTCTCGGAGTT- 3,
(6) SEQ ID NO: 5的信息 (6) Information of SEQ ID NO: 5
(i)序列特征 (i) Sequence characteristics
(A)长度: 32碱基 (A) Length: 32 bases
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(D)拓扑结构: 线性 (D) Topological structure: linear
(Π)分子类型: 寡核苷酸 (Π) Molecular type: Oligonucleotide
(xi)序列描述: SEQ ID NO : 5: (xi) Sequence description: SEQ ID NO: 5:
5,- CCCCCATGGATGAATAAGAGAGGGAAATATACA- 5,-CCCCCATGGATGAATAAGAGAGGGAAATATACA-
(7) SEQ ID NO: 6的信息 (7) Information of SEQ ID NO: 6
(i)序列特征 (i) Sequence characteristics
(A)长度: 33碱基
(B)类型: 核酸 (A) Length: 33 bases (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(D)拓扑结构: 线性 (D) Topological structure: linear
(i i)分子类型: 寡核苷酸 (i i) Molecular type: Oligonucleotide
(xi)序列描述: SEQ ID NO : 6: (xi) Sequence description: SEQ ID NO: 6:
5'-CCCGGATCCAACGTTCTCTCGGAGTTCCCGTCT-3' 5'-CCCGGATCCAACGTTCTCTCGGAGTTCCCGTCT-3 '
(8) SEQ ID NO: 7的信息: (8) Information of SEQ ID NO: 7:
(i)序列特征: (i) Sequence characteristics:
(A)长度: 15个氨基酸 (A) Length: 15 amino acids
(B)类型: 氨基酸 (B) Type: Amino acid
(D)拓扑结构: 线性 (D) Topological structure: linear
(i i)分子类型: 多肽 (i i) molecular type: peptide
(xi)序列描述: SEQ ID NO: 7: (xi) Sequence description: SEQ ID NO: 7:
Met-Asn-Lys-Arg-Gly-Lys-Tyr-Thr-Thr-Leu-Asn-Leu-Glu-Glu-Lys
Met-Asn-Lys-Arg-Gly-Lys-Tyr-Thr-Thr-Leu-Asn-Leu-Glu-Glu-Lys